[online

Hazard statements (CLP)

H301, H302, H311, H312, H314, H317, H319, H331, H332, H360D, H373, H411, H412

Code

Statements

H301

Toxic if swallowed

Hazard statements

of the Globally Harmonized System of Classification and

2.2 Label elements

of Chemicals of the United Nations, 3-chloro-2-methylpropene is harmful if swallowed (H302) and if inhaled (H332), causes severe skin burns and eye damage (H314), may cause an allergic skin reaction (H317) and is toxic to aquatic life with long-lasting effects (H411) (ECHA, 2015). The United States National Toxicology Program (NTP) states that 3-chloro-2-methyl- propene “is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals” (NTP, 2014). The German Commission for the Investigation of Health

SECTION 2: Hazards identification

of Chemical Compounds in the Work Area (MAK) has designated 3-chloro-2-methylpropene as a substance for which there is some evidence of carcinogenicity (Category 3B) (MAK, 2015). 2. Cancer in Humans No data were available to the Working Group. 3. Cancer in Experimental Animals 3-Chloro-2-methylpropene was evaluated previously by an IARC Monographs Working Group (LARC, 1995) which concluded that there was limited evidence in experimental animals for its carcinogenicity. See Table 3.1 3.1 Mouse 3.1.1. Oral administration Groups of 50 male and 50 female B6C3F, mice (age, 8 weeks) were given technical-grade 3-chloro-2-methylpropene (two different lots; purity, 93% and > 95%, respectively, containing 5% and 3.6% dimethylvinylchloride [1-chloro-2- methylpropene, an IARC Group 2B carcinogen with sufficient evidence in experimental animals (LARC, 1995)], respectively) in corn oil by gavage at doses of 0, 100, or 200 mg/kg body weight (bw) on 5 days per week for 103 weeks (Chan et al., 1986; NTP, 1986). Mean body weights of the males given 200 mg/kg bw and of all treated females were significantly lower (by less than 10%) than those of vehicle controls throughout most of the study. Survival in the treated groups was not significantly lower than that in vehicle controls; the numbers of survivors at the end of the experi- ment (105 weeks) were: 26 male controls, 37 males at 100 mg/kg bw and 32 males at 200 mg/kg bw; and 37 female controls, 43 females at 100 mg/kg bw and 27 females at 200 mg/kg bw. In males, the incidence of forestomach squa- mous cell papilloma was: 3/49 controls, 19/49 at 100 mg/kg bw (P < 0.001 by the Fisher’s exact test); and 30/49 at 200 mg/kg bw (P < 0.001 by the Fisher’s exact test) (dose-related increase at P < 0.001 by the Cochran-Armitage trend test); that of squamous cell carcinoma was: 0/49 controls, 5/49 at 100 mg/kg bw (P = 0.028 by the Fisher’s exact test) and 7/49 at 200 mg/kg bw (P = 0.006 by the Fisher’s exact test) (dose-related increase at P = 0.008 by the Cochran-Armitage trend test); and that of forestomach squamous cell papilloma or carcinoma (combined) was: 3/49 controls, 24/49 at 100 mg/kg bw (P < 0.001 by the Fisher’s exact test) and 36/49 at 200 mg/kg bw (P < 0.001 by the Fisher’s exact test) (dose-related increase at P < 0.001 by the Cochran-Armitage trend test). The historical incidence of fores- tomach squamous cell papilloma or carcinoma (combined) in male mice at the study laboratory 105 901 Table 3.1 Studies of carcinogenicity with 3-chloro-2-methylpropene in experimental animals VI DY Species, Route Incidence of tumours animals Significance Comments SHdVYDONOW strain (sex) Purity Ageatstart Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving Mouse, Gavage Forestomach Principal strengths: GLP, large - B6C3F, (M) Purity, 2 93% Squamous cell papilloma: Trend test: P < 0.001 (Cochran-Armitage test) | number of animals per group, SLL 8 wk Corn oil 3/49, 19/49, 30/49 Incidental tumour test: P < 0.001 at 100 and multiple doses tested, and covered 105 wk 0, 100, 200 mg/kg bw 200 mg/kg bw (Fisher’s exact test) most of the lifespan NTP (1986) 5 days/wk for 103 wk Squamous cell carcinoma: Trend test: P = 0.008 (Cochran-Armitage test) 1-Chloro-2-methylpropene, 3.6-5% cemeue 0/49, 5/49, 7/49 Incidental tumour test: P = 0.028 and P = 0.006 26, 37, 32 at 100 and 200 mg/kg bw, respectively (Fisher’s exact test) Squamous cell papilloma or Trend test: P < 0.001 (Cochran-Armitage test) carcinoma (combined): Incidental tumour test: P < 0.001 at 100 and 3/49, 24/49, 36/49 200 mg/kg bw (Fisher’s exact test) Mouse, Gavage Forestomach Principal strengths: GLP, large B6C3F, (F) Purity, 2 93% Squamous cell papilloma: Trend test: P < 0.001 (Cochran-Armitage test) | number of animals per group, 8 wk Corn oil 0/50, 15/48, 29/44 Incidental tumour test: P < 0.001 at 100 and multiple doses tested, and covered 105 wk 0, 100, 200 mg/kg bw 200 mg/kg bw (Fisher’s exact test) most of the lifespan NTP (1986) 5 days/wk for 103 wk 1-Chloro-2-methylpropene, 3.6-5% Squamous cell carcinoma: NS 50/group Incidence of forestomach squamous 0/50, 1/48, 2/44 37, 43, 27 cell papilloma or carcinoma Squamous cell papilloma or Trend test: P < 0.001 (Cochran-Armitage test) (combined) in historical controls: carcinoma (combined): Incidental tumour test: P < 0.001 at 100 and study laboratory, 0/145; NTP 0/50, 16/48, 31/44 200 mg/kg bw (Fisher’s exact test) studies, 4/1027 Table 3.1 (continued) Species, Route Incidence of tumours Significance Comments strain (sex) Purity Ageatstart Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Mouse, BDF, Whole-body inhalation Forestomach Principal strengths of the study: (M) Purity, 95% Squamous cell papilloma: Trend test: P = 0.0392 (Peto’s test) GLP, large number of animals per 6 wk vapour 1/50, 0/49, 3/50, 4/50 group, multiple doses tested, and 104 wk 0, 50, 100, 200 ppm Squamous cell carcinoma: NS covered most of the lifespan Katagirietal. 6 h/day, 5 days/wk 0/50, 0/49, 1/50, 0/50 1-Chloro-2-methylpropene, 1.4% (2000) 50/grou Incidence of Harderian gland 10. 44, a 46 Cr ae adenoma in historical controls: Adenoma: NS 30/598 (5.0%), 2.0-10.0% (Ohnishi 3/50, 7/49, 9/50, 5/50 et al., 2013) Mouse, BDF, Whole-body inhalation Forestomach Principal strengths of the study: (F) Purity, 95% Squamous cell papilloma: Trend test: P = 0.0437 (Peto’s test) GLP, large number of mice per 6 wk vapour 1/50, 0/48, 5/50, 4/49 group, multiple doses tested, and 104 wk 0, 50, 100, 200 ppm Squamous cell carcinoma: NS covered most of the lifespan Katagiri et al. 6 h/day, 5 days/wk 1-Chloro-2-methylpropene, 1.4% 0/50, 0/48, 0/50, 0/49 (2000) 50/group Incidence of Harderian gland Harderian gland 29, 29, 31, 33 adenoma in historical controls: Adenoma: Trend test: P = 0.0056 (Peto’s test) and 29/849 (3.4%), 0-12.0% 0/50, 4/48, 7/50, 8/49 P= 0.0061 (Cochran-Armitage test) Incidence: P = 0.0101 and P = 0.0051 at 100 and 200 ppm, respectively (Fisher’s exact test) ZOL auadoudyjAyj}aw-7-OJ0]YD-€ 801 VI Table 3.1 (continued) DY Species, Route Incidence of tumours Significance Comments strain (sex) Purity Ageatstart Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals SHdVYDONOW Rat, F344/N — Gavage Forestomach 1-Chloro-2-methylpropene, 3.6-5% - (M) Purity, 2 93% Squamous cell papilloma: Trend test: P < 0.001 (Cochran-Armitage test) Incidence of renal tubular cell SLL 8 wk Corn oil 1/50, 5/50, 30/48 Incidental tumour test: P < 0.001 at 150 mg/kg | tumours, renal transitional cell 105 wk 0, 75, 150 mg/kg bw bw (Fisher’s exact test) tumours and urinary bladder NTP (1986) 5 days/wk for 103 wk Squamous cell carcinoma: NS tumours in historical corn oil- 50/group 0/50, 0/50, 2/48 vehicle controls at the study 30, 25, 17 Kid laboratory: 1/150, 0/150 and 0/150, idney respectively Renal tubular cell adenoma: NS Incidence of testicular (interstitial 0/50, 1/50, 0/49 cell) tumours in historical controls: Renal tubular cell NS 92.0 + 6.9%, study laboratory; adenocarcinoma: 90.4 + 5.7%, all NTP studies 0/50, 1/50, 1/49 Renal transitional cell NS carcinoma: 0/50, 0/50, 1/49 Urinary bladder Transitional cell papilloma: NS 0/48, 0/49, 1/46 Testis Interstitial cell tumours: Trend test: P < 0.001 (life-table test), P = 0.003 36/50, 43/50, 43/48 (incidental tumour test) and P = 0.015 (Cochran-Armitage test) Incidence: P = 0.009 (life-table test at 75 mg/kg bw), P < 0.001 (life-table test at 150 mg/kg bw), P = 0.012 (incidental tumour test at 150 mg/kg bw) and P = 0.025 (Fisher’s exact test at 150 mg/kg bw) Table 3.1 (continued) Species, Route Incidence of tumours Significance Comments strain (sex) Purity Ageatstart Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Rat, F344/N — Gavage Forestomach Principal strengths of the study: (F) Purity, 2 93% Squamous cell papilloma: Trend test: P < 0.001 (Cochran-Armitage test) GLP, large number of rats per group, 8 wk Corn oil 1/50, 1/50, 10/50 Incidental tumour test: P = 0.004 at 150 mg/kg multiple doses tested, and covered 105 wk 0, 75, 150 mg/kg bw bw (Fisher’s exact test) most of the lifespan NTP (1986) 5 days/wk for 103 wk Subcutis 1-Chloro-2-methylpropene, 3.6-5% 50/group Fibroma: NS Incidence of subcutaneous fibroma 31, 32, 26 0/50 2/50 4/50 in historical controls at study , laboratory: 2/150 Rat, F344/ Whole-body inhalation Thyroid gland Principal strengths of the study: DuCrj (M) Purity, 95% Follicular cell adenoma: Trend test: P = 0.00213 (Cochran-Armitage GLP, large number of animals per 6 wk Vapour 2/50, 0/50, 2/50, 6/50 test) group, multiple doses tested, and 104 wk 0, 50, 100, 200 ppm Follicular cell adenoma or Trend test: P = 0.0388 (Cochran-Armitage test) covered most of the lifespan MHLW. 6 h/day, 5 days/wk adenocarcinoma (combined): 1-Chloro-2-methylpropene, 1.4% {1998b) 50, 50, 50, 50 4/50, 4/50, 3/50, 10/50 39, 35, 33, 30 Rat, F344/ Whole-body inhalation Any tumour type Principal strengths of the study: DuCrj (F) Purity, 95% No significant increase NS GLP, large number of animals per 6 wk Vapour group, multiple doses tested, and 104 wk 0, 50, 100, 200 ppm covered most of the lifespan MHLW 6 h/day, 5 days/wk 1-Chloro-2-methylpropene, 1.4% (1998b) 50, 50, 50, 50 38, 40, 45, 44 bw, body weight; F, female; GLP, good laboratory practice; M, male; NS, not significant; wk, week 601 auadoudyjAyj}aw-7-OJ0]YD-€ IARC MONOGRAPHS - 115 and that in NTP studies was 2/147 and 7/1005, per week for 104 weeks (MHLW, 1998a; Katagiri respectively. Evidence of metastasis or invasion etal., 2000). The control mice were handled in the of other organs was observed in two males at same manner and exposed to clean air in similar 100 mg/kg bw and three males at 200 mg/kg bw. chambers. A significant decrease in body weight The incidence of forestomach epithelial hyper- was observed in all exposed groups. Survival in plasia was significantly increased in all treated the treated groups was slightly higher than that males. in the controls; the numbers of survivors at the In females, the incidence of forestomach end of the experiment were: 40/50 male controls, squamous cell papilloma was: 0/50 controls, 44/50 males at 50 ppm, 43/50 males at 100 ppm, 15/48 at 100 mg/kg bw (P < 0.001 by the Fisher’s and 46/50 males at 200 ppm; and 29/50 female exact test) and 29/44 at 200 mg/kg bw (P < 0.001 controls, 29/48 females at 50 ppm, 31/50 females by the Fisher’s exact test) (dose-related increase at at 100 ppm, and 33/49 females at 200 ppm. P < 0.001 by the Cochran-Armitage trend test); Histopathological evaluation revealed a that of forestomach squamous cell carcinoma significant positive trend in the incidence of was: 0/50 controls, 1/48 at 100 mg/kg bw and 2/44 forestomach squamous cell papilloma in males at 200 mg/kg bw; and that of forestomach squa- and females (P = 0.0392 and P = 0.0437 by Peto’s mous cell papilloma or carcinoma (combined) trend test, respectively). The incidence of squa- was: 0/50 controls, 16/48 at 100 mg/kg bw mous cell papilloma was 1/50 controls, 0/49 at (P < 0.001 by the Fisher’s exact test) and 31/44 50 ppm, 3/50 at 100 ppm, and 4/50 at 200 ppm at 200 mg/kg bw (P < 0.001 by the Fisher’s exact in males, and 1/50 controls, 0/48 at 50 ppm, 5/50 test) (dose-related increase at P < 0.001 by the at 100 ppm, and 4/49 at 200 ppm in females. A Cochran-Armitage trend test). The historical forestomach squamous cell carcinoma was also incidence of forestomach squamous cell papil- observed in one male mouse at 100 ppm. The inci- loma or carcinoma (combined) in female mice at dence of forestomach epithelial hyperplasia was the study laboratory andin NTP studies was 0/145 also significantly increased (P < 0.01) in males and 4/1027, respectively. Evidence of metastasis and females at 200 ppm. In addition, a signifi- or invasion of other organs was observed in one cant positive trend was found in the incidence female mouse at 200 mg/kg bw. The incidence of Harderian gland adenoma in female mice of forestomach epithelial hyperplasia was signif- (P = 0.0056 by Peto’s trend test, P = 0.0061 by the icantly increased in females at 200 mg/kg bw. Cochran-Armitage trend test) with an incidence [The strengths of this study included compli- of 0/50 controls, 4/48 at 50 ppm, 7/50 at 100 ppm ance with good laboratory practice, large (P = 0.0101 by the Fisher’s exact test) and 8/49 at numbers of animals, the evaluation of multiple 200 ppm (P= 0.0051 by the Fisher’s exact test). The dose levels and a duration that involved most of incidence in the groups exposed to 100 and 200 the lifespan.] ppm was higher than the maximum incidence (29/849, 3.4%; 0-12.0%) in historical controls at 3.1.2 Inhalation the institute performing this experiment using Groups of 50 male and 50 female BDF, female BDF, mice. The incidence of Harderian (C57BL/6NCrj x DBA/2NCrj) mice (age, gland adenoma in males was: 3/50 controls, 7/49 6 weeks) were exposed to technical-grade at 50 ppm, 9/50 at 100 ppm, and 5/50 at 200 ppm 3-chloro-2-methylpropene (purity, 95%, (historical control incidence in male BDF, mice: containing 1.4% 1-chloro-2-methylpropene) by 30/598 (5.0%); range, 2.0-10.0%; Ohnishi et al. whole-body inhalation at a concentration of 50, 2013). 100 or 200 ppm for 6 hours per day on 5 days 110 [The strengths of this study included compli- ance with good laboratory practice, large numbers of animals, the evaluation of multiple dose levels and a duration that involved most of the lifespan] 3.2 Rat 3.2.1 Oral administration Groups of 50 male and 50 female Fischer 344/N rats (age, 8 weeks) were given tech- nical-grade 3-chloro-2-methylpropene (two different lots; purity, 93% and > 95%, respectively, containing 5% and 3.6% dimethylvinylchloride [1-chloro-2-methylpropene, an IARC group 2B carcinogen with sufficient evidence in experi- mental animals (IARC, 1995)], respectively) in corn oil by gavage at doses of 0, 75, or 150 mg/kg bw on 5 days per week for 103 weeks (Chan et al., 1986; NTP, 1986). The mean body weight of the males given 150 mg/kg bw was significantly lower than that of the vehicle controls, beginning at week 10 of the study. The mean body weights of males given 75 mg/kg bw and of all treated females were comparable with those of the vehicle controls throughout the study. Survival was marginally reduced among males receiving 150 mg/kg bw; the numbers of survivors at the end of the experiment (105 weeks) were: 30 male controls, 25 males at 75 mg/kg bw and 17 males at 150 mg/kg bw; and 31 female controls, 32 females at 75 mg/kg bw, and 26 females at 150 mg/kg bw. A dose-related increase in the incidence of squamous cell papilloma of the forestomach was observed in both males and females (P < 0.001 by the Cochran-Armitage trend test): 1/50 male controls, 5/50 males at 75 mg/kg bw, 30/48 males at 150 mg/kg bw (P < 0.001 by the Fisher’s exact test), 1/50 female controls, 1/50 females at 75 mg/kg bw, and 10/50 females at 150 mg/kg bw (P= 0.004 by the Fisher’s exact test). Forestomach squamous cell carcinomas were observed in 2 of the male rats bearing forestomach squamous cell papillomas at 150 mg/kg bw but not in controls or animals given 75 mg/kg bw. Consequently, the incidence was significantly increased (with a significant positive trend) for papilloma and carcinoma (combined) in males at 150 mg/kg bw. The historical incidence of forestomach squa- mous cell papilloma at the study laboratory and in NTP studies was 0/147 and 5/1062 in male rats, and 1/150 and 5/1073 in female rats, respectively. The incidence of basal cell or epithelial hyper- plasia (combined) of the forestomach in groups of treated males and that of basal cell hyperplasia in groups of treated females was also increased. In addition to forestomach tumours in males, a significant increase and positive trend in the incidence (P < 0.001 by the life-table trend test, P = 0003 by the incidental tumour trend test, P=0.015 by the Cochran-Armitage trend test) of testicular interstitial cell tumours was observed: 36/50 controls, 43/50 at 75 mg/kg bw (P = 0.009 by the life-table test), and 43/48 at 150 mg/kg bw (P < 0.001 by the life-table test, P = 0.012 by the incidental tumour test, P = 0.025 by the Fisher’s exact test), which was within the historical control range. The historical incidence for this tumour at the study laboratory was 92.0% + 6.9% (138/150), and that in all NTP studies was 90.4% + 5.7% (985/1090). [The Working Group concluded that it was uncertain that these testicular tumours were related to treatment.] A non-significant increase in tumours was observed in the kidney and urinary bladder of male rats and in the subcutaneous tissue of female rats. One renaltubularcelladenoma was observed in one male given 75 mg/kg bw and renal tubular cell adenocarcinomas were observed in another male given 75 mg/kg bw and in one male given 150 mg/kg bw. At 150 mg/kg bw, a renal transi- tional cell carcinoma was observed in one male and a transitional cell papilloma was observed in the urinary bladder of another. No renal tubule tumours, renal transitional cell tumours or urinary bladder tumours were observed in male or female controls. The historical incidence 111 IARC MONOGRAPHS - 115 of renal tubular cell tumours and renal transi- controls, 35 at 50 ppm, 33 at 100 ppm and 30 at tional cell tumours in corn oil vehicle-control 200 ppm. Survival in the treated females tended male rats at the study laboratory was 1/150 and to be higher than that in controls; the numbers 0/150, respectively, and that in NTP studies was of female survivors at the end of the experiment 4/1091 and 1/1091 (1 transitional cell papilloma), were: 38 controls, 40 at 50 ppm, 45 at 100 ppm respectively; that of urinary bladder tumours and 44 at 200 ppm. Histopathological evaluation in male rats treated with corn oil vehicle was revealed a slightly increased incidence of thyroid 0/150 at the study laboratory and 0/1040 in NTP follicular cell adenoma and of thyroid follicular studies. [The Working Group concluded that cell adenoma or adenocarcinoma (combined) in it was uncertain that these kidney and urinary treated males. The incidence of thyroid follicular bladder tumours were related to treatment.| cell adenoma in males was 2/50 controls, 0/50 at Subcutaneous fibromas were observed in 2/50 50 ppm, 2/50 at 100 ppm, and 6/50 at 200 ppm females at 75 mg/kg bw and in 4/50 females at (significant positive trend at P = 0.00213 by the 150 mg/kg bw. No such subcutaneous tumour Cochran-Armitage trend test). The incidence was seen in control female rats. The historical of thyroid follicular cell adenoma or adenocar- incidence of subcutaneous fibroma in control cinoma (combined) in males was 4/50 controls, female rats at the study laboratory and in NTP 4/50 at 50 ppm, 3/50 at 100 ppm and 10/50 at 200 studies was 2/150 and 13/1095, respectively [the ppm (significant positive trend at P = 0.0388 by Working Group concluded that these subcuta- the Cochran-Armitage trend test). The histor- neous tumours may have been related to treat- ical control incidence of thyroid follicular cell ment] (NIP, 1986), adenoma in male Fischer 344 rats at the study [The strengths of this study included compli- laboratory was 8/899 (0.9%; range, 0-4%). No ance with good laboratory practice, large significant increase in the incidence of tumours numbers of animals, the evaluation of multiple was observed in treated female Fischer 344/ dose levels and a duration that involved most of DuCrj rats (MHLW, 1998b). [The strengths of the lifespan.] this study included compliance with good labo- ratory practice, large numbers of animals, the 3.2.2 Inhalation evaluation of multiple dose levels and a duration that involved most of the lifespan] Groups of 50 male and 50 female Fischer 344/ DuCrj rats (age, 6 weeks) were exposed to tech- nical-grade 3-chloro-2-methylpropene (purity, 4. Mechanistic and Other 95%, containing 1.4% 1-chloro-2-methylprop- ene) by whole-body inhalation at a concentration Relevant Data of 50, 100 or 200 ppm for 6 hours per day on 5 days per week for 104 weeks. The control rats 4.1 Absorption, distribution, were handled in the same manner and exposed metabolism, excretion to clean air in similar chambers. Growth rates were slightly suppressed in males treated with 4.1.1 Humans 100 or 200 ppm and in females treated with 200 No data were available to the Working Group. ppm compared with the respective controls. Survival in the treated males was slightly lower than that in controls; the numbers of male survivors at the end of the experiment were: 39 112 4.1.2 Experimental systems The toxicokinetics of 3-chloro-2-methyl- propene in experimental systems was reviewed in IARC Monographs Volume 63 (LARC, 1995). At that time, only one study was available (Ghanayem & Burka, 1987). The reported data indicated extensive absorption and rapid excre- tion in rats after a single dose or up to four daily doses of 150 mg/kg bw of 3-chloro-2-methyl- propene (stated purity, 93%; containing ~5% 1-chloro-2-methylpropene [dimethylvinyl chlo- ride] by oral gavage. Distribution to the tissues was rapid and the highest concentrations were found in the forestomach, liver and kidney. Excretion was primarily via the urine but large amounts were exhaled, partly as carbon dioxide. The major 3-chloro-2-methylpropene metab- olite in rat urine was characterized as N-acetyl-S- (2-methylpropenyl)cysteine, presumably formed by direct conjugation of glutathione with the allylic carbon of 3-chloro-2-methylpropene (see Fig. 4.1), followed by catabolism to the mercap- turate (Ghanayem & Burka, 1987). 4.2 Mechanisms of carcinogenesis The evidence on the key characteristics of carcinogens (Smith et al. 2016) concerning whether 3-chloro-2-methylpropene is geno- toxic, induces chronic inflammation and alters cell proliferation, cell death or nutrient supply is summarized below. 4.2.1 Genetic and related effects See Table 4.1 (a) Humans No data in exposed humans were available to the Working Group. Fig. 4.1 Structure of N-acetyl-S-(2-methyl- propenyl)cysteine, the major metabolite of 3-chloro-2-methylpropene in rat urine H,C O H3C S OH H3C \r NH O (b) Experimental systems 3-Chloro-2-methylpropene is considered to be an electrophilic agent. The genotoxicity of 3-chloro-2-methylpropene was reviewed in IARC Monographs Volume 63 (IARC, 1995). Most of the studies reported were conducted with compounds that were 90.7% pure (the remaining material being primarily 1-chloro-2- methylpropene). The test preparations were mutagenic to Salmonella typhimurium TA100 in the presence and absence of metabolic activa- tion and to strain TA1537 in the presence of an exogenous metabolic system. Single studies also reported the induction of somatic recombina- tion in Drosophila melanogaster, mutation at the Tk locus in mouse L5178Y lymphoma cells, and sister-chromatid exchange and chromosomal aberrations in Chinese hamster cells, but not micronucleus formation in the bone marrow of mice treated in vivo. An earlier study reported the induction of unscheduled DNA synthesis in HeLa cells by 3-chloro-2-methylpropene (purity, 100%; reported in Eder et al., 1982) ata minimum dose of 10-3 mol/L [90.55 g/mL] (Schiffmann et al. 1983). [The Working Group noted that no statis- tical analysis was presented] 113 IARC MONOGRAPHS - 115 Table 4.1 Genetic and related effects of 3-chloro-2-methylpropene Species End- Test Results Concentration Comments Reference point (LEC or HIC) Without With metabolic activation Human, DNA Unscheduled (+) NT 103 mol/L Stated purity, 100% Schiffmann HeLa cellline damage DNA synthesis [90.55 pg/mL] (Eder et al., 1982); no et al. (1983) statistical analysis presented; the lowest dose was one to two orders of magnitude higher than that for other structurally related compounds; lethal dose NR Drosophila Mutation Sex-linked + NA 4500 ppm Foureman melanogaster recessive lethal et al. (1994) mutations Drosophila Mutation Heritable - NA 5000 ppm Foureman melanogaster translocation et al. (1994) test Drosophila Mutation Somatic + NA 2.75 ug/L Chroust melanogaster mutation and (50% lethal et al. (2007) recombination concentration) test +, positive; -, negative; (+), weakly positive; HIC, highest ineffective concentration; LEC, lowest effective concentration; NA, not applicable; NT, not tested An additional study in Drosophila melano- toxicity and genotoxicity of these compounds, gaster (analysed purity, 93.3%) reported a positive consistent with the direct reactivity or bioacti- result for the induction of sex-linked recessive vation at the halogenated carbon (Chroust et al., lethal mutations in post-meiotic and meiotic 2007). germ cells of adult males fed 3-chloro-2-meth- ylpropene. The same test sample gave negative 4.2.2 Other mechanisms results for the induction of reciprocal translo- cations (Foureman et_al., 1994). 3-Chloro-2- (a) Humans methylpropene [purity not specified] also gave No data were available to the Working Group. positive results in the wing spot test in Drosophila melanogaster when administered by inhalation (b) Experimental systems at the 50% lethal concentration (Chroust et al., In a 13-week study on the toxicity of 2007). A quantitative structure-activity relation- 3-chloro-2-methylpropene (same material as ship multivariate analysis of a series of structur- that used in the 2-year bioassay), inflamma- ally similar halogenated aliphatic compounds, tion and necrosis of the liver were observed in including 3-chloro-2-methylpropene, indicated treated rats and mice, and necrosis of cortical that nucleophilic superdelocalizability of the tubules was observed in the kidney of mice. In halogen atom (calculated by quantum mechanics) the 2-year study, a dose-dependent increase in was a good structural parameter to predict the the incidence of forestomach inflammation was 114 observed in treated male and female mice. The incidence of inflammation in the nasal cavity and of nephropathy/nephrosis was higher in exposed groups than in the controls in male and female mice and rats (NTP, 1986). The incidence and severity of epithelial cell proliferation of the forestomach was increased in Fischer 344/N rats given 3-chloro-2-methyl- propene at 150 mg/kg bw, but not at 75 mg/kg bw, by gavage on 5 days per week for 2 weeks (Ghanayem et al., 1986). A strong association between early forestomach mucosal cell prolif- eration and forestomach neoplasia was observed in treated rats and mice in the 2-year carcino- genicity bioassay conducted by the NTP, in which 3-chloro-2-methylpropene (technical grade, containing 5% dimethylvinyl chloride) was administered by gavage (Chan et al., 1986; NTP, 1986). In the nasal cavity, eosinophilic exudate asso- ciated with atrophy of the olfactory epithelia, respiratory metaplasia of the olfactory epithelia and olfactory gland, and eosinophilic changes in the respiratory and olfactory epithelia were observed in male and female BDF, mice exposed to 3-chloro-2-methylpropene (stated purity, 95%) at 0, 50, 100, or 200 ppm on 5 days per week for 104 weeks (Katagiri et al., 2000). 4.3 Data relevant to comparisons across agents and end-points 3-Chloro-2-methyl propene was not tested by the Tox21 and ToxCast™ research programmes of the government of the USA (Kavlock et al., 2012; Tice et al., 2013). Analyses of other compounds evaluated in this volume with high-throughput screening results are presented in the Monograph on 1-bromopropane in the present volume. 4.4 Susceptibility to cancer No data were available to the Working Group. 4.5

Other hazards

No additional data were available to the Working Group. 5. Summary of Data Reported 5.1 Exposure data 3-Chloro-2-methylpropene is available commercially at purities ranging from 95% (technical grade) to 98%, with dimethylvinyl chloride (1-chloro-2-methylpropene) as an impu- rity. 3-Chloro-2-methylpropene is an important intermediate in the production of pesticides, is used as a fumigant for seeds and is also an inter- mediate in the manufacture of plastics and other organic chemicals. Apart from a single study of environmental exposure published in 1982, no data on environmental or occupational exposure were available. 5.2 Human carcinogenicity data No data were available to the Working Group. 5.3 Animal carcinogenicity data Technical-grade 3-chloro-2-methylpropene was tested for carcinogenicity by oral adminis- tration (gavage) in one study in male and female mice and in one study in male and female rats, and by whole-body inhalation in one study in male and female mice and in one study in male and female rats. In the study in mice treated by gavage, tech- nical-grade 3-chloro-2-methylpropene caused a significantly increased incidence (with a signif- icant positive trend) of forestomach squamous cell papilloma, forestomach squamous cell carci- noma, and forestomach squamous cell papilloma or carcinoma (combined) in males and that of forestomach squamous cell papilloma and 115 IARC MONOGRAPHS - 115 forestomach squamous cell papilloma or carci- predominates, although large amounts are noma (combined) in females. exhaled, partly as carbon dioxide. In the study in mice treated by inha- With respect to the key characteristics of lation, administration of technical-grade human carcinogens, there is strong evidence 3-chloro-2-methylpropene resulted in a signifi- that 3-chloro-2-methylpropene is genotoxic, and cantly increased positive trend in the incidence these effects can operate in humans. 3-Chloro- of forestomach squamous cell papilloma in 2-methylpropene induced gene mutation in males and females. It also caused a significantly bacterial and mammalian cells, chromosomal increased incidence (with a significant positive aberrations and sister-chromatid exchange in trend) of adenoma of the Harderian gland in mammalian cells, and genetic crossing-over (or females. recombination) and sex-linked recessive lethal In the study in rats treated by gavage, tech- mutation in post-meiotic and meiotic germ cells nical-grade 3-chloro-2-methylpropene caused a in Drosophila melanogaster. Only one study of significantly increased incidence (with a signif- DNA damage in human (HeLa) cells in vitro was icant positive trend) of forestomach squamous available, but the results were positive. cell papilloma and forestomach squamous cell There is moderate evidence that 3-chloro-2- papilloma or carcinoma (combined) in males methylpropene induces chronic inflammation. and that of forestomach squamous cell papil- 3-Chloro-2-methylpropene induced inflamma- loma in females. Treated female rats also devel- tion in the liver and nasal cavity in rats and mice, oped subcutaneous fibromas that may have been and inflammation in the forestomach in male related to treatment. and female mice. In the study in rats treated by inhalation, There is weak evidence that 3-chloro-2- exposure to technical-grade 3-chloro-2-methyl- methylpropene alters cell __ proliferation. propene resulted in a significant positive trend Proliferation of the epithelial cells of the in the incidence of follicular cell adenoma and forestomach was increased in rats exposed to of follicular cell adenoma or adenocarcinoma 3-chloro-2-methylpropene. (combined) of the thyroid gland in males. No There were few data on the other key char- significant increase in the incidence of tumours acteristics of carcinogens (alters DNA repair or was observed in female rats. causes genomic instability, induces epigenetic alterations, induces oxidative stress, is immuno- suppressive, modulates receptor-mediated effects 5.4 Mechanistic and other relevant or causes immortalization). data 3-Chloro-2-methylpropene is considered to be an electrophilic agent. The main urinary 6. Evaluation metabolite is a mercapturate conjugate. No data were available on the toxicokinetics of 6.1 Cancer in humans 3-chloro-2-methylpropene in humans. In rats given 3-chloro-2-methylpropene by oral admin- There is inadequate evidencein humans forthe istration, the compound is rapidly absorbed, carcinogenicity of 3-chloro-2-methylpropene. distributed (primarily to the forestomach, liver and kidney) and excreted. Urinary excretion 116 6.2 Cancer in experimental animals There is sufficient evidence in experimental animals for the carcinogenicity of techni- cal-grade 3-chloro-2-methylpropene. 6.3 Overall evaluation Technical-grade 3-chloro-2-methylpropene is possibly carcinogenic to humans (Group 2B). References Chan PC, Haseman JK, Boorman GA, Huff J, Manus AG, Cardy RH (1986). Forestomach lesions in rats and mice administered 3-chloro-2-methylpropene by gavage for two years. Cancer Res, 46(12) 6349-52. PMID 3779651 Chroust K, Pavlova M, Prokop Z, Mendel J, Bozkova K, Kubat Z et al. (2007). Quantitative structure-activity relationships for toxicity and genotoxicity of halogen- ated aliphatic compounds: wing spot test of Drosophila melanogaster. Chemosphere, 67(1) 152-9. doi 10.1016/j chemosphere.2006.09.020 PMID:17113125 ECHA (2015). Pre-registered substance 3-chloro-2-meth- ylpropene, Information on chemicals. Helsinki, Finland: European Chemicals Agency. Available from: http://echa.europa.eu/information-on-chemicals. Eder E, Neudecker T, Lutz D, Henschler D (1982). Correlation of alkylating and mutagenic activities of allyl and allylic compounds: standard alkylation test vs. kinetic investigation. Chem Biol Interact, 38(3):303- doi 10.1016/0009-2797(82)90060-6 PMID 7037213 Foureman P, Mason JM, Valencia R, Zimmering S (1994). Chemical mutagenesis testing in Drosophila. X. Results of 70 coded chemicals tested for the National Toxicology Program. Environ Mol Mutagen, 23(3):208- 27. doi 10.1002/em.2850230310 PMID 8162896 Ghanayem BI, Burka LT (1987). Comparative metabolism and disposition of 1-chloro- and 3-chloro-2-methylpro- pene in rats and mice. Drug Metab Dispos, 15(1):91-6. PMID:2881764 Ghanayem BI, Maronpot RR, Matthews HB (1986). Association of chemically induced forestomach cell proliferation and carcinogenesis. Cancer Lett, 32(3):271-8. doi:10.1016/0304-3835(86)90179-5 PMID:3768853 HSDB (2014). 3-Chloro-2-methyl-l-propene. Report. CASRN: 563-47-3. Hazardous Substances Data Bank [online database]. United States National Library of Medicine. Available from: http://toxnet.nlm.nih.gov/. IARC (1995). Dry cleaning, some chlorinated solvents and other industrial chemicals. IARC Monogr Eval Carcinog Risks Hum, 63 1-551. Available from http:// monographs. iarc.fr/ENG/Monographs/vol63/index. php. PMID:9139128 IFA (2015). GESTIS database on hazardous substances: 3-chloro-2-methylpropene. Information system on other end-points. A study showed that workers with GSTT1 null genotype are more suscep- Occupational exposure to N,N-dimethyl- tible to N,N-dimethylformamide exposure-as- formamide induced liver injury in exposed sociated abnormal liver function (Luo et_al., workers (Wrbitzky, 1999; Wang et _al., 2014). 2005). Two other studies showed no associations Exposure to N,N-dimethylformamide induced between polymorphism of CYP2E] and the levels various symptoms, such as abdominal pain, (Nomiyamaetal.,2001b) orhalf-lives (Nomiyama nausea, vomiting or diarrhoea, as well as jaun- et_al., 2001c) of urinary N-methylformamide. dice and disulfiram-type reaction (Potter, 1973; A total of 66 male workers exposed to Fiorito etal., 1997). Serum aspartate aminotrans- N,N-dimethylformamide were investigated (Luo ferase (AST), alanine aminotransferase (ALT) et al., 2005). Compared with the GSTT1-positive (ALT > AST generally) and y-glutamyl transpep- genotype workers, the adjusted odds ratioand 95% tidase were increased (Potter, 1973; Nomiyama confidence intervals for abnormal liver function et_al., 2001d). HBV carrier status and increased test were 4.41 (95% CI, 1.15-16.9) for the GSTT1 body mass index had synergistic effects with null genotype workers. Compared with the N,N-dimethylformamide exposure in causing N,N-dimethylformamide low-exposure group liver abnormalities (Luo et al., 2001). A patient with GSTTI1-positive genotype workers, the odds who was exposed to N,N-dimethylformamide by ratio (adjusted for hepatitis B virus (HBV) status) a suicide attempt showed elevation in AST, ALT, of abnormal liver function test was 12.38 (95% CI, and total bilirubin and decrease in prothrombin 1.04-146.9) for the N,N-dimethylformamide time (Buylaert et al., 1996). high-exposure group with GSTT1-null genotype In experimental systems, there were workers. Multiplicative interactions between many studies in rats (e.g. Tanaka, 1971; Kim N,N-dimethylformamide exposure and GSTTI & Chung, 2013), mice (e.g. Craig et al., 1984; genotype were demonstrated. Rui et al., 2011), hamsters (Ungar et al., 1976), There was no_ significant difference cats or rabbits (Massmann, 1956) exposed to between slopes of single linear regression 158 N,N-dimethylformamide by inhalation (Craig et al., 1984; Senoh et al., 2003), inhalation and in drinking-water (Ohbayashi et al., 2008), gavage (Rui et al., 2011), and intraperitoneal injection (Ungar et al., 1976; Kim & Chung, 2013), which showed hepatotoxicity (Malley et al., 1994; Senoh et al., 2003). The main histopathological changes were centrilobular hepatocellular necrosis (Ungar et al., 1976; Kim & Chung, 2013), centri- lobular hepatocellular hypertrophy (Tanaka 1971; Ruietal., 2011), massive necrosis associated with centrilobular fibrosis (Senoh et _al., 2003), focal necrosis (Senoh et al., 2003), centrilobular single cell necrosis (Malley et al., 1994; Senoh et_al., 2003), and lipofuscin/hemosiderin accu- mulation in Kupffer cells (Malley et al., 1994). Serum sorbitol dehydrogenase (Malley et_al., 1994; Lynch et al., 2003), ALT (Tanaka, 1971; Rui et.al., 2011), AST (Tanaka, 1971; Rui et al., 2011), lactate dehydrogenase (Janaka, 1971; Ruiet al., 2011), cholesterol (Lynch et al., 2003; Senoh et al., 2003), phospholipid (Senoh et al., 2003), and total bile acids (Lynch et al., 2003) were increased. In rodents, exposure to N,N-dimethyl- formamide by inhalation (Kimmerle & Machemer, 1975), gavage (Saillenfait et al., 1997), in drinking-water (Fail et al., 1998), or by epicu- taneous administration (Hansen & Meyer, 1990) reduced fertility (Hansen & Meyer, 1990), and the number of fetuses (Hansen & Meyer, 1990), decreased maternal weight (Hansen & Meyer, 1990; Saillenfait et al., 1997) and fetal weight (Hansen & Meyer, 1990; Saillenfait et al., 1997) as well as inducing skeletal variations (Saillenfait et.al., 1997) or malformations (Fail et al., 1998). 5. Summary of Data Reported 5.1 Exposure data N,N-Dimethylformamide is a chemical witha high production volume that is mainly used in the manufacture of acrylic fibres, and in the synthet- ic-leather industry. N,N-Dimethylformamide is also used as a solvent in the electronics industry, in pesticide formulations, and as a component of paint strippers. Exposures of the general population have been reported in China among residents living near synthetic-leather factories. N,N-Dimethylformamide has been detected in the air in the vicinity of a waste site in the USA, and in environmental samples of air and water in Japan. Occupational exposures to N,N-dimethylformamide have been measured among workers employed in acrylic-fibre and synthetic-leather industries. 5.2 Human carcinogenicity data Evidence on the carcinogenicity of N,N- dimethylformamide in humans was available from studies of aircraft-repair workers, leather- tannery workers, and chemical manufacturing workers. One study described a cluster of three cases of cancer of the testes among 153 workers repairing F4 Phantom jet aircraft in the USA. These workers participated in or worked in proximity to a single process that involved uncontrolled exposure to N,N-dimethylformamide. The finding of the cluster investigation motivated a further study at a different aircraft-repair facility, where one unit repaired the F4 Phantom aircraft (also with exposure to N,N-dimethylformamide) and one unit repaired other aircraft (without exposure to N,N-dimethylformamide). In the exposed group, four cases of cancer of the testes were observed, compared with none in the unexposed group. 159 IARC MONOGRAPHS - 115 Shortly thereafter, a cluster of three cases significant increase in the incidence of hepato- of cancer of the testes was observed in workers cellular adenoma, hepatocellular carcinoma, and who used N,N-dimethylformamide at a leather hepatocellular adenoma, hepatocellular carci- tannery in New York state, USA. A subsequent noma or hepatoblastoma (combined), in males case-control study of cancer of the testis in the and females in all dose groups, with a signifi- county where the tannery was located, which cant positive trend. There was also a significant included the 3 original cases and 7 others, increase in the incidence of hepatoblastoma in supported the existence of a cluster among males. Multiple hepatocellular adenomas and leather workers. carcinomas were found in the liver of exposed A cohort study in one plant producing acrylic mice. In the second study by inhalation in mice, fibres in the USA, and a subsequent case-control no significant increase in tumour incidence was study including that plant and three others, all reported in any dose group. of which used N,N-dimethylformamide and In a 2-year GLP study in rats treated by inhal- other chemicals, found 11 cases of cancer of ation, exposure to N,N-dimethylformamide the testes, of which three had been exposed to caused a significant positive trend in the inci- N,N-dimethylformamide, with no observed dences of hepatocellular adenoma, hepato- excess. It is likely that the exposures to cellular carcinoma, and hepatocellular adenoma N,N-dimethylformamide in these manufac- or carcinoma (combined), in males and females; turing plants were lower than those of leath- the incidences of hepatocellular adenoma and er-tanning and aircraft-repair workers. hepatocellular carcinoma were significantly The data from studies of aircraft-repair increased in males and females at the highest workers and leather workers provided evidence dose. Multiple occurrences of hepatocellular for a positive association between exposure to tumours were found in the livers of exposed N,N-dimethylformamide and cancer of the rats. In the second study by inhalation in rats, testes; however, chance and confounding by no significant increase in tumour incidence was other occupational exposures could not be ruled reported in any dose group. out. In the GLP study of inhalation plus oral administration (combined) in male rats, N,N- dimethylformamide caused a significant increase 5.3 Animal carcinogenicity data in the incidences of hepatocellular adenoma, N,N-dimethylformamide was tested for and hepatocellular adenoma or carcinoma carcinogenicity in two studies by whole-body (combined) in the groups treated by inhalation inhalation in male and female mice, two studies plus oral administration, oral administration by whole-body inhalation in male and female rats, only, and inhalation only. It also caused a signif- and one study by whole-body inhalation plus oral icant increase in the incidence of hepatocellular (drinking-water) administration (combined) in carcinoma in one group treated by oral adminis- male rats. The Working Group determined that tration only and in all groups treated by inhala- one study by oral administration, one study by tion plus oral administration. subcutaneous injection, and one study by intra- peritoneal injection in rats were inadequate for the evaluation. In a 2-year study that complied with good laboratory practice (GLP), treatment of mice with N,N-dimethylformamide by inhalation caused a 160 5.4 Mechanistic and other relevant data With respect to the key characteristics of human carcinogens, there is strong evidence that N,N-dimethylformamide is metaboli- cally activated, including in exposed humans. N,N-Dimethylformamide is readily absorbed after inhalation, dermal, and oral exposure. Several urinary metabolites, including the mercapturic acid, have been identified in humans. Carbamoylated adducts formed from methyl isocyanate and/or another carbamoylating meta- bolic intermediate of N,N-dimethylformamide have been identified in globin (both lysine and N-terminal valine adducts). A carbamoy- lated cytosine adduct has also been detected in vivo in the urine of occupationally exposed humans. Additionally, formaldehyde is a prob- able metabolite from oxidative demethylation of N,N-dimethylformamide. There is strong evidence that N,N-dimethyl- formamide induces oxidative stress. A study of occupationally-exposed workers showed a distinct increase in the activity of super- oxide dismutase in blood compared with the controls. Experimental studies in vivo and in vitro confirmed that N,N-dimethylformamide is capable of increasing oxidative stress in rodents, as well as in human liver, leukaemia and colon carcinoma cells. There is strong evidence that N,N-dimethyl- formamide alters cell proliferation. No data were available in exposed humans. Experimental studies in vitro have shown an anti-prolifera- tive and differentiating effect in human cancer cells, while it induced cell proliferation in normal human and rodent liver cells. There is moderate evidence that N,N- dimethylformamide is genotoxic. Chromosomal and DNA damage has been observed in several studies in occupationally exposed humans, but results were equivocal. The results of studies of genotoxicity in various experimental systems in vivo and in vitro were mostly negative or inconclusive. There is weak evidence that N,N-dimethyl- formamide modulates receptor-mediated effects. So far there is little mechanistic evidence that N,N-dimethylformamide exerts any toxic effects through a specific receptor in humans or in experimental systems. Some experimental studies did report perturbations of the receptors for estrogen, transforming growth factor 6, and epidermal growth factor. There were few data on other key characteris- tics of carcinogens (alters DNA repair or causes genomic instability, induces epigenetic altera- tions, induces chronic inflammation, is immu- nosuppressive, or causes immortalization). 6. Evaluation 6.1 Cancer in humans There is limited evidence in humans for the carcinogenicity of N,N-dimethylformamide. A positive association has been observed between exposure to N,N-dimethylformamide and cancer of the testes. 6.2 Cancer in experimental animals There is sufficient evidence in experi- mental animals for the carcinogenicity of N,N-dimethylformamide. 6.3 Overall evaluation N,N-Dimethylformamide is probably carcino- genic to humans (Group 2A). 161 IARC MONOGRAPHS - 115 References Brindley C, Gescher A, Ross D (1983). Studies of the metabolism of dimethylformamide in mice. Chem Biol Interact, 45(3) 387-92. doi 10.1016/0009- Alwis KU, Blount BC, Britt AS, Patel D, Ashley DL 2797(83)90085-6 PMID:6883579 (2012). 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Hazardous Ingredients

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No.: 68-12-2 Degradation: Dimethylamine can be released Chem. Abstr. Serv. Name: N,N-Dimethyl- from N,N-dimethylformamide and, in the formamide presence of nitrogen oxides, carcinogenic IUPAC Systematic Name: N,N-Dimethyl- nitrosamines can be formed. Other potential formamide degradation products are ammonia, carbon monoxide, carbon dioxide, amines and Synonyms: N,N-Dimethylmethane amide, formaldehyde. formic acid dimethyl amide, N-formyl- dimethylamine Density (at 20 °C): 0.95 g/cm} (LEA, 2015) Acronym: DMF Octanol/water partition coefficient: log K,,,, -1.01 (HSDB, 2015) Melting point: —61 °C (LFA, 2015) 1.1.2 Structure and molecular formula, and Boiling point: 153 °C (LEA, 2015) relative molecular mass Vapour pressure (at 20 °C): 0.377 kPa (2.83 mm Hg) (IFA, 2015) \ < Vapour density: 2.51 (air = 1) (HSDB, 2015) N / Solubility: Entirely soluble in water at 20 °C H3C H (IFA, 2015) Molecular formula: C,H,NO Flammable limits: Lower explosion limit: Relative molecular mass: 73.09 2.2 vol. %; upper explosion limit: 16 vol. % (IFA, 2015) Flash point: 58 °C (LEA, 2015) 119 IARC MONOGRAPHS - 115 Ignition temperature: 440 °C (LFA, 2015) USA remained constant between 1986 and 2002, Conversion factor (101 kPa, 20 °C): and was reported to be between 50 and 100 million 1 ppm = 3.04 mg/m (IFA, 2015). pounds [approximately 20 000 and 45 000 tonnes] (HSDB, 2015). The annual production and import volume of nine USA companies was about 50 1.2 Production and use million pounds [~25 000 tonnes] (HSDB, 2015) and the total annual production volume in the 1.2.1 Production European Union ranged from 50 000 to 100 000 N,N-Dimethylformamide is predominantly tonnes in 2000 (SCOEL, 2006). No production produced in a single-step reaction between volumes could be traced for Asia, but more than dimethylamine and carbon monoxide under 300 suppliers of N,N-dimethylformamide could pressure at high temperatures and in the pres- be identified globally, including > 200 in Asia, ence of basic catalysts such as sodium meth- > 60 in the USA, and > 30 in the European Union (ChemBook, 2015). oxide. The crude product contains methanol and N,N-dimethylformamide with increased purity (up to 99.9%) is obtained by multiple distillations 1.2.2 Use (HSDB, 2015). Alternatively, it can be produced N,N-Dimethylformamide is used predom- by a two-step process in which methyl formate is inantly as an aprotic solvent in the manufac- prepared separately and, in a second step, reacts ture of polyacrylonitrile fibres, and trends in with dimethylamine under similar conditions its production parallel those of the polyacrylic as those described for the single-step reaction. fibre industry (HSDB, 2015). It is also used in the No catalysts are involved in the process (HSDB manufacture of high quality polyurethane and 2015). polyamide coatings (e.g. for leather or artificial N,N-Dimethylformamide is listed as a high leather fabrics), which are otherwise difficult to production volume chemical by the Organisation solubilize, and where a solvent with a slow rate of for Economic Co-operation and Development evaporation is needed. N,N-Dimethylformamide (OECD), indicating that this chemical is is commonly used as a solvent in the electronics produced or imported at levels greater than 1000 industry, in pesticides, in industrial paint-strip- tonnes per year in at least one member country ping applications, and as a reaction and crystal- or region (OECD, 2004, 2009). The OECD 2007 lizing solvent in the pharmaceutical industry. list of high production volume chemicals was It has limited use as a selective solvent for the compiled on the basis of submissions from eight separation of aliphatic hydrocarbons such as the member countries (including Australia, Canada, extraction of acetylene or butadiene from hydro- Japan, and the USA) in addition to the list carbon streams (HSDB, 2015). provided by the European Union (OECD, 2009). N,N-Dimethylformamide is also listed as a high production volume chemical in the USA 1.3. Measurement and analysis by the Environmental Protection Agency (EPA) Multiple methods exist for the analysis of indicating that > 1 million pounds [~450 tonnes] N,N-dimethylformamide and its metabolites were produced in or imported into the USA in in air, water, urine, and blood. The methods 1990 and/or 1994 (HSDB, 2015). are largely based on gas chromatography (GC) The annual production volume of N,N- and high-performance liquid chromatography dimethylformamide (excluding imports) in the (HPLC) with various detection systems such as 120 flame ionization, nitrogen-sensitive, or mass-sen- sitive detection. N,N-Dimethylformamide is most commonly measured in air by adsorbing on a silica gel and analysis by GC/flame ionization detection. The level of detection is approximately 0.05 mg per sample in a 15-L air sample or higher at a flow rate of 0.01-1 L/min (NIOSH, 1994), Several diffusive sampling devices have been described for exposure assessment of N,N-dimethylformamide in the air of the work- place (Tanaka et al., 2002; Baglioni et al., 2007). Because of the potential for dermal uptake, exposure to N,N-dimethylformamide should be measured by biomonitoring. Major metabo- lites in urine are N-hydroxymethyl-N-methyl- formamide (HMMEF), N-methylformamide and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) (Gescher, 1993). HMMEF degrades at injection temperatures > 250 °C, which allows the determination of total N-methylformamide in biological samples (Kawai et al., 1992). An HPLC method also has been described that has a pre-heat phase to convert HMMF to N-methylformamide. The reported detection limit was 0.5 mg/L (Tranfo et al., 1999). As an alternative, HMMEF can be converted chemi- cally to N-methylformamide in the presence of potassium carbonate during sample preparation (Mraz & Turecek, 1987). The limits of detec- tion are usually approximately 1 mg/L for total N-methylformamide and sufficient for exposure assessment in N,N-dimethylformamide-exposed workers (Mraz et al., 1987). Recently, a method for simultaneously analysing total N-methylformamide and AMCC by capillary GC and nitrogen-selective detector with an injector temperature > 250 °C has been described (Kafferlein_ & Angerer, 2005). Under these conditions, the limits of detection were 1 mg/L of urine for total N-methylformamide and 0.5 mg/L of urine for AMCC. The measurement of AMCC in urine can also form part of a multi-analysis of various mercapturic acids of organic compounds rather than measuring AMCC alone (Schettgen et al., 2008; Schettgen, 2010; Alwis et al., 2012). [The Working Group noted that AMCC can also be detected in the urine of the general population as a result of its endogenous production in the body (Kafferlein & Angerer, 1999).] 1.4 Occurrence and exposure 1.4.1 Natural occurrence N,N-Dimethylformamide is not known to occur as a natural product. 1.4.2 Environmental occurrence Industrial releases of N,N-dimethylform- amide into the air appear to be considerably larger than those into other environmental media. N,N-Dimethylformamide is expected to exist almost entirely in the vapour phase in ambient air. When emitted into air, most of the N,N-dimethylformamide released remains in that compartment where it is degraded by chem- ical reactions with hydroxyl radicals; its half-life in the air is estimated to be in the range of few days. Some atmospheric N,N-dimethylform- amide can reach the aquatic and terrestrial envi- ronment, presumably during periods of rain. Releases into the water or soil are expected to be followed by relatively rapid biodegradation (half- life, 18-36 hours) (IPCS, 2001; HSDB, 2015), Measurements of N,N-dimethylformamide in air and water are presented in Table 1.1. (a) Air In Lowell, MA, USA, N,N-dimethylform- amide was detected in the air over an aban- doned chemical waste reclamation plant (mean, 7 ug/m;3), a neighbouring industry (> 150 ug/m?), and a residential area (24 ug/m?) (Amster et al. 1983; cited in IPCS, 2001). In ambient air samples collected in the north-eastern USA in 1983, levels 121 CCL Table 1.1 Environmental exposure to N,N-dimethylformamide VI DY Country Duration Sites/situation DMF exposure Reference DMF in air NMF in urine DMF in surface water (ug/L) (ug/m}3) (mg/g creatinine) No. Mean SD Range No. Mean SD No. Mean SD __ Range China l-year “Unpolluted” area in 3662 98.3 109.7 4.4-678.3 Wang et al.(2014) follow-up Longwan SHdVYDONOW Cross- Near synthetic A 25 297.5 95 25 7.7 6.2 - sectional leather factories in B 39 430 122.5 39s «6.7 2.7 Sar C 22 180 0 SLL 22002415 «7 D 23 565 516.2 23 23.4 24.9 E 24 270 84.9 24 1.8 0.8 Japan Cross- All over Japan 105 0.092< 0.016-0.49 474 0).27¢ ND-0.53 Ministry of the sectional

Environmental hazards

Japan (2012) USA North-eastern ND-14 Kelly et al. 1994) USA Massachusetts/ 7- > 150 Amster et al. (1983) polluted area cited in IPCS (2001) « Monitoring daily throughout 2008 > Three samples were measured from 35 sites in Japan. N, N-Dimethylformamide was detected in all samples © Geometric mean 4 One sample was measured from 47 sites in Japan. N,N-Dimethylformamide was below the detection limit (0.019 ug/L) in 10 samples out of 47 DMF, N,N-dimethylformamide; ND, not detected; NMF, N-methylformamide; SD, standard deviation of N,N-dimethylformamide ranged from not detected to 14 ug/m? (Kelly et al., 1994). Airborne concentrations of N,N-dimethyl- formamide, measurednearsyntheticleather facto- ries in China where it is used, ranged from 180 to 565 ug/m}. In the same region, but away from the factories, mean concentration in 366 samples was 98.3 ug/m} (Wang et al., 2014). Airborne concen- trations of N,N-dimethylformamide measured at 35 sites all over Japan in 2008 ranged from 0.016 to 0.49 ug/m? (Ministry of the Environment Japan, 2012). (b) Water Concentrations of N,N-dimethylformamide in water from rivers, lakes, and bays were measured throughout Japan and, in a total of 47 samples from 47 sites, ranged from not detected to 0.53 ug/L (Ministry of the Japan, 2012) 1.4.3 Exposure of the general population A study on biological monitoring in the general population living in areas near synthetic leather factories in China investigated the consistency between the concentration of N,N- dimethylformamide in outdoor air and levels of urinary N-methylformamide (Wang et al., 2014). The mean concentration of urinary N-methyl- formamide ranged from 1.5 to 23.4 mg/g creatinine. Under the most intensive expo- sure (> 450 ug/m? of N,N-dimethylformamide in the air), the maximum value of urinary N-methylformamide was 41.03 mg/g creati- nine. The correlation between urinary N-methylformamide and air sampling was strong (P < 0.01) with a coefficient of 0.80 (Wang et al., 2014; Table 1.1). According to the 1981-83 United States National Occupational Exposure Survey, as many as 125 000 workers in the USA were potentially exposed to N,N-dimethylformamide (NOES 1997). Exposures to N,N-dimethylformamide were assessed in workplaces by both air monitoring and biomonitoring (urinary N-methylform- amide) beginning in the 1970s (Table 1.2). 1.4.5 Exposure assessment in epidemiological studies In a retrospective cohort study (Chen et al., 1988a, b), at an acrylic-fibre production plant in the USA, exposure to N,N-dimethylformamide was assessed semi-quantitatively by an “exposure Miyauchi H, Tsuda Y, Minozoe A, Tanaka S, Arito H, Tsukahara T et al. (2014). Occupational exposure to N,N-dimethylformamideinthesummerand winter. 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Evaluation of current biological exposure index for occupational N, N-dimethylformamide exposure from synthetic leather workers. J Occup Environ Med, 46(7) 729-36. doi 10.1097/01.jom.0000131795.88947.45 PMID:15247813 Wrbitzky R (1999). Liver function in workers exposed to N,N-dimethylformamide during the prod- uction of synthetic textiles. Int Arch Occup Environ 170 N,N-DIMETHYL-P-TOLUIDINE 1. Exposure Data 1.1. Identification of the agent 1.1.1 Nomenclature Chem. Abstr. Serv. Reg. No.: 99-97-8 Chen. Abstr. Serv. Name: N,N-Dimethy]l- para-toluidine IUPAC Systematic Name: N,N,4-Trimethyl- aniline Synonyms: 4-Dimethylaminotoluene, N,N-dimethyl-4-methylaniline, p,N,N- trimethylaniline, N,N,4-trimethylbenzen- amine Acronyms: DMPT 1.1.2 Structure and molecular formula, and relative molecular mass ls HC N \ CH, Molecular formula: C,H,,N Relative molecular mass: 135.21 1.1.3 Physical and chemical properties Description: Light yellow to brown, oily fluid with a characteristic amine-like odour (IFA, 2015) Density (at 20 °C): 0.94 g/cm? (IEA, 2015) Octanol/water partition coefficient (P): log K,,, 2.81 (HSDB, 2015) Melting point: -15 °C (IFA, 2015) Boiling point: 211 °C (HSDB, 2015) Volatility: Vapour pressure, 0.01 kPa [0.178 mm Hg], at 20 °C (IFA, 2015) Vapour density: 4.66 (air = 1) at 20 °C (IFA, 2015) Solubility: Insoluble to moderately soluble in water (650 mg/L at 37 °C) (ECHA, 2015) Stability: Lower explosion limit, 1.2 vol. %; upper explosion limit, 7.0 vol. % (IFA, 2015) Flash point: 83 °C (LFA, 2015) Ignition temperature: 425 °C (LEA, 2015) Conversion factor (at 101 kPa, 20 °C): 1 ppm = 5.62 mg/m? (IFA, 2015) 1.2 Production and use 1.2.1 Production (a) Production methods Multiple methods exist for the manufacture of N-alkyltoluidines, including N,N-dimethy]l- p-toluidine, such as acid-catalysed alkylation of unalkylated toluidines with lower unbranched alcohols or ether. Another method involves reductive alkylation with lower aldehydes or ketones using metal catalysts under hydrogen pressure (Bowers, 1996). 171 IARC MONOGRAPHS - 115 (b) Production volume Table 1.1 Annual production volume of N,N- N,N-Dimethyl-p-toluidine was listed as a dimethyl-p-toluidine in the USA, 1986-2014 high production volume chemical in the USA, indicating that > 1 million pounds [approxi- Year Production range (pounds) mately 454 tonnes] were produced or imported 1986 10 000-500 000 [~4.5-227 tonnes] between 1990 and 1994 annually (HSDB, 2015). 1990 10 000-500 000 [~4.5-227 tonnes] Annual United States production volumes 1994 > 1 million-10 million [~ > 454-4536 tonnes] of N,N-dimethyl-p-toluidine from 1998 > 1 million-10 million [~ > 454-4536 tonnes] 1986 to 2014 2002 > 500 000-1 million [~ > 227-454 tonnes] are given in Table 1.1. With annual production 2006 > 1 million- 10 million [~ > 454-4536 tonnes] reported to be 1-10 million pounds [approxi- 2014 > 1 million-10 million [~ > 454-4536 tonnes] mately 454-4536 tonnes] in 2014, it remains a Compiled by the Working Group from non-confidential Chemical high production volume chemical in the USA Data Reporting information (EPA, 2011, 2015; HSDB, 2015) (HSDB, 2015). The total tonnage band per annum is estimated to be between 10 and 100 tonnes in preparation was carried out by solid-phase the European Union (ECHA, 2015). Globally, extraction. more than 130 suppliers of N,N-dimethyl-p- Air monitoring of N,N-dimethyl-p-toluidine toluidine could be identified in 2016 with about has been described as part of NIOSH Method two thirds (> 80) located in Asia (ChemicalBook 2002 (aromatic amines) using gas chromatog- Inc., 2016). raphy and flame ionization detection (NIOSH 1994). Sample preparation includes sorption on 1.2.2 Use silica gel at a sampling rate of 1 L/minute or less, and the estimated limit of detection is 0.01 mg N,N-Dimethyl-p-toluidine is used as an per sample. accelerator in the preparation of dental materials No analytical procedures were found for and bone cements to activate the polymerization the determination of N,N-dimethyl-p-toluidine reaction or when curing methyl methacrylate specifically in the (air, water), monomers. The concentrations in these prepa- food, urine or blood, with the exception of the rations are usually between 0.5% and 3% (NTP, determination of its N-oxides in enzyme reac- 2012). It has also been used for the preparation tion mixtures (ie. liver microsomes) using of acrylic denture material for the past 50 years high-performance liquid chromatography with (HSDB, 2015). In addition, N,N-dimethyl-p- ultraviolet detection (Seto & Guengerich, 1993), toluidine is an important ingredient of industrial N,N-Dimethyl-p-toluidine may be determined glues and artificial fingernail preparations, when by current methods for the determination of short setting times are needed, and is also used as monocyclic aromatic amines in blood or urine an intermediate in the manufacture of dyes and (Richter _& Branner, 2002; Weiss & Angerer, pesticides (NTP, 2012). 2002; Lamani et al., 2015). 1.3. Measurement and analysis 1.4 Occurrence and exposure N,N-Dimethyl-p-toluidine in dental mate- rial was analysed by high-performance liquid 1.4.1 Natural occurrence chromatography with ultraviolet detection N,N-Dimethyl-p-toluidine does not occur (Shintani et_al. 1993; Stea et al. 1997). Sample naturally. 172 1.4.2 Environmental occurrence In the N,N-dimethyl-p- toluidine is broken down rapidly in air. It travels through soil and may volatilize from moist soil and water surfaces (HSDB, 2015). 1.4.3 Occupational exposure Potential widespread human exposure to N,N-dimethyl-p-toluidine can occur in occupa- tional settings related to its use in bone cements, dental prostheses, industrial glues, and artificial fingernails. Surgeons, surgical staff, dentists, dental technicians, nail salon operators, and users of industrial glues may receive significant exposure to N,N-dimethyl-p-toluidine by inhal- ation or through the skin (NTP, 1999). The National Occupational Exposure Survey, which was conducted by the National Institute for Occupational Safety and Health (NIOSH) between 1981 and 1983, estimated that 62 720 workers (among whom 27 118 were women) were potentially exposed to N,N-dimethyl-p- toluidine in the workplace (NIOSH, 1990). In 2014, three companies in the USA manufactured N,N-dimethyl-p-toluidine (HSDB, 2015). A NIOSH investigation of air sampling from a nail salon measured only trace amounts of N,N-dimethyl-p-toluidine (Kronoveter, 1977). No other measurements of occupational expo- sure were identified. 1.4.4 Exposure of the general population Sniffing glues, some of which contain 1-7% of the compound (Misiak & Scheffler, 2003; 3M Company, 2004), could possibly result in exposure to N,N-dimethyl-p-toluidine (Neumark et al., 1998; Wu et al., 2008; Marsolek et al., 2010). Due to its use as polymerization acceler- ator for the manufacture of bone cement and dental materials, N,N-dimethyl-p-toluidine has been identified in a variety of commonly used bone cements at concentrations of about et_al., 1997). Exposure from bone cement is thought to have been responsible for sensitiza- tion in some patients (Haddad et al., 1996). Two cases of the accidental ingestion of arti- ficial fingernail solutions containing unspecified concentrations of N,N-dimethyl-p-toluidine have been reported. In the first case, a child aged 16 months drank 15 mL of solution (estim- ated to contain about 60 mg of N,N-dimethyl- p-toluidine or 6 mg/kg body weight [bw]) and on admission to hospital, methaemoglobin was 43% (normal value, < 2%) (Potter et al., 1988). In the second case, a baby aged 5 months ingested 30 mL of solution and after 1 hour, methaemo- globin was 11%. The children recovered after the administration of methylene blue and oxygen (Kao et al., 1997). 1.4.5 Exposure assessment and biological markers No studies to assess exposure in humans by measuring N,N-dimethyl-p-toluidine or its metabolites in the blood or urine were available to the Working Group. 1.5 Regulations and guidelines No specific occupational exposure limit has been reported for N,N-dimethyl-p-toluidine, but this chemical can cause methaemoglobinaemia. The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area established guidance values for methaemoglobin-forming amino and nitro compounds in terms of an umbrella regula- tion. Methaemoglobin values > 1.5% indicate that workers are exposed to aromatic amino and nitro compounds, and the Commission has recommended a level of 5% as a “ceiling” (Leng & Bolt, 2006). [The magnitude of exposure to N,N-dimethyl-p-toluidine leading to this level has not yet been specified.] 173 IARC MONOGRAPHS - 115 According to the risk phrases of the Globally at 6 mg/kg bw, 27/50 at 20 mg/kg bw (P < 0.05) Harmonized System of

2.1 Classification of the substance or mixture

committee” comprising 15 employees. They classified all jobs into three categories: “low” exposure (no direct contact with liquids, workplace concen- trations of N,N-dimethylformamide in air consistently below 10 ppm and no odour of N,N-dimethylformamide), “moderate” exposure (intermittent contact with liquids containing morethan5%N,N-dimethylformamideandwork- place concentrations of N,N-dimethylformamide in air occasionally (more than once a week) above 10 ppm) and “high” exposure (frequent contact with liquids containing more than 5% N,N-dimethylformamide, workplace concen- trations of N,N-dimethylformamide in air often above 10 ppm and breathing protection often required for 15-60 min). Monitoring data on N,N-dimethylformamide were not available for the period; exposure was estimated for 1950-70 and exposure intensity for each job was assumed to be constant during this time period. In a nested case-control study in four plants (Walrath et_al., 1989), a much more detailed exposure assessment was performed. Data on air measurements of N,N-dimethylformamide 123 vel Table 1.2 Occupational exposure to N,N-dimethylformamide VI DY Country Workplace Airborne exposure Urinary concentration Reference DMF (mg/m?) total NMF (mg/L) Plants Industry Type of work No. Mean Range No. Mean Range (No.) Personal air sampling China 7 Leather production 43 27.7 Cai et al. (1992) SHdVYDONOW Polyurethane production 65 11.9 - Shoe sole production 17 2.1 SLL Laboratory A 23 1.2 Laboratory B 59 0.6 Leather printing 52 7.6 Resin production 59 1.8 Germany 1 Polyacrylic fibre 63 5.3> ND-485.7. 92 13.1 ND-108.7 _Kafferlein et al. (2000) Germany 1 Polyacrylic fibre Total 118 12.5 ND-115.2) 125 4,7>« 0.4-62.3° Wrbitzky & Angerer Wet-spinning 30 22.2 0.9-115.2 30 8.9% 0,4-54,0° (1998) Dry-spinning 25 19.5 2.4-112.2 28 6.7>« 0.9-62.3¢ Finishing 51 4.3 ND-41.6 55 3.0b« 0.6-19.9¢ Dyeing 12 7.6 0.3-29.8 12 5.5% — 0,.8-17.2¢ Italy 2 Synthetic leather 100 22.0 8.0-58.0 Cirla et al. 1984) Italy 1 Synthetic leather 125 13.5 0.4-75.2 125 17.14 1.5-114.2 — Imbrianietal. (2002) Japan 1 Synthetic resin Blending 3 7.042 6.1-9.1 Sakai et al. 1995)f Kneading 3 14.6% — 6.1-24.3 Extruding 3 14.94¢ 12.2-18 Ageing 3 4, 3c¢ 3.0-9.1 Total 10 24.7° Japan 1 Synthetic resin Summer 128 5.24 128 4,14 Miyauchiet al. (2014) Winter 142 3.04 142 1.4 Table 1.2 (continued) Country Workplace Airborne exposure Urinary concentration Reference DMF (mg/m?) total NMF (mg/L) Plants Industry Type of work No. Mean Range No. Mean Range (No.) Republic of 15 Fibre coating 57 14.9 0.3-48.9 57 17.54 0.4-97.9 Yang et al. (2000) Korea Synthetic leather 108 25.84 4.0-168.7 108 32.84 0.8-235.8 Paint 13 3.54 0.3-22.5 13 4.24 1.2-12.7 Synthetic fibre 116 2.44 0.3-32.2 116 8.34 1.2-91.4 Synthetic leather 4 2.14 0.6-11.6 4 7.84 0.4-54.9 Synthetic fibre 2 0.54 0.3-1.0 2 3.74 Fibre coating ll 3.74 0.5-21.0 ll 4,44 0.4-19.5 Synthetic leather 19 13.04 2.1-49.2 19 15.84 4.7-65.4 Paint 5 1.24 0.3-8.1 5 3.24 0.4-10.8 Republic of 9 Synthetic leather 116 26.84 143 47.54 Kim et al. (2004) Korea Taiwan, China 1 Synthetic leather and 176 35.3 0.3-263.3 Luo et al. (2001) resin Taiwan, China 1 Synthetic leather 12 34.7 12 17.9 Chang et al. (2004a) Taiwan, China 4 Various Total 75 4.64 0.1-58.9 75 0.474 0.03-104.4 Chang et al. (2004b) Synthetic acrylic fibre 23 Pale 0.1-17.5 23 0.094 —-0.03-3.20 factory Synthetic leather factory 8 19,94 10.5-58.9 8 4.954 1.32-21.01 A Synthetic acrylic leather 24 13.34 2.4-50.7 24 14.34 4.9-104.4 factory B Circuit board factory 20 1.74 0.3-29.9 20 0.054 —-0.03-1.20 Taiwan, China 4 Synthetic leather 59 12.44 1.6-155.3 20.84 1.35-178.6 Wang etal. (2004) Taiwan, China 1 Synthetic leather 65 34.9 2.2-104.8 Chang et al. (2005b) Taiwan, China 1 Synthetic leather 13 32.2 20.2-104.8 13 13.8> 7.5-73.7 Shieh et al. (2007) SCL SPILULWOJ|AYJOWIG-N'‘N 9CL Table 1.2 (continued) VI DY Country Workplace Belgium 1 Synthetic acrylic fibre Airborne exposure Mixer 54 32.7 Urinary concentration Reference Lauwerys et al. SHdVYDONOW DMF (mg/m?) total NMF (mg/L) Plants Industry Type of work No. Mean Range No. Mean Range (No.) Air sampling in the workplace Spinning 54 13.4 (1980) - China 2 Synthetic resin Resin line 12 15.04 5.4-30.3 He et al. (2010) SLL Control line 4 Pas 2.1-4.7 Synthetic leather Mixing 3 46.74 42.5-51.2 Wet process 9 49,14 13.1-199.8 Dry process 6 or 14.9-72.4 Laboratory 7 ja 1.1-7.7 Product 3 4,34 1.3-17.2 inspection Administration 4 ese ND-2.2 Italy 1 Synthetic leather Washing 10 21.54 5-40 Fiorito et al. 1997) Production 22 18.74 2-35 4 Post-shift urine samples > Median © mg/g creatinine ¢ Geometric mean * Stationary sampling f Temperature of the injector port < 250 °C for biomonitoring/method unclear DMF, N,N-dimethylformamide; ND, not detected; NMF, N-methylformamide and urinary concentrations of N-methylforma- mide were used whenever possible. For plant A, 167 personal N,N-dimethylformamide samples (1979-86), 107 urinary N-methyl- formamide samples (1975-79) and 14 N,N- dimethylformamide area samples (1975-80) were available. For plants B and C, respectively, 2916 and 2718 personal or area air samples of N,N-dimethylformamide (1974-86) were avail- able; ~61 000 urinary (1980-86) and ~12 000 air N-methylformamide measurements (1975-86) were used for the exposure assessment. For plant D, 2361 N,N-dimethylformamide samples (1974-86) and 2037 N-methylformamide samples (1978-1986) were available. Two estimates of 8-hour time-weighted average (TWA) exposure to N,N-dimethylformamide (average and peak exposure) were assigned to each job title based on the measurements; however, the final exposure classification was similar to the classification for the cohort study described in Chen et al. (1988a). Jobs were ranked as having “low” (1.0-< 2.0 ppm of N,N-dimethylformamide in air), “moderate” (2.0-< 10.0 ppm) and “high” (© 10.0 ppm) exposure. Controls were ranked as “none” or “present” if not exposed or exposed to < 1 ppm of N,N-dimethylformamide in air, respectively. Contrary to the retrospective cohort study, a job could be ranked differently over time because of the use of time-resolved monitoring data. 1.5 Regulations and guidelines Depending on the country, 8-hour TWA occupational exposure limits have been set at 15 mg/m? (5 ppm) or 30 mg/m? (10 ppm) for N,N-dimethylformamide in the air (GESTIS 2015). For example, the permissible exposure limit issued in the USA by the Occupational Safety and Health Administration is 30 mg/m’, whereas the threshold limit value recommended by the Scientific Committee for Occupational Exposure Limits of the European Union is 15 mg/m? (SCOEL, 2006; GESTIS, 2015). Safety and Health Administration label N,N- dimethylformamide with a skin notation (SCOEL, 2006; OSHA, 2016). As a consequence, biological limit values, in terms of measuring metabolites of N,N-dimethylformamide in urine, have been recommended to take into account all routes of exposure (inhalation, dermal, oral). The Scientific Committee for Occupational Exposure Limits recommends a_ biological limit value of 15 mg N-methylformamide per litre urine in end-of-shift samples, based on the association between exposure to 5 ppm airborne N,N-dimethylformamide and excre- tion of N-methylformamide in urine at the end of shift (GSCOEL, 2006). Several countries of the European Union list N,N-dimethylformamide as a reproductive toxicant. For example, in Germany, N,N-dimethylformamide is categorized in group B of reproductive toxicants because embryonic or fetal damage cannot be excluded if women are exposed to the compound during pregnancy, even when the respective threshold limit values are observed (DFG, 2015). According to the risk phrases of the Globally Harmonized System of and Labelling of Chemicals of the United Nations, N,N-dimethylformamide is harmful if inhaled (H332) and if skin contact occurs (H312). It also can cause serious eye irritation (H319) and may damage fertility or the unborn child (H360D) (ECHA, 2016). N,N-Dimethylformamide has recently been categorized as a group 4 carcinogen by the Commission for the Investigation of Health Hazards at the Workplace in Germany (DEG, 2015). EPA (1990) promulgated a reference concen- tration for N,N-dimethylformamide in the air of 0.03 mg/m. 127 IARC MONOGRAPHS - 115 2. Cancer in Humans repairs, the men with a diagnosed cancer (five seminomas and two embryonal cell cancers) had long histories of aircraft repair before their 2.1 Aircraft repair diagnosis. See Table 2.1 Naval air rework facilities (dedicated to mili- The human carcinogenicity of N,N-dimethyl- tary aircraft repair) presented opportunities for formamide was questioned by Ducatman et al. repair workers to be exposed to surface coatings (1986) beginning with an investigation ofa cluster and associated emulsifiers and surfactants of three cases of testicular germ cell cancer that including “Teflon” paints and dyes, solvents and occurred between 1981 and 1983. The three metals (Ducatmanetal., 1986). Arelatively uncon- testicular cancers (< 1 expected) occurred among trolled process that was unique to F4 Phantom 153 white civilian repairmen who were exposed repair at the two facilities involved open air to N,N-dimethylformamide while repairing the spraying of an 80% N,N-dimethylformamide exterior surfaces and exterior electrical compo- solution onto in-situ electrical cables in quanti- nents of the F4 Phantom jet aircraft at a single ties sufficient to dissolve the elastomeric surface United States naval air rework facility in North coatings of the cables, with the goal of performing Carolina, USA. The finding led to an evaluation needed maintenance on the bare cables in place. of testicular cancer in two groups of civilian The process was open and no specific ventilation aircraft repair workers, which was reported in the was employed: it was reported to involve substan- same paper. The two groups of workers were at a tial inhalation and dermal exposure. At the time United States naval air rework site in California. of the investigation, the process had been out of Similar to the North Carolina workers, the first use for more than a decade. A formal exposure of these groups also repaired the F4 Phantoms model was not created. Among the seven cases among other naval aircraft and were exposed to at both facilities (age range, 30-46 years), three N,N-dimethylformamide, while workers in the reported direct responsibility for the setting up second group performed re-work on a variety of and use of this process and the others recalled naval aircraft, but not the F4 Phantoms, and were that they were exposed in proximity. [The not exposed to N,N-dimethylformamide. At the Working Group was aware that this group of California facility, four of the 680 men with a military aircraft mechanics had respiratory and history of working on F4 phantoms were diag- dermal exposure to N,N-dimethylformamide. nosed with testicular germ cell cancers. The men Limitations of the study included the lack of in this groups of exposure workers had an esti- quantitative exposure measurements and the mated 19 040 person-years of experience from fact that follow-up for cancer incidence included 1970 to 1983, and that finding represented only the years when the workers were employed.| an apparent increase of fourfold [4.21; 95% The above findings in civilian repairmen confidence interval (CI), 1.15-10.78] in inci- working for the United States Navy motivated an dence compared with national incidence rates evaluation of testicular cancer hospitalizations of (P < 0.02). [It was not clear whether the expected United States active duty military personnel from numbers reported in the paper were age-ad- 1974 to 1979 (Garland et al., 1988). This study justed.] In contrast, none of the 446 men in the found 143 incident cases of testicular cancer in unexposed population (who did not repair the currently active duty personnel. No excess risk F4 Phantom) had a diagnosis of testicular germ was observed overall but, among aviation support cell cancer. Within the North Carolina and technicians, the standardized incidence ratio California populations involved in F4 Phantom was 6.2 (95% CI, 1.9-13.0; 5 cases). [The Working 128 Table 2.1 Cohort studies of cancer and exposure to N,N-dimethylformamide Reference, location Population size, Organ site Exposure Exposed __ Risk estimate Covariates Comments enrolment/follow-up description, exposure category or cases/ (95% CI) controlled period assessment method level deaths Ducatman et al. (1986) 680 exposed, 446 not Testis Allexposed 4 [4.21 (1.15-10.78)] NR Strengths: the investigation Naval air rework facility exposed; male civilian workers was prompted by a specific North Island (the 2nd employees at a US naval air hypothesis related to a facility investigated, rework facility cluster investigation at which did not contain Exposure assessment a previous facility; there the original cluster),CA, method: direct was a nested case-control USA observation; onsite survey, comparison to men who had 1970-1983 linkage to local cancer similar jobs but never used registry, outreach to local the same process physicians and hospitals, Limitations: statistical and review of medical comparison was to records national SEER data; only active workers included; the implicated exposure process had stopped before the investigation, and no exposure model was created CDC (1989) 80 male workers; males Testis Allworkers 3 40.5 (8.1-118.4) Age, sex, Exposure monitoring was Fulton County, NY, USA employed in the finishing in the period, performed after the relevant 1 January 1975 to 31 line of a leather tannery cluster location exposure had ceased; a December 1987 from 1975-1987 plant historical survey attempted Exposure assessment to assess the previous method: environmental history of high exposure by monitoring; medical interrogating the presence of record confirmed cases alcohol intolerance during and linkage to the New the period of the DMF York State cancer registry operation Strengths: additional follow-up of Fulton County testicular cancer cluster Limitations: statistical calculations include the initial cancer cluster; the exposure monitoring did not include the process of interest 6cL SPILULWOJ|AYJOWIG-N'‘N O€L VI Table 2.1 (continued) DY Reference, location Population size, Organ site Exposure Exposed __ Risk estimate Covariates Comments SHdVYDONOW enrolment/follow-up description, exposure category or cases/ (95% CI) controlled period assessment method level deaths Chen et al. (1988a) 3859 total: 2530 DMF Upper All DMF 11 [1.67 (0.83-2.98)] Age, sex, SRRs were generally Fibre-producing only; 1329 to DMF and aerodigestive cohort period elevated, but not statistically industrial facility, USA —_ acrylonitrile; and 1130 tract: buccal significant for the DMF-only 1 January 1956 to 31 with exposures to neither. cavity & cohort December 1984 Data are tabulated for the — pharynx Strengths: cancer incidence - cohort with any exposure Allcancers All DME 88 [0.92 (0.73-1.13)] study; exposure matrix SLL to DMF; male workers combined cohort Limitations: exposure with potential exposure Prostate AIlLDME 10 [1.92 (0.92-3.54)] measurements only after to DMF between 1950 and cohort 1970 Malignant All DMF 7 [1.55 (0.56-3.21)] Exposure assessment melanoma cohort method: assessment by employee committee; Lung All DMF 21 [0.97 (0.60-1.48)] corporate cancer registry, cohort eens. Testis AIDMF 1 [0.59 (0.01-3.28)] Upper Low/ 5 [4.17 (1.35-9.72)] aerodigestive moderate tract: buccal DMF cavity & High DMF 6 [3.0 (1.10-6.5)] pays a 4 [3.64 (0.99-9.31)] DMF exposure > 5 yrs 7 [3.18 (1.28-6.56)] DMF exposure Table 2.1 (continued) Reference, location Population size, Organ site Exposure Exposed __ Risk estimate Covariates Comments enrolment/follow-up description, exposure category or cases/ (95% CI) controlled period assessment method level deaths Chen et al. (1988b) See incidence study by All cancers All DMF 75 [0.98 (0.77-1.23)| Age, sex, Same population and Fibre-producing Chen et al. (1988a) combined cohort period, methods as Chen et al. industrial facility, USA | Male workers with DMF only 38 [0.95 (0.67-1.30)| location (1988a) 1 January 1950 to 31 potential exposure to DMF cohort Strengths: local worker December 1982 between 1950 and 1970 Upper All DME 3 [1.88 (0.39—4.48)] comparison group Exposure assessment aerodigestive cohort Limitations: mortality only; method: assessment by tract: buccal DMFonly 2 [2.5 (0.30-9.0)] no testicular cancer deaths employee committee; cavity & cohort were reported (mortality is corporate mortality data pharynx not the optimal end-point Lung AILDME 33 [1.24 (0.85-1.74)] for investigating this cancer) cohort Lung DMF-only 19 [1.40 (0.85-2.20)] cohort CI, confidence interval; DMF, N,N-dimethylformamide; NR, not reported; SEER, Surveillance, Epidemiology and End Results; SRR, standardized relative risk; US, United States; yr, year LEL SPILULWOJ|AYJOWIG-N'‘N IARC MONOGRAPHS - 115 Group considered it unlikely that active duty reported in the Levin et al. (1987) report were aviation support technicians in general would included, as well as an additional seven cases. have had significant exposure to the unique A group of 129 male controls of similar age process involving N,N-dimethylformamide.] who had other cancer diagnoses from the same registry was used. Five of the 10 cases had histo- ries of leather-related employment, resulting in 2.2 Leather workers an odds ratio of 5.8 (95% CI, 1.5-22.0) (CDC, See Table 2.1 and Table 2.2 1989). [The Working Group noted that the inclu- Levin et_al. (1987) reported a cluster of sion a posteriori of an initially detected disease three cases of testicular germ cell cancer in cluster within a small population calculation can workers who worked on a spray line in a leather contribute to an expected outcome of an elevated tannery (Fulton County, NY, USA) where standardized incidence ratio.] N,N-dimethylformamide was used. They were Workers from the index tannery workplace aged 25-36 years at the time of diagnosis, which were also enrolled in a prospective testicular occurred between 1982 and 1984, and had cancer screening programme. The National worked for 8-14 years on the spray line before Institute of Occupational Safety and Health diagnosis. Other than N,N-dimethylformamide investigators reported that 51 out of 83 workers and “dyes”, this report did not discuss other participated and, as of 1990, no additional potential exposures. cases of testicular cancer had been found In a further investigation, a team including (Calvert et al., 1990). personnel from the United States National Institute of Occupational Safety and Health 2.3 Chemical manufacturing performed air sampling of the spray process for partially tanned hides at the same facility (CDC, 2.3.1 Cohort study 1989), At the time of the investigation, the facility had ceased to use N,N-dimethylformamide in See Table 2.1 A major chemical manufacturer conducted the tanning process and therefore none was retrospective cohort cancer incidence (Chen etal., detected in air sampling. This investigation estab- 1988a) and cancer mortality (Chen et al., 1988b) lished that N,N-dimethylformamide had been studies of workers at an acrylic-fibre production used historically in the spraying process. The plant in the USA whose employment provided exposures identified included dyes, pigments, exposure to N,N-dimethylformamide alone surface coatings and _ solvents, including (2530 workers), acrylonitrile alone (16 workers), N,N-dimethylformamide. Sampling after the neither (1130 workers) or both (1329 workers). N,N-dimethylformamide process had stopped The two groups with any exposure to detected several glycol ethers, including 2-but- N,N-dimethylformamide were then combined oxyethanol, at up to 10.9 ppm. A cohort study into a single historical cohort (3859 workers). of testicular cancer in 80 workers in the index Monitoring data were reportedly unavailable tannery found a standardized incidence ratio for most of the historical period (1950-1970) of 40.5 (95% CI, 8.1-118.4) compared with state Surveillance, Epidemiology and End Results data. considered in these reports, and exposures were In addition, this report featured a case-control classified as “low”, “moderate” or “high” by a study, performed at the county level, of testicular committee of workers. Diagnostic information cancer cases in male residents aged 20-54 years on cancer was obtained from the manufactur- from January 1974 to March 1987. The three men er’s internal cancer registry and was therefore 132 Table 2.2 Case-control studies of cancer and exposure to N,N-dimethylformamide Reference, Population size, Organ site Exposure Exposed Riskestimate | Covariates Comments location description, exposure category or cases/deaths controlled enrolment/ assessment method level follow-up period CDC (1989) Cases: 10; New York _ Testis Leather 5 5.8 (95% CI, Date, year of Strengths: added Fulton County, State cancer registry tannery 1.5-22.0) diagnosis information (two NY, USA Controls: 129; New workers additional cases) to earlier Testicular cancer York State cancer cluster investigation cases from registry Limitations: the original county-level Exposure assessment cluster was included in the analysis of New method: based on case numerator York State cancer _ recorded usual registry data occupation at diagnosis Walrath et al. Cases: 39 buccal Liver/ Minimum NR 6.10 (90% CI, Age, sex, plant, Strengths: improved (1989) cavity & pharynx, hepatocellular latency 10 yrs 0.38-72.00) pay class description of JEM for this Four industrial 6 liver, 43 prostate, carcinoma employer facilities of one 11 testis, 39 skin; Liver/ > 10 yr NR 2.74 (90% CI, Age, study site, Limitations: exposure employer employer cancer hepatocellular duration 0.16-47.3) pay class, yearof | Monitoring data not 1956-1985 registry carcinoma diagnosis, age at available for the earlier Controls: 276; other diagnosis years: extrapolations were workers from cohort Upper All categories 15 0.89 (90% CI, Age, sex, plant, made Exposure assessment aerodigestive 0.35-2.29) pay class method: expert — tract: buccal assessment; existing cavity & monitoring data were pharynx ch aracterized by job Prostate All categories 17 1.47 (90% CI, Age, sex, plant, title, for mean and 0.66-3.30) ay class peak DMF; a facility ; , " pay history was developed Malignant Allcategories 16 1.70 (90% CI, Age, sex, plant, for each of four melanoma 0.52-5.51) pay class facilities Testis Allcategories 3 0.99 (90% CI, Age, sex, plant, of exposure 0.22-4.44) pay class Testis Moderate 2 11.60 (90% CI, Age, study site, exposure 0.47-286) pay class, year of only diagnosis, age at diagnosis CI, confidence interval; DMF, N,N-dimethylformamide; JEM, job-exposure matrix; NR, not reported; SEER, Surveillance, Epidemiology and End Results; US, United States; yr, year cel SPILULWOJ|AYJOWIG-N'‘N IARC MONOGRAPHS - 115 limited to the time of employment. Person-years not appear to pertain to the aircraft repair and were calculated from first exposure or first leather operations previously described.] employment from 1956 to 1984. Comparisons of corporate registry data were then made to 2.3.2 Case-control study national Surveillance, Epidemiology and End Results cancer registry data for 1973-1977. See Table 2.2 Thirty-four incident cases of all types of The same chemical manufacturer subse- cancer were recorded in the cohort of wage quently performed a broader case-control study workers exposed to N,N-dimethylformamide on exposure to N,N-dimethylformamide among only (Chen et_al., 1988a). Only one case of male active employees at four of its manufac- turing facilities including the one studied by testicular cancer was found compared with Chen et al. (1988a, 1988b), from 1956 to 1985, 1.7 expected [standardized relative _ risk, based on cancer cases reported to the company 0.59; 95% CI, 0.01-3.28] in the cohort exposed cancer registry (Walrath et al., 1989). The study to N,N-dimethylformamide only. Buccal cavity population included only cases identified among and pharyngeal cancers (combined) were signif- currently employed workers. For each case, two icantly increased (8 observed, 1 expected [stand- controls from the entire cohort were matched to ardized relative risk, 8.00; 95% CI, 3.45-15.76]) the case worker based on year of employment, inthecohortexposedtoN,N-dimethylformamide year of birth, sex, plant, and payroll/salary class. only, but not in the cohort exposed to both The study provided a more detailed exposure N,N-dimethylformamide and acrylonitrile. No assessment than that used in the preceding dose-response relationship was observed related cancer incidence and mortality studies (Chen to the classified exposure level. et_al., 1988a, 1988b), including measurements Chen et al. (1988b) used the same methods of N,N-dimethylformamide in the air and its to study mortality from all causes and cancer in urinary metabolites. Average and peak expo- the same population. Compared with internal sures were considered. When data were not company rates, non-statistically significant available, an extrapolation was made using excesses of buccal cavity and pharyngeal cancers exposure data from later years. The exposure (combined) and lung cancer were reported (Chen classifications were more comprehensive but et al., 1988a). [The exposure characterization for ultimately similar to those used in the cohort this N,N-dimethylformamide-manufacturing study (Chen et al., 1988a): “low”, “medium”, cohort was more complete than that in the “high” and none. The geometric mean concen- previously cited studies in aircraft repair and tration of N,N-dimethylformamide in the air leather work and provides an impression ranged from 1 to 2 ppm [3-6 mg/m] for jobs in that exposures were better controlled than the low exposure group to > 10 ppm [30 mg/m?] those for aircraft and leather workers. The for jobs in the high exposure group. Air concen- N,N-dimethylformamide solution at the manu- trations > 50 ppm [149 mg/m] were possible in facturing sites is reported to have generally been the high exposure category. Mantel-Haenszel up to 5% in concentration and intermittently and multiple logistic regression methods were > 5% —- less concentrated than the 80% solutions used to calculate odds ratios. The odds ratio used by aircraft repairmen and leather workers - for testicular cancer for ever being exposed was andtheobservationthatrespiratorsweresupposed 0.99 (90% CI, 0.22-4.44; 3 exposed cases). to have been used if atmospheric concentrations Odds ratios for testicular cancer by level of were > 10 ppm of N,N-dimethylformamide does exposure were 0.86 (90% CI, 0.09-8.56) for low 134 and 11.6 (90% CI, 0.47-286; 2 exposed cases, 2 exposed controls) for moderate exposure. No cases occurred in the high exposure cate- gory. The same data set was also considered with regard to different durations of exposure. The odds ratio for testicular cancer was 1.28 (90% CI, 0.14-7.12) for those with < 10 years exposure and no cases occurred among those with = 10 years exposure. Buccal cavity and phar- yngeal cancer were not found to be significantly increased when all facilities were combined, nor were any other studied cancer sites. [The Working Group noted that interpretation of these studies was limited by the small numbers of testicular cancer cases and by the restriction to cases occurring among active workers.| 3. Cancer in Experimental Animals N,N-Dimethylformamide was first reviewed by the IARC Monographs Working Group (IARC, 1989) when it had been tested for carcinogenicity by oral administration and by subcutaneous injection in one rat strain (Druckrey et al., 1967) and by intraperitoneal injection in another strain (Kommineni, 1972). All of these studies were judged to be inadequate for an evaluation and were still considered to be inadequate by the current Working Group because of the small number of animals, the short duration of treatment and the incom- plete reporting of the results. N,N-Dimethyl- formamide was subsequently reviewed by a second Working Group (IARC, 1999) when it had also been tested for carcinogenicity by inha- lation in one study in mice and one study in rats, with no significant increase in tumour incidence (Malley et al., 1994). Studies that were judged to be adequate for an evaluation (including studies released since the previous IARC Monographs) are summa- rized below. 3.1 Mouse See Table 3.1 Inhalation Groups of 78 male and 78 _ female Crl:CD-1 (ICR) BR mice (age, 55 days) were exposed to N,N-dimethylformamide (purity, 99.9%) by whole-body inhalation at concen- trations of 0, 25, 100, and 400 ppm for 6 hours per day on 5 days per week for 18 months. Five male and five females per group were killed at 2 weeks, 3 months and 12 months to evaluate cell proliferation in the liver. Survival in all exposure groups was similar to that of controls. Higher body weights and body-weight gain were observed in the male and female mice exposed to 100 and 400 ppmcompared withcontrolsand were consid- eredtobecompound-related. Themalesexposedto 100 and 400 ppm and females exposed to 400 ppm had higher liver weights relative to body weight at necropsy. No significant increase in tumour incidence was observed in any dose group. All treated males had significant increases in centrilobular hepatocellular hypertrophy and hepatic single cell necrosis. Centrilobular hepa- tocellular hypertrophy was also significantly increased in the females exposed to 100 and 400 ppm and hepatic single cell necrosis in all treated females (Malley etal., 1994). [The Working Group noted the short duration of the study.] In a well-conducted study that complied with good laboratory practice, groups of 50 maleand 50 female Crj:BDF, mice (age, 6 weeks) were exposed to N,N-dimethylformamide (purity, > 99.8%) by whole-body inhalation at concentrations of 0, 200, 400, and 800 ppm for 6 hours per day on 5 days per week for 104 weeks. No significant difference in survival was observed between the exposed groups and the controls. The survival rate of females exposed to 800 pm decreased margin- ally (but not significantly) after the 78th week because of the development of liver tumours. 135 9EL Table 3.1 Studies of carcinogenicity with N,N-dimethylformamide in mice and rats VI DY Study design Route Incidence of tumours Significance Comments Species, strain (sex) Purity Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals SHdVYDONOW Full carcinogenicity Inhalation Liver or testis Principal limitations: short duration of the study - Mouse, Crl:CD-1 Purity, 99.9% No increase in tumour NS 5 males per group were killed at 2 wks, 3 mo, SLL (ICR) BR (M) NA incidence and 12 mo, for evaluation of cell proliferation in 55 days 0, 25, 100, 400 ppm the liver 18 mo 6 h/day, 5 days/wk Malley et al. (1994) 78, 78, 78, 78 56%, 68%, 60%, 59% Full carcinogenicity Inhalation Liver or mammary gland Principal limitations: short duration of the study Mouse, Crl:CD-1 Purity, 99.9% No increase in tumour NS 5 males and 5 females per group were killed at (ICR) BR (F) NA incidence 2 wks, 3 mo, and 12 mo, for evaluation of cell 55 days 0, 25, 100, 400 ppm proliferation in the liver 18 mo 6 h/day, 5 days/wk Malley et al. (1994) 78, 78, 78, 78 68%, 57%, 62%, 76% Full carcinogenicity Inhalation Liver Principal strengths: well-conducted study that Mouse, Crj:BDF1 (M) Purity, > 99.8% Hepatocellular adenoma: Positive trend: P < 0.01, Peto’s appears to have complied with GLP 6 wks NA 6/50, 36/50*, 41/49*, 41/50* test; 104 wks 0, 200, 400, 800 ppm *P < 0.01, Fisher exact test Senoh etal. (2004) 6 h/day, 5 days/wk 50, 50, 50, 50 Hepatocellular carcinoma: Positive trend: P < 0.01, Peto’s 37, 33, 37, 40 2/50, 12/50*, 16/49*, 16/50* test; *P < 0.01, Fisher exact test Hepatoblastoma: 0/50, 13/50*, *P < 0.01, Fisher exact test 7149*, 4/50 Hepatocellular adenoma, Positive trend: P < 0.01, Peto’s hepatocellular carcinoma or test; hepatoblastoma (combined): *P < 0.01, Fisher exact test 8/50, 42/50*, 46/49*%, 44/50* Table 3.1 (continued) Study design Route Incidence of tumours Significance Comments Species, strain (sex) Purity Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Inhalation Liver Principal strengths: well-conducted study that Mouse, Crj:BDF1 (F) Purity, > 99.8% Hepatocellular adenoma: Positive trend: P < 0.01, Peto’s appears to have complied with GLP 6 wks NA 1/49, 42/50*, 47/50*, 48/49* test; 104 wks 0, 200, 400, 800 ppm *P < 0.01, Fisher exact test Senoh etal. (2004) 6 h/day, 5 days/wk 49, 50, 50, 49 Hepatocellular carcinoma: Positive trend: P < 0.01, Peto’s 29, 30, 21, 22 3/49, 25/50", 32/50*, 35/49* test; *P < 0.01, Fisher exact test Hepatoblastoma: 0/49, 0/50, NS 4/50, 0/49 Hepatocellular adenoma, Positive trend: P < 0.01, Peto’s hepatocellular carcinoma or test; hepatoblastoma (combined): *P < 0.01, Fisher exact test 3/49, 45/50*, 49/50*, 49/49* Full carcinogenicity Inhalation Liver, testis or mammary gland Principal limitations: survival for all dose Rat, Crl:CD BR (M) Purity, 99.9% No increase in tumour NS groups and controls was < 45% 47 days NA incidence 5 males per group were killed at 2 wks, 3 mo, 2 yrs 0, 25, 100, 400 ppm and 12 mo for evaluation of cell proliferation Malley et al. (1994) 6 h/day, 5 days/wk in the liver; 10 males per group were killed for 87, 87, 87, 87 interim pathological evaluation at 12 mo 27%, 34%, 40%, 44% Full carcinogenicity Inhalation Liver or mammary gland Principal limitations: survival for all dose Rat, Crl:CD BR (F) Purity, 99.9% No increase in tumour NS groups and controls was less than 40% 47 days NA incidence Five females per group were killed at 2 2yr 0, 25, 100, 400 ppm wks, 3 mo, and 12 mo for evaluation of cell Malley et al. (1994) 6 h/day, 5 days/wk proliferation in the liver; 10 females per group 87, 87, 87, 87 were killed for interim pathological evaluation 35%, 23%, 19%, 39% at 12 mo LEL SP!WEWOJ|AYJOWIG-N'‘N 8El VI Table 3.1 (continued) DY Study design Route Incidence of tumours Significance Comments Species, strain (sex) Purity Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals SHdVYDONOW Full carcinogenicity Inhalation Liver Principal strengths: well conducted study that - Rat, F344/DuCrj (M) Purity, > 99.8% Hepatocellular adenoma: Positive trend: P < 0.01, Peto’s appeared to comply with GLP SLL 6 wks NA 1/50, 3/50, 13/50*, 20/50* test; The authors stated that multiple occurrences 104 wks 0, 200, 400, 800 ppm *P < 0.01, Fisher exact test of hepatocellular tumours were found in the Senoh et al. (2004) 6 h/day, 5 days/wk liver of DMF-exposed rats, in contrast to the 50, 50, 50, 50 occurrence of a single tumour in the liver of the Hepatocellular carcinoma: Positive trend: P < 0.01, Peto’s 42, 38, 40, 37 animals of the control group 0/50, 1/50, 0/50, 24/50* test; *P < 0.01, Fisher exact test Hepatocellular adenoma or Positive trend: P < 0.01, Peto’s carcinoma (combined): 1/49, test; 4/50, 13/50*, 33/50* *P < 0.01, Fisher exact test Full carcinogenicity Inhalation Liver Principal strengths: well-conducted study that Rat, F344/DuCrj (F) Purity, > 99.8% Hepatocellular adenoma: Positive trend: P < 0.01, Peto’s appeared to comply with GLP 6 wks NA 1/49, 1/50, 6/50, 16/50* test; The authors stated that multiple occurrences 104 wks 0, 200, 400, 800 ppm *P < 0.01, Fisher exact test of hepatocellular tumours were found in the Senoh et al. (2004) 6 h/day, 5 days/wk liver of DMF-exposed rats, in contrast to the 50, 50, 50, 50 occurrence of a single tumour in the liver of the Hepatocellular carcinoma: Positive trend: P < 0.01, Peto’s 42, 38, 38, 30 animals of the control group 0/49, 0/50, 0/50, 5/50* test; *P < 0.05, Fisher exact test Hepatocellular adenoma or Positive trend: P < 0.01, Peto’s carcinoma (combined): 1/49, test; 1/50, 6/50, 19/50* *P < 0.01, Fisher exact test d, day; DMF, N,N-dimethylformamide; F, female; GLP, good laboratory practice; M, male; mo, month; NA, not applicable; NS, not significant; ppm, parts per million; wk, week; yr, year Body weights of all treated males and of females exposed to 800 ppm were decreased by more than 10% compared with controls. The incidence of hepatocellular adenoma (males: 6/50 controls, 36/50 at 200 ppm, 41/49 at 400 ppm, and 41/50 at 800 ppm; females: 1/49 controls, 42/50 at 200 ppm, 47/50 at 400 ppm, and 48/49 at 800 ppm), hepatocellular carcinoma (males: 2/50 controls, 12/50 at 200 ppm, 16/49 at 400 ppm, and 16/50 at 800 ppm; females: 3/49 controls, 25/50 at 200 ppm, 32/50 at 400 ppm, and 35/49 at 800 ppm) and hepatocellular adenoma, hepato- cellularcarcinomaorhepatoblastoma(combined) weresignificantlyincreasedinallgroupsoftreated males and females (P < 0.01, Fisher’s exact test ) in a dose-related manner (P < 0.01, Peto’s trend test). There was also a significant increase in the incidence of hepatoblastoma in males exposed to 200 and 400 ppm (0/50 controls, 13/50* at 200 ppm, 7/49* at 400 ppm and 4/50 at 800 ppm; * P < 0.01, Fisher’s exact test). The inci- dence of hepatoblastoma in females was 0/49 controls, 0/50 at 200 ppm, 4/50 at 400 ppm and 0/49 at 800 ppm. Multiple hepato- cellular adenomas and carcinomas were found in the livers of treated mice, and the tumour tissues often occupied almost all areas of the entire liver. No N,N-dimethylformamide- related neoplastic or non-neoplastic lesions were found in any other organ except the liver (Senoh et al., 2004). [The strengths of this study included the use of multiple doses, a large number of animals per group and the testing of two sexes.| 3.2 Rat 3.2.1 Inhalation See Table 3.1 Groups of 87 male and 87 female Crl:CD BR rats (age, 47 days) were exposed to N,N-dimethylformamide (purity, 99.9%) by whole-body inhalation at concentrations of 0, 25, per week for 2 years. Five male and five females per group were killed at 2 weeks, 3 months, and 12 months to evaluate cell proliferation in the liver, and 10 males and 10 females per group were killed for interim pathological evaluation at 12 months. Exposure to the highest concen- tration reduced body-weight gain in both sexes. Survival in all treated groups was similar to that of controls; however, survival in all treated groups and controls was less than 45%. The males treated with 100 and 400 ppm and females treated with 400 ppm had higher liver weights relative to body weight at necropsy. No signifi- cant increase in tumour incidence was observed in any treated group. The incidence of minimal to mild centrilobular hepatocellular hyper- trophy was increased in males and females treated with 100 and 400 ppm (Malley et al., 1994). [The Working Group noted the poor survival of all groups, including controls.] In a well-conducted study that complied with good laboratory practice, groups of 50 male and 50 female Fischer 344/DuCrj rats (age, 6 weeks) were exposed to N,N-dimethylformamide (purity, > 99.8%) by whole-body inhalation at concentrations of 0, 200, 400, and 800 ppm for 6 hours per day on 5 days per week for 104 weeks. Survival rates of treated males did not differ significantly from those of controls. Survival of the females treated with 800 ppm was significantly decreased compared with that of controls after the 9th week. Body weights of the males and females treated with 400 and 800 ppm were decreased by more than 10% compared with their respective controls. Exposure to N,N-dimethylformamide caused a dose-related increase (P < 0.01, Peto’s trend test) in the incidence of hepatocellular adenoma, hepatocellular carcinoma and hepa- tocellular adenoma or carcinoma (combined) in males and females. The incidence of hepato- cellular adenoma was significantly increased in male rats exposed to 400 and 800 ppm (1/50 controls, 3/50 at 200 ppm, 13/50” at 400 ppm 139 IARC MONOGRAPHS - 115 Table 3.2 Incidence of hepatocellular tumours in male F344/DuCrlCrj rats exposed to N,N- dimethylformamide by inhalation and/or in drinking-water Parameter Inhalation (ppm) 0 200 400 Drinking-water (ppm) 0 800 1600 0 800 1600 0 800 1600 Number of rats examined 50 Surviving animals 41 34 40 36 41 37 43 38 Adenoma? 1 6* 8* 15* 28** 45** 26* 43** 46** Carcinoma? 0 4* 1 6** 14** 2 12** 14** Adenoma or carcinoma 1 6* 12* 16* 30** 46** 26* 45** 47°* (combined) « Number of tumour-bearing animals * Significantly different from untreated control group, at P <0.05 by chi-square test ** Significantly different from untreated control group, each drinking-water-alone group and each inhalation-alone group with matching concentrations, respectively, at P < 0.05 by chi-square test Adapted from Ohbayashiet al. (2009), J Toxicol Sci, 34(1):53-63. and 20/50* at 800 ppm; *P < 0.01, Fisher's In a well-conducted study that complied exact test) and in females exposed to 800 ppm with good laboratory practice, groups of (1/49 controls, 1/50 at 200 ppm, 6/50 at 400 ppm 50 male Fischer 344/DuCrlCrj rats (age, 6 weeks) and 16/50* at 800 ppm; *P < 0.01, Fisher’s exact were exposed to N,N-dimethylformamide test). The incidence of hepatocellular carcinoma (purity, > 99.5%) by whole-body inhalation was significantly increased in both sexes exposed at concentrations of 0, 200, and 400 ppm for to 800 ppm (males: 0/50 controls, 1/50 at 200 6 hours per day on 5 days per week, and given ppm, 0/50 at 400 ppm, and 24/50* at 800 ppm; N,N-dimethylformamide-formulated — drink- females: 0/49 controls, 0/50 at 200 ppm, 0/50 ing-water at 0, 800, or 1600 ppm (w/w) ad libitum at 400 ppm and 5/50** at 800 ppm; *P < 0.01, for 24 hours per day on 7 days per week for 104 **P < 0.05, Fisher’s exact test). Multiple occur- weeks. No significant difference in survival rences of hepatocellular tumours were found was found between the untreated control in the liver of exposed rats and no multiplicity group and the two inhalation-only groups, the of hepatocellular tumours was observed in the two oral administration-only groups or the control groups. No N,N-dimethylformamide- four combined exposure groups. The terminal related neoplastic or non-neoplastic lesions were body weight was significantly decreased in the found in any other organ except the liver (Senoh inhalation-only group, both oral administra- etal., 2004). [The strengths of this study included tion-only groups and all four combined expo- the use of multiple doses, a large number of sure groups compared with controls. Exposure animals per group, and testing in males and to N,N-dimethylformamide caused a signif- females.| icant increase in the incidence of hepatocel- lular adenoma, and hepatocellular adenoma or 3.2.2 Inhalation and drinking-water carcinoma (combined) in the four combined (combined) exposure groups, the two oral administra- tion-only groups and the two inhalation-only See Table 3.2 groups compared with the untreated controls, a 140 significant increase in the incidence of hepato- cellular carcinoma in the four combined expo- sure groups and in the oral administration-only group exposed to 1600 ppm compared with the untreated controls, and a significant increase in the incidence of hepatocellular adenoma, hepatocellular carcinoma and _ hepatocellular adenoma or carcinoma (combined) in the four combined exposure groups compared with each of the oral administration-only and inhala- tion-only groups with matching concentrations (see Table 3.2 for tumour incidences and statis- tics). The incidence of hepatocellular tumours induced by the combined exposures tended to exceed the dose-response relationship that would be expected under the assumption that the incidence of hepatocellular tumours induced by the single-route exposures through inhala- tion and ingestion are additive [the authors also stated that the combined exposures were found to produce multiple occurrences of hepato- cellular adenomas compared with the single- route exposures but did not show these data] (Ohbayashi et al., 2009). [The strengths of this study included the use of multiple doses and a large number of animals per group. The Working Group noted that only one sex was used and that no mention or discussion was made of any observations in any tissue or organs other than the liver.] 4. Mechanistic and Other Relevant Data 4.1 Absorption, distribution, metabolism, excretion 4.1.1 Humans (a) Absorption, distribution, and excretion N,N-Dimethylformamide is readily absorbed after inhalation, dermal and oral expo- sure. The absorption and elimination of N,N-dimethylformamide was studied after inha- lation of vapour (Mraz & Nohova, 1992a), and after percutaneous absorption of both vapour and liquid (Mraz & Nohova, 1992b). In the inha- lation study (Mraz & Nohova, 1992a), retention of N,N-dimethylformamide in the lungs was 90% in 10 volunteers (5 men and 5 women) exposed for 8 hours to concentrations of 10, 30, or 60 mg/m:>. Aftera single exposure, for the metabo- litesthataccountedfor49% ofthedose, thehalf-lives of urinary excretion and urinary recoveries were: N-methylformamide, 4 hours (22%); formamide, 7 hours (13%); and AMCC, 23 hours (13%). A small amount (0.3% of the dose) of unchanged N,N-dimethylformamide was also detected with a half-life of excretion of 2 hours. With repeated exposure to 30 mg/m? for 8 hours per day on 5 consecutive days, a significant accumulation of AMCC was observed with an equilibrium being reached on day 4. [The Working Group noted that N-methylformamide and formamide, which were determined by gas chromatography, were at least in part formed from corresponding hydroxymethyl derivatives by thermal decom- posure (formaldehyde release) in the hot sample chamber of the gas chromatograph.] After a 4-hour exposure to N,N-dimethyl- formamide vapour (50 mg/m), 13-36% of the total dose (depending on air humidity and temperature) was absorbed through the skin in 8 volunteers (3 men and 5 women). Absorption 141 IARC MONOGRAPHS - 115 of liquid N,N-dimethylformamide was studied of actual occupational exposure by Wang et al. (i) by dipping one hand up to the wrist for 2, 10, (2007) at airborne concentrations below 10 ppm. 15, and 20 minutes; and (ii) by a patch experi- Internal exposure determined by urinary ment, in which N,N-dimethylformamide was N-methylformamide was monitored in two placed on a patch of teflon foil, which was then groups of occupationally exposed workers across attached to the forearm of the volunteers for 8 a working week to determine the total body hours. The rate of percutaneous absorption in burden. Twenty-five workers in a synthetic the dipping experiment was 9.4 + 4 mg/cm? per leather factory and 20 workers in a copper lami- hour (mean + standard deviation (SD); n = 4). In nate circuit board factory were recruited (Chang the patch experiment, the urinary excretion of et al., 2005a). The average airborne concentra- metabolites was not delayed compared with the tion of N,N-dimethylformamide was similar for short-term dipping experiments, but the metab- both groups (about 4 ppm) but dermal expo- olites amounted to only half of those excreted sure to N,N-dimethylformamide of the synthet- after inhalation at the same absorbed dose ic-leather workers was significantly higher. A levels (Mraz & Nohova, 1992b). A similar value significant pattern of linear accumulation was of percutaneous absorption rate (11 mg/cm? found across a 5-day work cycle for synthetic per hour), based on in-vitro experiments on leather workers but not for copper laminate circuit excised human skin, was reported (Bortsevich, board workers. [The Working Group noted that 1984). The same study showed that aqueous dermal exposure to N,N-dimethylformamide N,N-dimethylformamide is poorly absorbed; over 5 consecutive days can result in its accumu- < 1% in 4 hours was absorbed from 15% aqueous lation in the body.] N,N-dimethylformamide, whereas absorption In a recent study of the percutaneous absorp- of pure liquid N,N-dimethylformamide was tion of N,N-dimethylformamide in 193 occupa- 51% in 4 hours (Bortsevich, 1984). Absorption tionally exposed workers, the mean breathing of N,N-dimethylformamide vapour through zone concentrations in both summer and the skin was further evaluated in an experi- winter were below the occupational exposure ment on 13 male volunteers exposed for 4 hours limit of 10 ppm. However, the urinary levels of through whole-body exposure (but inhaling N-methylformamide and AMCC were 2.6 and clean air via a respirator) as well as lung-only 1.6 times higher, respectively, in the summer (at inhalation (at intervals of 96 hours or more) 34.1 + 2.6 °C; mean + SD) than in the winter (at to N,N-dimethylformamide at concentrations 17.1 + 4.8 °C; mean + SD) (Tsuda et al., 2014). below 10 ppm. Based on the comparison of excreted amounts of N-methylformamide, it was (b) Metabolism estimated that the skin and the lung absorption In an early GC study by Kimmerle & Eben amounted to 40.4% and 59.6%, respectively. (1975a), N-methylformamide and formamide The biological half-life of urinary N-methylform- were reported to be major urinary metabo- amide after dermal exposure (4.75 + 1.63 hours) lites. However, further studies showed that was significantly longer than that after respira- these metabolites were actually HMMF and tory exposure (2.42 + 0.63 hours) (Nomiyama N-hydroxymethylformamide (HMF), respec- et_al., 2001a). tively, which released formaldehyde under An even higher contribution of skin absorp- the conditions of GC (in the sample chamber) tion amounting to 71% of the total dose of N,N- (Brindley _et_al., 1983; Scailteur_et_al., 1984; dimethylformamide was found under conditions Kestell et al., 1986). AMCC was identified for the first time in the urine of a volunteer who inhaled 142 an unspecified dose of N,N-dimethylformamide (Mraz & Tureéek, 1987). [The Working Group noted that the above mentioned data indicated the formation of elec- trophilic metabolites, namely methyl isocyanate and/or another N-methylcarbamoylating species and formaldehyde.] More recently, a liquid chromatography- tandem mass spectrometry method was developed for simultaneous determination of HMMEF, N-methylformamide, and AMCC and was applied to the analysis of 13 urine samples taken from workers occupationally exposed to N,N-dimethylformamide. The molar excretion ratio of HMMF N-methylformamide AMCC was approximately 4 1 1. HMMEF was confirmed as the most abundant urinary metabolite in humans (Sohn et al., 2005), Interactions of N,N-dimethylformamide with ethanol were also studied. In an early study, four volunteers were exposed to N,N-dimethylformamide at concentrations of 50-80 ppm [152-243 mg/m3] for 2 hours with or without preceding oral administration of ethanol. Persons who received ethanol excreted slightly elevated urinary concentrations of N,N-dimethylformamide during the first 4 hours and lower blood levels of N-methylformamide (Eben & Kimmerle, 1976). A haemoglobin adduct of methyl isocyan- ate, a proposed metabolic intermediate of N,N-dimethylformamide and N-methylform- amide, with N-terminal valine (N-(N-methyl- carbamoyl)valine) was identified in the blood of 35 workers in the polyacrylic fibre industry exposed to N,N-dimethylformamide. The exposure was assessed by measuring urinary N-methylformamide which was between 1.3 and 46.5 mg/L (mean, 17.0 mg/L; median, 9.8 mg/L). The methyl isocyanate adduct was released by modified Edman degradation and converted to 3-methyl-5-isoprolylhydantoin, the concentra- tions of which in globin samples ranged from 26.1 to 412.0 nmol/g of globin (Kafferlein & Angerer, 2001). More recently, a new lysine adduct, N,-(N-methylcarbamoyl)lysine, was identified in globin samples of humans occu- pationally exposed to N,N-dimethylformamide (Mraz et al., 2006). 4.1.2 Experimental systems (a) Absorption, distribution, and excretion The toxicokinetics of N,N-dimethylform- amide in rats, mice and cynomolgus monkeys was extensively studied by Hundley et al. (1993a, b). Male and female cynomolgus monkeys were exposed by whole-body inhalation to N,N-dimethylformamide at concentrations of 30, 100, or 500 ppm [90, 300, or 1500 mg/m?] for 6 hours per day on 5 days per week for 13 consec- utive weeks. Disproportionate increases were observed in N,N-dimethylformamide plasma area under the concentration curve (AUC) values of 19-37-fold in male monkeys and 35-54-fold in females as the atmospheric concentrations increased fivefold from 100 to 500 ppm. Plasma half-lives of N,N-dimethylformamide were 1-2 hours and those of N-methylformamide (actually HMMF + N-methylformamide) were significantly longer (4-15 hours). Plasma N-methylformamide concentrations exceeded those of N,N-dimethylformamide 0.5 hour after the beginning of each exposure. HMMF formed 56-95% of the urinary metabolites, regardless of exposure level and duration of the study (Hundley et al., 1993b). The AUC values and peak plasma levels for N,N-dimethylformamide in rats and mice following a single exposure to 500 ppm (Hundley et al., 1993a) were substantially greater than the respective values in monkeys after a similar exposure. In contrast, the enhance- ment of metabolism by repeated exposure to N,N-dimethylformamide at 500 ppm, which was observed in rats and mice, was not clearly demon- strated in monkeys (Hundley et al., 1993b). In another study, cynomolgus monkeys were exposed to N,N-dimethylformamide at 500 ppm 143 IARC MONOGRAPHS - 115 [1500 mg/m] for 6 hours per day on 5 days per formaldehyde under physiological conditions to week for 2 weeks either by head-only or whole- produce N-methylformamide. In early studies, body inhalation (one monkey per exposure N-methylformamide was identified by GC as a route), plasmasamplestaken0.5-18hoursafterthe major metabolite in rats and dogs (Kimmerle first exposure showed N,N-dimethylformamide & Eben, 1975b). Further metabolic oxidation of AUC values that were three times higher in the N-methylformamide leads to HMF, which has monkey exposed by whole-body inhalation, indi- been identified as a minor urinary metabolite cating considerable absorption by non-inhalation in rats (Tulip et al., 1989). Unequivocal evidence route(s). The same comparison of plasma samples exists that HMF is formed after the metabolism taken after the final (10th) exposure revealed an of N-methylformamide in rats and mice (Tulip N,N-dimethylformamide AUC value that was et al., 1986; Threadgill et_al., 1987; Cross et al., sixfold for the monkey exposed by whole-body 1990). In a close analogy with HMMF, HMF inhalation (Hurtt et al., 1991). undergoes thermal decomposition to formamide Rapid absorption was observed in pregnant when analysed by GC; therefore, GC data on Sprague-Dawley rats treated on gestation days 12 formamide actually usually include HMF and and 18 with a single oral dose of 100 mg/kg bw formamide (Mraz & Tureéek, 1987; Mraz et al. [4C]-N, N-dimethylformamide. The radioactivity 1987; Mraz & Nohova, 1992a). in plasma peaked within 1 hour after treatment. The mercapturic acid AMCC is formed by a A major part of the dose (60-70%) was excreted in toxicologically relevant pathway via glutathione the urine and 3-4% in the faeces. Levels of radio- (GSH) conjugation with a presumed electrophilic activity in embryonic and fetal tissues were nearly N-methylcarbamoylating intermediate, probably equal to those in maternal plasma up to 8 and methyl isocyanate or its chemical equivalent 24 hours, respectively, but were higher thereafter. formed by oxidation of N-methylformamide and/ In lactating rats treated with a single oral dose or HMME. The GSH conjugate, S-(N-methyl- of 100 mg/kg bw [!4C]-N,N-dimethylformamide carbamoyl)glutathione undergoes mercapturic on lactation day 14, N,N-dimethylformamide, acid pathway to yield AMCC, which is then HMME, and N-methylformamide were found in excreted in the urine. AMCC was found in the milk at concentrations equal to those in the the urine of rats, mice, Syrian hamsters (Mraz plasma (Saillenfait et al., 1997). et _al., 1989) as well as in human urine (Mraz & A single ethanol drinking episode signifi- Turecek, 1987; Mraz & Nohova, 1992a, b). The cantly enhanced the dermal absorption of N,N- pattern of N,N-dimethylformamide metabolites dimethylformamide in a study using the skin of is qualitatively the same in various rodent species ethanol-dosed and control Wistar rats to test the and in humans (Fig. 4.1). Experiments with liver penetration of N,N-dimethylformamide in vitro microsomes from rats and mice treated with (Brand et al., 2006). inducers of CYP2E1 and with CYP2E1 purified from rat and mouse liver microsomes indicated (b) Metabolism the pivotal role of CYP2E1 in the oxidation of The metabolism of N,N-dimethylformamide N,N-dimethylformamide to HMMF as well as in has been extensively reviewed by Gescher the formation of the key reactive intermediate, (1993). In both humans and animals, N,N- which was proposed to be methyl isocyanate dimethylformamide is primarily oxidized (Gescher, 1993; Mraz et al., 1993; Chieli et_al., by cytochrome P450 (CYP) 2El to HMME. 1995). The key role of CY P2E1 was later confirmed Although relatively stable in neutral or in an in-vitro study on N,N-dimethylformamide mildly acidic solutions, HMMF may release dealkylation. Among several forms of human 144 N,N-Dimethylformamide Fig. 4.1 Metabolism of N,N-dimethylformamide H DMF | CYP2El1 H,Cc7 | N O wet Ht HMMF Lo [CH,=O] (formaldehyde) H HO O UNv 0 CYP2E1 ye < HC a —— » [H,C—N=C=0] H HMF NMF Lo CH,=O HN O aa GS N H Formamide AMCC AMCC, N-acetyl-S-(N-methylcarbamoyl)cysteine; CYP2EI, cytochrome P450 2E1; DMF, N,N-dimethylformamide; GSH, glutathione; HMF, N-hydroxymethylformamide; HMMF, N-hydroxymethyl-N-methylformamide; MAP, mercapturic acid pathway; MIC, methyl isocyanate; NMF, N-methylformamide; SMG, S-(N-methylcarbamoyl)glutathione Adapted with permission from Gescher 1993) and Mraz et al. (1993) recombinant CYPs, namely, CYPIA1, 1A2, measurementsinexperimentsin mice. Theconver- 2B6, 2C10, 3A4 and 2E1, only CYP2E]1 oxidized sion of N-methylformamide to urinary AMCC N,N-dimethylformamide (Amato et al., 2001). and biliary S-(N-methylcarbamoyl)glutathione Methyl isocyanate or its chemically equivalent was found to be subject to large primary kinetic metabolite is formed via N-methylformamide isotope effects when hydrogen was replaced by as an intermediate, although HMMEF is also a deuterium in the formyl group (k,,/k, , 4.5 + 1.0 substrate of CYP2E1. However, the amount of and 7 + 2, respectively) indicating the cleavage S-(N-methylcarbamoyl)glutathione generated of the formyl C-H bond is the rate limiting step from HMMF in vitro was much smaller than that (Threadgill et al., 1987). The proposed mecha- formed from N-methylformamide under iden- nism of the key step in the metabolic activation of tical conditions (Mraz et al., 1993). The mech- N,N-dimethylformamide through the oxidation anism by which the key reactive intermediate of N-methylformamide is shown in Figure 4.2. is formed was studied by kinetic isotope effect Methyl isocyanate was also reported to be a main 145 IARC MONOGRAPHS - 115 Fig. 4.2 Proposed mechanism of generation of the key electrophilic carbamoylating species, methyl isocyanate H acoNN eo CYPRE! ie co Hec7NNc=0 H;C—N=C=0 H e NMF was -e", -H~ -e, -H” MIC, methyl isocyanate; NMF, N-methylformamide Adapted from Gescher (1993) product of N-methylformamide photo-oxidation The effects of the co-administration of with hydroxyl radicals under aerobic conditions ethanol and N,N-dimethylformamide on and the carbonyl centred radical was proposed the metabolism of N,N-dimethylformamide, as a precursor to methyl isocyanate based N-methylformamide and ethanol were inves- on quantum chemical calculations (Bunkan tigated in several early studies. Increases in et al., 2015). This observation represents indirect the concentrations of N,N-dimethylformamide, support for the above-mentioned mechanism N-methylformamide, ethanol or acetaldehyde (Fig. 4.2). in blood were observed after co-exposure. Adducts with globin at N-terminal valine These results were attributed to the inhibition and N.-lysine, N-(N-methylcarbamoyl)valine by N,N-dimethylformamide of the activity of and N,.-(N-methylcarbamoy]l)lysine, respectively, alcohol dehydrogenase observed both in vitro were identified in rats exposed to high doses and in vivo (Eben & Kimmerle, 1976; Hanasono of N,N-dimethylformamide (1000 mg/kg bw) et_al., 1977; Sharkawi, 1979) and of aldehyde (Mraz et al., 2004) as well as in humans exposed dehydrogenase observed in vivo (Elovaara et al. to N,N-dimethylformamide (Mraz et al., 2002, 1983). The effect of the competitive inhibition of 2006). As cysteine sulfhydryl groups are much CYP2E1, which seems to be very probable in the stronger nucleophiles than amino acid amine light of more recent observations (Mraz et al. groups, N-methylcarbamoylation is likely to 1993; Chieli et al., 1995), was not considered at occur also at the sulfhydryl groups of cysteine the time of these early studies. to yield reactive thiocarbamate species capable In conclusion, three metabolic events appear of transferring the N-methylcarbamoyl moiety to play key roles in N,N-dimethylformamide to other nucleophilic sites (transcarbamoy]l- toxicity: (i) N-methylcarbamoylation mediated ation). [The Working Group noted that, due by methyl isocyanate and/or its chemical equiv- to the reactivity of the thiocarbamate moiety, alents; (ii) free radical damage caused by carba- N-methylcarbamoylated cysteine residues may moyl radicals; and (iii) the probable formation have easily escaped detection in the analyses of of formaldehyde by oxidative demethylation adducted globin. The N-methylcarbamoylating (Fig. 4.1 and Fig. 4.2). The main enzyme respon- species, either methyl isocyanate or a N-methyl- sible for metabolic activation is CYP2E1. carbamoylated cysteine residue, may carbamoy- late nucleophilic sites in proteins and in the DNA.] 146 4.2 Mechanisms of carcinogenesis The evidence on the key characteristics of carcinogens (Smith et al, 2016), concerning whether N,N-dimethylformamide induces oxidative stress; alters cell proliferation, cell death, or nutrient supply; is genotoxic; or modulates receptor-mediated effects — is summarized below. 4.2.1 Oxidative stress (a) Humans (i) | Exposed humans N,N-Dimethylformamide-exposed Chinese workers (n = 104) and 101 controls were studied for oxidative and antioxidative status. The N,N-dimethylformamide concentration in workplace air was within the range of 3.3-12.4 mg/m. The N-methyl-carbamoylated haemoglobinadduct(NMHb)inbloodwaschosen as a biomarker measured as the Edman degra- dation product, 3-methyl-5-isopropylhydantoin (MVH). The MVH level in exposed workers was 19.69 + 12.52 mg/kg, and MVH was not detected in the control group. The activity of superoxide dismutase (SOD) in exposed workers (125.30 + 21.23 U/mL) was significantly higher than the control group (118.35 + 18.48 U/mL). However, the activity of SOD showed different trends with increasing MVH levels. When MVH < 24 mg/kg, the SOD activity increased with the increasing of MVH level. When MVH > 24 mg/kg, SOD activity decreased with increasing MVH level. No significant differences were observed in glutathione-S- transferase, malondialdehyde or 3-nitrotyrosine levels among the two groups. It was concluded that N,N-dimethylformamide exposure did not cause obvious lipid and/or protein peroxidative damage (Cheng et al., 2014). (ii) © Human cells in vitro HL-7702 normal human liver cells were used to study N,N-dimethylformamide-induced oxidative stress. Reactive oxygen species (ROS)- induced fluorescence was determined by flow cytometry using 2',7'-dichlorodihydrofluores- cein diacetate (DCFH-DA) and propidium iodide to identify viable cells. ROS levels increased with N,N-dimethylformamide dose (0-40 mM) to a maximum of ~1.5-foldin cells treated for 24 hours. The levels of 8-hydroxy-2’-deoxyguanosine meas- ured by enzyme-linked immunosorbent assay (ELISA) increased to ~1.5-fold after incubation with N,N-dimethylformamide at 40 and 100 mM for 24 hours (Wang et al., 2015). N,N-Dimethylformamide (60 mM) induced differentiation of human HL-60 promyelo- cytic leukaemia cells, and these cells acquired polymorphonuclear leukocyte functions and the ability to generate ROS when stimulated (Speier & Newburger, 1986). HL60cellsare known to contain CYP2E]1 (Nagaietal., 2002), which can metabolize N,N-dimethylformamide. Treatment of HL-60 cells with N,N-dimethylformamide resulted in a decline in total SOD, while catalase activity declined slightly. Oxidized glutathione (GSSG) reductaseactivityandreducedglutathione (GSH) and GSSG levels declined slightly over time. Concomitantly with the decrease in antioxidant enzymes during the course of N,N-dimethylformamide-induced _ differentia- tion of human HL-60 cells, treatment with phorbol myristate acetate (PMA, an ROS stim- ulating agent) increased the ability of the cells to generate hydrogen peroxide (Speier & Newburger, 1986). Atahigher dose, N,N-dimethylformamide (80 mM) induced granulocytic cell differentia- tion of human HL-60 promyelocytic leukaemia and PLB-985 human myeloid leukaemia cells. N,N-Dimethylformamide (80 mM) treatment increased glutathione peroxidase enzymatic activity, gene expression (mRNA) and protein levels in HL60 and PLB-985 cells (Shen et al., 1994). 147 IARC MONOGRAPHS - 115 N,N-Dimethylformamide (0.5%) induced The hepatic levels of reduced GSH and vitamin C granulocytic differentiation in PLB-985 human also decreased 55% and ~62%, respectively. The myelomonoblastic leukaemia cells after 6 days of levels of SOD, catalase, glutathione peroxidase, treatment. Further treatment with PMA (1 uM) reduced GSH, and vitamin C each decreased and formyl-methionyl-leucine phenylalanine 40-60% in the kidney (Jyothi et al., 2012). Ina (800 nM), ROS stimulating agents, induced ROS spectroscopic and modelling study, Kalyani etal. detected by nitroblue tetrazolium staining in the (2014) reported a destabilization of SOD, which cells (Katschinski et al., 1999). might explain the decreases in activity reported Both N,N-dimethylformamide and its metab- by Iyothi et al. (2012). olite N-methylformamide reduced total GSH ICR mice (both sexes) treated with levelsinaconcentration-response manner (0-200 N,N-dimethylformamide daily by gavage for mM) after incubation in DLD-1 Clone A human 90 days had significant increases in MDA levels coloncarcinoma cells (Cordeiro & Savarese, 1984). measured as TBARS in heart homogenates and Ina follow-up study using the same experimental in liver homogenates (at the 0.32, 0.63, and conditions, both reduced GSH and GSSG were 1.26 g/kg bw dose levels) compared with controls. measured separately. In concentration-response SOD activity levels decreased significantly in studies with both N,N-dimethylformamide and heart homogenates and liver homogenates (at N-methylformamide, it was found that that the the 0.63 and 1.26 g/kg bw dose levels) compared levels of GSSG remained relatively constant with controls. GSH levels decreased only in liver over 0-200 mM, while the levels of reduced homogenates at 0.32, 0.63, and 1.26 g/kg bw GSH decreased with increasing concentra- (Rui et al., 2011). tions of N,N-dimethylformamide or N-methyl- Male Sprague-Dawley rats were dosed formamide (Cordeiro & Savarese, 1986). with N-methylformamide or N,N-dimethyl- formamide by intraperitoneal injection (1 mL/ (6) Non-human mammalian systems kg bw). Only N-methylformamide depleted the (i) In vivo hepatic GSH level by about 30% 1 hour after N,N-Dimethylformamide treatment of injection. Twenty-four hours after the treatment, Sprague-Dawley rats after intraperitoneal injec- the GSH level returned to the control value tion (500 mg/kg bw per day for three consecu- (Scailteur & Lauwerys, 1984). tive days) had no effect on the hepatic levels of GSH levels were measured in liver, kidney, thiobarbituric acid reactive substances (TBARS). heart, lung and spleen tissues from male N,N-Dimethylformamide decreased the hepatic CBA/CA mice dosed with single doses of GSH content to 61% of control values (Kim et al. N-methylformamide (400 mg/kg bw), N-ethyl- 2010). formamide (495 mg/kg bw) or formamide (dose Malondialdehyde (MDA) levels (measured not reported) after 1 hour. N-Methylform- as TBARS) increased four- to ninefold in the amide, but not N-ethylformamide or formamide, liver, and four- to sevenfold in the kidney of decreased hepatic GSH levels by 59.8%. Of the male Wistar rats, 24 and 48 hours after intra- other tissues investigated only the kidneys peritoneal injection with a single dose of exhibited GSH _ depletion (21.6%). In a N,N-dimethylformamide (1.5 g/kg bw). The dose-response study, hepatic GSH levels activities of the hepatic antioxidant enzymes in male BALB/c mice decreased with decreased at both time-points: SOD (~50%), cata- increasing dosage of N-methylformamide lase (~50%), and glutathione peroxidase (~58%). (0-400 mg/kg bw) 1 hour after intra- 148 peritoneal injection of N-methylformamide (Gescher et al., 1982). N-Methylformamide (200 mg/kg bw) injected intraperitoneally into BALB/c mice depleted total hepatic GSH to 21% of control levels 2 hours after administration and induced hepatotoxicity. In CBA/CA and BDF1 mice, the same dose of N-methylformamide depleted total hepatic GSH levels to 53% of control levels and did not cause hepatotoxicity (Pearson et al., 1987). N-Methylformamide treatment of male BALB/c mice (single intraperitoneal injection of 50-200 mg/kg bw) induced a dose-dependent decrease in hepatic non-protein sulfhydryls (reduced GSH is the major non-protein sulfhydryl in cells). Higher doses (up to 800 mg/kg bw) did not induce a further decline. A time-course study revealed that 4 hours after a dose of 300 mg/kg bw, non-protein sulfhydryls were reduced to 25% of control values. N-Methylformamide did not significantly decrease the levels of hepatic non-protein sulfhydryls in male Sprague-Dawley rats 4 hours after an intraperitoneal injection of 1000 mg/kg bw (Tulip & Timbrell, 1988). Changes in gene expression were examined in the livers of male BALB/c mice dosed with a single intraperitoneal injection (300 mg/kg bw) of N-methylformamide. Comparison of the gene expression patterns of N-methylformamide versus saline control identified a series of signif- icantly altered genes associated with oxidative stress (e.g. upregulation of aldehyde oxidase 1 Aoxl, heat shock protein Hspbl, Hspa8s, and Hsp105) (Mutlib et al., 2006). N,N-dimethylformamide induced CYP2El protein levels in liver microsomes from male Sprague Dawley rats after intraperitoneal injec- tion of N,N-dimethylformamide at 0, 450, 900 or 1800 mg/kg bw once per day for 3 days. The activities of p-nitrophenylhydroxylase (a measure of CYP2E] enzymatic activity) in the N,N-dimethylformamide-treated groups were significantly higher than that of the control group after 3 days of treatment. Under the same experimental conditions there were no significant increases in CYP1A1 or CYP2B1/2 proteins or increases in their associated enzymes ethoxyre- sorufin-O-deethylase (EROD) or pentoxyresoru- fin-O-dealkylase (PROD) activities, respectively (Koh et al., 1999). The effects of N,N-dimethyl- formamide on the induction of CYP2E1 protein levels, p-nitrophenylhydroxylase, glutathione S-transferase and glutathione peroxidase activities were studied in the livers of male Sprague-Dawley rats after intraperitoneal injection of N,N-dimethylformamide at 0, 450, 900, or 1800 mg/kg bw once per day for 3 days. The levels of CY P2E]1 analysed by Western immunoblot increased and p-nitrophenylhy- droxylase activity increased ~3.7-fold after 3 days of treatment. Glutathione S-transferase levels increased slightly with N,N-dimethylformamide treatment while N,N-dimethylformamide had no effect on glutathione peroxidase levels (Kim & Chung, 2013). In another report, CYP2E]1 protein was induced ~1.5-fold in liver homogenates from rats treated with N,N-dimethylformamide (500 mg/kg bw daily for 3 days) (Kim _ & Kim 2011). It has been reported that CYP2E1 induc- tion can lead to the over production of ROS and oxidative stress (Caro & Cederbaum, 2004; Gonzalez, 2005). [A possible explana- tion for the ability of N,N-dimethylformamide to induce hepatotoxicity is the production of ROS by N,N-dimethylformamide through CYP2E1 induction and uncoupling of the cata- lytic cycle (release or escape of the superoxide anion radical during the course of the P450 catalytic cycle) (Gonzalez, 2005) combined with N,N-dimethylformamide and the depletion of GSH by N-methylformamide.] (ii) Invitro N-Methylformamide decreased total intra- cellular GSH levels in murine TLX5 lymphoma cells in a concentration-related fashion up to 160 mM after a 48-hour incubation (Bill et al., 1988) 149 IARC MONOGRAPHS - 115 (c) Non-mammalian systems given daily for 21 days, respectively. HCT-15 N,N-Dimethylformamide induced oxidative tumour growth was inhibited 65% by N,N-dimethylformamide compared with stress in the proteobacteria, Paracoccus sp. SKG. controls. In two independent experiments Glutathione S-transferase, catalase, and SOD with N,N-dimethylformamide, DLD-2 activities increased in a concentration—-response manner. Cellular levels of methionine as well as tumour growth was inhibited by 45 and 67%. protein levels ofthe enzyme methionine sulfoxide N-Methylformamide treatment produced 43 and 75% growth inhibition of HCT-15 reductase, which catalyses the reduction of both and DLD-2 tumours, respectively (Dexter free and protein-bound methionine sulfoxide et al., 1982). Nude mice (15 mice per group) were residues back to methionine (Ezraty et al., 2005) were increased (Kirankumar et al., 2014). subcutaneously injected with SHG-44 human [The Working Group noted that both glioma cells grown in the absence or presence N,N-dimethylformamide and N-methyl- of N,N-dimethylformamide. Pretreatment of SHG-44 cells with N,N-dimethylformamide formamide have been shown to alter indices of oxidative stress. N,N-Dimethylformamide reduced the growth rate of the xenografts (Li et al., 1997). induced ROS in human liver cells and increased CD-1 male nude mice (eight mice per group) malondialdehyde levels in the livers of mice were injected subcutaneously with human and rats. N,N-dimethylformamide altered the colon carcinoma HT-29 cells or HT-29 cells levels of glutathione peroxidase and SOD in grown in medium containing 1% (170 mM) human tumour cells and lowered the levels of hepatic GSH in the livers of mice and rats. N-methylformamide (designated HT29-NMF N-Methylformamide reduced the levels of cells) inducing cell differentiation. HT29-NMF cells had an increased latency period in the GSH in tumour cells and in the livers of mice appearance of palpable tumours compared with and rats and increased the expression of genes associated with oxidative stress in the livers of HT-29 cells and tumour weights were lower in mice carrying the HT29-NMF cell xenographs treated mice.] compared with mice carrying the HT29 cell xenographs, indicating slower growth induced 4.2.2 Altered cell proliferation or death by N-methylformamide (D’Agnano et al., 1992). (a) Humans (iii) Human cells in vitro (i) | Exposed humans N,N-Dimethylformamide has been shown No data were available to the Working Group. to inhibit the growth of human tumour cells in (ii) | Human tumour cells implanted into culture in many studies. experimental animals The growth of RKO colon carcinoma cells (Brattain et al., 1984) treated with N,N-Dimethylformamide and N-methyl- N,N-dimethylformamide (0.7%) resulted in formamide inhibited the growth of two an 88% reduction in cell number compared human colon cancer cell lines xenografted with untreated control cells (Zipfel et_al., 1993). into female nude mice. Mice received subcuta- Similarly, cell proliferation of MOSER human neous transplants of HCT-15 or DLD-2 human colon carcinoma cells, transforming growth colon cancer cells and were treated intraperi- factor-8 (TGF-6)-resistant MOSER human toneally with N,N-dimethylformamide or colon carcinoma cells and HCT 116 human N-methylformamide, 2219 and 374 mg/kg bw colon carcinoma cells was reduced by 1% 150 N,N-dimethylformamide; there was essentially minimal cell growth for each cell line (Mulder et al., 1988). DLD-1 and HCT-15 human colon carci- noma cell lines and several subclones of DLD-1 cells (Clone A, Clone D) were treated with N,N-dimethylformamide (0.8%) which induced morphological changes in the cells and alterations in their growth properties. N,N-Dimethylformamide also induced differ- entiation and maturation to a less malig- nant phenotype (Dexter et al. 1979; Dexter & Hager, 1980). Growth in the presence of N,N-dimethylformamide also caused a marked reduction in the tumorigenicity of the cells in vivo. While 20 of 20 nude mice injected with DLD-1 cells developed tumours in 10-14 days, only one nude mouse out of ten inoculated with N,N-dimethylformamide-treated DLD-1 cells developed a tumour in the third month after the injection (Dexter et al., 1979; Dexter & Hager, 1980). N-Methylformamide increased the doubling times of DLD-1 Clone A cells in culture in a concentration-response fashion (Dibner et _al. 1985). N-Methylformamide (1%, 170 mM) treat- ment of human HT-29 colon carcinoma cells induced cell differentiation and reduced cell proliferation. N-Methylformamide treatment induced a G,/G, phase accumulation, with a higher percentage of treated cells in G,/G, phase compared with controls (D’Agnano et al., 1992). N,N-Dimethylformamide (0.25, 0.5, 0.75, and 1%) produced a concentration-dependent inhib- itory effect on cell proliferation in monolayers of human glioma SHG-44 cells Li et al. (1997). The percentage of DNA fragmentation and apoptotic nuclei increased in cultures of human colonic carcinoma HT-29 cells after treatment with N,N-dimethylformamide (1%) for 72 hours (Heerdt et al., 1996). The effects of N,N-dimethylformamide on hyperthermia-induced apoptosis, DNA frag- mentation and cell cycle effects were studied in PLB-985 human myelomonoblastic leukaemia cells. Hyperthermia (42 or 43 °C, 1 hour) induced apoptosis and DNA fragmentation in PLB-985 cells and led to a decrease in the number of G,/G, cells as determined by flow cytometry. N,N-Dimethylformamide (0.5%) treatment of these cells caused differentiation along the gran- ulocytic pathway and prevented the induction of apoptotic death (Katschinski et al., 1999). In the normal human liver cell line, HL-7702, N,N-dimethylformamide (0, 50, 100, 150, and 200 mM) for 12 hours increased the apop- totic rate of hepatocytes in a concentration-re- lated manner as measured by flow cytometry. N,N-Dimethylformamide induced a_ steady decrease of the expression of the anti-apoptotic Bcl-2 protein shown by Western blotting and its level was lower than the control group. The expression of the pro-apoptotic Bax protein showed no significant differences between the different N, N-dimethylformamide concentration groups. Increasing the N,N-dimethylformamide concentration decreased the ratio of Bcl-2/ Bax; at 200 mM this ratio was significantly lower than that of the controls. Procaspase-3 protein was observed at 150 and 200 mM, which demonstrated that there was active caspase-3 (Lu et al., 2008). (6) Non-human mammalian systems (i) Invivo Dose related increases in cell proliferation were seen in male F344/DuCrlCrj rats (SPF) exposed to N,N-dimethylformamide by both inhalation (0, 200, or 400 ppm for 6 hours per day, 5 days per week, for 4 weeks) and in drink- ing-water (0, 800, 1600, or 3200 ppm for 24 hours per day, 7 days per week, for 4 weeks). Maximal induction up to 9.7-fold was reported at the 200 ppm inhalation/3200 ppm oral dose level. Rats treated with N,N-dimethylformamide only by inhalation or only by oral dosing also showed 151 IARC MONOGRAPHS - 115 modest dose-related increases in cell prolifera- growth rate upon cessation of the treatment tion (Ohbayashi et al., 2008). (Iwakawa etal., 1987b). Inasimilar study, CH,Hf/ The effects of N,N-dimethylformamide Kam mice were injected intramuscularly with a on several hepatic enzymes associated with single-cell suspension of the FSA fibrosarcoma. apoptosis were investigated in Sprague- Treatment with N-methylformamide (300 mg/kg Dawley rats after intraperitoneal injections of bw) only slightly inhibited the tumour growth 500 mg/kg bw per day for 3 consecutive days. when administered for 8 days (Iwakawa et al. N,N-Dimethylformamide induced a marginal 1987). increase in the ratio of the protein levels of Bax Changes in gene expression associated with to Bcl-xL (an apoptosis inhibitor), and had no cellproliferation were reportedinthelivers ofmale effects on poly (ADP-ribose) polymerase (PARP) BALB/c mice dosed with a single intraperitoneal cleavage or caspase-3 activity (Kim et al., 2010). injection (300 mg/kg bw) of N-methylformamide Fourteen of 17 CE/J mice receiving intra- (Mutlib et al., 2006). peritoneal injections of murine rhabdomyosar- (ii) Invitro coma cells (pre-cultured in media containing N,N-Dimethylformamide (1%, 170 mM) N,N-dimethylformamide (1%) for 10 days) did not develop tumours after 6 months. This was decreased the growth of both murine rhabdo- compared with 21 mice receiving intraperitoneal myosarcoma cells (Dexter, 1977), AKR-2B mouse injections of untreated rhabdomyosarcoma cells, embryo fibroblast cells and their methylcholan- which died of tumour between 11 and 31 days threne-transformed counterpart AKR-MCA (Dexter, 1977). cells in culture (Hoosein et al., 1988). The N-Methylformamide(43to170mM) decreased growth of murine M5076 ovarian sarcoma cells injected intramuscularly into the growth of TLX5 murine lymphoma cells female BDF, mice was inhibited by daily intra- and changed the distribution of these cells in the peritoneal injections of N-methylformamide cell cycle determined by flow cytofluorimetry, (25, 50, 100, or 200 mg/kg bw) for 17 days. In increasing the proportion of G, cells and reducing similar types of studies, formamide (200 mg/kg the proportion of cells in the S and G,/M phases. bw) or N-methylformamide (200 mg/kg bw) N-Methylformamide (0-160 mM) also inhibited administered to female BDF, mice daily during the ability of TLX5 cells to grow in soft agar (Bill the course of 17 days markedly reduced the et.al., 1988). tumour volume of M5076 tumours. Daily dosing N-Methylformamide (0.5 or 1%) reduced the with N-methylformamide (300 mg/kg bw) or clonogenicity of MCA-K (mammary carcinoma), formamide (300 mg/kg bw) for 9 days by intra- butnot FSA (fibrosarcoma) or HCA-I cells (hepato- peritoneal injections reduced murine sarcoma carcinoma) in vitro (Iwakawa et al., 1987b). 180 tumour volume in female BDF, mice injected One study reported that N,N-dimethyl- with this tumour (Gescher et al., 1982). formamide increased apoptosis. Treatment of N-Methylformamide (300 mg/kg bw) admin- immortalized Rat-1 fibroblasts in monolayer istered intraperitoneally daily for 6 days slowed with N,N-dimethylformamide at 50-175 mM the growth of FSA fibrosarcoma, and HCA-I for 24 hours induced cell death by apoptosis with hepatocarcinoma tumour xenographs and early apoptotic changes observed in the cells, cell totally inhibited the growth of the MCA-K shrinkage with nuclear condensation, and the formation of membrane and DNA fragmenta- mammary carcinoma tumour xenographs in CH,Hf/Kam mice. However, the effects were tion (Boyle & Hickman, 1997). transient; tumours resumed their pretreatment 152 [The Working Group noted that N,N- dimethylformamide and N-methylformamide have been used in preclinical cancer chem- otherapeutic trials. Experimental studies showed that N,N-dimethylformamide and N-methylformamide inhibited the growth of cancer cells in culture, and cancer cell lines implanted into mice. In these studies, cancer cells were cultured in the presence of N,N-dimethylformamide — or N-methylformamide and injected into mice, or mice carrying cancer xenographs were dosed with N,N-dimethylformamide or N-methyl- formamide. N,N-Dimethylformamide also altered the malignant phenotype of cancer cells and reduced cell growth in vitro. One study in rats showed that N,N-dimethylformamide increased hepatic cell proliferation. N,N-Dimethyl- formamide increased apoptosis in tumour cells and in a normal human liver cell line.] 4.2.3 Genetic and related effects (a) Humans (i) | Exposed humans See Table 4.1 In the analytical study of Hennebriider & Angerer (2005), urine samples taken from male workers exposed to N,N-dimethylformamide vapours (exposure level not given) were analysed by a sensitive LC/MS method for DNA adducts. N*-(N-methylcarbamoyl)cyto- sine (N4-NMCC), a DNA adduct, which was already known to be formed from MIC (Segal et al., 1989), a probable metabolic intermediate of N,N-dimethylformamide, was detected. In 10/32 urine samples collected from occupation- ally exposed subjects N4-NMCC was detected in concentrations of 31-172 ng/L (detection limit was 8 ng/L). No N4-NMCC was detected in urine samples taken from 24 subjects with no record of N,N-dimethylformamide exposure (Hennebriider & Angerer, 2005). tail extent, Olive tail moment, and tail DNA/ total DNA (%) were reported by Chen (2004) in workers who were occupationally exposed to N,N-dimethylformamide (6-7 years of exposure) as compared with controls (P < 0.05). Changes in mitochondrial DNA in blood leukocytes, namely, a common deletion AmtDNA*77 and mtDNA copy numbers, were reported in a study on 13 male synthetic- leather factory workers. Exposure to N,N-dimethylformamide was followed by air sampling (median, 10.59 ppm; range, 6.65-34.38 ppm), as well as by urine analysis for the biomarkers “NMF” (actually HMMF+NMF; median, 13.77 mg/L; range, 7.47-73.64 mg/L) and AMCC (median, 40.70 mg/L; range, 6.76-442.24 mg/L). The values found in subjects exposed to N,N-dimethylformamide were signif- icantly higher than those in controls, which were matched by age, seniority, smoking, and alcohol drinking habits. Moreover, both param- eters appeared to be exposure-dependent when subjects exposed to N,N-dimethylformamide at concentrations higher and lower than 10 ppm (permissible exposure limit) were compared. The frequencies of AmtDNA*77 were significantly elevated also in subjects with high urinary levels of AMCC (2 40 mg/L). On the other hand elevated mtDNA copy number was found in subjects with relatively high urinary “NMF” concentra- tions (= 15 mg/L) considered as a short-term marker of exposure to N,N-dimethylformamide (Shieh et al., 2007). Rates of sister-chromatid exchange were determined in peripheral blood lympho- cytes of 22 non-smoking women exposed to N,N-dimethylformamide working in a synthet- ic-leather manufacturing plant for 1.1-9.9 years. The exposed group was divided into three subgroups according to the exposure level, which was 0.3, 0.7, and 5.8 ppm (time-weighted average, TWA) in the low, intermediate, and high expo- sure group, respectively. A co-exposure by low 153 VSL Table 4.1 Genetic and related effects of N,N-dimethylformamide in exposed humans VI DY Biological sample Description of exposed and _End-point Test Results Agent, dose Comments Reference SHdVYDONOW controls (LED or HID) Urine 32 male workers in acrylic DNA damage N'-(N- + DMF Hennebriider fibre industry and 24 control methylcarbamoyl) & Angerer from general population cytosine by LC-ESI- (2005) MS/MS Peripheral blood = Workers with 6-7 yrs of DNA damage DNA strand breaks — + DMF Chen (2004) cells exposure to DMF (comet assay) (P < 0.05) - Leukocytes 13 male synthetic leather DNA damage End-point deletion + DMF Smoking taken into Shieh et al. SLL factory workers; 13 AmtDNA*”, account (2007) control subjects from the mitochondrial administrative department mtDNA copy of the same factory number Lymphocytes 22 non-smoking women; Chromosomal Sister-chromatid + DMF + toluene, — Exposure-related Seiji et al. synthetic leather production, damage exchange (P< 0.01) 0.7 ppm (TWA) effect; co-exposure (1992) 3 exposure groups; 22 sex-, with toluene in age- and residence-matched the intermediate controls exposure group Lymphocytes 26 male viscose rayon plant DNAand UDS, chromosomal (+) DMF + Effect of smoking Major et al. workers; 26 matched controls chromosomal aberrations, (P<0.01) acetonitrile separated; effects (1998) and 6 industrial controls damage aneuploidy, sister- of DMF and (administrative workers) chromatid exchange acrylonitrile not separated [synergism is likely] Lymphocytes 40 occupationally exposed Chromosomal (+) DMF, 150 mg/m? — Smoking not Koudela & workers, 31 unexposed damage aberrations P<0.05 (TWA) reported Spazier (1981) persons Lymphocytes 56 workers in printed circuit- Chromosomal Sister-chromatid (-) DMF + No association Cheng et al. board manufacture; no damage exchange epichlorohydrin, with DMF (1999) unexposed controls 33.6 + 3.9 ppm, exposure but long-term strong association with exposure to epichlorohydrin Lymphocytes 29 workers synthetic leather © Chromosomal Sister-chromatid (-) DMF, 34.6£6.3 Smokingand GST Cheng etal. manufacture; no unexposed damage exchange ppm, long-term genotypes taken (1999) controls into account +, positive; (+) or (-), positive or negative in a study with limited quality; DMF, N,N-dimethylformamide; GST, glutathione S-transferases; HID, highest ineffective dose; LC-ESI-MS/MS, liquid chromatography and electrospray ionization mass spectrometry; LED, lowest effective dose; NMF, N-methylformamide; TWA, time-weighted average; UDS, unscheduled DNA synthesis; yr, year concentrations of toluene (0.9 ppm, TWA) was reported for the intermediate exposure group but not for other groups. Age-, sex-, and resi- dence-matched controls from the same factory (administrative staff) were used. A dose-de- pendent increase in sister-chromatid exchanges was found in the exposed group (rates of 8.26, 7.23, and 5.67 for the high, intermediate, and low exposure subgroup, respectively) and the mean of exposed group was significantly higher than that of controls (P < 0.01). However, the mean sister-chromatid exchange rate value in the low exposure group appeared to be lower than that in controls (P < 0.05) (Seiji et al., 1992), Two other field studies in humans exposed to N,N-dimethylformamide in vivo found increases in chromosomal aberrations (Major et al., 1998; Koudela & Spazier, 1981), aneuploidy, sister-chro- matid exchange, and unscheduled DNA synthesis (Major et al., 1998). [The Working Group noted that the genotoxic effects observed may not be related to N,N-dimethylformamide exposure because of co-exposure with high concentrations of acrylonitrile (Major et al. 1998), and because no data were available on smoking and co-expo- sure with numerous other chemicals occurring at the workplace (Koudela & Spazier, 1981).] Cheng et al. (1999) studied sister-chromatid exchange in peripheral blood lymphocytes of 85 plant workers occupationally exposed to N,N-dimethylformamide and/or epichloro- hydrine. Airborne N,N-dimethylformamide concentrations during the work-shifts ranged from 1 to 83 ppm (TWA). An association between sister-chromatid exchange frequencies and epichlorohydrin exposure levels, but not N,N-dimethylformamide exposure levels, was found. No unexposed control group was used and the exposure history of the subjects studied was not reported. (ii) © Human cells in vitro See Table 4.2 In vitro, N,N-dimethylformamide gave negative results in a human hepatocyte primary culture DNA repair assay based on autoradio- graphic detection of unscheduled DNA synthesis at concentrations up to 10 mM (McQueen et al., 1988). Positive results were reported when phospho- rylated histone H2AX (yH2AX) was used as a biomarker of double-strand breaks in human liver cell DNA treated with N,N-dimethylformamide (1.5-100 mM) (Xuan et al., 2008; Wang et al., 2015). The levels of 8-hydroxy-2’-deoxyguanosine were increased 1.5-fold in human liver cells HL-7702 exposed to N,N-dimethylformamide at 40 and 100 mM (Wang et al., 2015). Negative results for N,N-dimethylformamide (up to 10 mM, 731 ug/mL) were obtained ina study of genotoxic insult evaluated by GreenScreen HC GADD45a-GFP genotoxicity assay (Hastwell et_al., 2006). No increase in the frequency of sister-chro- matid exchange was observed in human lymphocytes incubated in vitro with N,N-dimethylformamide at concentrations up to 1.1 M for 24, 48, or 72 hours (Antoine et al., 1983). (b) Experimental systems See Table 4.3 Studies on genetic and related effects in various experimental systems in vivo (e.g. rats, mice, and Syrian hamsters) and in vitro (including human lymphocytes, rat and mouse hepatocytes, mouse lymphoma cells, Chinese hamster ovary cells, yeast, Salmonella typhimu- rium, and Escherichia coli) were comprehensively reviewed in a previous IARC Monograph, when the data on N,N-dimethylformamide were eval- uated by the Working Group in 1999. At the time, the results were mostly negative or inconclusive, and did not provide evidence supporting a geno- toxic mechanism (IARC, 1999). For instance, N,N-dimethylformamide gave negative results 155 IARC MONOGRAPHS - 115 Table 4.2 Genetic and related effects of N,N-dimethylformamide in human cells in vitro Species Cell type End-point Test Results Dose Reference (LED or HID) Human Hepatocytes DNA damage UDS - 10 mM McQueen et al. [731 g/mL] (1988) Human Hepatocytes DNA damage yH2AX + 1.56 mM Xuan et al. (2008) [114 wg/mL] Human Liver HL-7702 cells DNA damage yH2AX + 6.4mM Wang et al. (2015) [468 g/mL] Human Liver HL-7702 cells DNA damage 8-OHdG (ELISA) + 40 mM Wang et al. (2015) [2924 ug/mL] Human Lymphoblastoid TK6 = DNA damage GADD45a-GFP - 10 mM Hastwell et al. cell line assay [731 g/mL] (2006) Human Lymphocytes Chromosomal Sister-chromatid - 11M Antoine et al. 1983) damage exchange [80 400 pg/mL] +, positive; -, negative; ELISA, enzyme-linked immunosorbent assay; GADD 45a, growth arrest and DNA damage-inducible 45a gene; GFP, green fluorescent protein; HID, highest ineffective dose; LED, lowest effective dose; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; UDS, unscheduled DNA synthesis for micronuclei in mice (Antoine et al., 1983) and Formaldehyde, a necessary product of in the white/whitet eye mosaic test in Drosophila N,N-dimethylformamide demethylation, and (Vogel & Nivard, 1993). MIC, a likely metabolic intermediate of Recent studies of genotoxicity in vitro N,N-dimethylformamide, can also cause chro- have shown predominantly negative results. mosomal damage (Goswami, 1986; Tice et_al., N,N-Dimethylformamide administered by 1987; IARC, 2006). gavage at the dose levels of 50, 100, and 200 mg/kg bw once per day for 14 consecutive 4,2.4 Receptor-mediated effects days to rats significantly increased lymphocyte comet tail, average tail length, and tail moment (a) Humans (Xing et al., 2014). However, negative results for Sperm motility was reduced in 12 workers N,N-dimethylformamide at concentrations up to exposed to N,N-dimethylformamide in a 257 mM were obtained in an RAD54-GFP assay synthetic-leather factory compared with on yeast (Saccharomyces cerevisiae). RAD54 is a 8 non-exposed sociodemographically matched DNA repair gene induced by a variety of DNA controls (Chang et al., 2004a). Sperm motility lesions above the constitutive level (Knight et al., was related to urinary N-methylformamide level, 2007). N,N-Dimethylformamide (4% v/v) was but not airborne N,N-dimethylformamide. also not mutagenic in the umu-test on Salmonella In occupationally exposed workers, effects of typhimurium TA1535 (Degirmenci et al., 2000). N,N-dimethylformamide on coagulation were In a study on the combined action of probably related to change on the membrane N,N-dimethylformamide and hydrogen peroxide receptor of platelets and to a modification in in the presence of Cu(II), N,N-dimethylforma- phospholipid components. A follow-up exper- mide (0.5-4%) markedly enhanced DNA iment in vitro with platelets indicated that strand breaks and 8-hydroxy-2'-deoxyguano- effects on aggregation were most likely induced sine induced by 50 uM H,O, and 20 uM Cu(II) by epinephrine and adenosine diphosphate (Midorikawa et al., 2000). (Imbriani et al., 1986). 156 N,N-Dimethylformamide Table 4.3 Genetic and related effects of N,N-dimethylformamide in experimental systems Species, strain End-point Test Results Dose Reference (sex), tissue (LED or HID) Rat, Wistar (F), DNA damage DNA strand + 50 mg/kg bw Xing et al. (2014) lymphocytes breaks (comet) gavage; 1 x/day, 14 days Mouse Balb/C (M), Chromosomal Micronuclei - 2000 mg/kg bw Antoine et al. bone marrow damage i.p. dosing 30h (1983) after treatment Drosophila, Cross | Mutation Somatic mutation —- 10 mM Vogel & Nivard C-1, strains y and and recombination [731 g/mL] (1993) Ww test (SMART) Saccharomyces DNA damage RAD54-GFP assay - 257 mM Knight et al. (2007) cerevisiae GenTO1/ [18 800 g/mL] GenC01 (control strain) Salmonella DNA damage umu-test NT 4% (v/v), tested Degirmenci et al. typhimurium with (but not (2000) TA1535 without) metabolic activation +, positive; -, negative; bw, body weight; GFP, green fluorescent protein; F, female; HID, highest ineffective dose; i.p., intraperitoneal; M, male; NT, not tested; RAD54, gene coding for a DNA repair/recombination protein; v/v, volume per volume In human colon carcinoma and other cell was able to interfere with ER-binding kinetics lines, N,N-dimethylformamide — stimulated and could cause receptor inactivation on mitogenesis via the epidermal growth factor the binding site directly or the ER-estradiol (EGF) receptor and increased TGF-B receptors, complex (Sasson & Notides, 1988). In addition, with results variable across cell type (Levine N,N-dimethylformamide was also able to act et _al., 1985a, b; Levine et al., 1989; Levine & as a weak ER agonist in fish (Ren et al., 1996). Chakrabarty, 1992). N,N-dimethylformamide N,N-Dimethylformamide also influenced the sex also significantly induced the number of uroki- ratio and the gonadosomatic index in rainbow nase receptors in a human colon carcinoma cell trout (van den Hurk & Slof, 1981). line (Boyd et al., 1988). 4.2.5 Other mechanisms (b) Experimental systems In the polyclonal human ovarian adeno- A 13-week toxicity study in female and carcinoma cell line HOC-7, male 0.5% cynomolgus N,N- monkeys treated with dimethylformamide prolonged the cell doubling N,N-dimethylformamide at 30-500 ppm by inha- time, downregulated c-Myc protein expression lation showed a slight trend towards increasing and caused development of the HOC-7 cells to estrous cycle (Hurtt et al., 1992). a less malignant cell phenotype (Somay et al., Some experimental studies did report 1992). In addition, N,N-dimethyl-formamide has receptor perturbations at relatively high (in also been found to induce the EGF receptor, but the range of 7 to 15%) dose levels. While using the overall inhibitory effect on c-Myc expression N,N-dimethylformamide to increase the solu- by N,N-dimethylformamide has been suggested bility of estrogen receptor (ER) agonists, it to dominate its eventual anti-proliferative effect was observed that N,N-dimethylformamide in these HOC-7 cells (Grunt et al., 1993), 157 IARC MONOGRAPHS - 115 4.3 Data relevant to comparisons model for N,N-dimethylformamide and N-methylformaldehyde in *1C homozygotes and across agents and end-points *1D-hetero- and -homozygotes, in a study of the For all compounds evaluated in the present *1C/*1D CYP2E]1 polymorphism in 20 male and volume, including N,N-dimethylformamide, 24 female workers (Nomiyama et al., 2001b). analyses of high-throughput screening data A total of 123 workers exposed to generated by the Tox21 and ToxCast™ research N,N-dimethylformamide were investigated for programmes of the government of the USA genotypes of CYP2E1 and half-lives of urinary (Kavlock et_al., 2012; Tice et_al., 2013) are N-methylformamide (Nomiyama et al., 2001c). presented in the Monograph on 1-bromopropane The workers comprised 77 cl homozygotes, 45 in the present volume. c2 heterozygotes, and 1 c2 homozygotes. The half-lives of cl homozygotes were not signifi- cantly different from those of the c2 heterozy- 4.4 Susceptibility to cancer gotes and there were no differences between No studies with cancer as an outcome that N-methylformamide half-lives for subjects with investigated susceptibility were available to the or without the c2 allele. Working Group. There were susceptibility studies concerning 4.5 and and 26/50 at 60 mg/kg bw (P < 0.05); hepato- Labelling of Chemicals of the United Nations, cellular carcinoma (multiple): 7/50 controls, 7/50 N,N-dimethyl-p-toluidine is harmful if is swal- at 6 mg/kg bw, 16/50 at 20 mg/kg bw (P < 0.05) lowed (H301), is in contact with skin (H311), or is and 22/50 at 60 mg/kg bw (P < 0.01); hepato- inhaled (H331). N,N-Dimethyl-p-toluidine may cellular carcinoma (including multiple): 22/50 also cause damage to organs through prolonged controls (P for trend, 0.002), 25/50 at 6 mg/kg and repeated exposure (reproductive organs after bw, 30/50 at 20 g/kg bw and 36/50 at 60 mg/kg oral exposure) (H373) and is harmful to aquatic bw (P = 0.005); hepatocellular adenoma or carci- life, with long-lasting effects (H412) (ECHA noma (combined): 38/50 controls (P for trend, 2015). 0.005), 44/50 at 6 mg/kg bw, 47/50 at 20 mg/kg bw (P= 0.010), and 48/50 at 60 mg/kg bw (P = 0.006); and hepatoblastoma: 1/50 controls, 5/50 at 2. Cancer in Humans 6 mg/kg bw, 10/50 at 20 mg/kg bw (P= 0.005) and 8/50 at 60 mg/kg bw (P = 0.021). The incidence No data were available to the Working Group. of hepatocellular adenoma, hepatocellular carci- noma or hepatoblastoma (combined) was 38/50 controls (P for trend, 0.006), 45/50 at 6 mg/kg bw, 3. Cancer in Experimental Animals 48/50 at 20 mg/kg bw (P = 0.004), and 48/50 at 60 mg/kg bw (P= 0.006). The historical incidence for hepatoblastoma in studies by gavage in corn See Table 3.1 oil was 14/350 (4.0% + 2.8%; range, 0-8%) and for all routes was 61/1149 (5.3% + 7.1%; range, 3.1 Mouse 0-34%) and that for hepatocellular adenoma, hepatocellular carcinoma or hepatoblastoma Groups of 50 male and 50 female B6C3F,/N (combined) in studies by gavage in corn oil was mice (age, 5-6 weeks) were given N,N-dimethyl- 242/350 (69.1% + 8.0%; range, 58-78%) and for p-toluidine (purity, > 99%) by gavage in corn oil all routes was 852/1149 (74.2% + 11.5%; range, at doses of 0 (control), 6, 20, or 60 mg/kg bw on 5 52-92%). days per week for 105 weeks. Survival of females In females, treatment-related increases at 60 mg/kg bw was significantly lower than in the incidence of hepatocellular tumours that of the group receiving the vehicle control. were observed, including that of hepatocel- The mean body weights of males and females lular adenoma (multiple): 2/50 controls, 6/50 at at 60 mg/kg bw were > 10% lower than those of 6 mg/kg bw, 29/50 at 20 mg/kg bw (P < 0.01) and the vehicle controls after week 89 and week 65, 35/50 at 60 mg/kg bw (P < 0.01); hepatocellular respectively. The decreased survival of females at adenoma (including multiple): 17/50 controls (P 60 mg/kg bw, and the decrease in body-weight for trend, < 0.001), 19/50 at 6 mg/kg bw, 37/50 at gain in males and females at 60 mg/kg bw were 20 mg/kg bw (P< 0.001), and 44/50 at 60 mg/kg bw attributed to the development of treatment-re- (P < 0.001); hepatocellular carcinoma (multiple): lated tumours (NTP, 2012; Dunnick et al., 2014). 1/50 controls, 3/50 at 6 mg/kg bw, 5/50 at 20 mg/kg In males, treatment-related increases in bw, and 19/50 at 60 mg/kg bw (P < 0.01); hepa- hepatocellular tumours were observed in males, tocellular carcinoma (including multiple): 6/50 including increases in the incidence of hepato- controls (P for trend, < 0.001), 13/50 at 6 mg/kg cellular adenoma (multiple): 17/50 controls, 19/50 bw (P = 0.049), 18/50 at 20 mg/kg bw (P = 0.002), 174 Table 3.1 Studies of carcinogenicity in experimental animals treated with N,N-dimethyl-p-toluidine by gavage in corn oil Species, strain (sex) Purity Incidence of tumours Significance Comments Age at start Dose regimen Duration No. of animals at start Reference No. of surviving animals Mouse, B6C3F,/N (M) Purity, > 99% Liver GLP study 5-6 wks 0, 6, 20, 60 mg/kg bw Hepatocellular adenoma (multiple): Trend: NR 105 wks 5 days/wk for 105 wks 17/50, 19/50, 27/50*, 26/50* *P< 0.05 NTP (2012) 50/group Hepatocellular adenoma (including multiple): 29/50, NS ee 34/50, 37/50, 36/50 Hepatocellular carcinoma (multiple): Trend: NR 7/50, 7/50, 16/50*, 22/50** *P< 0.05 “*P< 0.01 Hepatocellular carcinoma (including multiple): Trend: P = 0.002 22/50, 25/50, 30/50, 36/50* *P = 0.005 Hepatocellular adenoma or carcinoma (combined): Trend: P = 0.005 38/50, 44/50, 47/50*, 48/50** *P = 0.010 **P = 0.006 Hepatoblastoma: *P = 0.005 1/50, 5/50, 10/50*, 8/50** *P = 0.021 Hepatocellular adenoma, hepatocellular carcinoma Trend: P = 0.006 or hepatoblastoma (combined): *P = 0.004 38/50, 45/50, 48/50*, 48/50** **P = 0.006 Mouse, B6C3F,/N (F) Purity, > 99% Liver GLP study 5-6 wks 0, 6, 20, 60 mg/kg bw Hepatocellular adenoma (multiple): Trend: NR 105 wks 5 days/wk for 105 wks 2/50, 6/50, 29/50*, 35/50* *P< 0.01 NTP (2012) 50/group Hepatocellular adenoma (including multiple): Trend: P < 0.001 43, 40, 39, 32 17/50, 19/50, 37/50*, 44/50* *P < 0.001 Hepatocellular carcinoma (multiple): Trend: NR 1/50, 3/50, 5/50, 19/50* *P< 0.01 Hepatocellular carcinoma (including multiple): Trend: P < 0.001 6/50, 13/50*, 18/50**, 31/50*** *P = 0.049 *P = 0.002 oP < 0.001 Hepatocellular adenoma or carcinoma (combined): — Trend: P< 0.001 20/50, 25/50, 42/50*, 45/50* *P < 0.001 Hepatoblastoma: Trend: P = 0.007 0/50, 1/50, 0/50, 4/50* *P = 0.044 GLI sulpIn|oy-d-|AYyJ@UWIG-N‘N 9OLL VI Table 3.1 (continued) DY Species, strain (sex) Purity Incidence of tumours Significance Comments SHdVYDONOW Age at start Dose regimen Duration No. of animals at start Reference No. of surviving animals Mouse, B6C3F,/N (F) Hepatocellular adenoma, hepatocellular carcinoma, Trend: P< 0.001 5-6 wks or hepatoblastoma (combined): *P < 0.001 105 wks 20/50, 26/50, 42/50*, 45/50* NTP (2012) Lung - (cont.) Alveolar/bronchiolar adenoma: Trend: P< 0.001 SLL 2/50, 4/50, 8/50*, 12/50** *P = 0.039 **P < 0.001 Alveolar/bronchiolar carcinoma: NS 0/50, 1/50, 2/50, 1/50 Alveolar/bronchiolar adenoma or carcinoma Trend: P < 0.001 (combined): *P = 0.021 2/50, 5/50, 9/50*, 13/50** **P < 0.001 Forestomach Squamous cell papilloma: Trend: P = 0.037 1/50, 5/50, 6/50”, 7/50** *P = 0.049 **P = 0.017 Squamous cell carcinoma: NS 0/50, 1/50, 0/50, 0/50 Squamous cell papilloma or carcinoma (combined): *P = 0.049 1/50, 6/50, 6/50*, 7/50** **P = 0.017 Rat, F344 (M) Purity, > 99% Liver GLP study 6-7 wks 0, 6, 20, 60 mg/kg bw Hepatocellular adenoma: NS The incidence of thyroid 104 wks 5 days/wk for 104 wks 0/50, 0/50, 1/50, 1/50 follicular cell adenoma or NTP (2012) 50/group Hepatocellular carcinoma: Trend: P< 0,001 Carcinoma (combined) at 37, 37, 31, 21 0/50, 0/50, 1/50, 6/50* *P — 0.009 60 mg/kg bw was outside Hepatocellular adenoma or carcinoma (combined): — Trend: P< 0.001 that of the rag Or tne ole 0/50, 0/50, 2/50, 6/50* *P = 0.009 (all routes, 0-6%) Nasal cavity Transitional epithelium adenoma: Trend: P < 0.001 0/50, 3/49, 2/50, 11/49* *P< 0.001 Transitional epithelium carcinoma: Trend: P = 0.033 0/50, 0/49, 0/50, 2/49 Transitional epithelium adenoma or carcinoma Trend: P < 0.001 (combined): *P< 0.001 0/50, 3/49, 2/50, 13/49* Table 3.1 (continued) Species, strain (sex) Purity Incidence of tumours Significance Comments Age at start Dose regimen Duration No. of animals at start Reference No. of surviving animals Rat, F344 (M) Thyroid 6-7 wks Follicular cell adenoma: NS 104 wks 1/50, 0/49, 1/50, 3/49 NTP (2012) Follicular cell carcinoma: NS (cont.) 0/50, 2/49, 1/50, 2/49 Follicular cell adenoma or carcinoma (combined): NS 1/50, 2/49, 2/50, 4/49 Rat, F344 (F) Purity, > 99% Liver GLP study 6-7 wks 0, 6, 20, 60 mg/kg bw Hepatocellular adenoma: Trend: P = 0.044 The incidence of transitional 105 wks 5 days/wk for 105 wks 0/50, 1/50, 1/50, 3/49 epithelium adenoma of the NTP (2012) 50/group Hepatocellular carcinoma (multiple): nasal cavity at 60 mg/kg bw NS 33, 42, 33, 23 0/50, 0/50, 0/50, 1/49 was outside that of the range for this tumour in historical Hepatocellular carcinoma (including multiple): Trend: P < 0.001 controls (corn oil gavage 0/50, 0/50, 0/50, 4/49* *P=0.041 studies, 0%; all routes, 0-2%) Hepatocellular adenoma or carcinoma (combined): Trend: P < 0.001 0/50, 1/50, 1/50, 7/49* *P = 0.003 Nasal cavity Transitional epithelium adenoma: NS 0/50, 1/49, 0/50, 2/49 bw, body weight; F, female; GLP, good laboratory practice; M, male; NR, not reported; NS, not significant; wk, week LLL sulpIn|oy-d-|AYyJ@UWIG-N‘N IARC MONOGRAPHS - 115 and 31/50 at 60 mg/kg bw (P < 0.001); hepato- papilloma: 1/50 controls (P for trend, 0.037), cellular adenoma or carcinoma (combined): 5/50 at 6 mg/kg bw, 6/50 at 20 mg/kg (P = 0.049), 20/50 controls (P for trend, < 0.001), 25/50 at and 7/50 at 60 mg/kg bw (P = 0.017); and fores- 6 mg/kg bw, 42/50 at 20 mg/kg bw (P < 0.001), tomach squamous cell papilloma or carcinoma and 45/50 at 60 mg/kg bw (P < 0.001); and hepa- (combined): 1/50 controls, 6/50 at 6 mg/kg bw, toblastoma: 0/50 controls (P for trend, 0.007), 6/50 at 20 mg/kg bw (P = 0.049) and 7/50 at 1/50 at 6 mg/kg bw, 0/50 at 20 mg/kg bw, and 60 mg/kg bw (P = 0.017). The historical inci- 4/50 at 60 mg/kg bw (P = 0.044). The incidence dence of forestomach squamous cell papilloma of hepatocellular adenoma, hepatocellular carci- in studies of gavage in corn oil was 12/348 noma or hepatoblastoma (combined) was 20/50 (3.5% + 1.5%; range, 2-6%) and for all routes was controls (P for trend, < 0.001), 26/50 at 6 mg/kg 22/1198 (1.8% + 1.7%; range, 0-6%) and that of bw, 42/50 (P < 0.001) at 20 mg/kg bw, and 45/50 at forestomach squamous cell papilloma or carci- 60 mg/kg bw (P< 0.001). The historical incidence noma (combined) in studies of gavage in corn oil for hepatoblastoma in studies on gavage in corn was 12/348 (3.5% + 1.5%; range, 2-6%) and for all oil was 1/347 (0.3% + 0.8%; range, 0-2%) and for routes was 23/1198 (1.9% + 1.6%; range, 0-6%). all routes was 4/1195 (0.3% + 0.8%; range, 0-2%) One female mouse given 6 mg/kg bw had a fores- and that for hepatocellular adenoma, hepatocel- tomach squamous cell carcinoma (0/50 controls, lular carcinoma or hepatoblastoma (combined) 1/50 at 6 mg/kg bw, 0/50 at 20 mg/kg bw, and in studies on gavage in corn oil was 91/347 0/50 at 60 mg/kg bw). The historical incidence for (26.2% + 12.7%; range, 8-40%) and for all routes forestomach squamous cell carcinoma in studies was 444/1195 (37.2% + 22.9%; range, 6-82%). In on gavage in corn oil was 0/348 and that for all addition, an increase was observed in the inci- routes was 1/1198 (0.1% + 0.4%; range, 0-2%). dence of lung alveolar/bronchiolar adenoma: 2/50 Treatment-related non-neoplastic lesions controls (P for trend, < 0.001), 4/50 at 6 mg/kg were seen in the liver, nose, lung, olfactory lobe bw, 8/50 at 20 mg/kg bw (P = 0.039), and 12/50 and spleen in males and females. In females, at 60 mg/kg bw (P < 0.001); and alveolar/bron- increases in non-neoplastic lesions of the fores- chiolar adenoma or carcinoma (combined): 2/50 tomach, bone marrow and mesenteric lymph controls (P for trend, < 0.001), 5/50 at 6 mg/kg node were also observed (NTP, 2012). [The bw, 9/50 at 20 mg/kg bw (P = 0.021), and 13/50 strength of this study was that it was a 2-year at 60 mg/kg bw (P < 0.001). The historical inci- cancer bioassay in males and females with a large dence for alveolar/bronchiolar adenoma or carci- number of animals per group, multiple doses, and noma (combined) in studies on gavage in corn in compliance with good laboratory practice.] oil was 23/346 (6.7% + 3.2%; range, 2-12%) and for all routes was 100/1196 (8.4% + 4.3%; range, 3.2 Rat 2-22%). A few alveolar/bronchiolar carcinomas were found in treated groups (0/50 controls, 1/50 Groups of 50 male and 50 female Fischer at 6 mg/kg bw, 2/50 at 20 mg/kg bw and 1/50 344/N rats (age, 6-7 weeks) were given at 60 mg/kg bw). The incidence for this tumour N,N-dimethyl-p-toluidine (purity, > 99%) by was not significant and was within the historical gavage in corn oil at doses of 0 (control), 6, 20, or control range, which was 7/346 (2.0% + 2.0%; 60 mg/kg bw on 5 days per week for 104 weeks range, 0-4%) in studies on gavage in corn oil (males) or 105 weeks (females). At termination and 44/1196 (3.7% + 3.3%; range, 0-14%) for all after 2 years, survival in male rats at 60 mg/kg studies. Significant increases were also observed bw was significantly lower than that in controls, in the incidence of forestomach squamous cell which was attributed to the development of 178 treatment-related tumours. Mean body weights of males and females at 60 mg/kg bw were > 10% lower than those in the vehicle-control group after weeks 61 and 33, respectively (NTP, 2012; Dunnick et al., 2014). In males, treatment-related increases in the incidence of hepatocellular tumours included those of hepatocellular carcinoma: 0/50 controls (P for trend, < 0.001), 0/50 at 6 mg/kg bw, 1/50 at 20 mg/kg bw, and 6/50 at 60 mg/kg bw (P= 0.009); and hepatocellular adenoma or carcinoma (combined): 0/50 controls (P for trend, < 0.001), 0/50 at 6 mg/kg bw, 2/50 at 20 mg/kg bw, and 6/50 at 60 mg/kg bw (P = 0.009). The incidence of hepatocellular adenoma was 0/50 controls, 0/50 at 6 mg/kg bw, 1/50 at 20 mg/kg bw, and 1/50 at 60 mg/kg bw. The historical incidence of hepato- cellular carcinoma was 0/299 in studies of gavage in corn oil and 5/1249 (0.4% + 1.0%; range, 0-4%) for all routes. Increases were also observed in the incidence of nasal cavity transitional epithelium adenoma: 0/50 controls (P for trend, < 0.001), 3/49 at 6 mg/kg bw, 2/50 at 20 mg/kg bw, and 11/49 at 60 mg/kg bw (P < 0.001); and nasal cavity transitional epithelium adenoma or carcinoma (combined): 0/50 controls (P for trend, < 0.001), 3/49 at 6 mg/kg bw, 2/50 at 20 mg/kg bw, and 13/49 at 60 mg//kg bw (P < 0.001). The historical incidence for nasal cavity transitional epithelium adenoma indicated that they have not previously been observed in historical controls (studies of gavage in corn oil 0/299; all routes 0/1248). The incidence of nasal cavity transitional epithelium carcinoma was not significantly increased by pairwise comparison, although 2 males given 60 mg/kg bw had these tumours (0/50 controls, 0/49 at 6 mg/kg bw, 0/50 at 20 mg/kg bw, and 2/49 at 60 mg/kg bw; P for trend, 0.033); no histor- ical control data were available for this tumour type. There was a non-significant increase in thyroid gland follicular cell adenoma or carci- noma (combined) (1/50 controls, 2/49 at 6 mg/kg bw, 2/50 at 20 mg/kg bw, and 4/49 at 60 mg/kg bw), although the incidence at 60 mg/kg bw was outside of the incidence range for this tumour in historical controls. The incidence for thyroid gland follicular cell adenoma or carcinoma (combined) in the historical controls was 9/299 (3.0% + 2.1%; range, 0-6%) in studies on gavage in corn oil and 23/1239 (1.9% + 2.2%; range, 0-6%) for all routes. A non-significant increase in the incidence of thyroid gland follicular cell carcinoma was observed (0/50 controls, 2/49 at 6 mg/kg bw, 1/50 at 20 mg/kg bw, and 2/49 at 60 mg/kg bw), for which the historical control incidence was 3/299 (1.0% + 1.7%; range, 0-4%) in studies on gavage in corn oil and 10/1239 (0.8% + 1.5%; range, 0-4%) for all routes. In females, the incidence of hepatocellular adenoma was significantly increased according to trend statistics (P = 0.044): 0/50 controls, 1/50 at 6 mg/kg bw, 1/50 at 20 mg/kg bw, and 3/49 at 60 mg/kg bw; and that of hepatocellular carcinoma was significantly increased at the highest dose: 0/50 controls, 0/50 at 6 mg/kg bw, 0/50 at 20 mg/kg bw, and 4/49 at 60 mg/kg bw (P = 0.041). A significant increase in the inci- dence of hepatocellular adenoma or carcinoma (combined) was also observed: 0/50 controls (P for trend, < 0.001), 1/50 at 6 mg/kg bw, 1/50 at 20 mg/kg bw, and 7/49 at 60 mg/kg bw (P = 0.003). The historical incidence of these tumours was 1/300 (0.3% + 0.8%; range, 0-2%) in studies with administration by gavage in corn oil, and 12/1200 (1.0% + 1.6%; range, 0-4%) for all routes. A non-significant increase in the incidence of nasal cavity transitional epithelium adenoma was also observed (0/50 controls, 1/49 at 6 mg/kg bw, 0/50 at 20 mg/kg bw, and 2/49 at 60 mg/kg bw), although the incidence in females given the highest dose was outside the historical range for this tumour, which was 0/299 in studies on gavage in corn oil, and 1/1196 (0.1% + 0.4%; range, 0-2%) for all routes. Treatment-related non-neoplastic lesions were seen in the liver and nose of males and females; non-neoplastic lesions also occurred in the spleen, kidney, and bone marrow of males 179 IARC MONOGRAPHS - 115 and females. In addition, significant increases at these sites was > 1. Male rats excreted about in forestomach and mesenteric lymph node 60% of the total dose in urine within 6 hours of non-neoplastic lesions were found in male rats receiving a dose of 2.5 mg/kg bw, whereas male (NTP, 2012). [The strength of this study was and female rats receiving 25 mg/kg bw excreted that it was a 2-year cancer bioassay in males about half as much (i.e. about 30%). However, and females, with a large number of animals per cumulative excretion in the urine was similar group, multiple doses, and in compliance with for both doses at the 24-hour time-point. These good laboratory practice.] results indicated initial saturation of absorption and/or elimination of the higher dose. The data on urinary excretion for 2.5 and 25 mg/kg bw 4. Mechanistic and Other were similar over time for male mice. Female Relevant Data mice appeared to excrete less of the higher dose in the urine than males; however, this difference 4.1 Absorption, distribution, was probably due to poor recovery of radiolabel in the urine of one or more individuals. All of metabolism, excretion the other data on disposition in female mice were 4.1.1 Absorption, distribution, and excretion similar to those of males. [The Working Group noted that the data from groups of male rats and (a) Humans mice that received 250 mg/kg bw by gavage were No data were available to the Working Group. compromised by acute toxicity.] Cumulative excretion of the dose in the high-dose rats (b) Experimental systems amounted to only ~70%, possibly as a result of [U-“C]-Labelled N,N-dimethyl-p-toluidine the initial acute toxicity. Most of the unexcreted was rapidly absorbed after a single oral admin- radiolabel was present in the gastrointestinal istration of 2.5, 25 or 250 mg/kg bw to male and tract at 24 hours. In a supplemental experiment female Fischer 344 rats and B6C3F, mice (four reported to the National Toxicology Program, animals/treatment group) (Dix et _al., 2007). the absorption and excretion of a high dose were Absorption of the dose was nearly complete nearly complete at 72 hours after treatment, with based on the data on excretion and their simi- about 2% of the total dose detected in tissues larity to those obtained from groups of rats and the gastrointestinal tract (NTP, 2012). The and mice injected intravenously. Cumulative data for male mice receiving 250 mg/kg bw in excretion amounted to about 90% in the urine this study were not reported here due to death and 4% in the faeces 24 hours after gavage. or unresolved morbidity associated with all indi- N,N-Dimethyl-p-toluidine-derived radioactivity viduals in the treatment group. appeared to have been widely distributed to the tissues. At the terminal 24-hour time-point, the 4.1.2 Metabolism assayed tissues, including blood, liver, kidney, (a) Humans skin, muscle and adipose tissue, contained 2-5% of the total dose. In rats, the highest concentra- No data were available to the Working Group. tion of radioactivity was detected in the liver, followed by the kidney and urinary bladder. The (6b) Experimental systems lung and liver contained the highest concen- See Figure 4.1 trations in mice. The tissue-to-blood ratio of As reported by Kim et al. (2007), the metabo- N,N-dimethyl-p-toluidine-derived radioactivity lism of N,N-dimethyl-p-toluidine was rapid and 180 N,N-Dimethyl-p-toluidine Fig. 4.1 Known and postulated metabolites of N,N-dimethyl-p-toluidine in rats O H;C. CH; CH HO, N HN N ‘CH; [CH,0] P450 CH, CH; O Non N,N-Dimethy]-p-toluidine N-Methy1-p-toluidine O p-(N-Acetylhydroxyamino)hippuric acid FMO | | P450 P450 [CH,0] Alcohol dehydrogenase Aldehyde dehydrogenase N-Acetyltransferase Aralkyl-CoA:Glycine N-Acyltransferase O Hc # cn; N JOH gp HN N NH ad ———_> CH; CH, N,N-Dimethy1-p-toluidine N-oxide p-Methylphenylhydroxylamine p-Nitrosoluene p-Quinone imine methide Adapted from Kim et al. (2007) and Dunnick et al. (2014), with permission of the publisher, SAGE Publications, Inc FMO, flavin-containing monooxygenase; P450, cytochrome P450; coA, coenzyme A extensive in rats in a study conducted by Dix et_al., 2007; Dunnick et_al., 2014). The forma- et al. (2007). The major radiolabelled metabo- tion of p-methylphenylhydroxylamine may lead lite in rat urine was identified as p-(N-acetylhy- to the oxidation of haemoglobin (Potter et al. droxyamino)hippuric acid that was catalysed by 1988) or DNA binding (Marques et _al., 1997). cytochrome P450, alcohol and aldehyde dehy- N,N-Dimethyl-p-toluidine N-oxide, considered drogenases, N-acetyl transferase and aralkyl-co- to be catalysed primarily by flavin-containing enzyme A:glycine-N-acyltransferase. The urine monooxygenases, was also excreted in urine, as also contained N-methyl-p-toluidine, an oxida- was a small amount of parent N,N-dimethyl-p- tive product of N,N-dimethyl-p-toluidine, and a toluidine (Kim et al., 2007). precursor of the glycine conjugate. Intermediates in this pathway are postu- lated to include p-methylphenylhydroxylamine and an electrophilic p-quinone imine methide (4-methylene-2,5-cyclohexadiene-1-imine) (Kim 181 IARC MONOGRAPHS - 115 Table 4.1 Genetic and related effects of N,N-dimethyl-p-toluidine in non-human mammals in vivo Species, strain Tissue End-point/test Results Dose (LED/HID) Reference (sex) Rat, Sprague- Liver DNA damage/ + 4 mg/kg bw, gavage and ip. Taningher et al. Dawley (M) DNA strand breaks (1993) Rat, Sprague- Liver DNA damage/ + 60 mg/kg bw, gavage, 4 days NTP (2012) Dawley (M) DNA strand breaks Mouse, Balb/C (M) Liver DNA damage/ + 1 mg/kg bw, gavage and ip. Taningher et al. DNA strand breaks (1993) Mouse, B6C3F, (M) Blood DNA damage/ - 75 mg/kg bw x 4 days, gavage NTP (2012) leukocytes, liver DNA strand breaks Mouse, B6C3F, (M Peripheral blood Chromosomal damage/ - 125 mg/kg bw per day for NTP (2012) & F) micronucleus formation 3 mo; gavage Mouse, B6C3F, (M) Peripheral blood Chromosomal damage/ - 75 mg/kg bw x 4 days, gavage NTP (2012) micronucleus formation +, positive; -, negative; +, equivocal (variable responses in several experiments within an adequate study); bw, body weight; F, female; HID, highest ineffective dose; i.p., intraperitoneal; LED, lowest effective dose; M, male; mo, month 4.2 Mechanisms of carcinogenesis DNA fragmentation was modestly increased by the alkaline elution test (Taningher et al., 1993). The evidence on the key characteristics of The alkaline comet assay gave negative results carcinogens (Smith et al. 2016), concerning in liver cells or blood leukocytes of male B6C3F, whether N,N-dimethyl-p-toluidine is genotoxic mice treated by gavage once daily for 4 days at and alters cell proliferation, cell death, and 30-75 mg/kg bw. In contrast, male Sprague- nutrient supply, is summarized below. Dawley rats given daily doses of 60 mg/kg bw for 4 days had a small but statistically significant 4.2.1 Genetic and related effects (P = 0.024) increased percentage of tail DNA in the liver cells compared with the group adminis- (a) Humans tered the corn-oil vehicle only (NTP, 2012). No data in exposed humans were available to Neither the frequencies of micronucleated the Working Group. In cultured human lympho- erythrocytes nor the percentage of circulating cytes, the bone cement polymethyl methacrylate, reticulocytes were altered in the peripheral containing N,N-dimethyl-p-toluidine (2.6% w/w) blood of male or female B6C3F,/N mice treated and hydroquinone (75 + 15 ppm), significantly by gavage with N,N-dimethyl-p-toluidine, increased the frequency of micronucleus forma- suggesting that neither chromosomal damage tion. However, the components of the mixture nor bone marrow toxicity were induced in mice were not tested individually (Bigatti et al., 1994). within the dose range and time periods tested (NTP, 2012). (b) Experimental systems (ii) Invitro See Table 4.1, Table 4.2, and Table 4.3 N,N-Dimethyl-p-toluidine showed both (i) In vivo aneugenic and clastogenic activity in a concen- In the livers of male BALB/c mice and male tration—-related manner in the absence of meta- Sprague-Dawley rats given N,N-dimethyl-p- bolic activation in Chinese hamster V79 cells toluidine by gavage or intraperitoneal injection, (Taningher et al., 1993). 182 N,N-Dimethyl-p-toluidine Table 4.2 Genetic and related effects of N,N-dimethyl-p-toluidine in non-human mammalian cells in vitro Test system, tissue End-point/ Results Agent, concentration Comments Reference test (LEC/HIC) Chinese hamster, Chromosomal = + Camphorquinone/DMPT DMPT alone was not Liet al. (2007) CHO-K1 ovary cell damage/ (equimolar), tested; antioxidants had line micronucleus 0-1 mM preventive effects formation Chinese hamster, Chromosomal = + 9-Fluorenone/DMPT DMPT alone was not Liet al. (2008) CHO-K]1 ovary cell damage/ (equimolar), tested; antioxidants had line micronucleus 0-0.5 mM preventive effects formation Chinese hamster Chromosomal = + DMPT, Dose-dependent Taningher et al. lung, V79 cells damage/ 0.3 mM

Response

0.3-1.2 mM) (1993) micronucleus formation +, positive; DMPT, N,N-dimethyl-p-toluidine; HIC, highest ineffective concentration; LEC, lowest effective concentration A concentration-related increase in micronu- inhibitory concentration value was also approxi- clei was observed in the CHO-K1 ovary cell line mately 4 mM] (Nomura et al., 2006). exposed to camphorquinone and N,N-dimethyl- Exposure to camphorquinone in association p-toluidine or 9-fluorenone and N,N-dimethyl-p- with N,N-dimethyl-p-toluidine induced DNA toluidine (P < 0.05), with or without visible light strand breaks in PhiX-174 RF double-stranded irradiation (Liet al., 2007, 2008) [N,N-dimethyl- plasmid DNA (Pagoria et al., 2005; Winter et al., p-toluidine was not tested alone]. 2005; Lee et al., 2007) [N,N-dimethyl-p-toluidine N,N-Dimethyl-p-toluidine was margin- was not tested alone]. ally positive in Salmonella typhimurium strain TA100 in the presence and absence of metabolic 4.2.2 Altered cell proliferation or death activation, and in strain TA104 in the presence of metabolic activation, but not in TA98 with (a) Humans or without metabolic activation. The compound No data in exposed humans were available to was not mutagenic in plate incorporation tests the Working Group. (Miller et al., 1986). N,N-Dimethyl-p-toluidine did not induce Negative results were reported with or without cytotoxicityin cultured humanoralkeratinocytes metabolic activation in Salmonella typhimurium (OKF6/TERT 2), and simultaneous treatment strains TA97, TA98, TA100 and TA1535 and in with camphorquinone and N,N-dimethyl-p- Escherichia coli WP2 uvrA/pKM101 (Janingher toluidine did not affect apoptosis (Volk et_al. et al., 1993; NTP, 2012). 2014). When tested in a bioluminescent test for When assessed in human gingival fibroblasts bacterial genotoxicity using dark mutants in vitro, N,N-dimethyl-p-toluidine (500 uM) of a marine luminous bacterium (Vibrio induced a significant and sustained accumula- fischeri M169) and a 24-hour exposure period, tion of cells in the GO/GI stage of the cell cycle N,N-dimethyl-p-toluidine showed considerable compared with controls at 24 hours, implying genotoxic activity at 4 mM [the half maximal that the growth inhibitory effect resulted from the arrest of DNA replication. The pattern of cell 183 IARC MONOGRAPHS - 115 Table 4.3 Genetic and related effects of N,N-dimethyl-p-toluidine in bacterial mutation tests Species Strain End-point/ Results Concentration Comments Reference test (LEC/HIC) Without With metabolic activation Salmonella TA98 Mutation/ - 10 uL (of Negative in plate Miller et al. typhimurium TA100 reverse + 300 ug/uL)/ incorporation tests (1986) TA104 mutation _ + plate (spot test) Salmonella TA97, TA98, Mutation/ - 70 pg/plate Taningher typhimurium TA100 reverse et al. (1993) mutation Salmonella TA97, TA98, Mutation/ - 1000 pg/plate NTP (2012) typhimurium TA100, reverse TA1535 mutation Salmonella TA98, Mutation/ = - - 1500 pg/plate NTP (2012) typhimurium TA100 reverse mutation Escherichia. WP2uvrA/ Mutation/ - 1500 pg/plate NTP (2012) coli pKM101 reverse mutation Vibrio M169 Mutation/ = + NT 4mM Bioluminescence Nomura et al. fischeri other assay (2006) +, positive; -, negative; +, equivocal (variable responses in several experiments within an adequate study); DMPT, N,N-dimethyl-p-toluidine; HIC, highest ineffective concentration; LEC, lowest effective concentration; NT, not tested death was consistent with necrosis ( Masukietal. 4.3 Data relevant to comparisons 2007). across agents and end-points In human osteoblastic cells exposed to different bone-cement extracts, a significant N,N-Dimethyl-p-toluidine was not tested by correlation was found between the concentra- the Tox21 and ToxCast™ research programmes of tion of N,N-dimethyl-p-toluidine present in the government of the USA (Kavlock et al., 2012; the extracts (up to 1 ug/100 mL) and a delay Tice et al., 2013). Analyses of other compounds in cell-replication cycle (Stea et al., 1997). [The evaluated in this volume with high-throughput Working Group noted that the extracts did not screening data are presented in the Monograph contain N,N-dimethyl-p-toluidine only.] on 1-bromopropane in the present volume. (b) Experimental systems 4.4 Susceptibility to cancer In rat polymorphonuclear leukocytes, N,N-dimethyl-p-toluidine increased cytotox- No data were available to the Working Group. icity in a concentration-dependent manner (Liso etal., 1997). 184 to dental materials containing N,N-dimethyl-p-toluidine has been reported (Tosti et al., 1990; Verschueren & Bruynzeel, 1991). Positive reactions to N,N-dimethyl-p-toluidine were found in seven cases, all of whom showed rapid onset of aseptic loosening of total hip replacements, out of 70 patients (Haddad et al., 1996). 4,5,2 Experimental systems Ina chronic study, N,N-dimethyl-p-toluidine caused methaemoglobinaemia in F344/N rats after administration by gavage (NIP, 2012; Dunnick et _al., 2014). Metabolic activation to p-methylphenylhydroxylamine was _ inferred from the presence of p-(N-acetylhydroxyamino) hippuric acid in the urine of rats given the compound (Kim et al., 2007; see Section 4.1.2 ). Macrocytic regenerative anaemia, methaemoglo- binaemia, and increased Heinz body production was seen in rats (and to a lesser extent in mice) in a subchronic gavage study. Bone marrow hyper- plasia occurred in exposed rats, but not in mice (NTP, 2012; Dunnick et al., 2014). 5. Summary of Data Reported 5.1 Exposure data N,N-Dimethyl-p-toluidine is a chemical with a high production volume that is used worldwide as an accelerator in the preparation of dental materials, bone cements, industrial glues and artificial-fingernail preparations, and also as an intermediate in the production of dyes and pesti- cides. Chemical-production workers, surgeons, surgical staff, dentists, dental technicians, nail- salon operators, and users of industrial glues may be exposed to N,N-dimethyl-p-toluidine by inhalation and through the skin. Exposure among the general population may occur from dental material, bone cement, sniffing glue and fingernail solutions. No quantitative data on exposure or occupational exposure limits were identified. 5.2 Human carcinogenicity data No data were available to the Working Group. 5.3 Animal carcinogenicity data N,N-Dimethyl-p-toluidine was tested for carcinogenicity by oral administration (gavage) in one good laboratory practice (GLP) study in male and female mice and one GLP study in male and female rats. In mice, N,N-dimethyl-p-toluidine caused a significantly increased incidence (with a signif- icant positive trend, except for hepatoblastoma in males) of hepatocellular carcinoma, hepa- toblastoma, hepatocellular adenoma or carci- noma (combined), and hepatocellular adenoma, hepatocellular carcinoma or hepatoblastoma (combined) in males, and _ hepatocellular adenoma, hepatocellular carcinoma, hepato- blastoma, hepatocellular adenoma or carci- noma (combined), and hepatocellular adenoma, 185 IARC MONOGRAPHS - 115 hepatocellular carcinoma or hepatoblastoma 5.4 Mechanistic and other relevant (combined) in females. It also caused a signif- data icantly increased incidence of alveolar/bron- chiolar adenoma of the lung (with a significant In rats and mice, N,N-dimethyl-p-toluidine positive trend), alveolar/bronchiolar adenoma or is rapidly absorbed after oral administration. carcinoma (combined) of the lung (with a signif- N,N-Dimethyl-p-toluidine and/or its metabo- icant positive trend), forestomach squamous cell lites are distributed widely to the tissues and the papilloma (with a significant positive trend) and highest concentrations are detected in the liver forestomach squamous cell papilloma or carci- and kidney of rats and lung and liver of mice. noma (combined) in female mice. N,N-Dimethyl-p-toluidine is rapidly metabol- In rats, N,N-dimethyl-p-toluidine caused a ized and excreted primarily in the urine. The significantly increased incidence (with a signif- major metabolic pathway leading to the forma- icant positive trend) of hepatocellular carci- tion of p-(N-acetylhydroxyamino)hippuric acid noma, hepatocellular adenoma or carcinoma in rats includes putative reactive intermediates. (combined), adenoma of the transitional epithe- The fate of N,N-dimethyl-p-toluidine has not lium of the nasal cavity and adenoma or carci- been determined in humans. noma (combined) of the transitional epithelium With respect to the key characteristics of of the nasal cavity in males. A significant positive human carcinogens, there is moderate evidence trend was observed in the incidence of carcinoma that N,N-dimethyl-p-toluidine is electrophilic of the transitional epithelium of the nasal cavity or can be metabolically activated, is genotoxic, in males. Increases in the incidence of follicular or alters cell proliferation, cell death or nutrient cell adenoma or carcinoma (combined) of the supply. thyroid gland in males were not significant by the A putative reactive intermediate of pairwise or trend tests, although the incidence of N,N-dimethyl-p-toluidine, p-methylphenyl- these tumours at the highest dose exceeded the hydroxylamine, was implicated in the formation range for historical controls for these tumours. of methaemoglobinaemia in a case of uninten- A significant positive trend was observed in the tional (human) oral exposure to N,N-dimethyl- incidence of hepatocellular adenoma, hepato- p-toluidine. N-Hydroxylated arylamines are cellular carcinoma and hepatocellular adenoma capable of forming DNA adducts. The formation or carcinoma (combined) in females. The inci- of an electrophilic p-quinone imine methide as dences of hepatocellular carcinoma and hepa- a result of the metabolism of N,N-dimethyl-p- tocellular adenoma or carcinoma (combined) in toluidine is plausible. females given the highest dose were significantly A significant increase in the frequency of increased compared with controls. The incidence micronucleus formation was observed in cultured of adenoma of the transitional epithelium of the human lymphocytes treated with the bone nasal cavity in females given the highest dose cement polymethyl methacrylate containing exceeded the range for historical controls for this N,N-dimethyl-p-toluidine. N,N-Dimethyl- tumour. p-toluidine exhibited aneugenic and clas- togenic activity in Chinese hamster V79 cells. N,N-Dimethyl-p-toluidine gave negative or marginally positive results in other assays, including mutation in Salmonella and DNA strand breaks in rats and mice. 186 The cell-replication cycle was delayed in human osteoblastic cells exposed to bone-cement extracts containing N,N-dimethyl-p-toluidine. N,N-Dimethyl-p-toluidine had a growth-inhibi- tory effect in human gingival fibroblasts in vitro and caused cytotoxicity in rat polymorphonu- clear leukocytes, but not in cultured human oral keratinocytes. There were few other data on other key char- acteristics of carcinogens (alters DNA repair or causes genomic instability, induces epigenetic alterations, induces oxidative stress, induces chronic inflammation, is immunosuppressive, modulates receptor-mediated effects, or causes immortalization). 6. Evaluation 6.1 Cancer in humans There is inadequate evidence in humans for the carcinogenicity of N,N-dimethyl-p-toluidine. 6.2 Cancer in experimental animals There is sufficient evidence in experi- mental animals for the carcinogenicity of N,N-dimethyl-p-toluidine. 6.3 Overall evaluation N,N-Dimethyl-p-toluidine is possibly carcino- genic to humans (Group 2B). References 3M Company (2004). 3M™ Scotch-Weld™ Instant Adhesive Surface Activator. 3M Material safety data sheet. St Paul (MN), USA: 3M Company. Bigatti MP, Lamberti L, Rizzi FP, Cannas M, Allasia G (1994). In vitro micronucleus induction by polymethyl methacrylate bone cement in cultured human lympho- cytes. Mutat Res, 321(3):133-7. doi:10.1016/0165- 1218(94)90037-X PMID:7513063 Bowers JS Jr (1996). Toluidines. In: Elvers B, Hawkins S editors. Ullmann’s Encyclopedia of Industrial Chemistry. Volume A27 5th ed. New York, USA VCH Publishers; pp. 159-61. ChemicalBook Inc (2016). Chemical Book [online data- base]. 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Substance use disorders among inhalant users: results from the National Epidemiologic Survey on alcohol and related conditions. Addict Behav, 33(7) 968-73. doi 10.1016/j addbeh.2008.02.019 PMID:18403132 189 HYDRAZINE Hydrazine was considered by the IARC Monographs Working Group in 1973, 1987, and 1998 (IARC, 1974, 1987, 1999). New data have since become available and these have been taken into consideration in the present evaluation. 1. Exposure Data Description: Colourless, fuming, oily liquid with a penetrating ammonia-like odour; odour threshold, 3-9 mg/m? 1.1. Identification of the agent Note: Anhydrous hydrazine contains less 1.1.1 Nomenclature than 37% water by mass; hydrazine hydrate [CAS No.: 7803-57-8] corresponds to a 64% Chem. Abstr. Serv. Reg. No.: 302-01-2 or less aqueous solution of hydrazine (HSDB Chem. Abstr. Serv. Name: Hydrazine 2010) IUPAC Systematic Name: Hydrazine Boiling point: 113.5 °C Synonyms: Levoxine, nitrogen hydride, Melting point: 2 °C diamide, diamine, anhydrous hydrazine, Vapour density: 1.1 (air = 1) hydrazine base. Solubility: 282.0 g/100 g in water at 25 °C; miscible with methyl, ethyl, propyl, 1.1.2 Structural and molecular formulae, and and isobutyl alcohols; insoluble in chloro- form and diethyl ether relative molecular mass Volatility: Vapour pressure, 14.4 mm Hg at 25 °C H,N-NH, Octanol/water partition coefficient (P): log Ko —2.07 at 25 °C Molecular formula: H,N, Flash point: 52 °C Relative molecular mass: 32.05 Explosive limits: Upper, 100%; lower, 1.8% by volume in air 1.1.3. Chemical and physical properties of the Stability: Hydrazine is stable when stored, pure substance but may cause hazardous reactions when in contact with strong oxidizing agents or acids. From IPCS (1987a), ECHA (2011), NIOSH Contact with cellulose or cotton textiles, (2014), HSDB (2010) especially at elevated temperatures, may 191 IARC MONOGRAPHS - 115 result in ignition. Vapours may form explo- Union (ECHA, 2011). The following suppliers of sive mixtures with air when the substance is hydrazine in 2016 were identified: 27 suppliers in heated above its flash point. China, 26 in the USA, 5 in the United Kingdom, Decomposition: Forms nitrogen oxides, 3 in Germany, 2 in Japan, and 1 in each of 5 other ammonia, and hydrogen countries; production volumes were not reported (ChemicalBook, 2016). Conversion factor: 1 ppm = 1.31 mg/m? at 25 °C (ii) | Hydrazine hydrate Production capacity estimates for hydra- zine hydrate in 1988 were: USA, 25 000 tonnes; 1.2 Production and use France, 10 000 tonnes; Germany, 10 000 tonnes; 1.2.1 Production Japan, 5000 tonnes; and United Kingdom, 3000 tonnes (Schirmann, 1989). (a) Production process By 2011, hydrazine hydrate was manufac- Most hydrazine is produced by a variation of tured in China, France, Germany, Japan, and the the Raschig process, where ammonia is oxidized Republic of Korea, and possibly in the Russian using alkaline hypochlorite with or without the Federation and the USA. The worldwide market presence of a ketone such as acetone or butanone. demand for hydrazine hydrate was then about It can also be made by the oxidation of ammonia 80 000-90 000 tonnes per year (ECHA, 2011). by hydrogen peroxide in the presence of butanone The following suppliers of hydrazine hydrate and an oxygen-transfer agent (Schiessl, 2003). in 2016 were identified: 178 suppliers in China, Anhydrous hydrazine is made by breaking 27 in the USA, 11 in the United Kingdom, 6 in the hydrazine-water azeotrope with aniline. Japan, 4 in Germany, and 1 in each of 5 other The aniline-water vapour condenses and phase countries; production volumes were not reported separates. The water layer, contaminated with a (ChemicalBook, 2016). small amount of aniline and hydrazine, flows toa biological treatment pond. The aniline and anhy- 1.2.2 Use drous hydrazine are separated in a final column In its hydrated form, hydrazine (solutions (Schiessl, 1995), with concentrations ranging from 0.01% to 100%) (b) Production volume is used in multiple applications. It is used to synthesize pharmaceuticals, agrochemicals, and (i) | Anhydrous hydrazine chemical blowing agents; as a stabilizing agent of In 2009, hydrazine was produced by three aromatic amines for manufacture of paints, inks, manufacturers worldwide (SRI, 2009; as cited in and organic dyes; and as a reagent for the treat- NTP, 2014), Hydrazine was then available from 27 ment of nuclear reactor waste. Hydrazine is used suppliers (ChemSources, 2009; as cited in NTP, as a monomer in polymerizations, mostly for 2014). USA imports of “hydrazine and hydroxyl- polyurethane coatings and adhesives; as a corro- amine and their salts” generally increased sion inhibitor in water treatment, mainly for the between 1989 and 2008, to a maximum of 235 removal of dissolved oxygen; for pH adjustment 000 tonnes in 1999 (USITC, 2009 as cited in in the feed water of boilers; and for the removal NTP, 2014). of solids from steam generators, notably in Anhydrous hydrazine was not manufactured nuclear and thermal power-generation plants. in the European Union in 2011, and less than 10 Oxygen scavenging of steam by hydrazine may tonnes per year was imported to the European 192 also be carried out in paper mills, steel manufac- ture, and chemical production. It is also used as a reducing agent in the deposition of metals (e.g. nickel, chromium, tin, and precious metals); in plastics and glass manufacture; for recovery of precious and basic metals from metallic salt solu- tions; and as effluent sand in the purification of chemical reagents. Hydrazine is also a laboratory chemical reagent (ECHA, 2011). The anhydrous form (at high purity levels, > 90%) is used as a propellant for aerospace vehi- cles (e.g. satellite propulsion and upper stages of satellite launchers); as a fuel in military power units for the F-16 fighter jet aircraft; and in gas generators for submarine rescue systems (ECHA 2011). 1.3. Measurement and analysis Hydrazine compounds are highly soluble in water and can be measured by spectrophotom- etry (HSDB, 2010). Sampling and analysis methods for hydra- zine in a variety of matrices are listed in Table 1.1. Other hydrazines, such as 1,1- and 1,2-dimethyl- hydrazine, may interfere with these methods. In addition, direct-reading papers or indi- cating tubes, based on colorimetric methods, are available commercially, with reported limits of detection of 65 ug/m:} for tapes and 330 ug/m for tubes (IPCS, 1987a). More recently, an optical probe for hydrazine in air has been described. It uses colorimetric, fluorescent, and chemiluminescent outputs, and has a limit of detection of 3.2 ppb (0.1 uM) (Cui et al., 2014). 1.4 Occurrence and exposure 1.4.1 Natural occurrence Hydrazine is naturally produced by the algae Azotobacter agile as a result of nitrogen fixation (ATSDR, 1997), and in tobacco plants (Choudhary & Hansen, 1998). Production and use of hydrazine may result in its release to the environment. It has been detected at low levels in wastewater samples (HSDB, 2010). 1.4.3 Occupational exposure The National Occupational Exposure Survey estimated that 60 490 workers (including 2841 women) in the USA were potentially exposed to hydrazine between 1981 and 1983 (NIOSH 1997). National estimates on exposure were not available from other countries. Hydrazine is usually handled in closed systems, but fugitive emissions may occur. Table 1.2 presents data on occupational expo- sures for hydrazine. Data suggested that open manual transfer of hydrazine hydrate results in exposure to hydrazine of < 0.02 mg/m, and that exposure is lower when closed transfer is used (HSE, 2006). Exposures of 0.014-0.35 mg/m? (maximum, 1.18 mg/m?) have been measured in hydrazine hydrate production, and 0.29-2.59 mg/m? in a rocket propellant facility where hydrazine was handled (Cook et al., 1979; IPCS, 1987a). Refilling F-16 aircraft with hydrazine resulted in exposures as high as 10.5 mg/m’, but they were usually < 0.13 mg/m? (Christensen, 1978; IPCS, 1987a). Exposure during a spillage of hydrazine was measured as 800 mg/m? (Suggs et al., 1980). Hydrazine hydrate and acetylhydrazine were separately measured, as a result combined at 0.8660 umol/g creatinine (range, not detected to 14.20 umol/g creatinine) in the urine of prod- uction workers in five factories who had been exposed to hydrazine at 0.014 mg/m? (range, not detected to 0.262 mg/m) (Nomiyama et al., 1998a). 1.4.4 Exposure of the general population No data were available to the Working Group. 193 v6L Table 1.1 Selected methods of analysis of hydrazine in various matrices VI DY Sample matrix Sample preparation Assay Limit of detection Reference SHdVYDONOW method Air Collect in bubbler with hydrochloric acid; neutralize with sodium hydroxide; SP 0.9 ug/sample NIOSH (1984) derivatize with dimethyl-aminobenzaldehyde; dilute with glacial acetic acid [NIOSH method 3503] Air Adsorb on sulfuric acid-coated silica gel; elute with water; derivatize with GC/FID 0.002 mg/m? NIOSH (1977a) 2-furaldehyde; extract with ethyl acetate [NIOSH method 248] Air Collect in bubbler with hydrochloric acid; derivatize with phosphomolybdic acid SP - 0.02 mg/m? NIOSH (1977b) [NIOSH method S143] SLL Air Collect in micro-impinger containing acetone and glacial acetic acid GC/NSD 5 ug/m? Holtzclaw et al. (1984) Air Collect on sulfuric acid coated Gas Chrom R; desorption with water [Method HPLC 1.6 wg/m? OSHA (1980a) ORG-20] Air Collect on sulfuric acid-treated glass fibre filters; extract with buffered EDTA LC/UV 0.076 ug/m? OSHA (1980b) disodium solution; derivatize with benzaldehyde [Method ORG-108] Air Method 1 (filter sampling; recommended for personal sampling up to 2 hours): HPLC/UV 0.002 ppm HSE (2014) collect on filters treated with phosphoric acid in acetonitrile solution; extract in sulfuric acid; derivatize in benzaldehyde in methanol; add sodium tetraborate buffer solution Air Method 2 (impinge sampling; recommended for long-term static sampling): collect HPLC/UV 0.002 ppm HSE (2014) in midget impinger containing dilute sulfuric acid; derivatize in benzaldehyde in methanol; add sodium tetraborate buffer solution Water Acidify with hydrochloric acid; derivatize with p-dimethyl-aminobenzaldehyde SP 5 pg/L ASTM (1991) Water Derivatize with vanillin in ethanol; acidify with sulfuric acid SP 0.065 ppm Amlathe & Gupta (1988) Water Derivatize with 5-chlorosalicylaldehyde in acidified aqueous solution (ethanol/ Flat570nm 0.08uM Chen et al. (2008) water/acetic acid = 30/66/4) Water Derivatize with acetone; extract with dichloromethane; dry with anhydrous GC/MS 0.70 ng/L Davis & Li (2008) sodium sulfate; concentrate by evaporation Water Derivatize with propyl chloroformate and pyridine; solid-phase microextraction GC/MS 4.4ng/L Gionfriddo et al. (2014) Water Derivatize with ortho-phthalaldehyde, extract with methylene chloride GC/MS Surface water, Oh et al. (2013) 0.002 ug/L; drinking- water, 0.007 pg/L Soil Extract with sulfuric acid; derivatize with 2,4-pentanedione GC/TID 0.1 ppm Leasure & Miller (1988) Food Extract with L-ascorbic acid; derivatize with 2-nitrobenzaldehyde; clean-up on GC/ECD 10 ppb Wright (1987) alumina column Table 1.1 (continued) Sample matrix Sample preparation Assay Limit of detection Reference method Food Derivatize with pentafluorobenzoyl chloride; extract with methylene chloride GC/MS 0.01 ppm Rutschmann & Buser (1991)? Tobacco & Derivatize with pentafluorobenzaldehyde; enrich with thin-layer chromatography; © GC/ECD 0.1 ng/cigarette Liu et al. (1974) tobacco smoke extract with ether Smokeless Derivatize with pentafluorobenzaldehyde; extract with hexane GC/MS 10 ng/g smokeless McAdam etal. tobacco (2015) Urine Precipitate protein with hydrochloric acid and ammonium sulfate, GC/NPD 8 pmol Preece et al. extract lipids with methylene chloride, derivatize aqueous fraction with (1992b)+ pentafluorobenzaldehyde, extract with ethyl acetate Urine Extract with methylene chloride, discard extract; derivatize aqueous fraction with GC/NPD 0.05 ng/mL Timbrell & p-chlorobenzaldehyde; extract with methylene chloride; dry and dissolve in ethyl Harland (1979) acetate Urine Deproteinate with trichloroacetic acid; derivatize with vanillin in ethanol; acidify | SP 0.065 ug/mL Amlathe & Gupta with sulfuric acid (1988) Plasma/Urine None/Dilute with deionized water Ion-exchange 8 ng/sample Fiala & Kulakus HPLC/ECD (1981) Plasma, liver Precipitate residual protein with hydrochloric acid and ammonium sulfate, GC/MS ~20 nmol/mL Preece et al. tissue extract lipids with methylene chloride, derivatize aqueous fraction with (1992b)+ pentafluorobenzaldehyde, extract with chloroform Serum, liver/ _— Acidify; derivatize with p-dimethylaminobenzaldehyde in ethanol SP 0.025 ug/sample Alvarez de Laviada brain tissue et al. (1987) Serum Treat with trichloroacetic acid; centrifuge; derivatize supernatant with SP 0.05 ng/mL Reynolds & p-dimethylaminobenzaldehyde in ethanol Thomas (1965) Living cells Probe was synthesized from n-(3-dimethylaminopropyl)-N-ethylcarbodiimide NMR/MS ~2 uM Qian et al. (2014) hydrochloride added to resorufin and 4-bromobutyric acid in dichloromethane; evaporated; purified and added to buffered cells a As cited in ATSDR (1997) ECD, electrochemical detection; EDTA, ethylenediaminetetraacetic acid; FID, flame ionization detector; Fl, fluorescence; GC, gas chromatography; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; NPD, nitrogen-phosphorus detector; NSD, nitrogen selective detection; SP, spectrophotometry; TID, thermionic ionization detector; UV, ultraviolet S6L QUIZeIPAH IARC MONOGRAPHS - 115 Table 1.2 Occupational exposures to hydrazine Country Job/process Numbers Air concentration Reference of people (mg/m?) exposed Typical Exceptional USA F-16 fighter jet refilling including nitrogen NR < 0.13 6.6-10.5 Christensen (1978) depressurization, catalyst purge, poppet valve replacement USA F-16 fighter jet applications 32 0.04-0.05 IPCS (1987a) USA Production 100-800 =< 0.13 0.13-0.268 IPCS (1987a) USA Production < 1100 < 0.35 < 1.18 IPCS (1987a) USA Derivative manufacture <25 < 0.13 ~0.13 IPCS (1987a) NR Evaporation from liquid spill NR 4 IPCS (1987b) NR At site of leakage NR 800 Suggs et al. (1980) USA Rocket propellant-handling facility NR 0.29-2.59 Cook et al. (1979) 0.013 (inside RPE) USA Rocket testing 10-100 0.01-0.02 0.14> IPCS (1987a) NR Process stream sampling NR 0.05-0.35 1.19 ATSDR (1997) Japans Production in 5 factories NR 0.014 ND-0.262 | Nomiyamaetal. (1998b) United Manual processing and distributing hydrazine NR 0.0045 HSE (2006) Kingdom: hydrate solutions, open transfer (45-60 min) NR 0.0096 Manual processing and distributing hydrazine 0.013 hydrate solutions, open transfer (41 min) NR Manual processing and distributing hydrazine NR 0.002 hydrate solutions, closed transfer (15 min) NR 0.003 4 Short-term samples during specific operations > Level measured during aeration of the wastewater holding period ° The data from Japan and the United Kingdom are for hydrazine hydrate, the others for hydrazine; note that exposures are all measured as hydrazine, but the processes involved handling of hydrazine hydrate ND, not detected; NR, not reported; RPE, respiratory protection equipment Compiled by the Working Group with data reported by ECHA (2011) 1.4.5 Exposure assessment in epidemiological In a retrospective cohort study in aerospace studies workers in southern California, USA, which used interviews, historical facility reports, and Workers were commonly also exposed to walkthrough surveys, job titles were discussed other carcinogens, which made it difficult to and classified into a time-dependent semi-quan- attribute identified risks specifically to hydra- titative job-exposure matrix (JEM) with respect zine (Cammarano et al., 1984; Wald et al., 1984; to hydrazine compounds (e.g. hydrazine, Zhao et al., 2005; Krishnadasan et al., 2007). In 1-methylhydrazine, and 1,1-dimethylhydrazine) a nested case-control study of 6107 aerospace (Morgenstern & Ritz, 2001; Zhao et _al., 2005; workers and 4607 workers enrolled in a radiation Ritz et al., 2006). The JEM was used to classify monitoring programme, 87% of those exposed jobs held for at least 6 or 12 months (depending to hydrazine were also exposed to trichloro- on the job) into low, medium, or high exposure. ethylene, while levels of radiation were not Morgenstern & Ritz (2001) stated that the cate- reported (Krishnadasan et al., 2007). gories reflect the probability, not the amount, 196 of hydrazine exposure, but this was not further clarified (Morgenstern & Ritz, 2001); however, subsequent reports referred to the classification in terms of intensity of exposure (Zhao et al. 2005; Ritz et al., 2006); any employee involved in working hands-on with rocket engines or in fuel production and testing was assumed to have been exposed to hydrazine. For example, rocket test-stand mechanics were assigned to the highest intensities of exposure because they probably had the greatest contact with rocket engines (Ritz et al., 2006). Each job title was assigned to one of four categories of presumptive exposure (high, medium, low, or unexposed) for each chemical reflecting the relative intensity of that exposure in each of three periods: the 1950-1960s, the 1970s, and the 1980-1990s. A cumulative expo- sure score for each worker in the cohort was calculated by summing across all employment periods. Thus, each job received an intensity score (0 to 3, from unexposed to highly exposed) that was multiplied by the number of years in the job and then summed. In separate analyses, to take account of the fact that more hydrazine was used in the 1960s, scores of 1 for low, 4 for medium, and 9 for high intensities were used for that decade, retaining the original scores for all other decades (Ritz et _al., 2006). In addition to hydrazine, there was exposure to other known carcinogens at the facility, including trichloro- ethylene, polyaromatic hydrocarbons, mineral oils, and benzene. In Ritz et al. (2006), a new JEM was derived assessing these exposures, and this information was used to adjust the hydra- zine effect estimates for potential confounding by co-exposure to other chemicals (Ritz et al. 2006). In a subsequent nested case-control study of cancer of the prostate identified through cancer registries, five controls per case were randomly selected, matched on age at start date of employ- ment, and cohort (radiation or aerospace). For cases and controls, 69% had no identified exposure to hydrazine; 21% of cases and 24% of controls were classified as having had low or moderate exposure; and 11% of cases and 7% of controls were classified as having had high expo- sure (Krishnadasan et al., 2007). A separate and subsequent study at the same aerospace facility used company records to iden- tify test-stand mechanics, including those moni- tored for radiation. These workers were paid hourly, unlike most of the test-stand workers (instrument mechanics, inspectors and engi- neers) who were salaried. The mechanics who were paid hourly were thought to be most likely to have been “hands-on” and hence to have had a higher probability of exposure to hydrazine and trichloroethylene. Hydrazine was present in rocket fuel at some but not all rocket-engine test stands. The electronic personnel files did not iden- tify on which rocket stands the mechanics had worked, so the authors used company telephone numbers to identify a minority of the test-stand mechanics who were thought to have worked on stands that used hydrazine. The assignments were validated by “information gathered from walkthrough surveys at operating and closed test stands with knowledgeable personnel who were involved with engine tests over the years, discus- sions with over 100 long-term employees (both retired and active), and review of medical records of workers, which often identified the test stands and the chemicals used” (Boice et al., 2006). Ritz et al. (1999) had been unable to link workers to specific test stands using company records, but former employees stated that many workers frequently changed work locations. In addition, leakage of fuel was thought to be a common source of exposure (Ritz et al., 1999). In a separate study of a cohort of workers from a site in the east Midlands, United Kingdom, that manufactured hydrazine in open tanks and vessels, the exposure assessment was weaker. No measurements of the concentration of hydrazine in air had been made, exposure esti- mates were derived by the simulation of spillages and calculations using data on the saturated 197 IARC MONOGRAPHS - 115 vapour pressure of hydrazine. This suggested Table 1.3 International limit values for that maximum levels of 100 ppm were possible occupational exposure to hydrazine (Wald et al., 1984). Exposures were estimated to have been 1-10 ppm in the general plant area, Country Limit value, and up to 100 ppm close to the hydrazine storage 8 hours short-term vessels (Wald et_al., 1984; Morris et al., 1995). ppm mg/m? ppm mg/m? Those directly involved, for at least 6 months, in Australia 0.01 0.013 the manufacture of hydrazine or its derivatives, Austria 0.10 0.130 0.40 0.52 or its use as a raw material, were classified as Belgium 0.01 0.013 having had high exposure. Moderate exposure Canada, Ontario 0.01 was assigned to those with only an incidental Canada, Quebec 0.10 0.130 presence in the hydrazine-manufacturing area (Province) for most of their career, e.g. fitters and engineers. Denmark 0.01 0.013 0.02 0.026 Finland 0.01 0.013 0.054 0.072 Their exposure was estimated to have probably France 0.10 0.100 been “< 0.5 or 1 ppm for most of their employ- Germany (AGS) 0.017 0.0225 0.034» 0.044a> ment”. The remainder of the cohort were thought 0.0017 0.0022¢ to have had “little or no exposure”. In the risk Hungary 0.13 analysis, the years at each exposure level were Ireland 0.01 Latvia calculated on an individual basis (Morris et al. 0.10 New Zealand 1995). 0.01 0.013 People’s Republic of 0.06 0.138 China 1.5 Regulations and guidelines Poland 0.05 Republic of Korea 0.10 0.05 0.06 Singapore Hydrazine is classified as 0.10 0.13 a carcinogen in Spainé 0.10 0.13 accordance with Article 57 (a) of the Registration, Switzerland 0.10 0.13 Evaluation, Authorisation and Restriction of USA, NIOSH 0.03¢ 0.04¢ Chemicals (REACH) regulations (EC-1907/2006; USA, OSHA 1.00 1.30 ECHA, 2011). Hydrazine is included in the list United Kingdom 0.02 0.03 0.10 0.13 of harmonized classification and labelling of 4 15 minutes average value hazardous substances as carcinogen category 1B > Workplace exposure concentration corresponding to the proposed tolerable cancer risk (ECHA, 2011). © Workplace exposure concentration corresponding to the proposed According to the European Chemicals preliminary acceptable cancer risk 4 Skin, sensitivity Agency (ECHA), the following Globally © Ceiling limit value Harmonized System of Classification and AGS, German Committee on Hazardous Substances (Ausschuss fiir Labelling of Chemicals (GHS) hazard statements Gefahrstoffe); NIOSH, National Institute for Occupational Safety and Health; OSHA, Occupational Safety and Health Administration apply: H301 “toxic if swallowed”; H311 “toxic From the GESTIS Substance Database (IFA, 2015) in contact with skin”; H314 “causes severe skin burns and eye damage”; H317 “may cause an The United States Environmental Protection allergic skin reaction”; H331 “toxic if inhaled”; Agency (EPA) Integrated Risk Information H350 “may cause cancer”; H400 “very toxic to System classifies hydrazine as a B2 carcinogen, a aquatic life”; H410 “very toxic to aquatic life with probable human carcinogen based on sufficient long lasting effects”; and H226 “flammable liquid evidence of carcinogenicity in animals. The inha- and vapour” (ECHA, 2016), lation unit risk rate was stated as 4.9 x 10-3 per ug/m3 based ona linearized multistage procedure 198 “extra risk model.” The oral cancer slope factor was stated as 3.0 (mg/kg per day)-! [these values are applicable to lifetime exposure] (EPA, 2016). Hydrazine was listed as a chemical that may cause cancer in 1988, under California’s prop- osition 95, The Safe Drinking-water and Toxic Enforcement Act 1986 (QEHHA, 2014). The American Conference of Governmental Industrial Hygienists (ACGIH) recommended an 8-hour time-weighted average (TWA) of 0.013 mg/m? (0.01 ppm) as the threshold limit value for occupational exposures to hydrazine in workplace air, and have categorized it as an A2 “suspected human carcinogen” (ACGIH, 1997). Other 8-hour TWA workplace limits vary from the proposed preliminary acceptable cancer risk value of 0.0017 ppm [0.0022 mg/m?] in Germany, to the Occupational Safety and Health Administration (OSHA) value of 1.3 mg/m} (IEA, 2015) in the USA. Several countries have also set short-term exposure limits (STELs) varying between 0.02 and 0.4 ppm [0.026-0.52 mg/m?] (IFA, 2015; Table 1.3). The Agency for Toxic Substances and Disease Registry of the Centers for Disease Control and Prevention (CDC) in the USA has set a minimal risk level (MRL) of 0.004 ppm [0.00524 mg/m}] for inhalation exposures of intermediate dura- tion (ATSDR, 2016). 2. Cancer in Humans See Table 2.1 Wald et al. (1984) studied 427 men in the east Midlands, United Kingdom, who were potentially exposed to hydrazine at a hydrazine-production plant between 1945 and 1971 (exposure at this plant ended in 1971), with at least 6 months of employment during this period. Workers were divided into presumed high, moderate, and low exposure groups. High exposure was estimated to be 1-10 ppm, moderate was < 1 ppm, and low exposure was little or no exposure. Follow-up went to 1982 in this original report and was extended in subsequent reports by Morris et al. (1995, 2015). The most recent follow-up until 2012 identified 205 deaths (Morris et al., 2015). Mean length of exposure was 6.8 years and mean follow-up was 32 years. There was no statistically significant excess risk for any cancer. The relative risk (RR) of mortality for cancer of the lung was 0.72 (95% confidence interval, CI, 0.42-1.15; 17 deaths), and no significant or close to significant excess of lung cancer was seen at any exposure level or duration (RR for high exposure group, 1.22; 95% CI, 0.45-2.67). There was no marked excess of cancer of the digestive system for all men combined (RR, 1.02; 95% CI, 0.65-1.53; 23 deaths) nor for any exposure level or dura- tion group (RR for high exposure, 0.44; 95% CI, 0.05-1.58). Few men were in the high exposure category, and there were only six lung cancers and two digestive cancers in this group. There was an overall deficit of all-cause mortality (standardized mortality ratio, SMR, 0.77), and the authors noted that these workers were not allowed to smoke at work due to the explosive nature of hydrazine. [The Working Group noted that this cohort was of limited value for the evaluation of hydra- zine since there were few deaths among men judged to have high exposure, while other expo- sure groups were exposed only incidentally or were judged not to have been exposed.] Ritz etal. (1999) studied a cohort of 6107 male workers who worked at the Santa Susana Field Laboratory rocket testing facility (Rocketdyne) near Los Angeles, California, USA. Workers had to have been employed before 1980 and worked at least 2 years, and not to have been exposed to radiation (i.e. no evidence of monitoring). Mortality follow-up went until 1994; no data on cancer incidence were reported. The average length of follow-up was 29 years. Exposure assess- ment was based on job title, and a JEM was devel- oped to estimate exposure depending on job title 199 007 Table 2.1 Cohort studies of cancer and exposure to hydrazine VI DY Reference, Population size, description, Organ site | Exposure No. of Risk estimate Covariates Comments SHdVYDONOW location, exposure assessment method category or exposed (95% CI) controlled enrolment/ level cases/ follow-up period deaths Morris et al. 427 male hydrazine-production Lung All 17 0.72 (0.42-1.15) Age, sex, Mean employment, 6.8 yrs; (2015) workers employed for = 6 mo period mean follow-up, 32 yrs; United Kingdom, Exposure assessment method: High a 1.22 (0.45-2.67) English referent rates east Midlands JEM; exposure divided into high Digestive All 23 1.02 (0.65-1.53) Strengths: well-defined cohort - 1947-2012 (regular, 1-10 ppm), moderate system Limitations: limited number of SLL (incidental, < 0.5-1 ppm), and Hich 5 0.57 7 men with high exposure little or no exposure; Pp 5 linkage & to 8 ‘57 (0.07-2.08) national mortality database Ritz etal. (2006) 6044 for mortality; 5049 for Lung, Moderate 37 1.24 (0.78-1.96) Pay type Results presented for 20-yr lag; USA, California incidence; employed for=>2 yrs = mortality High 36 1.67 (0.99-2.83) | (categorical), some overlap with Boice et al. 1960-2001 in 1960-1980 in a rocket-testing Trend-test P-value: 0.031 time (2006), more rocket testing facility since first mechanics assumed exposed Exposure assessment method: employment _ than in Boice et al., workers JEM; 56% had no/low exposure, Colorectum, Moderate 12 0.83 (0.35-1.95) (continuous), worked in earlier years than in 26-28% had moderate exposure, mortality High 10 1.55 (0.61-3.90) other possible Boice et al., during a time of 17% had high exposure; semi- Trend-test P-value: 0.481 carcinogens _ likely higher exposure due to quantitative ranking based on (TCE, PAHs, more rocket testing estimated intensity of exposure; mineral oils) Strengths: long follow-up, high exposure defined as likely Lung, Moderate 22 1.18 (0.62-2.24) semi-quantitative exposure direct contact with hydrazine; incidence High 26 2.49 (1.28-4.86) assessment, cancer incidence linkage to state cancer registries Trend-test P-value: 0.003 data (California and eight and National Death Index adjoining states) as well as Colorectum, Moderate 28 1.75 (0.93-3.30) mortality incidence High 19 2.16 (1.02-4.59) Limitations: lack of direct Trend-test P-value: 0.041 measurement of hydrazine levels; some lack of cancer ascertainment expected Table 2.1 (continued) Reference, Population size, description, Organ site | Exposure No. of Risk estimate Covariates Comments location, exposure assessment method category or exposed (95% CI) controlled enrolment/ level cases/ follow-up period deaths Boice et al. (2006) 8372; worked 26 moat rocket- = Lung Mortality: Age, sex, Overlap with Ritz et al. (2006), USA, California testing facility in 1948-1999 All workers 215 0.89 (0.78-1.02) period but many fewer rocket-testing 1948-1999 Exposure assessment method: Male 63 1.07 (0.82-1.37) mechanics classified as JEM; analyses of subset of 1651 test-stand exposed; includes more recent test-stand mechanics of whom pee etnnes workers than Ritz et al.; recent 315 were judged to have had . Male 15 1.45 (0.81-2.39) workers likely to have lower likely hydrazine exposure, with ee exposures another 205 judged to have had hani Strengths: long follow- possible but unlikely hydrazine eaneueel up; extensive exposure ; with likely exposure; linkage to state death ae hydrazi characterization based on ws ; ydrazine registries and the national death ; SrOOSAG assignment to categories index ; Limitations: no cancer Colorectum Mortality: eee Allworkers 70 0.97 (0.75-1.22) Male 19 1.10 (0.66-1.71) test-stand mechanics Male 5 1.67 (0.54-3.89) test-stand mechanics with likely hydrazine exposure CI, confidence interval; JEM, job-exposure matrix; PAHs, polycyclic aromatic hydrocarbons; TCE, trichloroethylene; TWA, time-weighted average LOC QUIZeIPAH IARC MONOGRAPHS - 115 and year of employment. Work history records high-exposure group, mortality from cancer of were available from the company. Inventories of the lymphoid and haematopoietic tissues (RR hydrazine over time were also available for 1955- for HYD-6, 2.83; 95% CI, 1.22-6.56; and RR for 1994. Workers were assigned by expert judgment HYD-24, 1.42; 95% CI, 0.54-3.72; 15-year lag), to categories of no, low, medium, and high prob- and bladder and kidney cancer (RR for HYD-6, ability of exposure to hydrazine, without refer- 1.65; 95% CI, 0.59-4.56; and RR for HY D-24, 1.80; ence to intensity of exposure; the low exposure 95% CI, 0.63-5.12) was also increased. Limited group was small and results for this group were data on smoking for 295 workers showed no asso- not reported. Hydrazine used in rocket fuel can ciation between smoking and estimated occu- exist as three variants: hydrazine itself, 1-methyl- pational exposure to hydrazine. Furthermore, hydrazine, and 1,1-dimethylhydrazine, but relative risks for a group of smoking related-can- these could not be separated and exposure was cers other than lung showed no positive trends analysed generically as “hydrazine” irrespective with exposure. of the particular variant. Two sets of analyses Ritz et al. (2006) updated the earlier study were conducted, based on minimum length of (Ritzetal., 1999) on this cohort for mortality until duration of exposure (6 months or 24 months: 2001. A few workers from the previous cohort HYD-6 or HYD-24, respectively). In some anal- were found not to have been eligible, resulting yses, the exposure variable was high exposure by in a cohort size of 6044. Cancer incidence decade, as it was thought that intensity changed between 1988 and 2000 was also ascertained in over time. No quantitative estimates of hydra- California and eight other states of the USA for zine exposure intensity were given. Exposure 5049 workers who were alive as of the beginning was analysed as a time-dependent variable, using of 1988. Death certificate information indicated highest exposure at any time, and retaining that cancer incidence would have approximately that highest exposure category if subsequent 89% complete coverage using only these states exposure was lower. Cancer of the lung was of during the follow-up. Exposure assessment was primary interest in the analysis, but data for updated via walkthrough surveys, interviews cancers of the lymphoid and haematopoietic with company personnel, and review of records. tissues (ICD-9, 200-208), bladder and kidney, Each job title was assigned to one of four catego- upper aerodigestive tract (ICD-9, 140-141 and ries (no exposure, low, moderate, high) in three 161) and pancreas were also reported. Covariates periods (1950-1969, 1970-1979, and 1980-1999). included in internal analyses were age, time since Employees with direct exposure to rocket engines hire, and pay grade, and lags of 0, 10, and 15 years or fuel production and testing were presumed to were used. Results shown were similar for both have been exposed to hydrazine. Exposure inten- duration of exposure groups (HYD-6, n = 1053; sity was scored annually 0-3 for each job (3 being HYD-24, n = 827). Relative risks for cancer of highest exposure), based on the four categories the lung for high exposure with a 15-year lag above, and then summed across employment were 1.93 (95% CI, 1.27-2.93) or 2.10 (95% CI, years to derive a cumulative exposure score. 1.36-3.25) for high exposure within the HYD-6 Further details of exposure assessment are avail- and HYD-24 groups, respectively. No elevation able in Zhao et al. (2005). Cox regression was in risk of cancer of the lung was seen for medium conducted with calendar time as the time vari- exposure. Analyses of high exposure (using the able. Pay grade, age, time since hire, and expo- HYD-6 group) by decade showed that excesses of sures to trichloroethylene or mineral oils were cancers of the lung, and lymphoid and haemato- included in all models. Cumulative exposure poietic tissues were greatest in 1960-1969. In the 202 scores were divided into three semiquantitative categories, low (< 3), medium (3 to < 12), and high (> 12) for analyses; 56% (3401) of workers were judged to have had no/low exposure, 26% (1593) moderate exposure, and 17% (1050) high exposure. During follow-up, 2117 workers had died (35%) and 691 incident cancers were found. Mean duration of employment was 16 years. Rate ratios for cancer of the lung with no lag for mortality (194 deaths) were 1.00, 1.46 (95% CI, 0.96-2.22), and 1.49 (95% CI, 0.94-2.35) for no/ low, moderate, and high exposures, respectively (P for trend, 0.065), and for incidence (92 cases) the RRs were 1.00, 1.15 (95% CI, 0.60-2.20), and 2.31 (95% CI, 1.21-4.43) for the same exposure categories (P for trend, 0.007). With 20 years lag, these were respectively 1.00, 1.24 (95% CI, 0.78-1.96), and 1.67 (95% CI, 0.99-2.83) (P for trend, 0.031) for mortality, and 1.00, 1.18, (95% CI, 0.62-2.24), and 2.49 (95% CI, 1.28-4.86) (P for trend, 0.003) for incidence. For cancer of the colorectum, the relative risks for incidence (90 cases) were 1.00, 1.64 (95% CI, 0.86-3.11), and 2.09 (95% CI, 1.02-4.31) (P for trend, 0.043) for no lag, and 1.00, 1.75 (95% CI, 0.93-3.30), and 2.16 (95% CI, 1.02—4.59) (P for trend, 0.041) for 20 years lag. There was evidence for positive trends for cancer of the kidney (17 deaths, 16 incident cases) and pancreas (39 deaths, 21 cases), but these did not attain statistical significance. No trends were seen with “NHL and leukaemia” for either mortality or incidence (0 and 20-year lags), in contrast to the earlier mortality follow-up. Limited data on smoking for 200 workers showed no association between smoking and estimated occupational exposure to hydrazine. Furthermore, relative risks for all smoking related-cancers other than lung showed no positive trends with exposure. [The Working Group noted that this cohort was particularly informative because it is rela- tively large, with semi-quantitative exposure assessment, and included cancer incidence as well as mortality.] Krishnadasan_et_al. (2007) conducted a nested case-control study of 362 cases of cancer of the prostate and 1805 controls, matched on date of first employment and age at diagnosis. The study was nested within two cohorts, 4607 radiation-exposed Rocketdyne workers and the same 6107 rocket-testing Rocketdyne workers studied by Ritz et al. (2006). Exposures to hydra- zine, trichloroethylene, polycyclic aromatic hydrocarbons, benzene, and mineral oil were evaluated. Incident cases of prostate cancer were ascertained in California and eight other states of the USA, as in Ritz et al. (2006). Based ona JEM, odds ratios (20-year lag) for those with high and low exposure to hydrazine, versus no exposure, were 0.84 (95% CI, 0.48-1.5) and 0.75 (95% CI, 0.50-1.1), respectively (P for trend, 0.30). [This study was largely uninformative, given the earlier data from Ritz et al. (2006), where no trends for prostate cancer were observed for hydrazine exposure among the rocket-testing workers.] Boice et al. (2006) studied a group of 8372 Rocketdyne workers who worked for 6 months or more at the Santa Susana Field Laboratory rocket-testing facility from 1948 onward, and had potential exposure to hydrazine. Women were included among the rocket-testing facility workers, representing 15% of the cohort. Also included were 182 test-stand mechanics who were exposed to radiation. Analyses were limited to mortality, with no incidence data. Mortality comparisons were made using California referent rates, as well as referent rates based on 32 979 other Rocketdyne workers who did not do rocket testing and had no potential exposure to hydra- zine. Internal comparisons were also conducted. Follow-up of test facility workers was conducted until 1999 and identified 2251 deaths (27% of the cohort). Among the 8372 rocket-testing workers, 1651 (20%) were test-stand mechanics, judged to have potential exposure to hydrazine. Of these however, Boice et al. (2006) estimated that only 203 IARC MONOGRAPHS - 115 315 (30%) were likely to have been exposed to employment, showed the highest lung cancer hydrazine, while 205 others were judged to have mortality risk (RR, 1.40; 95% CI, 0.80-2.47) in possible but unlikely exposure to hydrazine, and the group exposed 1960-1969 (n = 1454), with the remainder were judged to have had no expo- an excess (RR, 1.29; 95% CI, 0.70-2.40) also seen sure to hydrazine. These definitions were based in those exposed before 1960 (n = 984). Some on job title and other company records. Some internal analyses using Cox regression were test-stand mechanics were also classified as being also conducted among test-stand mechanics by exposed to trichloroethylene, with 121 of these length of employment and likelihood of hydra- thought to have been exposed to both hydrazine zine exposure (likely, possible, none). These and trichloroethylene. analyses, in which follow-up time was the time Cancer mortality analyses of all rocket- variable, and which controlled for year of birth, testing workers compared with California popu- year of hire, pay grade (hourly or salaried), and lation rates were unremarkable and not signif- exposure to trichloroethylene, did not show any icantly different from the null for any specific significant trends for either lung cancer or colon type of cancer analysed, including lung (SMR, cancer. A smoking survey carried out among 0.89; 95% CI, 0.78-1.02). All-cause mortality was about 75 hourly-paid and 75 salaried test-facility also decreased (SMR, 0.83; 95% CI, 0.80-0.86). workers, found that about 60% of hourly workers Analyses by duration of employment also showed had ever smoked versus 40% salaried workers, no significant excesses for any type of cancer for suggesting the importance of controlling for workers with longest duration of employment. pay grade. [The Working Group noted that Analyses of male test-stand mechanics paid Boice et al. (2006) is an informative study with hourly also did not show significant excesses for relatively large number of deaths and extensive any cancer type, nor were there any significant exposure characterization by assigned catego- excesses for those with > 5 years employment. ries. The Working Group further noted that The standardized mortality ratio for lung cancer while the two cohorts of Rocketdyne workers was 1.07 (95% CI, 0.82—1.37) based on California (Boice et al., 2006; Ritz et al., 2006) had impor- referent data; the standardized mortality ratio tant overlap, they were different populations. for lung cancer for > 5 years employment was As noted above, the cohort studied by Boice 1.06 (95% CI, 0.66-1.60). For the 315 test-stand et al. (2006) hada shorter minimum employment workers estimated to have likely hydrazine period and included women and workers hired exposure, the standardized mortality ratio for in both earlier and later years. Furthermore, Ritz all cancers was 1.09 (95% CI, 0.75-1.52). The et al. (2006) considered all test-stand mechanics standardized mortality ratio for lung cancer in to have been exposed, while Boice et al. (2006) this group was 1.45 (95% CI, 0.81-2.39). Findings considered only a minority to have had likely or for deaths for other specific cancers were unre- possible exposures based on presumed test-stand markable and based on small numbers. Internal assignment. However, as pointed out in Zhao analyses of likely exposed, possibly exposed, et_al. (2005), test-stand mechanics frequently versus not exposed to hydrazine for test-stand rotated among different test stands, making the workers showed no evidence of a positive trend classification used by Boice et al. (2006) impre- with increasing likelihood of exposure (analyses cise. Furthermore, Ritz et al. (2006) included controlled for pay grade, hourly vs salaried). incidence data, while Boice et al. (2006) included Analyses of test-stand mechanics by decade of only mortality data. Hence, few results in the exposure, comparing those with 3 or more years two studies are directly comparable. It is of note employment with those with less than 3 years that test-stand mechanics exposed before 1960 204 (RR for lung cancer, 1.29) and during the 1960s (RR, 1.40), with = 3 years employment versus < 3 years employment in Boice et al. (2006) may be roughly comparable to the workers with high and moderate exposure with a 20-year lag in Ritz etal. (2006) (lung cancer mortality for those with moderate and high exposure, lung cancer RR, 1.24 and 1.67, respectively). These groups, across the two studies, showed a moderate excess of lung cancer. It should also be noted that in Boice et al. (2006), the group with likely or possible expo- sure had a lung cancer rate ratio of 1.45 versus the non-exposed. Finally, there was a notable increase in incidence of lung cancer in the Ritz et al. (2006) study in the group with high expo- sure and a 20-year lag (RR, 2.49).] 3. Cancer in Experimental Animals Hydrazine or hydrazine salts were previously reviewed by the IARC Monographs Working Group (Volume 4, IARC, 1974; Supplement 7, LARC, 1987; and Volume 71, [ARC, 1999). The Working Group concluded that there was suffi- cient evidence in experimental animals for the carcinogenicity of hydrazine (IARC, 1999). In the present Monograph, the Working Group evalu- ated the studies of carcinogenesis in experimental animals that had been reviewed previously, and also reviewed any new studies published since the earlier review. Hydrazine was found to cause tumours of the lung in mice with an incidence of 100% (Biancifiori & Ribacchi, 1962), and was then subsequently used in the laboratory to study formation of tumours of the lung in mice, rats, and hamsters. Most of the studies in mice in the present Monograph were single-dose studies designed to investigate possible mitigating agents for hydrazine, or mechanisms of lung-tumour induction by hydrazine. See Table 3.1 Mouse 3.1.1 Gavage A group of 22 female BALB/c mice (age, 8 weeks) was given hydrazine sulfate in water as 251 daily gavage doses at 1.13 mg per mouse during 46 weeks. By the end of the experiment (46 weeks), the mice had 100% incidence [P < 0.0001] of pulmonary adenoma [the majority] or carci- noma. A group of 216 untreated control female mice allowed to live their full lifespan (up to 121 weeks) had no pulmonary tumours (Biancifiori & Ribacchi, 1962). [While a high incidence of pulmonary tumours was reported in treated mice versus controls, the study lacked experimental details and had a poor experimental design. For example, the initial number of treated mice was not given and no vehicle controls were included.| In a follow-up study, Biancifiori et al. (1963) treated a group of 84 female BALB/c mice (age, ~50 days) with hydrazine sulfate at 1.13 mg per day in water by gavage for up to 310 days. About three mice were killed every 10th day. The incidence of pulmonary tumours for the groups killed between day 200 and 240 was 15/15 (100%), between day 250 and 290 was 13/15 (87%), between day 300 and 340 was 14/14 (100%), and between day 350 and 380 was 8/8 (100%). [No information was provided if controls were used. While high incidences of pulmonary tumours were reported in treated mice, this study lacked experimental details and had a limited experi- mental design. The study was inadequate for the evaluation. | Groups of 21 male and 21 female CBA/Cb/ Se mice (age, 8 weeks) were treated daily with hydrazine sulfate at a total dose of 283 mg in water by gavage for 36 weeks, and then main- tained for their lifetime (Biancifiori et al., 1964). Groups of 37 male and 47 female CBA/Cb/Se mice served as untreated controls. Groups of 10 male and 10 female BALB/c mice (age, 8 weeks) were treated daily with hydrazine sulfate at a total 205 90¢ Table 3.1 Studies of carcinogenicity with hydrazine in experimental animals VI DY Study design Route Incidence or multiplicity Significance Comments Control animals were held for their SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Mouse, BALB/c (F) Hydrazine sulfate (purity, NR) Adenoma or carcinoma lifetime - 8 wks Water (combined): While a high incidence (100%) of SLL 46 wks 0 (untreated), 1.13 mg/dose 0/166, 22/22* *[P < 0.0001] pulmonary tumours [mainly adenoma] Biancifiori & Ribacchi 251 treatments over 46 wks was reported in treated mice vs (1962) 216, NR “controls” (0%), the lack of experimental 166, 22 detail and poor experimental design (e.g. failure to report number of mice in treated group, single dose used, no discussion of pathology) made the study difficult to evaluate Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Pulmonary adenoma or of experimental details; single dose used; (M) Water carcinoma (combined): small number of animals in exposure 8 wks 0 (untreated), 283 mg of total dose Incidence: 1/37, 16/21* *[P < 0.0001] group; exposed for only 36 wks; no Lifetime “Daily” gavage for 36 wks Multiplicity: 1, 3 discussion of clinical signs Biancifiori et al. (1964) 37, 21 Liver NA Hepatoma [hepatocellular tumours]: Incidence: 4/37, 13/21* *[P < 0.0001] Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Pulmonary adenoma or of experimental details; single dose used; (F) Water carcinoma (combined): small number of animals in exposure 8 wks 0 (untreated), 283 mg of total dose Incidence: 4/47, 19/21* *[P < 0.0001] group; exposed for only 36 wks; no Lifetime “Daily” gavage for 36 wks Multiplicity: 1, 6 discussion of clinical signs Biancifiori et al. (1964) 47, 21 Liver NA Hepatoma [hepatocellular *[P< 0.0001] tumours]: 2/47, 15/21* Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, BALB/c (M) Hydrazine sulfate (purity, NR) Pulmonary adenoma: of experimental details; single dose used 8 wks Water Incidence: 6/22, 7/8* *[P < 0.006] for very short time; small number of Lifetime 0 (untreated), 32 mg -— total dose Multiplicity: 1.0, 2.4 animals in exposure and control groups; Biancifiori et al. (1964) “Daily” gavage for 4 wks exposed for only 4 wks; no discussion of 22, 10 clinical signs NA Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, BALB/c (F) Hydrazine sulfate (purity, NR) Pulmonary adenoma: of experimental details; single dose used 8 wks Water Incidence: 5/23, 8/10* *[P < 0.003] for very short time; small number of Lifetime 0 (untreated), 32 mg — total dose Multiplicity: 1.0, 2.9 animals in exposure and control groups; Biancifiori et al. (1964) “Daily” gavage for 4 wks exposed for only 4 wks; no discussion of 23, 10 clinical signs NA Full carcinogenicity Gavage Liver Principal limitations: use of single dose; Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Hepatocellular adenoma *[P < 0.0001] short duration of exposure (M) Water or carcinoma (combined): Same data reported in Biancifiori et al. 8 wks 0 (untreated), 1.13 mg/dose 4/37, 13/21* (1964) Lifetime 1 x /day for 36 wks Lung Severi & Biancifiori 37, 21 Adenoma or carcinoma *[P < 0.0001] (1968) NA (combined): 1/37, 16/21* Full carcinogenicity Gavage Lung Principal limitations: use of single dose; Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Adenoma or carcinoma *[P < 0.0001] short duration of exposure (F) Water (combined): 4/47, 19/21* Same data reported in Biancifiori et al. 8 wks 0 (untreated), 1.13 mg/dose Liver (1964) Lifetime 1 x /day for 36 wks Hepatocellular adenoma *[P < 0.0001] Severi & Biancifiori 47, 21 or carcinoma (combined): (1968) NA 2/47, 15/21* Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, BALB/c/Cb/Se Hydrazine sulfate, “pure” Adenoma or carcinoma of experimental design; small number of (M+F, combined) Water buffered with sodium (combined): animals; no discussion of clinical signs, Newborn bicarbonate Incidence: 3/20, 20/20* *[P < 0.0001] survival or body weights Up to 425 days 0, 17 mg - total dose Multiplicity: 1, 10 Milia et al. (1965) 1 x /day for 60 days Total tumours: 3, 200 20, 20 NR, NR LOC QUIZeIPAH 80C VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Principal limitations: experimental Mouse, Swiss (F) Hydrazine sulfate (purity, NR) Adenoma or carcinoma *P < 0.001 details poorly described; single sex; short - Age NR Distilled water (combined): 8/79, 6/13* duration of exposure; poor survival in SLL Up to 40 wks 0 (untreated), 0.25 mg/dose treated group Roe et al. 1967) 1 x /day, 5 days/wk for 40 wks 85, 25 42,4 Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Pulmonary tumours: 2/26, NA of experimental details; no discussion of (M) Water 3/25, 4/25, 3/26, 5/25, 5/23 pathology or clinical observations and Age NR 0.14 (intact virgin), 0.14 no concurrent controls Lifetime (gonadectomized), 0.28 (intact virgin), Study to investigate a possible hormonal Biancifiori (1969) 0.28 (gonadectomized), 0.56 (intact effect on the formation of tumours by virgin), 0.56 (gonadectomized) mg 1 x hydrazine sulfate /day for a total of 150 doses (over 25 wks) 26, 25, 25, 26, 25, 23 NA Full carcinogenicity Gavage Lung Principal limitations: poor description Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Pulmonary tumours: NA of experimental details; no discussion of (F) Water 10/25, 2/25, 16/25, 6/25, pathology or clinical observations, and Age NR 0.14 (intact virgin), 0.14 21/24, 7/25 no concurrent controls Lifetime (gonadectomized), 0.28 (intact virgin), Study to investigate a possible hormonal Biancifiori (1969) 0.28 (gonadectomized), 0.56 (intact effect on the formation of tumours by virgin), 0.56 (gonadectomized) mg 1 x hydrazine sulfate /day for a total of 150 doses (over 25 wks) 25, 25, 25, 25, 24, 25 NA Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Principal limitations: experimental Mouse, Hydrazine sulfate (purity, NR) Pulmonary tumours: 1/10, *[P < 0.05] details poorly described; inadequate (BALB/c x DBA/2)F, 2% aqueous sodium bicarbonate 13/28* (CDF,) (F) number of controls; no discussion of 0, 41.6 mg total dose clinical observations or pathology 7-8 wks 1 x /wk for 8 wks 28-33 wks 10, 28 Kelly et al. (1969) 10, 26 Full carcinogenicity Gavage Liver Principal limitations: short duration of Mouse, CBA/Cb/Se M Hydrazine sulfate (purity, NR) Hepatoma [hepatocellular *[P< 0.003] exposure; lack of vehicle controls 8 wks Water tumours]: 3/30, 1/26, 7/25, **[P< 0.0001] Hepatomas are mainly hepatocellular Lifetime 0 (untreated), 0.14, 0.28, 0.56, 12/25*, 15/25** carcinomas Biancifiori (1970a) 1.13 mg/day 150 daily doses over 25 wks 30, 26, 25, 25, 25 NA Full carcinogenicity Gavage Liver Principal limitations: short duration of Mouse, CBA/Cb/Se Hydrazine sulfate (purity, NR) Hepatoma [hepatocellular *[P< 0.0001] exposure; lack of vehicle controls (F) Water tumours]: 1/29, 0/25, 2/25, Hepatomas are mainly hepatocellular 8 wks 0 (untreated), 0.14, 0.28, 0.56, 16/24*, 15/24* carcinomas Lifetime 1.13 mg/day Biancifiori (1970a) 150 daily doses over 25 wks 29, 25, 25, 24, 24 NA Full carcinogenicity Gavage Lung Principal limitations: no discussion of Mouse, BALB/c/Cb/ Hydrazine sulfate (purity, NR) Pulmonary adenoma or *[P < 0.05] body-weight gain, or survival; lack of Se (M) Water carcinoma (combined): TP < 0.008] vehicle controls 8 wks 0 (untreated), 0.14 (21 mg total), 0.28 6/25, 13/24*, 15/24**, TP < 0.004] Lifetime (42 mg total), 0.56 (84 mg total), 1.13 17/26***, 20/22****, errrTP < 0.0001] Biancifiori (1970b) (170 mg total), 1.13 (32 mg total) mg/ 17/20**** dose 28 or 150 doses over 4 or 25 wks 25, 24, 24, 26, 25, 20 NA 602 QUIZeIPAH OLC VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Principal limitations: no discussion Mouse, BALB/c/Cb/ Hydrazine sulfate (purity, NR) Pulmonary adenoma or +P < 0.02] of clinical signs, body-weight gain, or - Se (F) Water carcinoma (combined): *TP < 0.0001] survival; lack of vehicle controls SLL 8 wks 0 (untreated), 0.14 (21 mg total), 0.28 1/25, 8/25*, 17/19**, Lifetime (42 mg total), 0.56 (84 mg total), 1.13 19/25**, 20/22**, 15/20** Biancifiori (1970b) (170 mg total), 1.13 (32 mg total) mg/ dose 28 or 150 doses over 4 or 25 wks 25, 25, 19, 25, 22, 20 NA Full carcinogenicity Gavage Lung Principal limitations: poor description of Mouse, BALB/c/Cb/ Hydrazine sulfate (purity, NR) Adenoma or carcinoma experimental details including duration Se (F) Water (combined) of actual exposure; single dose used; 8 wks 0 mg (intact virgins), 1.13 mg (intact Incidence: 1/25, 20/22*, *[P < 0.0001] no discussion of body-weight gain, or Lifetime virgins), 0 mg (breeders), 1.13 mg 2/25, 25/25*, 7/26, 15/25** *TP < 0.05] survival; lack of vehicle controls Biancifiori (1970c) (breeders), 0 mg (gonadectomized), Multiplicity: 1, 3, 1, 14, 1,5 Study to test the effect of ovarian 1.13 mg (gonadectomized) stimulation on pulmonary tumours Total tumours: 1, 60, 1, 150 daily doses of 1.13 mg/dose induced by hydrazine sulfate in BALB/c/ 201, 1, 50 25, 22, 25, 25, 26, 25 Cb/Se (BALB/c) mice NA Full carcinogenicity Gavage Lung Principal limitations: poor description of Mouse, C3Hb/Cb/Se Hydrazine sulfate (purity, NR) Adenoma or carcinoma (P< 0.01] experimental details including duration (M) Water (combined): 0/25, 7/27*, of actual exposure; single dose used; 8 wks 0 mg (virgin), 1.13 mg (virgin), 1/24, 1/25 no discussion of body-weight gain, or Lifetime 0 mg (gonadectomized), 1.13 mg survival; lack of vehicle controls Biancifiori (1971) (gonadectomized) - total dose Tumours were mainly lung adenomas 150 daily doses of 1.13 mg/dose (170 mg total dose) 25, 27, 24, 25 NA Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Gavage Lung Principal limitations: poor description of Mouse, C3Hb/Cb/ Hydrazine sulfate (purity, NR) Adenoma or carcinoma *[P < 0.0004] experimental details including duration Se (F) Water (combined): 1/25, 13/27%, * TP < 0.01] of actual exposure; single dose used; 8 wks 0 mg (virgin), 1.13 mg (virgin), 3/25, 5/26, 3/23, 12/31** no discussion of body-weight gain, or Lifetime 0 mg (gonadectomized), 1.13 mg survival; lack of vehicle controls Biancifiori (1971) (gonadectomized), 0 mg (breeders), Tumours were mainly lung adenomas 1.13 mg (breeders) — total dose 150 daily doses of 1.13 mg/dose (170 mg total dose) 25, 27, 25, 26, 23, 31 NA Carcinogenicity with Gavage Lung Principal limitations: no histological other modifying Hydrazine sulfate, “analytical grade” Incidence: 1/47, 22/29*, *[P < 0.0001] description of tumours; used only factors Water 11/18*, 26/33*, 17/31*, one dose; reported combined tumour Mouse, Swiss (M+F, 0, 1.1 mg; 1.1 mg+1.1 mg L-arginine; 13/18*, 16/21* incidence for males and females; no combined) 1.1 mg+1.1 mg pyridoxine clinical signs, body weights or mortality 10 wks hydrochloride; 1.1 mg+1.1 mg folic acid, information reported Up to 15 mo 1.1 mg+1.1 mg L-arginine+L-sodium Study on the effect of antioxidants on the Maru & Bhide (1982) glutamate; 1.1 mg+1.1 mg L-sodium formation of lung tumours in mice by glutamate+pyridoxine hydrochloride hydrazine sulfate mg/mouse/day 1 x /day, 5 days/wk for up to 15 mo 60, 60, 60, 60, 60, 60, 60 47, 29, 18, 33, 31, 18, 21 Full carcinogenicity Drinking-water Lung Principal limitations: use of a single dose Mouse, Swiss (M) Hydrazine sulfate, Fisher certified ACS Adenoma or *[P < 0.0001] 6 wks Water adenocarcinoma Lifetime 0 mg, 0.74 mg average daily (combined): 11/110, 25/50* Toth (1969) consumption Daily/ad libitum in drinking-water 110, 50 NA LLC QUIZeIPAH CLC VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Drinking-water Lung Principal limitations: use of a single dose Mouse, Swiss (F) Hydrazine sulfate, Fisher certified ACS Adenoma or *[P < 0.0001] - 6 wks Water adenocarcinoma SLL Lifetime 0 mg, 0.65 mg average daily (combined): 14/110, 24/50* Toth (1969) consumption Daily/ad libitum in drinking-water 110, 50 NA Full carcinogenicity Drinking-water Lung Principal limitations: use of a single dose Mouse, A/J (M) Hydrazine sulfate (purity, NR) Adenoma or *[P < 0.0001] 6 wks Water adenocarcinoma *TP < 0.005] Up to 48 wks 0 mg/L, 0 mg/L + 1% L-arginine-L- (combined): 12/20, 11/20, Yamamoto & glutamate (in the diet), 325 mg/L, 38/38*, 34/37** Weisburger (1970) 325 mg/L + 1% L-arginine-L-glutamate (in the diet), ad libitum in drinking- water 20, 20, 38, 37 NR Full carcinogenicity Drinking-water Haematopoietic and Principal limitations: limited description Mouse, NMRI (M) Hydrazine hydrate (purity, 99.3%) lymphoid tissues of histopathology 5-6 wks Water Malignant lymphoma: NS 2 yrs 0, 2, 10, 50 ppm 4/50, 13/50, 6/50, 4/50 Steinhoff et al. (1990) ad libitum 50, 50, 50, 50 NR Full carcinogenicity Drinking-water Lung Principal limitations: limited description Mouse, NMRI (F) Hydrazine hydrate, 99.3% Lung tumours (benign): *P < 0.05 of histopathology 5-6 wks Water 6/50, 6/50, 9/50, 15/50* 2 yrs 0, 2, 10, 50 ppm Haematopoietic and lymphoid tissues Steinhoff et al. (1990) ad libitum Malignant lymphoma: NS 50, 50, 50, 50 19/50, 31/50, 20/50, 19/50 NR Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Drinking-water Liver Principal strengths: GLP study Mouse, Crj:BDF,(M) — Hydrazine monohydrate (purity, 100%) Haemangioma or NS 6 wks Deionized water haemangiosarcoma 2 yrs 0, 20, 40, 80 ppm (combined): 3/50, 1/50, Matsumoto et al. ad libitum, 7 days/wk 6/50, 0/50 (2016) 50, 50, 50, 50 Hepatocellular adenoma NS (increase) 31, 30, 36, 40 or carcinoma (combined): 34/50, 24/50, 15/50, 10/50 Hepatocellular adenoma: NS (increase) 17/50, 12/50, 8/50, 6/50 Hepatocellular carcinoma: NS (increase) 21/50, 14/50, 9/50, 4/50 Full carcinogenicity Drinking-water Liver Principal strengths: GLP study Mouse, Crj:BDF, (F) Hydrazine monohydrate (purity, 100%) Haemangioma or P< 0.05 by Peto trend Authors reported that hepatic 6 wks Deionized water haemangiosarcoma test haemangiomas in female mice were 2 yrs 0, 40, 80, 160 ppm (combined): 1/50, 2/50, observed in three animals in the Matsumoto et al. ad libitum, 7 days/wk 1/50, 4/50 laboratory historical control data, which (2016) 50, 50, 50, 50 Haemangioma: 0/50, 0/50, P< 0.01 by Peto trend consisted of 899 female Crj:BDF, mice 26, 37, 29, 23 (3/899, 0.3%) 1/50, 3/50 test Haemangiosarcoma: 1/50, NS 2/50, 0/50, 1/50 Hepatocellular adenoma P< 0.01 by Peto trend or carcinoma (combined): test, *P < 0.05 by 7/50, 8/50, 3/50, 17/50* Fisher’s exact test Hepatocellular adenoma: 5/50, 6/50, 2/50, 14/50* P< 0.01 by Peto trend test, *P < 0.05 by Fisher’s exact test Hepatocellular carcinoma: 2/50, 2/50, 1/50, 4/50 P< 0.01 by Peto trend test ELC QUIZeIPAH VLC VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Intraperitoneally Lung Principal limitations: poor description Mouse, BALB/c/Cb/Se Hydrazine sulfate (“pure”) Adenoma or carcinoma of experimental design; small number of - (M+F, combined) Water (combined): animals per group, use of a single dose; SLL Newborn 0, 19 mg - total dose Incidence: 2/20, 20/20* *[P < 0.0001] no discussion of clinical signs, survival Up to 270 days 1 x /day for 90 days Multiplicity: 1, 5 or body weights Milia et al. (1965) 20, 20 Total tumours: 2, 100 NR, NR Full carcinogenicity Intraperitoneally Lung Principal limitations: experimental Mouse, Hydrazine sulfate (purity, NR) Pulmonary tumours: 1/9, NS details poorly described, inadequate (BALB/c x DBA/2)F, 2% aqueous sodium bicarbonate 6/30 number of controls, no discussion of (CDF,) (M) 0, 20.8 mg total dose clinical observations or pathology 7-8 wks 1 x /wk for 8 wks 28-33 wks 9, 30 Kelly et al. (1969) 8, 30 Full carcinogenicity Intraperitoneally Haematopoietic and Principal limitations: experimental Mouse, strain NR Hydrazine (purity, NR) lymphoid tissues details poorly described, use of a single (M+F, combined) Physiological saline Reticular-cell sarcoma or *[P < 0.0001] dose, short exposure time, no discussion Age NR 0, 400 mg/kg bw myeloid leukaemia: 0/60, of clinical observations or pathology, 313 days 16 injections over 46 days 13/34* poor description and discussion of Juhasz et al. (1966) 60, 60 tumour incidences NR, 34 Carcinogenicity with Intraperitoneally Lung Principal limitations: experimental other modifying Hydrazine sulfate (purity, NR) Adenoma: 9/75, NR, NR, details poorly described; no discussion factor Water 5/18 of clinical observations or pathology Mouse, C57BL/6 0 (no irradiation), 0 (irradiated), 95 Haematopoietic and (M+F, combined) (irradiated), 95 (no irradiation) mg/ lymphoid tissues 6-8 wk kg bw Leukaemia: NR, 2/25, 4/29, [NS] Up to 62 wks Single 400 R total-body irradiation 0/33 Mirvish et al. 1969) followed by 10 weekly injections 2 75, 29, 40, 36 NR, NR, NR, NR Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Inhalation Lung Principal limitations: single sex and low Mouse, C57BL/6 (F) Hydrazine (purity, 99.8%) Adenoma: 4/378, NR, NR, -*[P.< 0.05] doses used; tumour incidence for some 7 wks Air 12/379* exposure groups not reported 27 mo 0, 0.05, 0.25, 1.0 ppm Vernot et al. (1985) 6 h/day, 5 days/wk for 1 yr 400, 400, 400, 400 NR, NR, NR, NR Full carcinogenicity Gavage Lung Principal limitations: use of single dose, Rat, Cb/Se (M) Hydrazine sulfate (purity, NR) Pulmonary adenoma or short duration of exposures 8 wks Water carcinoma (combined): See also Biancifiori et al. (1966) Lifetime 0, 18 mg/dose 0/28, 3/14* *[P < 0.05] Severi & Biancifiori 1 x /day for 68 wks en (1968) 28> lle Malignant tumours: NA 0/28, 4/13* *[P < 0.01] Full carcinogenicity Gavage Lung Principal limitations: use of a single Rat, Cb/Se (F) Hydrazine sulfate (purity, NR) Pulmonary adenoma or dose, short duration of exposures 8 wks Water carcinoma (combined): See also Biancifiori et al. (1966) Lifetime 0, 12 mg/dose 0/22, 5/18* *(P < 0.02] Severi & Biancifiori 1 x /day for 68 wks (1968) 22,18 NA Full carcinogenicity Drinking-water Liver Rat, Wistar (M) Hydrazine hydrate (purity, 99.3%) Hepatocellular adenoma: 6 wks Water 0/50, 1/49, 1/50, 4/49* *P<0.01 cietime Vank tibia, 30 eh drinki Hepatocellular carcinoma: einho ohr ad libitum in the drinking-water 1988) 50, 50, 50, 50 0/50, 0/49, 1/50, 0/49 NS NA GLC QUIZeIPAH 9OLC VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Drinking-water Liver One of the hepatocellular carcinoma Rat, Wistar (F) Hydrazine hydrate (purity, 99.3%) Hepatocellular adenoma: may not have been a primary tumour - 6 wks Water 0/50, 0/50, 0/50, 4/47* *P< 0.01 SLL citetime & Moh opie 30 oe drinki Hepatocellular carcinoma: einho ohr ad libitum in the drinking-water 1988 50, 50, 50. 50 0/50, 0/50, 0/50, 3/47* *P<0.01 NA Full carcinogenicity Drinking-water Liver Principal strengths: GLP study Rat, F344/DuCrj (M) Hydrazine monohydrate (purity, 100%) Hepatocellular adenoma or 6 wks Deionized water carcinoma (combined): 2 yrs 0, 20, 40, 80 ppm 0/50, 0/50, 0/50, 4/50 P< 0.01 by Peto trend Matsumoto et al. ad libitum, 7 days/wk test (2016) 50, 50, 50, 50 Hepatocellular adenoma 37, 39, 44, 39 0/50, 0/50, 0/50, 3/50 P< 0.01 by Peto trend test Hepatocellular carcinoma 0/50, 0/50, 0/50, 1/50 NS Testis Interstitial cell tumours: 37/50, 45/50*, 43/50, 44/50 P< 0.05 by Peto trend test, *P < 0.05 by Fisher’s exact test Full carcinogenicity Drinking-water Liver Principal strengths: GLP study Rat, F344/DuCrj (F) Hydrazine monohydrate (purity, 100%) Hepatocellular adenoma or Authors reported that hepatocellular 6 wks Deionized water carcinoma (combined): carcinoma was observed in only one 2 yrs 0, 20, 40, 80 ppm 1/50, 0/50, 3/50, 6/50 P <0.01 by Peto trend animal in the laboratory historical Matsumoto et al. ad libitum, 7 days/wk test control data, which consisted of 898 (2016) 50, 50, 50, 50 female F344/DuCrj rats (1/898, 0.1%) Hepatocellular adenoma: 40, 39, 44, 29 1/50, 0/50, 3/50, 4/50 P< 0.05 by Peto trend test Hepatocellular carcinoma: 0/50, 0/50, 0/50, 4/50 P <0.01 by Peto trend test Table 3.1 (continued) Study design Route Incidence or multiplicity Significance Comments Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Inhalation Nose Rat, F344 (M) Hydrazine (purity, 99.8%) Adenoma (adenomatous 7 wks Air polyp): 30 mo 0, 0.05, 0.25, 1.0, 5.0 ppm 0/146, 2/96, 1/94, 9/97*, *P< 0.01 Vernot et al. (1985) 6 h/day, 5 days/wk for 1 yr 58/98* 150, 100, 100, 100, 100 Thyroid NR, NR, NR, NR, NR Carcinoma: 7/146, 6/96, 5/94, 9/97, ™~*P < 0.05 13/98** Full carcinogenicity Inhalation Nose Rat, F344 (F) Hydrazine (purity, 99.8%) Adenoma (adenomatous 7 wks Air polyp): 30 mo 0, 0.05, 0.25, 1.0, 5.0 ppm 0/145, 2/97, 0/98, 2/94, *P<0.01 Vernot et al. (1985) 6 h/day, 5 days/wk for 1 yr 28/95* 150, 100, 100, 100, 100 NR, NR, NR, NR, NR Full carcinogenicity Drinking-water Liver Principal limitations: study examined Hamster, Syrian Hydrazine sulfate (purity, > 99%) Hepatocellular carcinoma: the liver only golden (M) Water 0/31, 0/31, 4/34, 11/34* *(P < 0.0004] Study designed to investigate liver Age NR 0, 170, 340, 510 mg/L DNA methylation over a 2-yr hydrazine 2 yrs ad libitum exposure period to test the relationship Bosan et al. (1987) 40, 40, 40, 40 between DNA methylation and NR, NR, NR, NR carcinogenesis LIC QUIZeIPAH B8LC VI Table 3.1 (continued) DY Study design Route Incidence or multiplicity Significance Comments SHdVYDONOW Species, strain (sex) Agent tested (purity) of tumours Age at start Vehicle Duration Dose regimen Reference No. of animals at start No. of surviving animals Full carcinogenicity Drinking-water Liver Principal limitations: study examined Hamster, Syrian Hydrazine sulfate (purity, > 99%; ACS — Hepatocellular adenoma: the liver only - golden (M) grade) 0/25, 1/30, 4/43, 10/40* *[P < 0.005] Study designed to investigate SLL ae NR ree 340. 510 me/L Hepatocellular carcinoma: metiy ation status or wae cytosine mo , 170, 340, 510 mg uring the course of induction by FitzGerald & Shank ad libitum 0/25, 0/30, 1/43, 3/40 Tonsequent f hydrazine of liver cancer in hamsters en 25, 30, 43, 40 hepatocellular NR, NR, NR, NR adenoma or carcinoma (combined) is significantly increased (P < 0.005) in high- dose animals] Full carcinogenicity Inhalation Nose Hamster, Syrian Hydrazine (purity, 99.8%) Adenoma (adenomatous golden (M) Air polyp): 7 wks 0, 0.25, 1.0, 5.0 ppm 1/181, 0/154, 1/148, 16/160* *P<0.01 24 mo 6 h/day, 5 days/wk for 1 yr Thyroid Vernot et al. (1985) 200, 200, 200, 200 Parafollicular cell adenoma: NR, NR, NR, NR 0/145, 0/117, 0/127, 4/137 NS Colon Adenocarcinoma: 0/158, 0/146, 2/129, 3/139 NS bw, body weight; F, female; GLP, good laboratory practice; M, male; mo, month; NA, not applicable; NR, not reported; NS, not significant; ppm, parts per million; wk, week; yr, year dose of 32 mg in water by gavage for 4 weeks and held for their lifetime. Groups of 22 male and 23 female BALB/c mice served as untreated controls. Exposure of male and female CBA/Cb/Se mice to hydrazine sulfate caused a significant increase in the incidence of pulmonary adenoma or carci- noma (combined) [mainly adenoma] (males: 16/21 (76%) [P < 0.0001] versus 1/37 (3%) controls; females: 19/21 (90%) [P< 0.0001] versus 4/47 (9%) controls) and hepatoma [hepatocellular tumours] (males: 13/21 (62%) [P< 0.0001] versus 4/37 (11%) controls; females: 15/21 (71%) [P < 0.0001] versus 2/47 (4%) controls) in exposed mice. Exposure of male and female BALB/c mice to hydrazine sulfate caused a significant increase in the incidence of pulmonary adenoma or carcinoma (combined) [mainly adenoma] (males: 7/8 (88%) [P < 0.006] versus 6/22 (27%) controls; females: 8/10 (80%) [P < 0.003] versus 5/23 (22%) controls). [The data for the CBA/Cb/Se mice experiment were also reported in Severi & Biancifiori (1968). While high incidences of pulmonary and liver tumours were reported in exposed CBA/Cb/Se mice, and a high incidence of pulmonary tumours was reported in exposed BALB/c mice, the Working Group noted the poor experimental design, lack of experimental details and vehicle controls, use of a single dose, and small number of mice in treated groups (especially for the BALB/c groups).] A group of 20 male and female (combined) newborn BALB/c mice was given hydrazine sulfate in sodium bicarbonate (total dose of 17 mg) daily by gavage for 60 days and were then held for up to age 425 days. A group of 20 male and female (combined) mice served as vehicle controls. There was a 100% (20/20) [P < 0.0001] incidence of lung adenoma or carcinoma (combined) (carcinoma, 16%) in the treated group compared with a 15% incidence (3/20, adenoma only) in controls (Milia et al., 1965). [While a high incidence of pulmonary tumours was reported in treated mice versus controls, the Working Group noted the lack of experimental details, limited experimental design, and use of a single dose level.] A group of 25 female Swiss mice [age at start not reported] was given hydrazine sulfate at a dose of 0.25 mg per day by gavage in water on 5 days per week for up to 40 weeks. A group of 85 untreated female Swiss mice served as controls. The incidence of bronchiolo-alveolar adenoma or carcinoma (combined) was 6/13 (46%) (P < 0.001) in treated mice that survived for up to 60 weeks, versus 8/79 (10%) controls (Roe et al., 1967). [The Working Group noted that experimental details were poorly described, only one sex was used, the duration of exposure was short, and there was poor survival in the treated group.] In a study to investigate a possible hormonal effect on the formation of tumours by hydra- zine sulfate, Biancifiori (1969) treated groups of 23 to 26 intact virgin or gonadectomized male and female CBA/Cb/Se mice (age, 8 weeks) with hydrazine sulfate as 150 daily doses of 0.14, 0.28, or 0.56 mg (total doses of 21, 42, or 84 mg) by gavage over about 25 weeks. The mice were then held for their lifetime. No concurrent controls were reported. In the groups receiving the highest dose, the incidence of pulmonary tumours [histopathology not further specified] was 87% (21/24) in intact virgin females and 20% (5/25) in intact virgin males. In the groups receiving the intermediate dose, the incidence of pulmonary tumours was 56% (16/25) and 16% (4/25), respectively. In groups receiving the lowest dose, the incidence of pulmonary tumour was 40% (10/25) and 7% (2/26), respectively. In gonadectomized mice, the incidence of pulmo- nary tumours [mainly adenoma] was 28% (7/25) in females and 21% (5/23) in males at the highest dose, 24% (6/25) and 11% (3/26) at the interme- diate dose, and 8% (2/25) and 12% (3/25) at the lowest dose. [The Working Group noted that the study had limited description of experimental details (including histopathology), and lacked concurrent controls.| 219 IARC MONOGRAPHS - 115 A group of 30 female (BALB/c x DBA/2) F, held for their lifetime (Biancifiori, 1970b). Groups (CDF,) mice (age, 7-8 weeks) was given hydra- of 25 males and 25 females served as untreated zine sulfate (total dose, 41.6 mg) in aqueous 2% controls. Hydrazine sulfate significantly sodium bicarbonate by gavage once per week increased the incidence of pulmonary adenoma for 8 weeks, and then held for up to 33 weeks. or carcinoma (combined) [mainly adenoma] inall A group of 10 female mice served as vehicle dosed groups compared with controls. Tumour controls. Pulmonary tumours [not further spec- incidence for males was: 6/25 (24%), 13/24 (54%) ified] were observed in 13/28 (46%) [P < 0.05] [P < 0.05], 15/24 (62.5%) [P < 0.008], 17/26 (65%) treated mice compared with 1/10 (10%) controls [P < 0.004], 17/20 (85%) [P < 0.0001], 20/22 (91%) (Kelly et al., 1969). [While a high incidence of [P < 0.0001]; and for females was: 1/25 (4%), 8/25 pulmonary tumours was reported in treated (32%) [P < 0.02], 17/19 (89%) [P < 0.0001], 19/25 mice versus controls, the Working Group noted (76%) [P < 0.0001], 15/20 (75%) [P < 0.0001] and the lack of experimental details, limited descrip- 20/22 (91%) [P < 0.0001]; for untreated controls, tion of the histopathology, and the limited exper- and the groups treated with 21, 42, 84, 32, and imental design including the use of one sex, only 170 mg, respectively. [The Working Group noted 10 control animals, and a single dose.] the limited description of experimental details, Groups of 24-26 male and female CBA/Cb/Se including body-weight gain or survival, and the mice (age, 8 weeks) were treated with hydrazine lack of vehicle controls.] sulfate at a dose of 0.14, 0.28, 0.56, or 1.13 mg Groups of 22-25 intact virgin, breeders, or per day (total dose: 21, 42, 84, or 170 mg) by gonadectomized female BALB/c mice (age, 8 gavage in water 150 times over 25 weeks, and weeks) were treated with hydrazine sulfate at a then held for their lifetime (Biancifiori, 1970a). dose of 1.13 mg (total dose, 170 mg) by gavage in Groups of 30 males and 29 females served as water as 150 daily doses, and then held for their untreated controls. Exposure of male and female lifetime (Biancifiori, 1970c). Three groups of25-26 mice to hydrazine sulfate caused a dose-re- intact virgin, breeders, or gonadectomized mice lated significant increase in the incidence of served as untreated controls. Hydrazine sulfate hepatoma [mainly hepatocellular carcinoma] increased the incidence of pulmonary tumours (males: 3/30 (10%), 1/26 (4%), 7/25 (28%), 12/25 to 90% (20/22) [P < 0.0001, mainly adenoma] in (48%) [P < 0.003], 15/25 (60%) [P < 0.0001]; intact virgins versus 4% (1/25) in controls, 100% females: 1/29 (3%), 0/25, 2/25 (8%), 16/24 (66%) (25/25) [P < 0.0001; adenoma:carcinoma, about [P < 0.0001], 15/24 (62.5%) [P < 0.0001]; for 1:1] in breeders versus 8% (2/25) in controls, and untreated controls, and the groups at 21, 42, 84, 60% (15/25) [P < 0.05, mainly adenoma] in gona- and 170 mg, respectively]). Multiple pulmonary dectomized mice versus 27% (7/26) in controls. tumours were reported to be present in many [The Working Group noted the poor description treated animals, but data on tumour incidence of experimental details, including duration of were not provided. [The Working Group noted actual exposure, body-weight gain or survival, the lack of vehicle controls.] and the lack of vehicle controls and use of a single Groups of 20-26 male and 19-25 female dose.] BALB/c mice (age, 8 weeks) were treated with Biancifiori (1971) gave five groups of 25-31 hydrazine sulfate at a dose of 0.14, 0.28, 0.56, or 8-week-old male and female intact virgin, gona- 1.13 mg per day (total dose, 21, 42, 84, or 170 mg) dectomized, or breeder (female only) C3H/Cb/ by gavage in water for 150 daily doses over 25 Se mice, 150 daily doses of 1.13 mg/dose (170 mg weeks, or at a dose of 1.13 mg per day (total dose, total dose) of hydrazine sulfate in water by gavage 32 mg) for 28 daily doses over 4 weeks, and then over 25 weeks. The animals were then held for 220 their lifetime. Five groups of 23-25 intact virgin, gonadectomized or breeder animals served as untreated controls. Hydrazine sulfate increased the incidence of pulmonary tumours to 26% (7/27) [P< 0.01, mainly adenoma] in intact virgin males versus 0% (0/25) in controls, and to 48% (13/27) [P < 0.0004, mainly adenoma] in intact virgin females versus 4% (1/25) in controls. The incidence of pulmonary tumours was 4% (1/25) [adenoma] in gonadectomized males versus 4% (1/24) in controls, and 19% (5/26) in gonadec- tomized females versus 12% (3/25) in controls. Hydrazine sulfate increased the incidence of pulmonary tumours to 39% (12/31) [P < 0.01; adenoma:carcinoma, about 2:1] in female breeders versus 13% (3/23) in controls. [The Working Group noted the limited description of experimental details, including body-weight gain or survival, and the lack of vehicle controls, and use of a single dose.] In a study to investigate the effect of antioxi- dants on the formation of tumours of the lung in mice by hydrazine sulfate, groups of 30 male and 30 female Swiss mice (age, 10 weeks) were treated with hydrazine sulfate at daily doses of 0 (control) or 1.1 mg by gavage in water for 5 days per week for up to 15 months. Five groups of treated mice were also simultaneously treated with 1.1 mg per day of the following antioxidants: L-arginine, pyridoxine hydrochloride, folic acid, L-arginine + L-sodium glutamate, or L-sodium glutamate + pyridoxine hydrochloride. The experiment was terminated at 15 months, except for the control group, which was maintained for lifetime. In all of the six groups treated with hydrazine sulfate, the incidence of lung tumours [not further speci- fied] in males and females (combined) was signif- icantly increased and ranged from 17/31 (55%) to 26/33 (79%) [P < 0.0001, for all treated groups versus the control group], while the incidence in the control group was 1/47 (2%) (Maru & Bhide 1982). [The Working Group noted that this study used a single dose, reported combined tumour incidence for males and females, and reported no clinical signs, body weights, mortality informa- tion, or histological description of tumours.] In a study of perinatal exposure in Swiss mice in which hydrazine sulfate was used to induce lung tumours, exposed groups of male and female Swiss mice (parental generation) and groups of 6 (unexposed F,) or 22 males and females (combined) of their F, generation were treated with hydrazine sulfate (in distilled water) at 0 (unexposed F,) or 1.1 mg per day, 5 days per week, by gavage, at age 10-11 weeks, for their lifetime, respectively (Menon & Bhide 1983). Two groups of 10 (unexposed F,) or 35 males and females (combined) of the F, genera- tion, were also exposed or not (unexposed F,) to hydrazine sulfate transplacentally and through lactation. A group of 20 males and females served as untreated controls. To raise the F, progeny of hydrazine-treated females, dams received hydrazine (1.1 mg per day) from day 1 of gestation, and continued to receive hydrazine treatment through lactation until death. To raise the exposed F, progeny, F, males and females (combined) were treated with hydrazine from the age of 11 weeks for a period of 4 weeks, and were then kept for mating; pregnant F, females continued to receive hydrazine from day 1 of gestation through lactation until death. To raise the unexposed F, progeny, F, parents were of the untreated F, generation. The incidence of adenocarcinoma of the lung in the parental generation exposed to hydrazine sulfate was 72% (21/29; P< 0.05, X? test, versus 1/20 untreated controls) in females and 88% (30/34; P < 0.05, X? test, versus 1/20 untreated controls) in males. F, mice receiving hydrazine sulfate only during gestation and lactation, and subse- quently given distilled water from a young adult age showed a 50% (3/6; P < 0.05, X? test, versus 1/20 untreated controls) incidence of adenocarci- noma of the lung, while F, mice raised from the same stock of parents and receiving hydrazine sulfate at 1.1 mg per day showed a 90% (20/22; P < 0.05, X? test, versus 1/20 untreated controls) 221 IARC MONOGRAPHS - 115 incidence of adenocarcinoma of the lung. The increase in tumour incidence was observed in F, generation of mice, whose parents received male and female AKR or C3H mice exposed to distilled water during gestation and lactation, hydrazine sulfate. [The Working Group noted and then distilled water by gavage from a young the use of a single dose.] adult age (unexposed F, generation controls), In a study to see whether the formation oflung had a 10% (1/10) incidence of adenocarcinoma of tumours by hydrazine could be inhibited by argi- the lung, while the F, generation of mice, whose nine glutamate, groups of 20 or 37-38 male A/J parents received hydrazine sulfate by gavage for mice (age, 6 weeks) were given drinking-water 4 weeks and which were themselves exposed to containing hydrazine sulfate at a concentration hydrazine sulfate during gestation and lactation, of 0 or 325 mg/L and feed containing 0% or 1% showed a 45% (16/35; P < 0.05, X? test, versus 1/10 arginine glutamate for up to 48 weeks (Yamamoto unexposed F, generation controls) incidence of & Weisburger, 1970). Hydrazine sulfate caused a adenocarcinoma of the lung. [The Working significant increase [P < 0.0001] in the incidence Group noted that the study used only a single of lung adenoma or carcinoma (combined) (38/38; dose, reported combined tumour incidences for 100%) in exposed mice compared with control males and females, and did not report on clinical mice (12/20; 60%). Upon addition of arginine signs, body weights or mortality.] glutamate, the incidence of pulmonary tumours in hydrazine-treated mice was not significantly 3.1.2 Drinking-water affected, and arginine glutamate did not change the incidence of pulmonary tumours in control Toth (1969) reported on a study in three mice. [The Working Group noted the use of a strains of mice treated with hydrazine sulfate. single dose.] Groups of 110 (control) or 50 male and female Groups of 50 male and 50 female NMRI Swiss mice (age, 6 weeks) were given drink- mice (age, 5-6 weeks) were given drinking-water ing-water containing 0% or 0.012% [120 mg/L] containing hydrazine hydrate at a concentra- hydrazine sulfate (average daily dose, 0.74 mg tion of 0, 2, 10, or 50 ppm ad libitum for up to and 0.65 mg for males and females, respectively) 2 years. A significant reduction in body weight for their lifetime. Exposure of male and female was observed in groups at the highest dose. There Swiss mice to hydrazine sulfate caused a signif- was no difference in survival in any of the treated icant increase in the incidence of adenoma or groups or controls. There was a significant carcinoma (combined) of the lung: males, 25/50 increase (P< 0.05) in the incidence of benign lung (50%) [P < 0.0001] versus 11/110 (10%) controls; tumours in females at the highest dose (15/50) females, 24/50 (48%) [P < 0.0001] versus 14/110 compared with controls (6/50). There was no (13%) controls. Additionally, groups of 30 other significant increase in tumour incidence (control) or 40 male and female AKR mice (age, in any of the treatment groups compared with 6 weeks) were given drinking-water containing controls (Steinhoff et al., 1990). [The Working 0% or 0.012% hydrazine sulfate (average daily Group noted the limited description of the dose, 0.63 mg for males and females) for their pathology of the lesions observed in treated and lifetime, and groups of 30 (control) or 41 male control mice.| and 30 (control) or 40 female C3H mice (age, 6 Matsumoto et al. (2016) reported the results weeks) were given drinking-water containing 0% of a study that complied with good laboratory or 0.012% hydrazine sulfate (average daily dose, practice (GLP) in which groups of 50 male and 0.98 mg and 0.84 mg for males and females, 50 female Crj:BDF, mice (age, 6 weeks) were respectively) for their lifetime. No significant given drinking-water containing hydrazine 222 monohydrate at a concentration of 0, 20, 40, or 80 ppm (males), and 0, 40, 80, or 160 ppm (females) ad libitum for 2 years. Body weights of males at the intermediate and highest dose and of all groups of treated females were significantly decreased in a dose-related manner compared with controls. There was no significant difference in survival in any of the treated groups compared with controls, except in females at the lowest dose, in which survival was better than in controls. In females at the highest dose, hydrazine monohydrate caused a significant increase in the incidence of hepatocellular adenoma (14/50, 28%; P < 0.05) compared with controls (5/50, 10%), and a signif- icant increase in the incidence of hepatocellular adenoma or carcinoma (combined) (17/50, 34%; P < 0.05) compared with controls (7/50, 14%). There was a significant, dose-dependent positive trend (P < 0.01, Peto trend test) in the incidences of haemangioma of the liver, hepatocellular adenoma, hepatocellular carcinoma, and hepa- tocellular adenoma or carcinoma (combined) in treated female mice. There was also a significant, dose-dependent positive trend (P < 0.05, Peto trend test) for the incidence of haemangioma or haemangiosarcoma (combined) of the liver in females. There was no significant increase in the incidence of any tumours in males. [The strengths of this GLP study included the use of multiple doses, a high number of animals per group, and two sexes.] 3.1.3 Intraperitoneal injection Two groups of 20 newborn male and female (combined) BALB/c/Cb/Se mice were treated by intraperitoneal injection of hydrazine sulfate (in water) once a day for 90 days (0 or 19 mg total dose/mouse) and then held for up to 270 days (Milia et al., 1965). There was a 100% inci- dence (20/20) [P < 0.0001] of lung adenoma or carcinoma (combined) (carcinomas, 27%) in the treated group compared with a 10% inci- dence (2/20, adenomas only) in controls. [The Working Group noted the limited description of experimental design, small number of animals per group, use of only one dose, and absence of discussion of clinical signs, survival or body weights. ] Groups of 9 (control) or 30 male CDF, mice (age 7-8 weeks) were treated by intraperitoneal injection of saline (control) or hydrazine sulfate in 2% aqueous sodium bicarbonate once a week for 8 weeks (0 or 20.8 mg total dose/mouse) and then held for up to 33 weeks (Kelly et al., 1969). There was a non-significant increase in the inci- dence of pulmonary tumours [not further spec- ified] in 6/30 treated animals (20%) versus 1/9 (10%) in controls. [The Working Group noted the poor description of experimental details, the use of a single dose, the small number of controls, the short exposure time, and absence of discussion of clinical observations or pathology.] Groups of 30 male and 30 female (combined) white mice (strain and age not reported) [weight, ~20 g] were treated with hydrazine at a dose of 0 (control) or 400 mg/kg body weight (bw) in physiological saline in 16 intraperitoneal injec- tions administered over 46 days, and were then held for up to 313 days (Juhasz et al., 1966). It was reported that 13/34 (38%) mice surviving the treatment developed tumours of the haemato- poietic and lymphoid tissues (reticulum cell sarcoma or myeloid leukaemia) [P < 0.0001] between days 100 and 313 of the experiment. No reticulum cell sarcoma or myeloid leukaemia was reported in the 60 controls after 1 year. [The Working Group noted the poor description of experimental details, the use of a single dose, the high mortality in treated animals, the reported combined tumour incidences, the short expo- sure time, the absence of discussion of clinical observations or pathology, and the poor descrip- tion and discussion of tumour incidences.] In a study of hydrazine and a modifying factor (irradiation), groups of 29 or 40 male and female C57BL/6 mice (equal numbers of each sex per group) (age, 6-8 weeks) were exposed once to 223 IARC MONOGRAPHS - 115 400 R total-body irradiation and, starting 1 week Rat after irradiation, given intraperitoneal injections of aqueous hydrazine sulfate at a dose of 0 or 3.2.1 Gavage 95 mg/kg bw once per week for 10 weeks, and Groups of 14-28 male and female CB/Se rats then held for up to 62 weeks. A group of 36 mice were given “pure” hydrazine sulfate as a daily received no irradiation and weekly injections of gavage dose in water at 0, 12 (females only), or 18 hydrazine sulfate, and were also held for up to (males only) mg/day for 68 weeks, and then held 62 weeks. A fourth group of at least 75 animals for their lifetime. Pulmonary adenoma or carci- served as untreated controls. In the non-irradi- noma was observed in 3/14 (21%) [P< 0.05] treated ated groups, lung adenomas were observed in males and 5/18 (28%) [P < 0.02] treated females. 5/18 (28%) [not significant] mice treated with Malignant liver tumours were observed in 4/13 hydrazine sulfate compared with 9/75 (12%) (30%) [P < 0.01] treated males. No pulmonary untreated controls. The incidence of leukaemia or liver tumours were observed in control males was not increased in the irradiated/hydrazine (0/28) or females (0/22) (Biancifiori et al., 1966; treated group (4/29) compared with irradiat- Severi & Biancifiori 1968). [The Working Group ed-only controls (2/25) (Mirvish et al., 1969). [The noted the short duration of exposure, the small Working Group noted the use of a single dose, number of rats per exposure group, the use of a the limited description of experimental details, single dose, and the limited description of exper- the short exposure time, the lack of reporting of imental design and results.] clinical signs, body weights, and mortality infor- mation or histological description of tumours.] 3.2.2 Drinking-water 3.1.4 Inhalation Groups of 50 male and 50 female Wistar rats Groups of 400 female C57BL/6 mice (age, 7 were given drinking-water containing hydrazine weeks) were exposed to 0 (concurrent control), hydrate (purity, 99.3%) at a concentration of 0, 0.05, 0.25, or 1.0 ppm [0, 0.04, 0.2, and 0.8 mg/m}] 2, 10, or 50 ppm ad libitum for their lifetime. A anhydrous hydrazine (purity, 99.8%) by inhala- significant reduction in body weight was observed tion for 6 hours per day, 5 days per week, for 1 year, in males and females at the highest dose. There and then held for an additional 15 months. Deaths was no significant difference in survival in any during exposure were below 10% in all groups, of the treated groups or controls. There was a and exposed mice demonstrated significantly significant increase (P < 0.01) in the incidences of higher mortality than controls at the end of the hepatocellular adenoma in males at the highest experiment, but without any dose-response rela- dose (4/49) and females at the highest dose (4/47), tionship (controls, 72-79%; exposed, 84-87%). and of hepatocellular carcinoma in females at An increase in the incidence of lung adenoma the highest dose (3/47; one of the tumours may was reported in mice at the highest dose (12/379, not have been a primary tumour). The inci- 3%) [P < 0.05] versus controls (4/378, 1%). No dence of hepatocellular adenoma or carcinoma information on lung tumours was given for the (combined) in females at the highest dose (7/47) other exposure groups (Vernot et al., 1985). [The was significantly increased [P < 0.01]. No hepa- Working Group noted the low doses used and tocellular tumours were observed in the 50 male the lack of tumour data for the groups receiving and 50 female controls (Steinhoff & Mohr, 1988). the lowest and intermediate doses.] In a GLP study, groups of 50 male and 50 female F344/DuCrj rats (age, 6 weeks) were given drinking-water containing hydrazine 224 monohydrate at a concentration of 0, 20, 40, or 80 ppm ad libitum for 2 years (Matsumoto et al. 2016). Body weights of males and females at the intermediate and highest doses were significantly decreased in a dose-related manner compared with controls. Survival in females at the highest dose was significantly reduced compared with controls, while survival was similar to controls for all other groups of treated females and for all groups of treated males. Hydrazine monohy- drate in male rats caused a significant positive trend in the incidence of hepatocellular adenoma (0/50, 0/50, 0/50, 3/50; P < 0.01 by Peto trend test) and hepatocellular adenoma or carcinoma (combined) (0/50, 0/50, 0/50, 4/50; P < 0.01 by Peto trend test). In female rats, hydrazine mono- hydrate caused a significant positive trend in the incidence of hepatocellular adenoma (1/50, 0/50, 3/50, 4/50; P < 0.05 by Peto trend test), hepatocel- lular carcinoma (0/50, 0/50, 0/50, 4/50; P < 0.01 by Peto trend test), and hepatocellular adenoma or carcinoma (combined) (1/50, 0/50, 3/50, 6/50; P < 0.01 by Peto trend test). There was also a significant increase and positive trend in the incidence of interstitial cell tumours of the testis (37/50, 45/50*, 43/50, 44/50; *P < 0.05; P < 0.05 by Peto trend test). [The strengths of this GLP study included the use of multiple doses, a high number of rats per group, and two sexes.] 3.2.3 Inhalation Groups of 150 (controls) or 100 male and female F344 rats (age, 7 weeks) were exposed to anhydrous hydrazine (purity, 99%) at a concen- tration of 0 (control), 0.05, 0.25, 1.0, or 5.0 ppm [0, 0.04, 0.2, 0.8, or 4.0 mg/m?] by inhalation for 6 hours per day, 5 days per week for 1 year, and then held for an additional 18 months. Deaths during exposure were low in all groups (below 10%), and mortality was similar in all groups at the end of the study. A significant increase in the incidence of adenomatous polyps [adenoma] of the nose was reported in males exposed to the higher two doses (0/146, 2/96, 1/94, 9/97%*, 58/98*; *P < 0.01) and in females at the highest dose (0/145, 2/97, 0/98, 2/94, 28/95*; *P < 0.01). A significant increase in the incidence of thyroid carcinoma was also reported in males at the highest dose (7/146, 6/96, 5/94, 9/97, 13/98**; **P < 0.05) (Vernot et al., 1985), Groups of 100 male and 100 female F344 rats (age, 9-11 weeks) were exposed to anhydrous hydrazine (purity, 98.8%) at a concentration of 0, 75, or 750 ppm [0, 98 and 980 mg/m] by inhalation for 1 hour per day, 1 day per week, for 10 weeks, and then held for 24-30 months. Polypoid adenomas of the nose were found in 4/99 (4%, P < 0.05) males at the highest dose, and 6/95 (6%, P < 0.05) females at the highest dose. In addition, one nasal squamous cell carci- noma was reported in males. No nasal tumours were observed in 98 male and 98 female controls (Latendresse et al., 1995). [The Working Group noted that this study was inadequate for the evaluation because of inadequate exposure for a study of full carcinogenicity. ] 3.3. Hamster 3.3.1 Gavage Groups of 23-56 male and female (sex distri- bution not given) Syrian golden hamsters (age, 8 weeks) were treated with hydrazine sulfate (purity not reported) at a total dose of 0, 180, or 280 mg by gavage in water for 15 or 20 weeks, and then held for life (Biancifiori, 1970a). There was a significant decrease in survival in treated hamsters compared with controls. No informa- tion on body weight was provided. No signif- icant increase in the incidence of tumours was reported. A significant increase in cirrhosis of the liver was reported in both treatment groups, but not in the controls. [The Working Group noted that the study used a short duration of exposure, and was considered inadequate for the evalua- tion because of inadequate reporting of results in controls.] 225 IARC MONOGRAPHS - 115 3.3.2 Drinking-water a significant decrease in survival in all treated groups. The incidence of hepatocellular adenoma Groups of 50 male and 50 female Syrian was 0/25 (control), 1/30 (3%), 4/43 (9%), and 10/40 golden hamsters (age, 9 weeks) were given drink- (25%) [P < 0.005], and the incidence of hepato- ing-water containing hydrazine sulfate (purity cellular carcinoma was 0/25 (control), 0/30, 1/43 not reported) at a concentration of 0.012% (2%) and 3/40 (8%), respectively. [The Working [120 mg/L] for life. The average daily intake of Group noted that the incidence of hepatocellular hydrazine sulfate was 2.3 mg for both sexes. adenoma or carcinoma (combined) was signifi- There was a slight decrease in body weight, but no cantly increased [P < 0.005] in the group at the effect on survival in treated animals compared highest dose compared with controls.] with controls. No significant increases in the number of tumours produced were reported 3.3.3 Inhalation in treated hamsters. A non-significant increase in the incidence of polypoid adenoma of the Groups of 200 male Syrian golden hamsters caecum was reported in treated males (3/49, 6%) (age, 7 weeks) were exposed to anhydrous and females (4/45, 9%) compared with controls, hydrazine (purity, 98.8%) at a concentration of but statistics and tumour incidence in controls 0 (control), 0.25, 1.0, or 5.0 ppm [0, 0.2, 0.8 or were not provided (Toth, 1972). [The Working 4.0 mg/m?] by inhalation for 6 hours per day, Group considered that the study was inadequate 5 days per week, for 1 year, and then held for an for the evaluation because of the use of a single additional 1 year (Vernot et al., 1985). Exposure dose and because of the inadequate reporting of to hydrazine had no effect on body weights of results in controls.] hamsters in any treated group compared with Groups of 40 male Syrian golden hamsters controls. Deaths during exposure were high (age at start not given; weight, 50-60 g at 1 week in all exposure groups (32-33% versus 19% in before exposure) were given drinking-water controls), but mortality was similar in all groups containing hydrazine sulfate (purity, > 99%) at the end of the study (2 years). A significant at a concentration of 0 (control), 170, 340, or increase in the incidence of nose adenomatous 510 mg/L for 2 years (average doses, 0, 4.6, 8.3, polyps [adenoma] was reported in the group at and 10.3 mg/kg bw per day) (Bosan et al., 1987). the highest dose: 1/181 (1%) controls; 0/154; 1/148 There was a significant decrease in survival in all (1%); 16/160* (10%), *P < 0.01. A non-significant treated groups. Hepatocellular carcinomas were increase in the incidence of parafollicular cell observed in hamsters treated with the two higher adenoma of the thyroid gland was also reported doses of hydrazine sulfate after 78 weeks of expo- in the group at the highest dose (4/137, 3%, sure. The incidences of hepatocellular carcinoma versus 0/145 in controls), as was a non-significant were 0/31 (controls), 0/31, 4/34 (12%), and 11/34 increase in the incidence of adenocarcinoma of (32%; [P < 0.0004)). the colon in the groups at the intermediate and Groups of 25-40 male Syrian golden hamsters highest dose (2/129, 2% and 3/139, 2%, respect- (age at start not given; weight, 50-70 g at 1 week ively, versus 0/158 in controls) (Vernot et al. before exposure) were given drinking-water 1985). containing hydrazine sulfate (purity, > 99%) at a concentration of 0 (control), 170, 340, or 510 mg/L for up to 21 months (average doses calculated from consumption data: 0, 4.2, 6.7, and 9.8 mg/kg bw per day) (FitzGerald & Shank, 1996). There was 226 Mechanistic and Other Relevant Data Absorption, distribution, metabolism, excretion 4.1.1 Absorption, distribution, excretion (a) Humans Organ toxicity after accidental or intentional exposure to hydrazine demonstrated absorption into the systemic circulation and distribution to target tissues (Nagappan & Riddell, 2000; Kao et al., 2007). After occupational exposure, hydra- zine was absorbed and excreted in the urine (Nomiyama et al., 1998a). Exposure to the ther- apeutic drug isoniazid, containing a hydrazine group, resulted in excretion of hydrazine and its metabolites in the urine (Timbrell et al., 1977; Blair et_al., 1985; Donald et al., 1994; Preziosi, 2007). (6b) Experimental systems Absorption of hydrazine was rapid after either oral or dermal administration in exper- imental animals. Hydrazine was detected in femoral blood within 30 seconds after applica- tion of a dose of 3-15 mmol/kg bw to an area of shaved chest skin of anaesthetized dogs. The serum concentration of hydrazine peaked within the first hour for most doses, followed by a slow decline over a 6-hour holding period. Unchanged hydrazine was excreted in the urine. Mortality was high across the dosing range (Smith & Clark, 1972). When hydrazine hydrate (corresponding to hydrazine free base at 3-81 mg/kg bw) was given orally (gavage) to rats, hydrazine was at its greatest concentration in the plasma and liver within 30 minutes after dosing, with the excep- tion ofa peak in plasma concentration 90 minutes after administration of the highest dose (Preece et al., 1992a). The liver to plasma ratio of hydra- zine decreased with increasing dose, suggesting saturation of uptake by the liver. An effect on dose elimination was also observed, with about 40% of the lowest dose but less than 20% of the highest dose being excreted in the urine within 24 hours after administration. Dambrauskas & Cornish (1964) investigated the fate of hydrazine in rats given hydrazine hydrate at a dose of 60 mg/kg bw by subcutaneous injection. The dose was well distributed, with hydrazine detected in adipose, blood, brain, kidney, liver, lung, muscle, skin, and other tissues within 2 hours after dosing. About 13% of the total administered dose was recovered in the assayed tissues as unchanged hydrazine, with the highest concentration present in the kidney (41-56 ug/g). Hydrazine (8% of the total administered dose) was excreted in the urine. Matsuyama et al. (1983) provided additional evidence that hydrazine crosses the blood-brain barrier in rats. After intravenous injection, hydrazine was detected in the brain accompanied by an increase in gamma-amino- butyric acid (GABA) over a period of 10 hours. Hydrazine was rapidly absorbed and distributed to tissues when administered as hydrazine sulfate at a dose of 0.31 mmol/kg bw by subcutaneous injection in rats (Kaneo et al., 1984), Springer et al. (1981) recovered up to 75% of [!5N]-labelled hydrazine from single doses of 1 mmol/kg bw administered by various injection routes (intra- peritoneal, subcutaneous, intravenous) in rats. Up to 25% of the total administered dose was recovered as nitrogen (N,) in expired air within 48 hours. An additional 50% was excreted in the urine as unchanged hydrazine and acid-la- bile metabolite(s). The disappearance of intrave- nously administered hydrazine from blood was described as biphasic, with calculated half-lives of 0.74 and 27 hours. In mice, intraperitoneal administration of [!°N]-labelled hydrazine sulfate at 1 mmol/kg bw resulted in rapid distribution to tissues including blood, brain, liver, kidney, and lung (Nelson & Gordon, 1981). Clearance from tissues was extensive by 24 hours. About 30% of the administered dose was recovered as N, and 227 IARC MONOGRAPHS - 115 40% was recovered in the urine as hydrazine acetylated metabolites are species-dependent. and metabolites within 48 hours after injection Bollard et al. (2005) detected diacetylhydrazine (Nelson & Gordon, 1981). in the urine of rats and mice treated orally with Mice excreted about 50% of an administered hydrazine hydrochloride at a dose of 100 or dose of either 40 or 60 mg/kg bw as unchanged 250 mg/kg bw. In contrast, acetylhydrazine was hydrazine in the urine within 48 hours after detected in the urine in rats, but not in mice. This a subcutaneous injection. Less than 1.5% of result was attributed to higher activity of N-acetyl the administered hydrazine remained in each transferase in the mouse. Dogs, unlike rats and carcass at this time-point. The unrecovered dose mice, have little to no capacity to acetylate hydra- was assumed to be metabolites (Dambrauskas & zine (McKennis et al., 1959). This limitation may Cornish, 1964). have contributed to the prolonged elimination of hydrazine in dogs as reported by Smith & Clark 4.1.2 Metabolism (1972). Rabbits treated with hydrazine excreted diacetylhydrazine in the urine (McKennis et al. (a) Humans 1959). Kaneo et_al. (1984) demonstrated that Hydrazine was metabolized to acetylhydra- metabolism of hydrazine to acetylhydrazine in zine (monoacetylhydrazine) in exposed workers rats was reversible. Acetylhydrazine may give (Nomiyama et _al., 1998a). In male Japanese rise to a carbon-centred acetyl radical or carbo- workers, the rate of acetylation was dependent on cation, capable of binding to macromolecules or polymorphisms in N-acetyltransferase (NAT2), oxidation to carbon dioxide (CO,) (Sinha, 1987; with half-lives of about 2 and 4 hours in rapid Morike et al., 1996; Preziosi, 2007). Hydrazine and slow acetylators, respectively (Koizumi may also give rise to hydrazones (Preziosi, et al., 1998). Acetylhydrazine and diacetylhydra- 2007). The metabolite 1,4,5,6-tetrahydro-6- zine have been detected in the urine of subjects oxo-3-pyridazine carboxylic acid, a derivative receiving isoniazid (Ellard & Gammon, 1976; of oxoglutarate hydrazone, was found in the Timbrell et al., 1977). Oxidative metabolism of urine of rats and mice treated with hydrazine hydrazine was demonstrated in human micro- (Nelson & Gordon, 1981; Delaney & Timbrell, somes based on disappearance of the chemical 1995; Bollard et al., 2005). Hydrazine is oxidized over time from the incubation mixture; however, to N, in rats and mice (Nelson & Gordon, 1981; the rate of metabolism was slower than in rat Springer et al., 1981). Degradation of hydrazine microsomes (Jenner & Timbrell, 1995). A review to ammonia (NH,) may be possible, especially of isoniazid metabolism indicated that pathways in dogs (McKennis et_al., 1959; Colvin, 1969; of hydrazine biotransformation are similar in Preziosi, 2007). Oxidative metabolism of hydra- humans and other mammals (Preziosi, 2007). zine was catalysed primarily by cytochrome The metabolism of hydrazine is discussed in P450s (CYPs) in rat microsomes (Noda et_al. greater detail below. 1987; Jenner & Timbrell, 1995), Mixed function oxidases may also play a role (Jenner & Timbrell, (b) Experimental systems 1995). Results from incubation in rat hepatocytes Pathways of hydrazine metabolism in exper- suggested the involvement of CYP2E1, CYP2B1, imental animals include oxidation and acetyl- and CYP1A1/2 in the metabolism of hydrazine ation (Colvin, 1969; Fig. 4.1). Acetylhydrazine (Delaney & Timbrell, 1995). CYP activity was and/or diacetylhydrazine have been observed demonstrated in the metabolism of [!°N,]-[*C]- in the urine of animals treated with hydrazine. labelled acetylhydrazine to N, and CO, in the The presence and/or relative abundance of these rat (Morike et al., 1996). A hydrazine radical, 228 Hydrazine Fig. 4.1 Proposed metabolic scheme for hydrazine in mammals COOH oY NH O 1,4,5,6-Tetrahydro-6-oxo-3-pyridazine carboxylic acid H,O “YY COOH N—NH, N H3C N—-NH, COOH Pyruvic acid hydrazone Oxoglutaric acid hydrazone Pyruvic acid DK Oxoglutaric acid 0 H Ny HN NH) ——® HC“ “N-NH) ——> C7 ON Y H 4H CH, Hydrazine Acetylhydrazine Diacetylhydrazine [HN = NH] Diazene [NH;] H,N NB [H,N—NH)] Hydrazine radical O | oo | ° + CH, CH; . Covalent binding <= — Acetyl radical or carbocation CO, Adapted from Nelson & Gordon (1981), Noda et al. 1985), Delaney & Timbrell 1995), and Morike et al. (1996) 229 IARC MONOGRAPHS - 115 potentially giving rise to a diimide (diazene), was assay) depended on the route of administration. identified in microsomal incubations of hydra- DNA damage was found in the stomach, liver, and zine using a spin-trapping method (Noda et al. lungs of mice given hydrazine as a single intra- 1985). peritoneal dose at 100 mg/kg bw. When the same dose was administered orally, DNA damage was Mechanisms of carcinogenesis also found in the colon and brain (Sasaki et al., 1998). More recently, another study reported the The evidence on the "key characteristics" of induction of single-strand breaks and alkali-la- carcinogens (Smith et al., 2016) - concerning bile sites (comet assay) in primary lung cells from whether hydrazine is genotoxic, induces oxida- male rats as well as in the lungs of rats given a tive stress, alters cell proliferation, cell death or single oral dose of hydrazine (Robbiano et al. nutrient supply, and modulates receptor-medi- 2006). Lack of induction of sister-chromatid ated mechanisms — is summarized below. exchanges in bone marrow or liver of mice, and conflicting results on induction of micronuclei 4.2.1 Genetic and related effects in mouse bone-marrow cells (in one study out of See Table 4.1 and Table 4.2 three), were observed (LARC, 1999), Hydrazine induced DNA strand breaks in (a) Humans rat hepatocytes and unscheduled DNA synthesis No data on exposed humans were available to in mouse hepatocytes. There were conflicting the Working Group. results for the induction of gene mutations in One study reported the induction of single- mouse lymphoma L5178Y cells (one positive strand breaks and alkali-labile sites (comet assay) result and two negative, all in the absence of in human lung cells in vitro (Robbiano et al. exogenous metabolic activation). Hydrazine 2006). induced sister-chromatid exchanges and chro- mosomal aberrations in Chinese hamster ovary (b) Experimental systems cells, but gave negative results for the induction Considerable information was previously of chromosomal aberrations in rat liver RL1 cells reviewed by the IARC Monographs Working (IARC, 1999). Group regarding whether hydrazine is geno- Hydrazine was mutagenic in yeast and toxic in experimental systems (LARC, 1999). bacteria, induced DNA damage in bacteria, and Multiple studies identified N7-methylguanine caused somatic mutations in Drosophila (LARC, and O%-methylguanine in the livers of mice, rats 1999). (including neonates) and hamsters treated with hydrazine in vivo. The available data suggested 4.2.2 Oxidative stress that the DNA methylation mechanism involved (a) Humans reaction of hydrazine with endogenous formal- dehyde, followed by metabolism of the resulting No data in exposed humans were available to hydrazone to a methylating agent, most likely the Working Group. diazomethane.