NE Native Snowflower Extract Concentrate SB P3 Melaleuca alternifolia

Hazard statements (CLP)

H315, H319, H335

Code

Statements

H315

Causes skin irritation

Signal word

WARNING

Hazard statements

H315 Causes skin irritation H319 Causes serious eye irritation H335 May cause respiratory irritation

Prevention

P101 If medical advice is needed, have product container or label at hand. P103 Read label before use. P271 Use only outdoors or in a well-ventilated area. P261 Avoid breathing mist/vapour/spray. P272 Contaminated work clothing should not be allowed out of the workplace. P280 Wear protective gloves/protective clothing/eye protection/face protection.

Response

P302+P352 IF ON SKIN: Wash with plenty of soap and water. P332+P313 If skin irritation occurs: Get medical advice/attention. P362 Take off contaminated clothing and wash before reuse. P305+P351+P388 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P337+P313 If eye irritation persists: Get medical advice/attention. P304+P340 IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. P312 Call a POISON CENTRE or doctor/physician if you feel unwell.

Storage

P403+P233 Store in a well-ventilated place. Keep container tightly closed. P405 Store locked up.

Disposal

P501 Dispose of contents/container in accordance with local/national/international regulations.

2.1 Classification of the substance or mixture

POISONS SCHEDULE: Unscheduled HAZARDOUS CHEMICAL – NON-DANGEROUS GOODS: According to the WHS Regulations and the ADG Code; Globally Harmonized System of Classification and Labelling of Chemicals [GHS]; Regulation (EC) No 1223/2009 of the European Parliament and the Council of 30 November 2009 on cosmetic products (the ”Cosmetics Regulation” or the “Regulation”), Governing the composition, labelling and packaging of finished cosmetic products. CLASSIFICATION: Skin Corrosion/Irritant Category 2 Serious Eye Damage/Eye Irritation Category 2A Specific Target Organ Toxicity Single Exposure Category 3 LABEL ELEMENTS GHS LABEL ELEMENTS: SIGNAL WORD: WARNING HAZARD STATEMENT[S] H315 Causes skin irritation H319 Causes serious eye irritation H335 May cause respiratory irritation PRECAUTIONARY STATEMENT[S] PREVENTION: P101 If medical advice is needed, have product container or label at hand. P103 Read label before use. P271 Use only outdoors or in a well-ventilated area. P261 Avoid breathing mist/vapour/spray. P272 Contaminated work clothing should not be allowed out of the workplace. P280 Wear protective gloves/protective clothing/eye protection/face protection. RESPONSE: P302+P352 IF ON SKIN: Wash with plenty of soap and water. P332+P313 If skin irritation occurs: Get medical advice/attention. P362 Take off contaminated clothing and wash before reuse. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to P305+P351+P388 do. Continue rinsing. P337+P313 If eye irritation persists: Get medical advice/attention. P304+P340 IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. P312 Call a POISON CENTRE or doctor/physician if you feel unwell. STORAGE: P403+P233 Store in a well-ventilated place. Keep container tightly closed. P405 Store locked up. DISPOSAL: P501 Dispose of contents/container in accordance with local/national/international regulations. SECTION 3: COMPOSTIONAL INFORMATION ON INGREDIENTS SUBSTANCE CHEMICAL NAME CAS No EC [%w/w] Melaleuca alternifolia Leaf Extract 85085-48-9 285-377-1 Glycerine 56-81-5 200-289-5 34-55% Melaleuca alternifolia Leaf 85085-48-9 285-377-1 20-50% Water/Aqua 7732-18-5 231-791-2 14-24% Sodium Benzoate 532-32-1 208-534-8 <0.5% Citric Acid 77-92-9 201-069-1 <0.4% Potassium Sorbate 24634-61-5 246-376-1 <0.3% Cellular Extraction of Melaleuca alternifolia Leaf. Natural extract preserved with Sodium Benzoate; Citric Acid; Potassium Sorbate SECTION 4: FIRST AID MEASURES DESCRIPTION OF FIRST AID MEASURES EYE CONTACT: If this product comes into contact with the eye: 4 Wash out immediately with fresh running water; 4 Ensure complete irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids; 4 Seek medical attention without delay; if pain persists or recurs seek medical attention; 4 Removal of contact lenses after an eye injury should only be undertaken by skilled personnel. SKIN CONTACT: If skin contact occurs: 4 Immediately remove all contaminated clothing, including footwear; 4 Flush skin and hair with running water (and soap if available); 4 Seek medical attention in event of irritation. INHALATION: 4 If fumes or combustion products are inhaled remove from contaminated area; 4 Lay patient down. Keep warm and rested; 4 Prostheses such as false teeth, which may block airway, should be removed, where possible, prior to initiating first aid procedures; 4 Apply artificial respiration if not breathing, preferably with a demand value resuscitator, bag-valve mask device, or pocket mask as trained. Perform CPR if necessary; 4 Transport to hospital, or doctor, without delay. SWALLOWED: 4 Immediately give a glass of water; 4 First aid is not generally required. If in doubt, contact a Poisons Information Centre or doctor. INDICATION OF ANY IMMEDIATE MEDICAL ATTENTION AND SPECIAL TREATMENT NEEDED: Treat symptomatically. SECTION 5: FIRE FIGHTING MEASURES EXTINGUISHING MEDIA Water spray or fog; Foam; Dry chemical powder; BCF (where regulations permit). SPECIAL HAZARDS ARISING FROM THE SUBSTANCE FIRE INCOMPATIBILITY: Avoid contamination with oxidising agents i.e. nitrates, oxidising acids, chlorine bleaches, pool chlorine etc. as ignition may result. ADVICE FOR FIRE FIGHTERS FIRE FIGHTING: 4 Alert Fire Brigade and tell them location and nature of hazard; 4 Wear full body protective clothing with breathing apparatus; 4 Prevent, by any means available, spillage from entering drains or watercourse; 4 Use water delivered as a fine spray to control fore and cool adjacent area. FIRE/EXPLOSION HAZARD: 4 Combustible; 4 Slight fire hazard when exposed to heat or flame; 4 Heating may cause expansion or decomposition leading to violent rupture of CONTAINERS; 4 On combustion, may emit toxic fumes or carbon monoxide (CO); 4 Combustion products include; carbon dioxide (CO2) acrolein, other pyrolysis products typical of burning organic material. May emit poisonous fumes. May emit corrosive fumes. HAZCHEM: Not applicable. SECTION 6: ACCIDENTIAL RELEASE MEASURES PERSONAL PRECAUTIONS, PROTECTIVE EQUIPMENT AND EMERGENCY PROCEDURES See Section 8. ENVIRONMENTAL PRECAUTIONS See Section 12. METHODS OF MATERIAL FOR CONTAMINATION AND CLEAN UP MINOR SPILLS: 4 Remove all ignition sources; 4 Clean up all spills immediately; 4 Avoid breathing vapours and contact with skin and eyes; 4 Control personal contact with the substance, by using protective equipment. MAJOR SPILLS: 4 MODERATE HAZARD: Clear area of personnel and move upwind; 4 Alert Fire Brigade and tell them location and nature of hazard; 4 Wear breathing apparatus plus protective gloves. SECTION 7: HANDLING AND STORAGE PRECAUTIONS FOR SAFE HANDLING SAFE HANDLING: 4 Avoid all personal contact, including inhalation; 4 Wear protective clothing when risk of exposure occurs; 4 Prevent concentration in hollows and sumps; 4 DO NOT allow clothing wet with substance to stay in contact with the skin. OTHER INFORMATION: 4 Store in original containers; 4 Keep containers securely sealed; 4 No smoking, naked lights or ignition sources; 4 Store in a cool, dry, well-ventilated area. CONDITIONS FOR SAFE STORAGE, INCLUDING AND INCOMPATIBILITES SUITABLE CONTAINERS: Packaging as recommended by manufacturer; Check all containers are clearly labelled and free from leaks. STORAGE INCOMPATIBILITY: Avoid reaction with oxidising agents X: Must not be stored together; O: May be stored together with specific preventions; +: May be stored together Flammable Explosive Poison Oxidising Respiratory Warning Corrosive + X O + SECTION 8: EXPOSURE CONTROLS/PERSONAL PROTECTION CONTROL PARAMETERS The product is not classified. No control parameters are to be mentioned. EXPOSURE CONTROLS APPROPRIATE ENGINEERING CONTROLS: 4 Engineering controls are used to remove a hazard or place a barrier between the worker and the hazard. Well-designed engineering controls can be highly effective in protecting workers and will typically be independent of worker interactions to provide third high level of protection; 4 The basic types of engineering controls are; Process controls which involve changing the way a job activity or process is done to reduce the risk; 4 Enclosure and/or isolation of emission source which keeps a selected hazard ‘physically’ away from the worker and ventilation that strategically ‘adds’ and removes’ air in the work environment. PERSONAL PORTECTION: EYE AND FACE PROTECTION: 4 Safety glasses with side shield; 4 Chemical goggles; 4 Contact lenses may pose a special hazard; soft contact lenses may absorb and concentrate irritants. A written policy document, describing the wearing of lenses or restrictions on use, should be created for each workplace or task. SKIN PROTECTION: See Hand Protection below. HAND/FEET PROTECTION: 4 Wear chemical protective gloves, e.g. PVC; 4 Wear safety footwear or safety gumboots, e.g. Rubber; 4 The selection of suitable gloves does not only depend on the material, but also on further marks of quality, which vary from manufacturer to manufacturer; 4 Where the chemical is a preparation of several substances, the resistance of the glove material cannot be calculated in advance and has therefore to be checked prior to the application; 4 The exact break through time for substances has to be obtained from the manufacturer of the protective gloves and has to be observed when making a final choice; 4 Personal hygiene is a key element of effective hand care. BODY PROTECTION: See Other Protection below. OTHER: Overalls; PVC Apron; Barrier Cream. STANDARDS: The following Australian Standards will provide general advice regarding safety clothing and equipment: AS/NZS 1715: Respiratory Equipment AS 1161: Protective Gloves AS2919: Industrial Clothing AS1336/AS/NZS 1337: Industrial Eye Protection AS/NZS2210: Occupational Protective Footwear THERMAL HAZARDS: Not available SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES PHYSICAL/CHEMICAL PROPERTIES RESULT PHYSICAL/CHEMICAL PROPERTIES RESULT APPEARANCE: Mobile liquid BOILING POINT RANGE: Not available ODOUR: Characteristic FLAMMABILITY LIMITS: Not available COLOUR: Translucent yellow to brown AUTO-IGNITION TEMPERATURE: Not available TASTE: Not determined VAPOUR PRESSURE: No data available REFRACTIVE INDEX @20°C: 1.370 – 1.550 DENSITY: Not available SPECIFIC GRAVITY @20°C: 1.130 – 1.280 VISCOSITY, KINEMATIC: No data available WATER SOLUBILITY: Soluble OXIDISING PROPERTIES: Not oxidising FLASH POINT: 160°C EXPLOSIVE PROPERTIES: Not explosive EVAPORATION RATE: Non-volatile BULK DENSITY: Not applicable PH: 3.00 – 5.00 RELATIVE VAPOUR DENSITY: No data available MELTING/FREEZING POINT: Not available SECTION 10: STABILITY AND REACTIVITY REACTIVITY: See Section 7 CHEMICAL STABILITY: This product is chemically stable POSSIBILITY OF HAZARDOUS REACTIONS: See Section 7 CONDITIONS TO AVOID: See Section 7 INCOMPATIBLE MATERIALS: See Section 7 HAZARDOUS DECOMPOSITION PROUCTS: See Section 5 SECTION 11: TOXICOLOGICAL INFORMATION INFORMATION ON TOXICOLOGICAL EFFECTION INHALED: 4 The material can cause respiratory irritation in some persons. The body’s response to such irritation can cause further lung damage; 4 Not normally a hazard due to non-volatile nature of product. INGESTION: 4 Although ingestion is not thought to produce harmful effects (as classified under EC Directives), the material may still be damaging to the health of the individual, following ingestion, especially where pre-existing organ (e.g. liver, kidney (damage is evident; 4 Ingestion of large quantities may cause nausea, diarrhoea and vomiting. SKIN CONTACT: 4 The material may accentuate any pre-existing dermatitis condition; 4 Skin contact is not thought to have harmful health effects (as classified under EC Directives); the material may still produce health damage following entry though wounds, lesions or abrasions; 4 Open cuts abraded, or irritated skin should not be exposed to this material; 4 Entry into the blood stream, though, for example, cuts abrasions or lesions, following direct contact or after a delay of some time. Repeated exposure can cause contact dermatitis, which is characterised by redness, swelling and blistering. EYE: 4 Evidence exits, or practical experience predicts, that the material may cause eye irritation in a substantial number of individuals; Prolonged eye contact may cause inflammation characterised by a temporary redness of the conjunctiva (similar to windburn). CHRONIC: 4 Long term exposure to respiratory irritants may result in disease of the airways involving difficult breathing and related systemic problems; 4 Substance accumulation, in the human body, may occur and may cause some concern following or long-term occupational exposure. SCCNFP ALLERGENS ANNEX III – COSMETIC DIRECTIVE 2003/15/EC 7th Amendment Detection Limit 0.001% CONSTITUENT IFRA EFFA CAS EC RANGE Amyl Cinnamal: Yes No 122-40-7 204-541-5 Not detected Amyl Cinnamyl Alcohol: Yes No 101-85-9 202-982-8 Not detected Anise Alcohol: No Yes 105-13-5 203-273-6 Not detected Benzyl Alcohol: No Yes 100-51-6 202-859-9 Not detected Benzyl Benzoate: No Yes 120-51-4 204-402-9 Not detected Benzyl Cinnamate: No Yes 103-41-3 203-109-3 Not detected Benzyl Salicylate: No Yes 118-58-1 204-262-9 Not detected Cinnamal: Yes 104-55-2 203-213-9 Not detected Cinnamyl Alcohol: Yes 104-54-1 203-212-3 Not detected Citral: Yes 5392-40-5 226-394-6 Not detected Citronellol: No Yes 5392-40-5 203-375-0 Not detected Coumarin: No Yes 91-64-5 202-086-7 Not detected Eugenol: Yes 97-53-0 202-589-1 Not detected Farnesol: Yes 4602-84-0 225-004-1 Not detected Geraniol: No Yes 106-24-1 203-377-1 Not detected Hexyl Cinnamal: Yes No 101-86-0 202-983-3 Not detected Hydroxycitronellal: Yes No 107-75-5 203-518-7 Not detected Isoeugenol: Yes 97-54-1 202-590-7 Not detected Butylphenyl Methylpropional: Yes No 80-54-6 201-289-8 Not detected d-Limonene: Yes 5989-27-5 227-813-5 Not detected Linalool: Yes 78-70-6 201-134-4 Not detected Hydroxyisohexyl 3-Cyclohexene No 31906-04-4 250-863-4 Not detected Carboxaldehyde: Methyl 2-Octynoate: Yes No 111-12-6 203-836-6 Not detected 127-51-5/ Alpha-Isomethyl lonone: 204-846-3/ Yes No 90028-68-5 289-861-3 Not detected Evernia Prunastri Extract [Oakmoss]: Yes No 9000-50-4/ Not detected 6817-10-2 90028-67-4/ 289-860-8 Evernia Furfuracea Extract[Treemoss]: Yes No 68648-41-9 Not detected ADDITIONAL EFFA LISTED SENSITISERS & IFRA NOTIFIABLE SUBSTANCES Detection Limit 0.001% CONSTITUENT IFRA EFFA CAS EC RANGE No Additional Sensitisers: No Not allocated Not detected No Additional Notifiable Substances: No Not allocated Not detected SECTION 12: ECOLOGICAL INFORMATION TOXICITY: INGREDIENT ENDPOINT TEST DURATION [hr] SPECIES VALUE SOURCE Glycerin LC50 96 Fish >11mg/L 2 Glycerin EC50 96 Algae or other aquatic plants 77712.039mg/L 3 Glycerin ECO 24 Crustacea >500mg/L 1 Legend: Extracted from 1. IUCLID Toxicity Data 2. Europe ECHA Registered Substance – Eco toxicological Information – Aquatic Toxicity 3. EPIWIN Suite V3.12 – Aquatic Toxicity Data (Estimated) 4. US EPA, Ecotox database – Aquatic Toxicity Data 5. ECETOC Aquatic Hazard Assessment Data 6. NITE (Japan) – Bio concentration Data 7. METI (Japan) – Bio concentration Data 8. Vendor Data For Glycerin: Low Kow: -2.66 to -2.47, Atmospheric Fate: Glycerol is broken down in the air by hydroxyl radicals the half-life for this process is 6.8 hours. However, only a negligible amount of the substance will move to the atmospheric compartment. Terrestrial Fate: Only a negligible amount of Glycerin will move into the soil compartment, if released into the environment. Aquatic Fate: Glycerol is considered to be readily biodegradable in the aquatic environment. DO NOT discharge into sewer or waterways. PERSISTENCE AND DEGRADABILITY: 4 LOW persistence level Water/Soil/Air; 4 Use according to good working practice; pollution to soil, rivers and the ocean. BIO-ACCUMULATIVE POTENTIAL: 4 Glycerin: LOW (LogKOW = 1.76). MOBILITY IN SOIL: 4 Glycerin: HIGH (KOC = 1). SECTION 13: DISPOSAL CONSIDERATIONS WASTE TREATMENT METHODS PRODUCT/PACKAGING DISPOSAL: 4 Legislation addressing waste disposal requirements may differ by country, state and/or territory. Each user must refer to laws operating in their area. In some areas, certain wastes must be tracked; 4 A Hierarchy of Control seems to be common – the user should investigate: 4 Reduction; 4 Reuse; 4 Recycle; 4 Disposal [if all else fails]. 4 DO NOT allow wash water from cleaning or process equipment to enter drains. 4 It may be necessary to collect all wash water for treatment before disposal; 4 In all cases disposal to sewer may be subject to local laws and regulations and these should be considered first; 4 Where in doubt contact the responsible authority; 4 Recycle wherever possible or consult manufacturer for recycling options; 4 Consult State Land Waste Authority for disposal; 4 Bury or incinerate residue at an approved site; 4 Recycle containers if possible or dispose of in an authorised landfill. SECTION 14: TRANSPORT INFORMATION LABELS REQUIRED MARINE POLLUTANT: No HAZCHEM: Not applicable LAND TRANSPORT [AGD]: Not regulated for transport of Dangerous Goods AIR TRANSPORT [ICAO-IATA/DGR]; Not regulated for transport of Dangerous Goods SEA TRANSPORT [IMDG-Code/GGVSee]: Not regulated for transport of Dangerous Goods UN NUMBER: Not required PROPER SHIPPING NAME: Not required TECHNICAL SHIPPING NAME: Not applicable DG CLASS/SUBSIDARY RISK: Not applicable PACKAGING GROUP: Not allocated SPECIAL PRECAUTIONS: Not established HAZCHEM CODE: Not allocated SECTION 15: REGULATORY INFORMATION SAFETY, HEALTH AND ENVIRONMENTAL REGULATIONS/LEGISLATION SPECIFIC FOR THE SUBSTANCE OR MIXTURE The substance is not listed as a hazardous chemical under the following international agreements: 4 Montreal Protocol on Substances that Deplete the Ozone Layer; 4 Stockholm Convention on Persistent Organic Pollutants; 4 Rotterdam Convention on the Prior Informed Consent Procedure for Certain Hazardous Chemicals and Pesticides in International Trade; 4 Basel convention on the Control of Trans boundary Movements of Hazardous Wastes and their Disposal; 4 International Convention for the Prevention of Pollution from Ships (MARPOL); 4 Standard for the Uniform Scheduling of Medicines and Poisons (SUSMP); 4 Agriculture and Veterinary Chemicals Code Act 1994; 4 Australian Inventory of chemical Substances (AICS). SUBSTANCE CHEMICAL NAME Melaleuca alternifolia Leaf Extract NATIONAL INVENTORY COUNTRY STATUS üû Australian Inventory of Chemical Substances (AICS): AUSTRALIA ü Domestic Substances List (DSL): CANADA û Non-Domestic Substances List (NDSL): CANADA û Inventory of Existing Chemical Substances Produced for Imported to China (IECSC): CHINA ü European Chemicals Agency (ECHA-EINECS-ELINCS-NLP-COSING): EUROPE ü Japanese Existing and New Chemical Substances Inventory (ENCS): JAPAN û Korea Existing Chemicals Inventory (KECI): SOUTH KOREA û New Zealand Inventory (NZIoC): NEW ZEALAND ü Philippines Inventory of Chemicals and chemical Substances (PICCS): THE PHILLIPPINES ü Toxic Substances Control Act (TSCA): USA û Taiwan Chemical Substance Inventory (TCSI): TAIWAN û Vietnam National Chemical Database System VIETNAM û SECTION 16: ADDITIONAL INFORMATION QUALITY STATEMENT NATIVE EXTRACTS Pty Ltd specialises in the manufacture and supply of the highest quality, pure, naturally derived phyto-active compounds in hydrophilic extracts, seed oils and pure natural powders; for use in the Cosmetic, Pharmaceutical and Nutraceutical industries globally. Our company’s objective is to manufacture and supply the highest quality and purity of natural ingredients across multiple delivery formats that meet the application/formulation objectives and specifications of our customers. Our commitment to quality extends beyond our products and applies to our blends, services, workplace, environmental practices and partnership and relationships engaged with commercial growers and Indigenous communities. Any quality problems arising will be identified and solved with speed, technical efficiency and economy, stakeholder engagement – focusing our human and technical resources internally and externally to the prevention of quality deficiencies to meet our company goal of “right first time, every time”. The successful operation of our QMS relies on the cooperation, participation and engagement of our personnel across all areas of the company. Our commitment to quality underpins our continued success, the satisfaction of customers and staff, our pursuit to achieve new scientific discoveries and new benchmarks in performance ingredients. We are committed to improving our performance in every aspect of our business. NATIVE EXTRACTS will to provide high and consistent quality in Botanical extracts and naturally derived phyto-active ingredients, evolving the botanical extract from inferior processes and synthetic standardisation to the delivery of stable, active True to Nature phyto-activity, influencing new innovation in natural product development, new advances in consumer experiences, influencing the emergence of new primary industry partnerships, and participating in socially and environmentally responsible practices. Our commitment is to safety and accurate work to ensure our ingredients conform to various regulatory bodies locally and internationally and are safe to our customers, their clients and the environment. All work is done in conformance to NATIVE EXTRACTS’ QMS, the applicable technical and administrative operating policies and procedures of NATIVE EXTRACTS, legal and regulatory requirements, and specific customer requirements. Through front-line input and management leadership, we will continue to improve our people and processes to anticipate, meet, and exceed the needs of our customers. We support the continually improving quality of our customer's maintenance and other technical operations through the services we provide. ANIMAL TESTING NATIVE EXTRACTS Pty Ltd does not test raw materials on animals, neither initially nor as a routine test. The product suppliers for NATIVE EXTRACTS Pty Ltd do not test their products on animals, neither initially nor as a routine test. None of NATIVE EXTRACTS Pty Ltd finished extracts are tested on animals, either initially or as a routine test. MANUFACTURING PRODUCTS INGREDIENTS DISCLAIMER As the availability of ingredients and raw materials is not always certain whether due to changes in nature or otherwise, NATIVE EXTRACTS Pty Ltd reserves the right to substitute alternate ingredients/raw materials in the manufacture of its products in order to maintain supply to its customers. Customers should always refer to the ingredients label as affixed to each product or to specification sheets, which are current at all time of supply of the product. LABELLING DISCLAIMER NATIVE EXTRACTS Pty Ltd is a manufacturer of extracts. If you intend to re-label our products under your own name/brand for the purpose of on selling or retailing, we thoroughly recommend that you keep up to date with constant changing labelling laws. Please visit www.acco.gov.au or www.nicnas.gov.au. NATIVE EXTRACTS Pty Ltd cannot be held responsible for consequential loss/product recall due to incorrect labelling. DISCLAIMER This Safety Data Sheet was prepared according to: Safe Work Australia’s Code of Practice for the Preparation of Safety Data Sheets for Hazardous Chemicals, [Publication date: 23/12/2011] and Globally Harmonized System of Classification and Labelling of Chemicals (GHS) [NOHSC:1008(2004)]. The information contained in this Safety Data Sheet is obtained from current and reliable sources. NATIVE EXTRACTS Pty Ltd provides the information contained herein in good faith but makes no representation as to its comprehensiveness or accuracy. This Safety Data Sheet summaries our best current knowledge of the health and safety hazard information of the product but does not claim to be all-inclusive. This document is thus, intended only as a guide to the appropriate precautionary handling of the material by properly trained personnel using this product. Individuals receiving this information must exercise their independent judgment in determining its appropriateness for a particular purpose. As the ordinary or otherwise use(s) of this product is outside the control of NATIVE EXTRACTS Pty Ltd, no representation or warranty, expressed or implied, is made as to the effect(s) of such use(s), (including damage or injury), or the results obtained. NATIVE EXTRACTS Pty Ltd expressly disclaims responsibility as to the ordinary or otherwise use(s). Furthermore, nothing contained herein should be considered as a recommendation by NATIVE EXTRACTS Pty Ltd as to the fitness for any use. The liability of NATIVE EXTRACTS Pty Ltd is limited to the value of the goods and does not include any consequential loss. NATIVE EXTRACTS Pty Ltd shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. NATIVE EXTRACTS Pty Ltd shall not be responsible for any damage resulting from use of or reliance upon this information. The user of the product is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties. ACRONYMS LDLo stands for Lethal Dose Low, the minimum amount of a < Less than material which tests have shown will be lethal to a specified LDLo type of animal. This is normally quoted in mg.kg body weight. > Greater than Lt Litre °C Degrees Celsius Max. Maximum ACCC Australian Competition and Consumer Commission Mg Milligram ADG Australian Dangerous Goods Min. Minimum AICS Australian Inventory of Chemical Substances ml Millilitre AICS Australian Inventory of Chemical Substances M3 Cubic metre ACGIH American Conference of Government Industrial Hygienists mm Millimetre AS Australian Standards mm Hg Millimetre of Mercury BOD Biochemical Oxygen Demand N/A NA Not Applicable CAS Chemical Abstracts Service (Registry Number) The National Industry Chemicals Notification and NICNAS Assessment Scheme (AUSTRALIA) Cm3 Cubic centimetres The National Institute for Occupational Safety and Health NIOSH (USA) COD Chemical Oxygen Demand National occupational Health and Safety Commission NOHSC (AUSTRALIA) The European Commission database with information on Coslng n.o.s. Cosmetic Ingredients and Substances Not otherwise specified DG Dangerous Goods NZS New Zealand Standards EC European Commission NZloC New Zealand Inventory of Chemicals EC stands for the effective concentration. EC50 refers to the concentration of a toxicant, which includes a response halfway EC50 Organisation for Economic Co-operation and Development OECD between the baseline and maximum after a specified exposure (Test Method number) time European Inventory of Existing Commercial Chemical Substances EINECS OSHA (Identifying Number) The Occupational Safety and Health Administration (USA) EFFA European Flavour Association PEL Permissible Exposure Limit EU Europe/European Union Ppb Parts per billion g grams Ppm Parts per million The Globally Harmonised System of Classification and Labelling GHS RTECS of Chemicals The Registry of Toxic Effects of Chemical Substances GMO Genetically modified organism Scientific Committee on Cosmetic Products and non-Food SCCNFP Products (EUROPE) Emergency action code of numbers and letters that provide Hazchem Code SDS information to emergency services especially fire fighters Safety Data Sheet hr Hour STEL Short Term Exposure Limit The Safe Work Australia Hazardous Substances Information HSIS Subsp. System Subspecies HSNO Hazardous Substances Approval Code Subspecies Standard for the Uniform Scheduling of Medicine and Poisons (AUSTRALIA) TD stands for Toxic Dose. TD is the amount given all at IATA The International Air Transport Association once, which causes the untoward symptoms in the majority TD of persons, or in the majority of a group of test animals. This is normally quoted in mg/kg body weight. ICAO The International Civil Aviation Organisation TGA Therapeutic Goods Administration (AUSTRALIA) IFRA The International Fragrance Association TLV Threshold Limit Value IMDG International Maritime Dangerous Goods TWA Time Weighted Average INCI The International Nomenclature of Cosmetic Ingredients UK United Kingdom ISO International Organisation for Standardisation USA The United States of America Kg Kilograms µg Microgram LC stands for lethal concentration. LC50 is the concentration of a material in air which causes the death of 50% (one half) of a LC50 group of test animals. The material is inhaled over a set period of µl Micro litre time, usually 1 or 4 hours. This is normally quoted in mg/kg body weight. LD50 stands for Lethal Dose. This is the amount of a material, given all at once, which causes the death of 50% (one half) of a LD50 group of test animals. This is normally quoted in mg/kg body weight. DATA SOURCE AICS; Australian Code for the Transport of Dangerous Goods by Rail and Road; Globally Harmonized System of Classification and Labelling of Chemicals (GHS) [NOHSC:1008(2004)]; Work Safe Australia WHS Regulations; Coslng; Supplier Documentation; EFFA; HSIS; IATA Dangerous Goods Regulations; IFRA; IMDG Code; The International Cosmetic Ingredients Dictionary and Handbook; NICNAS; SUSMP; NZloC; NOHSC Australia. DOCUMENT PREPARED BY Vanessa Minnikin, Quality Assurance. Email: vminnikin@nativeextracts.com NE CONCENTRATE MANUFACTURING FLOWCHART MANUFACTURING CONCENTRATE FLOWCHART STEP 1: Raw material order is placed with approved supplier; Supplier provides organic certificate, CofA, STEP 2: Raw material other regulatory documentation; Supplier completes shipped to NATIVE Growers Product Statement-Starting Material, incuding EXTRACTS Pty Ltd Batch No STEP 3: Raw material received into STEP 4: Raw material is checked against NATIVE EXTRACTS Pty Ltd warehouse, regulatory documents for correct species, recorded, assigned GIN number, contamination and suitability for extraction placed in quarantine for QA inspection process STEP 5: PASS: Raw material is passed, QA sticker and stamped 'received', dated and STEP 6: When extract is signed; Raw material is coded into MYOB, organic status sticker assigned and placed required raw material is in appropriate area; brough to room temperature FAIL: Raw material is quarantined and either returned to supplier or destroyed; Non- in clean room facility conformance report is issued STEP 7: Raw material is STEP 8: Liquid extractant is added to STEP 9: Raw material is then packed into extraction vessel and vessel is closed and extracted under pressure in system and sealed sealed a closed system STEP 11: PASS: Extract dated, signed and added to MYOB STEP 10: Once extraction is completed, extract is pumped inventory; Updated TDS, SDS completed and released to out into clean sterile container, labeled and sealed; Placed warehouse for sale; in quarantine, samples taken for QA/QC testing; Extract is FAIL: Extract reworked or rejected; Disposed and non- 100% plant/fruit/bark extract conformance raised STEP 12: Raw material left over from extraction is STEP 13: Extraction vessel is removed from vessel and washed, sterilised and air placed in green waste bin dried for next extraction for garden mulch A range of commercially cultivated, organic and wild harvested sources grown without the use of pesticides and following environmental practice to meet eco-sustainable and or organic guidelines. Individual Datasheets, Specifications [TDS], CofA’s and Safety Data Sheets [SDS] are available on request. NE-REG-481 Version 1.0 /Volumes/SENIOR MANAGEMENT/1. QMS-ISO9001-2015/REGULATORY 450-519/NE-REG-481_NE Reviewed: Manufacturing [Con] Flowchart_v1.0_2018-01-25.docx Approved by DIRECTOR: 2018-01-25 Ó NATIVE EXTRACTS Pty Ltd Page 1 of 2 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 NE CONCENTRATE MANUFACTURING FLOWCHART DISCLAIMER: The information contained has been obtained from current and reliable sources. NATIVE EXTRACTS Pty Ltd is not responsible for any reliance readers place on the material in this document and readers rely on the information in this document at their own risk and we recommend efficacy testing to be done on the finished product formulation to determine if it meets your specific target performance. However, this information is not intended to constitute an ‘authoritative statement’ under the National Industrial Chemical Notification and assessment Scheme Australia and New Zealand rules and regulations. The range of compounds delivered in NATIVE EXTRACTS Pty Ltd products is only a guide as there will be fluctuations in the range available per batch as this is a natural product and reflects the nature of natural differences from harvest to harvest, source to source, batch to batch etc. NATIVE EXTRACTS Pty Ltd provides the information contained herein in good faith but makes no representation as to its comprehensiveness or accuracy. Individuals receiving this information must exercise their independent judgment in determining its appropriateness for a particular purpose. As the ordinary or otherwise use(s) of this product is outside the control of NATIVE EXTRACTS Pty Ltd, no representation or warranty, expressed or implied, is made as to the effect(s) of such use(s), (including damage or injury), or the results obtained. NATIVE EXTRACTS Pty Ltd expressly disclaims responsibility as to the ordinary or otherwise use(s). Furthermore, nothing contained herein should be considered as a recommendation by NATIVE EXTRACTS Pty Ltd as to the fitness for any use. The liability of NATIVE EXTRACTS Pty Ltd is limited to the value of the goods and does not include any consequential loss. NATIVE EXTRACTS Pty Ltd shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. NATIVE EXTRACTS Pty Ltd shall not be responsible for any damage resulting from use of or reliance upon this information. The user of the product is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties. Computer generated, released without signature. REVISION No DATE NATURE OF CHANGE APPROVED BY NE-REG-481 Version 1.0 /Volumes/SENIOR MANAGEMENT/1. QMS-ISO9001-2015/REGULATORY 450-519/NE-REG-481_NE Reviewed: Manufacturing [Con] Flowchart_v1.0_2018-01-25.docx Approved by DIRECTOR: 2018-01-25 Ó NATIVE EXTRACTS Pty Ltd Page 2 of 2 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 NSO OIL MANUFACTURING FLOWCHART MANUFACTURING OIL FLOWCHART STEP 1: Raw material order is placed with approved supplier; Supplier provides organic certificate, CofA, STEP 2: Raw material other regulatory documentation; Supplier completes shipped to NATIVE Growers Product Statement-Starting Material, incuding EXTRACTS Pty Ltd Batch No STEP 3: Raw material received into STEP 4: Raw material is checked against NATIVE EXTRACTS Pty Ltd warehouse, regulatory documents for correct species, recorded, assigned GIN number, contamination and suitability for extraction placed in quarantine for QA inspection process STEP 5: PASS: Raw material is passed, QA sticker and stamped 'received', dated and STEP 6: When oil is required, signed; Raw material is coded into MYOB, organic status sticker assigned and placed raw material is brought to in appropriate area; room temperature in clean FAIL: Raw material is quarantined and either returned to supplier or destroyed; Non- room facility conformance report is issued STEP 7: When oil is required STEP 9: Raw material is STEP 8: Vegetable oi <98% and <1-5% of raw material is exposed to cellular Vitamin E <0.5% is added to vessel packed into extraction extraction under presure in a sn vessel is closed and sealed system and sealed closed system STEP 11: PASS: Oil dated, signed and added to MYOB STEP 10: Once extraction is completed, oil is pumped out inventory; Updated TDS, SDS completed and released to into clean sterile container, labeled and sealed; Placed in warehouse for sale; quarantine, samples taken for QA/QC testing FAIL: Oil reworked or rejected; Disposed and non- conformance raised STEP 12: Raw material left over from extraction is STEP 13: Extraction vessel is removed from vessel and washed, sterilised and air placed in green waste bin dried for next extraction for garden mulch A range of commercially cultivated, organic and wild harvested sources grown without the use of pesticides and following environmental practice to meet eco-sustainable and or organic guidelines. Individual Datasheets, Specifications [TDS], CofA’s and Safety Data Sheets [SDS] are available on request. DISCLAIMER: The information contained has been obtained from current and reliable sources. NATIVE EXTRACTS Pty Ltd is not responsible for any reliance readers place on the material in this document and NE-REG503 Version 1.0 /Volumes/SENIOR MANAGEMENT/1. QMS-ISO9001-2015/REGULATORY 450-519/NE-REG-503_NSO Reviewed: Manufacturing Flowchart_v1.0_2019-05-31.docx Approved by DIRECTOR: 2019-05-31 Ó NATIVE EXTRACTS Pty Ltd Page 1 of 2 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 NSO OIL MANUFACTURING FLOWCHART readers rely on the information in this document at their own risk and we recommend efficacy testing to be done on the finished product formulation to determine if it meets your specific target performance. However, this information is not intended to constitute an ‘authoritative statement’ under the National Industrial Chemical Notification and assessment Scheme Australia and New Zealand rules and regulations. The range of compounds delivered in NATIVE EXTRACTS Pty Ltd products is only a guide as there will be fluctuations in the range available per batch as this is a natural product and reflects the nature of natural differences from harvest to harvest, source to source, batch to batch etc. NATIVE EXTRACTS Pty Ltd provides the information contained herein in good faith but makes no representation as to its comprehensiveness or accuracy. Individuals receiving this information must exercise their independent judgment in determining its appropriateness for a particular purpose. As the ordinary or otherwise use(s) of this product is outside the control of NATIVE EXTRACTS Pty Ltd, no representation or warranty, expressed or implied, is made as to the effect(s) of such use(s), (including damage or injury), or the results obtained. NATIVE EXTRACTS Pty Ltd expressly disclaims responsibility as to the ordinary or otherwise use(s). Furthermore, nothing contained herein should be considered as a recommendation by NATIVE EXTRACTS Pty Ltd as to the fitness for any use. The liability of NATIVE EXTRACTS Pty Ltd is limited to the value of the goods and does not include any consequential loss. NATIVE EXTRACTS Pty Ltd shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. NATIVE EXTRACTS Pty Ltd shall not be responsible for any damage resulting from use of or reliance upon this information. The user of the product is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties. Computer generated, released without signature. REVISION No DATE NATURE OF CHANGE APPROVED BY NE-REG503 Version 1.0 /Volumes/SENIOR MANAGEMENT/1. QMS-ISO9001-2015/REGULATORY 450-519/NE-REG-503_NSO Reviewed: Manufacturing Flowchart_v1.0_2019-05-31.docx Approved by DIRECTOR: 2019-05-31 Ó NATIVE EXTRACTS Pty Ltd Page 2 of 2 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 SECTION 1. IDENTIFICATION OF THE SUBSTANCE AND SUPPLIER PRODUCT IDENTIFER Product Name: NSO Snowflower Oil Botanical Name: Melaleuca alternifolia (and) Vitis vinifera Product Code: ANE0513 INCI Name: Vitis vinifera (Grape) Seed Oil (and) Melaleuca alternifolia (Tea Tree) Leaf Extract CAS: Not allocated EC: Not allocated REACH Restriction: Exempt from registration ex Annex V UN Number: Not required HS Code: 1515.90.94 RECOMMENDED USE OF THE CHEMICAL AND RESTRICTIONS OF USE Relevant identified uses: Cosmetic ingredient; Topical application; Not to be ingested Usage: 2.0 - 5.0% SUPPLIER DETAILS Name: NATIVE EXTRACTS Pty Ltd Address: 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA Telephone: +61 2 6686 5725 Email: enquiries@nativeextracts.com Website: www.nativeextracts.com EMERGENCY TELEPHONE NUMBERS [24/H/24H] – INTERNATIONAL CENTRES WITHIN YOUR COUNTRY AUSTRALIA: Poisons Information Centre 13 11 26 USA: Poison Control Centre 1-800-222-1222 GERMANY Federal Institute for Risk Assessment ITALY: National Institute of Health UNITED KINGDOM: National Poison Information Services OTHER COUNTRIES: Please contact relevant government services SECTION 2. HAZARDS IDENTIFIED CLASSIFICATION OF THE SUBSTANCE OR MIXTURE POISONS SCHEDULE: Unscheduled NON-HAZARDOUS CHEMICAL – NON-DANGEROUS GOODS: According to the WHS Regulations and the ADG Code; Globally Harmonized System of Classification and Labelling of Chemicals [GHS]; Regulation (EC) No 1223/2009 of the European Parliament and the Council of 30 November 2009 on cosmetic products (the ”Cosmetics Regulation” or the “Regulation”), Governing the composition, labelling and packaging of finished cosmetic products. CLASSIFICATION: Not applicable LABEL ELEMENTS GHS LABEL ELEMENTS: Not applicable SIGNAL WORD: Not applicable HAZARD STATEMENT[S] Not applicable PRECAUTIONARY STATEMENT[S] PREVENTION: Not applicable RESPONSE: Not applicable STORAGE Not applicable DISPOSAL: Not applicable SECTION 3: COMPOSTIONAL INFORMATION ON INGREDIENTS SUBSTANCE CHEMICAL NAME CAS No EC [%w/w] Vitis vinifera (Grape) Seed Oil 8024-22-4 / 84929-27-1 284-511-6 / - <98% Melaleuca alternifolia Leaf 85085-48-9 285-377-1 <1.0 – 5.0% Tocopherols [Mixed, low a-type] 1406-66-2 Not allocated <0.5% Cellular Extraction of manufactured in Australia Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 1 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 SECTION 4: FIRST AID MEASURES DESCRIPTION OF FIRST AID MEASURES EYE CONTACT: If this product comes into contact with the eye: 4 Wash out immediately with fresh running water; 4 Ensure complete irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids; 4 Seek medical attention without delay; if pain persists or recurs seek medical attention; 4 Removal of contact lenses after an eye injury should only be undertaken by skilled personnel. SKIN CONTACT: 4 First aid is not generally required; If in doubt, contact a Poisons Information Centre or doctor. INHALATION: 4 If fumes or combustion products are inhaled remove from contaminated area; 4 Lay patient down. Keep warm and rested; 4 Prostheses such as false teeth, which may block airway, should be removed, where possible, prior to initiating first aid procedures; 4 Apply artificial respiration if not breathing, preferably with a demand value resuscitator, bag-valve mask device, or pocket mask as trained. Perform CPR if necessary; 4 First aid is not generally . SWALLOWED: 4 Immediately give a glass of water; 4 First aid is not generally required; If in doubt, contact a Poisons Information Centre or doctor. INDICATION OF ANY IMMEDIATE MEDICAL ATTENTION AND SPECIAL TREATMENT NEEDED: Treat symptomatically. SECTION 5: FIRE FIGHTING MEASURES EXTINGUISHING MEDIA Water spray or fog; Foam SPECIAL HAZARDS ARISING FROM THE SUBSTANCE FIRE INCOMPATIBILITY: Not applicable. ADVICE FOR FIRE FIGHTERS FIRE FIGHTING: Use water delivered as a fine spray to control fore and cool adjacent area. FIRE/EXPLOSION HAZARD: Not applicable. HAZCHEM: Not applicable. SECTION 6: ACCIDENTIAL RELEASE MEASURES PERSONAL PRECAUTIONS, PROTECTIVE EQUIPMENT AND EMERGENCY PROCEDURES See Section 8. ENVIRONMENTAL PRECAUTIONS See Section 12. METHODS OF MATERIAL FOR CONTAMINATION AND CLEAN UP MINOR SPILLS: 4 Remove all ignition sources; 4 Clean up all spills immediately; 4 Avoid breathing vapours and contact with skin and eyes; 4 Control personal contact with the substance, by using protective equipment. MAJOR SPILLS: 4 MODERATE HAZARD: Clear area of personnel and move upwind; 4 Alert Fire Brigade and tell them location and nature of hazard; 4 Wear breathing apparatus plus protective gloves. SECTION 7: HANDLING AND STORAGE PRECAUTIONS FOR SAFE HANDLING SAFE HANDLING: 4 Avoid all personal contact, including inhalation; 4 Wear protective clothing when risk of exposure occurs; 4 Prevent concentration in hollows and sumps; 4 DO NOT allow clothing wet with substance to stay in contact with the skin. OTHER INFORMATION: 4 Store in original containers; 4 Keep containers securely sealed; 4 No smoking, naked lights or ignition sources; 4 Store in a cool, dry, well-ventilated area. CONDITIONS FOR SAFE STORAGE, INCLUDING AND INCOMPATIBILITES SUITABLE CONTAINERS: Packaging as recommended by manufacturer; Check all containers are clearly labelled and free from leaks. Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 2 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 STORAGE INCOMPATIBILITY: Avoid reaction with oxidising agents X: Must not be stored together; O: May be stored together with specific preventions; +: May be stored together Flammable Explosive Poison Oxidising Respiratory Warning Corrosive + X O + SECTION 8: EXPOSURE CONTROLS/PERSONAL PROTECTION CONTROL PARAMETERS The product is not classified. No control parameters are to be mentioned. EXPOSURE CONTROLS APPROPRIATE ENGINEERING CONTROLS: 4 Engineering controls are used to remove a hazard or place a barrier between the worker and the hazard. Well-designed engineering controls can be highly effective in protecting workers and will typically be independent of worker interactions to provide third high level of protection; 4 The basic types of engineering controls are; Process controls which involve changing the way a job activity or process is done to reduce the risk; 4 Enclosure and/or isolation of emission source which keeps a selected hazard ‘physically’ away from the worker and ventilation that strategically ‘adds’ and removes’ air in the work environment. PERSONAL PORTECTION: EYE AND FACE PROTECTION: 4 Safety glasses with side shield; 4 Chemical goggles; 4 Contact lenses may pose a special hazard; soft contact lenses may absorb and concentrate irritants. A written policy document, describing the wearing of lenses or restrictions on use, should be created for each workplace or task. SKIN PROTECTION: See Hand Protection below. HAND/FEET PROTECTION: 4 Wear chemical protective gloves, e.g. PVC; 4 Wear safety footwear or safety gumboots, e.g. Rubber; 4 The selection of suitable gloves does not only depend on the material, but also on further marks of quality, which vary from manufacturer to manufacturer; 4 Where the chemical is a preparation of several substances, the resistance of the glove material cannot be calculated in advance and has therefore to be checked prior to the application; 4 The exact break through time for substances has to be obtained from the manufacturer of the protective gloves and has to be observed when making a final choice; 4 Personal hygiene is a key element of effective hand care. BODY PROTECTION: See Other Protection below. OTHER: Overalls; PVC Apron; Barrier Cream. STANDARDS: The following Australian Standards will provide general advice regarding safety clothing and equipment: AS/NZS 1715: Respiratory Equipment AS 1161: Protective Gloves AS2919: Industrial Clothing AS1336/AS/NZS 1337: Industrial Eye Protection AS/NZS2210: Occupational Protective Footwear THERMAL HAZARDS: Not available SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES PHYSICAL/CHEMICAL PROPERTIES RESULT PHYSICAL/CHEMICAL PROPERTIES RESULT APPEARANCE: Viscous liquid WATER SOLUBILITY: Insoluble ODOUR: Characteristic FLASH POINT: <100°C [Closed cup] COLOUR: Yellow to green MELTING/FREEZING POINT: Not available TASTE: Not determined BOILING POINT RANGE: Not available REFRACTIVE INDEX @20°C: 1.450 – 1.490 VAPOUR PRESSURE: No data available SPECIFIC GRAVITY @20°C: 1.900 – 0.940 VAPOUR DENSITY: Not available PEROXIDE VALUE: 2.87 mEq/Kg VISCOSITY, KINEMATIC: No data available SOUABILITY: Soluble in vegetable oils Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 3 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 SECTION 10: STABILITY AND REACTIVITY REACTIVITY: Oxidation with atmospheric oxygen; Formation of free fatty acids. Stable under recommended handling and storage conditions; this material presents no CHEMICAL STABILITY: significant reactivity hazard. POSSIBILITY OF HAZARDOUS REACTIONS: Reacts with oxidants. CONDITIONS TO AVOID: Avoid heat, flames, sunlight and other sources of ignition. MATERIALS TO AVOID: Strong oxidising agents. INCOMPATIBLE MATERIALS: See Section 7 HAZARDOUS DECOMPOSITION PROUCTS: Product does not decompose with proper handling. SECTION 11: TOXICOLOGICAL INFORMATION INFORMATION ON TOXICOLOGICAL EFFECTION INHALED: Not expected to be an irritant. INGESTION: Not expected to be an irritant. SKIN CONTACT: Not expected to be an irritant. EYE: Not expected to be an irritant CHRONIC: Not expected to be an irritant. SCCNFP ALLERGENS ANNEX III – COSMETIC DIRECTIVE 2003/15/EC 7th Amendment Detection Limit 0.001% CONSTITUENT IFRA EFFA CAS EC RANGE Amyl Cinnamal: Yes No 122-40-7 204-541-5 Not detected Amyl Cinnamyl Alcohol: Yes No 101-85-9 202-982-8 Not detected Anise Alcohol: No Yes 105-13-5 203-273-6 Not detected Benzyl Alcohol: No Yes 100-51-6 202-859-9 Not detected Benzyl Benzoate: No Yes 120-51-4 204-402-9 Not detected Benzyl Cinnamate: No Yes 103-41-3 203-109-3 Not detected Benzyl Salicylate: No Yes 118-58-1 204-262-9 Not detected Cinnamal: Yes 104-55-2 203-213-9 Not detected Cinnamyl Alcohol: Yes 104-54-1 203-212-3 Not detected Citral: Yes 5392-40-5 226-394-6 Not detected Citronellol: No Yes 5392-40-5 203-375-0 Not detected Coumarin: No Yes 91-64-5 202-086-7 Not detected Eugenol: Yes 97-53-0 202-589-1 Not detected Farnesol: Yes 4602-84-0 225-004-1 Not detected Geraniol: No Yes 106-24-1 203-377-1 Not detected Hexyl Cinnamal: Yes No 101-86-0 202-983-3 Not detected Hydroxycitronellal: Yes No 107-75-5 203-518-7 Not detected Isoeugenol: Yes 97-54-1 202-590-7 Not detected Butylphenyl Methylpropional: Yes No 80-54-6 201-289-8 Not detected d-Limonene: Yes 5989-27-5 227-813-5 Not detected Linalool: Yes 78-70-6 201-134-4 Not detected Hydroxyisohexyl 3-Cyclohexene Carboxaldehyde: No 31906-04-4 250-863-4 Not detected Methyl 2-Octynoate: Yes No 111-12-6 203-836-6 Not detected 127-51-5/ Alpha-Isomethyl lonone: 204-846-3/ Yes No Not detected 90028-68-5 289-861-3 Evernia Prunastri Extract [Oakmoss]: Yes No 9000-50-4/ Not detected 6817-10-2 90028-67-4/ 289-860-8 Evernia Furfuracea Extract[Treemoss]: Yes No 68648-41-9 Not detected ADDITIONAL EFFA LISTED SENSITISERS & IFRA NOTIFIABLE SUBSTANCES Detection Limit 0.001% CONSTITUENT IFRA EFFA CAS EC RANGE No Additional Sensitisers: No Not allocated Not detected No Additional Notifiable Substances: No Not allocated Not detected SECTION 12: ECOLOGICAL INFORMATION ECO-TOXICITY: None established; Use according to good working practices; Avoid pollution to soil, rivers and the ocean. PERSISTENCE AND DEGRADABILITY: 4 LOW persistence level and readily biodegradable; During natural decomposition; 4 No dangerous products are developed; Use according to good working practice; pollution to soil, rivers and the ocean. BIO-ACCUMULATIVE POTENTIAL: None established. Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 4 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 MOBILITY IN SOIL: None established. SECTION 13: DISPOSAL CONSIDERATIONS WASTE TREATMENT METHODS PRODUCT/PACKAGING DISPOSAL: 4 Legislation addressing waste disposal requirements may differ by country, state and/or territory. Each user must refer to laws operating in their area. In some areas, certain wastes must be tracked; 4 A Hierarchy of Control seems to be common – the user should investigate: 4 Reduction; 4 Reuse; 4 Recycle; 4 Disposal [if all else fails]. 4 It may be necessary to collect all wash water for treatment before disposal; 4 In all cases disposal to sewer may be subject to local laws and regulations and these should be considered first; 4 Where in doubt contact the responsible authority; 4 Recycle wherever possible or consult manufacturer for recycling options; 4 Consult State Land Waste Authority for disposal; 4 Bury or incinerate residue at an approved site; 4 Recycle containers if possible, or dispose of in an authorised landfill. SECTION 14: TRANSPORT INFORMATION LABELS REQUIRED MARINE POLLUTANT: No HAZCHEM: Not applicable LAND TRANSPORT [AGD]: Not regulated for transport of Dangerous Goods AIR TRANSPORT [ICAO-IATA/DGR]; Not regulated for transport of Dangerous Goods SEA TRANSPORT [IMDG-Code/GGVSee]: Not regulated for transport of Dangerous Goods UN NUMBER: Not required PROPER SHIPPING NAME: Not required TECHNICAL SHIPPING NAME: Not applicable DG CLASS/SUBSIDARY RISK: Not applicable PACKAGING GROUP: Not allocated SPECIAL PRECAUTIONS: Not established HAZCHEM CODE: Not allocated SECTION 15: REGULATORY INFORMATION SAFETY, HEALTH AND ENVIRONMENTAL REGULATIONS/LEGISLATION SPECIFIC FOR THE SUBSTANCE OR MIXTURE The substance is not listed as a hazardous chemical under the following international agreements: 4 Montreal Protocol on Substances that Deplete the Ozone Layer; 4 Stockholm Convention on Persistent Organic Pollutants; 4 Rotterdam Convention on the Prior Informed Consent Procedure for Certain Hazardous Chemicals and Pesticides in International Trade; 4 Basel convention on the Control of Trans boundary Movements of Hazardous Wastes and their Disposal; 4 International Convention for the Prevention of Pollution from Ships (MARPOL); 4 Standard for the Uniform Scheduling of Medicines and Poisons (SUSMP); 4 Agriculture and Veterinary Chemicals Code Act 1994; 4 Australian Inventory of chemical Substances (AICS). SUBSTANCE CHEMICAL NAME Vitis vinifera (Grape) Seed Oil (and) Melaleuca alternifolia (Tea Tree) Leaf Extract NATIONAL INVENTORY COUNTRY STATUS üû Australian Inventory of Chemical Substances (AICS): AUSTRALIA û Domestic Substances List (DSL): CANADA û Non-Domestic Substances List (NDSL): CANADA û Inventory of Existing Chemical Substances Produced for Imported to China (IECSC): CHINA û European Chemicals Agency (ECHA-EINECS-ELINCS-NLP-COSING): EUROPE û Japanese Existing and New Chemical Substances Inventory (ENCS): JAPAN û Korea Existing Chemicals Inventory (KECI): SOUTH KOREA û New Zealand Inventory (NZIoC): NEW ZEALAND û Philippines Inventory of Chemicals and chemical Substances (PICCS): THE PHILLIPPINES û Toxic Substances Control Act (TSCA): USA û Taiwan Chemical Substance Inventory (TCSI): TAIWAN û Vietnam National Chemical Database System VIETNAM û Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 5 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 SECTION 16: ADDITIONAL INFORMATION QUALITY STATEMENT NATIVE EXTRACTS Pty Ltd specialises in the manufacture and supply of the highest quality, pure, naturally derived phyto-active compounds in hydrophilic extracts, seed oils and pure natural powders; for use in the Cosmetic, Pharmaceutical and Nutraceutical industries globally. Our company’s objective is to manufacture and supply the highest quality and purity of natural ingredients across multiple delivery formats that meet the application/formulation objectives and specifications of our customers. Our commitment to quality extends beyond our products and applies to our blends, services, workplace, environmental practices and partnership and relationships engaged with commercial growers and Indigenous communities. Any quality problems arising will be identified and solved with speed, technical efficiency and economy, stakeholder engagement – focusing our human and technical resources internally and externally to the prevention of quality deficiencies to meet our company goal of “right first time, every time”. The successful operation of our QMS relies on the cooperation, participation and engagement of our personnel across all areas of the company. Our commitment to quality underpins our continued success, the satisfaction of customers and staff, our pursuit to achieve new scientific discoveries and new benchmarks in performance ingredients. We are committed to improving our performance in every aspect of our business. NATIVE EXTRACTS will to provide high and consistent quality in Botanical extracts and naturally derived phyto-active ingredients, evolving the botanical extract from inferior processes and synthetic standardisation to the delivery of stable, active True to Nature phyto-activity, influencing new innovation in natural product development, new advances in consumer experiences, influencing the emergence of new primary industry partnerships, and participating in socially and environmentally responsible practices. Our commitment is to safety and accurate work to ensure our ingredients conform to various regulatory bodies locally and internationally and are safe to our customers, their clients and the environment. All work is done in conformance to NATIVE EXTRACTS’ QMS, the applicable technical and administrative operating policies and procedures of NATIVE EXTRACTS, legal and regulatory requirements, and specific customer requirements. Through front-line input and management leadership, we will continue to improve our people and processes to anticipate, meet, and exceed the needs of our customers. We support the continually improving quality of our customer's maintenance and other technical operations through the services we provide. ANIMAL TESTING NATIVE EXTRACTS Pty Ltd does not test raw materials on animals, neither initially nor as a routine test. The product suppliers for NATIVE EXTRACTS Pty Ltd do not test their products on animals, neither initially nor as a routine test. None of NATIVE EXTRACTS Pty Ltd finished extracts are tested on animals, either initially or as a routine test. MANUFACTURING PRODUCTS INGREDIENTS DISCLAIMER As the availability of ingredients and raw materials is not always certain whether due to changes in nature or otherwise, NATIVE EXTRACTS Pty Ltd reserves the right to substitute alternate ingredients/raw materials in the manufacture of its products in order to maintain supply to its customers. Customers should always refer to the ingredients label as affixed to each product or to specification sheets, which are current at all time of supply of the product. LABELLING DISCLAIMER NATIVE EXTRACTS Pty Ltd is a manufacturer of extracts. If you intend to re-label our products under your own name/brand for the purpose of on selling or retailing, we thoroughly recommend that you keep up to date with constant changing labelling laws. Please visit www.acco.gov.au or www.nicnas.gov.au. NATIVE EXTRACTS Pty Ltd cannot be held responsible for consequential loss/product recall due to incorrect labelling. DISCLAIMER This Safety Data Sheet was prepared according to: Safe Work Australia’s Code of Practice for the Preparation of Safety Data Sheets for Hazardous Chemicals, [Publication date: 23/12/2011] and Globally Harmonized System of Classification and Labelling of Chemicals (GHS) [NOHSC:1008(2004)]. The information contained in this Safety Data Sheet is obtained from current and reliable sources. NATIVE EXTRACTS Pty Ltd provides the information contained herein in good faith but makes no representation as to its comprehensiveness or accuracy. This Safety Data Sheet summaries our best current knowledge of the health and safety hazard information of the product but does not claim to be all-inclusive. This document is thus, intended only as a guide to the appropriate precautionary handling of the material by properly trained personnel using this product. Individuals receiving this information must exercise their independent judgment in determining its appropriateness for a particular purpose. As the ordinary or otherwise use(s) of this product is outside the control of NATIVE EXTRACTS Pty Ltd, no representation or warranty, expressed or implied, is made as to the effect(s) of such use(s), (including damage or injury), or the results obtained. NATIVE EXTRACTS Pty Ltd expressly disclaims responsibility as to the ordinary or otherwise use(s). Furthermore, nothing contained herein should be considered as a recommendation by NATIVE EXTRACTS Pty Ltd as to the fitness for any use. The liability of NATIVE EXTRACTS Pty Ltd is limited to the value of the goods and does not include any consequential loss. NATIVE EXTRACTS Pty Ltd shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. NATIVE EXTRACTS Pty Ltd shall not be responsible for any damage resulting from use of or reliance upon this information. The user of the product is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual property rights of third parties. ACRONYMS LDLo stands for Lethal Dose Low, the minimum amount of a < Less than LDLo material which tests have shown will be lethal to a specified type of animal. This is normally quoted in mg.kg body weight. > Greater than Lt Litre °C Degrees Celsius Max. Maximum ACCC Australian Competition and Consumer Commission Mg Milligram ADG Australian Dangerous Goods Min. Minimum AICS Australian Inventory of Chemical Substances ml Millilitre AICS Australian Inventory of Chemical Substances M3 Cubic metre ACGIH American Conference of Government Industrial Hygienists mm Millimetre AS Australian Standards mm Hg Millimetre of Mercury BOD Biochemical Oxygen Demand N/A NA Not Applicable CAS Chemical Abstracts Service (Registry Number) NICNAS The National Industry Chemicals Notification ants Assessment Scheme (AUSTRALIA) Cm3 Cubic centimetres NIOSH The National Institute for Occupational Safety and Health (USA) COD Chemical Oxygen Demand NOHSC National occupational Health and Safety Commission (AUSTRALIA) The European Commission database with information on Coslng Cosmetic Ingredients and Substances n.o.s. Not otherwise specified DG Dangerous Goods NZS New Zealand Standards EC European Commission NZloC New Zealand Inventory of Chemicals EC stands for the effective concentration. EC50 refers to the concentration of a toxicant, which includes a response EC50 halfway between the baseline and maximum after a specified Organisation for Economic Co-operation and Development (Test OECD Method number) exposure time European Inventory of Existing Commercial Chemical EINECS Substances (Identifying Number) OSHA The Occupational Safety and Health Administration (USA) EFFA European Flavour Association PEL Permissible Exposure Limit EU Europe/European Union Ppb Parts per billion g grams Ppm Parts per million The Globally Harmonised System of Classification and GHS Labelling of Chemicals RTECS The Registry of Toxic Effects of Chemical Substances Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 6 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 GMO Genetically modified organism SCCNFP Scientific Committee on Cosmetic Products and non-Food Products (EUROPE) Hazchem Code Emergency action code of numbers and letters that provide SDS Safety Data Sheet information to emergency services especially fire fighters hr Hour STEL Short Term Exposure Limit The Safe Work Australia Hazardous Substances Information HSIS System Subsp. Subspecies HSNO Hazardous Substances Approval Code Standard for the Uniform Scheduling of Medicine and Poisons Subspecies (AUSTRALIA) TD stands for Toxic Dose. TD is the amount given all at once, which IATA The International Air Transport Association causes the untoward symptoms in the majority of persons, or in the TD majority of a group of test animals. This is normally quoted in mg/kg body weight. ICAO The International Civil Aviation Organisation TGA Therapeutic Goods Administration (AUSTRALIA) IFRA The International Fragrance Association TLV Threshold Limit Value IMDG International Maritime Dangerous Goods TWA Time Weighted Average INCI The International Nomenclature of Cosmetic Ingredients UK United Kingdom ISO International Organisation for Standardisation USA The United States of America Kg Kilograms µg Microgram LC stands for lethal concentration. LC50 is the concentration of a material in air which causes the death of 50% (one half) LC50 of a group of test animals. The material is inhaled over a set µl Micro litre period of time, usually 1 or 4 hours. This is normally quoted in mg/kg body weight. LD50 stands for Lethal Dose. This is the amount of a material, given all at once, which causes the death of 50% LD50 (one half) of a group of test animals. This is normally quoted in mg/kg body weight. DATA SOURCE AICS; Australian Code for the Transport of Dangerous Goods by Rail and Road; Globally Harmonized System of Classification and Labelling of Chemicals (GHS) [NOHSC:1008(2004)]; Work Safe Australia WHS Regulations; Coslng; Supplier Documentation; EFFA; HSIS; IATA Dangerous Goods Regulations; IFRA; IMDG Code; The International Cosmetic Ingredients Dictionary and Handbook; NICNAS; SUSMP; NZloC; NOHSC Australia. DOCUMENT PREPARED BY Vanessa Minnikin, Quality Assurance. Email: vminnikin@nativeextracts.com Version: 2.1 Issued: 2018-09-25 Revision: Print Date: 18 April 2019 /Volumes/NATIVE EXTRACTS/2. NSO/3. SDS/1. WORD DOCMENTS/NSO Snowflower Oil ANE0513 SDS.docx Page 7 of 7 24 Kays Lane ALSTONVILLE NSW 2477 AUSTRALIA P +61 2 6686 5725 Literature review on tea tree oil Toxicity profiles for tea tree oil, constituents of tea tree oil and known oxidation products By Jesper Bo Nielsen, PhD November 2005 1 Contents CONTENTS 2 1 PREFACE 4 1.1 BACKGROUND 4 1.2 TERMS OF REFERENCE 4 2 EXECUTIVE SUMMARY 5 3 LITERATURE SEARCH STRATEGY 7 3.1 DATA SOURCES 7 3.2 SEARCH TERMS 7 4 SUMMARY OF TOXICITY PROFILES FOR TTO CONSTITUENTS 9 4.1 ACUTE TOXICITY 9 4.2 SKIN AND EYE IRRITATION 9 4.2.1 In vitro and animal data 9 4.2.2 Human data 10 4.3 SKIN SENSITIZATION 12 4.3.1 In vitro and animal data 12 4.3.2 Human data 12 4.4 DERMAL/PERCUTANEOUS ABSORPTION 16 4.5 REPEAT DOSE TOXICITY 18 4.6 MUTAGENICITY/CARCINOGENICITY 20 4.6.1 Bacterial assays 20 4.6.2 Tests with mammalian cells 21 4.7 REPRODUCTIVE TOXICITY 21 4.8 TOXICOKINETICS 22 4.9 DEGRADATION/OXIDATION PRODUCTS 24 4.10 PHOTOTOXICITY 24 5 TOXICITY PROFILE FOR TTO 25 6 TOXICITY PROFILES FOR INDIVIDUAL TTO CONSTITUENTS 33 6.1 TERPINEN-4-OL 33 6.2 γ-TERPINENE 35 6.3 α-TERPINENE 36 6.4 1,8-CINEOLE – EUCALYPTOL 38 6.5 TERPINOLENE 42 6.6 P-CYMENE 44 6.7 α-PINENE 47 6.8 α-TERPINEOL 49 6.9 AROMADENDRENE 51 6.10 δ-CADINENE 52 6.11 LIMONENE 53 6.12 SABINENE 58 6.13 GLOBULOL 59 6.14 VIRIDIFLOROL 60 6.15 DEGRADATION PRODUCTS 61 6.15.1 Thymol and carvone 63 2 6.15.2 Ascaridol / isoascaridol and 1,2,4-trihydroxymenthane 64 6.16 OTHER COMPONENTS OF INTEREST 65 6.16.1 Myrcene 65 6.16.2 Phellandrene 66 6.16.3 Caryophyllene 67 7 STRUCTURAL FORMULAS 68 8 REFERENCES 71 3 1 Preface 1.1 Background In 2003, Jesper Nielsen PhD provided RIRDC with a review of the data gaps that existed to demonstrate the toxicology and safety of tea tree oil. This review of data was measured against criteria to be satisfied in the following European regulatory arenas: Cosmetics, Pharmaceuticals, and biocides. The review clearly highlighted relevant data gaps and outlined weaknesses in existing data. Since that report in 2003, the SCCP has issued an opinion on Tea Tree Oil which has concluded that the committee has insufficient data to make an assessment on its safety. As a result of the previous literature review and the publication of the SCCP opinion, the Australian tea tree industry has together with the Australian government (through RIRDC) commissioned a literature search on the toxicity of individual tea tree oil components including potential products formed due to oxidation of the oil. The present report has therefore to be seen as a supplement to the initial report with focus on the toxicity profiles of the individual components and potential oxidation products of tea tree oil. The following Terms of Reference were developed. 1.2 Terms of Reference Using the data provided by RIRDC the review should critically evaluate the available literature and cover the following terms: 1) Acute toxicity 2) Skin and eye irritation 3) Skin and respiratory sensitization a. Skin sensitization/allergenicity i. Animal data ii. Human data iii. Rate of allergic reactions iv. Existence of subgroups with increased susceptibility v. Identification of causative agent/s 4) Dermal/percutaneous absorption 5) Repeat dose toxicity 6) Mutagenicity/genotoxicity 7) Carcinogenicity 8) Reproductive toxicity 9) Toxicokinetics 10) Phototoxicity Further, the review should try to identify an appropriate No Observed Adverse Effect Level (NOAEL) for tea tree oil, which could be used in the calculation of a Margin of Safety for tea tree oil. The level of detail used in the review should be sufficient to allow the SCCP evaluator to independently verify the conclusions. Studies not covered in the SCCP opinion should be highlighted to assist the SCCP. 4 2 Executive summary The present review is based on the publicly available literature and summarizes the toxicity profiles of the 14 individual constituents of TTO with an expected concentration in newly refined TTO above 0.5 % and five known oxidative degradation products from TTO. The purpose has been to supply SCCP and other regulatory agencies with an updated review of the relevant literature on the human toxicity of TTO and TTO constituents including suggestions for “No observed adverse effect levels” (NOAELs) for specified targets as well as an overall NOAEL for TTO. The review will also identify potential problems related to the use of TTO products and provide possible approached to be considered by the industry. • TTO products and formulations have generally, except for the neat products, been reported to be without significant risk for acute human toxicity. Oral exposure to neat TTO does, however, have a clear potential for servere human toxicity. • The known toxicokinetics indicate transport to the liver, hepatic biotransformation followed by renal elimination. The relatively short elimination half-lives expected on the basis of the presently known information on TTO constituents does not indicate significant accumulation over time of either parent compound or metabolites. • The NOAEL for irritative effects of TTO is expeted to be equal to or above 25% based on human studies and considering the experimental studies probably below 50%. • The allergic potential of freshly produced Melaleuca alternifolia oil is presumed to be low on healthy skin, whereas photoaged Melaleuca alternifolia oil must be considered to be a stronger sensitizer due to formation of oxidative degradation products. • The prevalence of positive findings following exposure of pre-sensitized dermatological patients in the clinical studies is generally around 0.4-0.6%. Thus TTO has probably a weak sensitizing potential among pre-sensitised people, though the present known number may be an overestimate due to problems with aged testing material and selection bias in some clinical studies. • Oxidative degradation products from TTO appear to possess a clear sensitizing potency. • The formation of oxidation products in TTO and TTO products need to be controlled. Whether this apparently technical problem can be dealt with during production, through addition of anti-oxidants, or through documented shelf-lives for the products is an issue that needs appropriate consideration. 5 • Several constituents of TTO do not cause toxicity themselves, but enhance the percutaneous penetration of other substances. • The relative occurrences of individual constituents of TTO differ between what is applied on the skin and what is absorbed. The penetration rates for those TTO constituents eventually penetrating the skin, i.e. terpinen-4-ol and α-terpineol (the least lipophilic) are relatively low. • Based on the available information on the repeat dose toxicity, the renal effects would have the lowest estimated NOAEL. Present data suggest a NOAEL of 510 mg/kg with a worst case scenario estimate of 117 mg/kg b.w. • Two TTO constituents (1,8-cineole and phellandrene) may act as weak promoters. There is no strong evidence that any of the TTO constituents are mutagenic. The carcinogenic mechanism explaining the gender and species specific renal tumors induced by limonene in male F344 rats is not seen in humans. Based on the available information, neither TTO nor its constituents are expected to pose any carcinogenic risk to humans. • Among constituents of TTO for which evidence of potential foetotoxicity is available, α-terpinene has the lowest estimate of a NOAEL (30 mg/kg bw) and the highest relative occurrence (9% on average) in TTO. Based on reproductive toxicity, a NOAEL for TTO can tentatively be set at 330 mg TTO/kg bw following oral exposure. • An overall NOAEL for TTO based on the presently available scientific information is based on the potential foetotoxicity of a TTO constituent and is estimated at 330 mg/kg b.w. A margin of safety estimate for dermal use of TTO products based on this value would need to incorporate the fraction of an applied dose absorbed and the actual concentration of TTO in the product besides an estimate of the amount of TTO applied on the skin. 6 3 Literature search strategy A search was made for each of the relevant components/products in scientific literature databases and on the internet. These search results were then combined with various keywords to limit the results to information relating to the toxicity of the components. 3.1 Data sources The following literature databases were searched in August 2005: • Medline (via UWA library) • Biological Abstracts (via UWA library) • Agricola (via UWA library) • Scopus (http://www.scopus.com/scopus/search/form.url) • Web of knowledge (including Current Contents) (via UWA library) • PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) • Ingenta (http://www.ingentaconnect.com/) Documents and data were also sourced from the internet. In particular, the following websites were searched; • National Toxicology Program (http://ntp-server.niehs.nih.gov/) • Toxnet (http://toxnet.nlm.nih.gov/) • IPCS Intox databank (http://www.intox.org/databank/index.htm) Data was requested from the Research Institute for Fragrance Materials (RIFM). Reports were generated for many of the components. These were reviewed and any additional references added to the Endnote database. RIFM also has unpublished reports on the dermal irritation and sensitisation capacity of several components of tea tree oil (eg. α-pinene). These reports have not been available for this review. Bibra Information Systems Ltd. has published toxicity profiles for the following compounds; Eucalyptol (1991), Terpinolene (1993), Linalool (1995), α- and γ- terpinene (1992), α-terpineol (2001), α-pinene (2002) and α-phellandrene (1993). These profiles are generally short summaries of published literature at the time of publication. As most reports are of older data, we have refrained from including them in our review. 3.2 Search terms The present review is based on the publicly available literature and summarizes the toxicity profiles of the 14 individual constituents of TTO with an expected concentration in newly refined TTO above 0.5 % given by the official ISO-norm for TTO (Table 1). Besides these 14 constituents, the review includes toxicity profiles on five known oxidative degradation products from TTO. For a thorough review of the toxicological profile of TTO, the reader is referred to a review on TTO toxicity for RIRDC (Nielsen 2003) or a recently published review article (Hammer et al. 2005). Reading and understanding toxicological profiles requires appreciation of the difference between hazard and risk. Thus, exposure to a hazardous chemical may occur without any significant health risk, given that the exposure/dose is 7 sufficiently low. During the discussion of potential risks associated with exposure to the individual constituents of TTO, the concentrations of these constituents in the oil and in products will be considered. Table 1. Main constituents of TTO with expected range and average percentages for premium grade TTO. The main constituents of TTO are terpenes (C10); sesquiterpenes (C15) constitutes only a small fraction. Constituent Min-Max (%) Average (%) Terpinen-4-ol 37 – 45 41.0 γ-terpinene 10 – 28 19.0 α-terpinene 5.0 – 13 9.0 p-cymene 0.5 – 12 6.0 1,8-cineole 3.0 – 7.0 5.0 α-terpineol 1.5 -8.0 4.8 δ-cardinene Traces – 8.0 4.0 Aromadendrene Traces – 7.0 3.5 α-pinene 1.0 – 6.0 3.5 Terpinolene 1.5 – 5.0 3.3 Limonene 0.5 – 4.0 2.3 Sabinene Traces – 3.5 1.7 Globulol Traces – 3.0 1.5 Viridiflorol Traces – 1.5 0.7 Data on components was searched for using the following component names: terpinen-4-ol, terpinene, 1,8-cineole, eucalyptol, terpinolene, cymene, pinene, terpineol, aromadendrene, cadinene, limonene, sabinene, globulol, and viridiflorol. Searches were also conducted for the autoxidation products ascaridol(e), isoascaridol(e) and 1,2,4-trihydroxymenthane. Alternate names and synonyms such as terpinenol, carvomenthenol, and eucalyptol were also used. Search results for each component were combined with search results for each of the terms listed in Table 2. Additional search terms such as NOAEL and Draize were also used. Terms were truncated so that permutations of each search term would be identified. Table 2. Terms used to search for data relating to the toxicity of Tea tree oil components (asterisk indicates a wildcard) Allerg* Hepato* Poison* Carcino* Irrit* Rat Chronic Metaboli* Sedat* Embryo* Mutagen* Sensiti* Foeto/Feto* Nephro* Teratogen* Genotox* Neuro* Toxic* 8 4 Summary of toxicity profiles for TTO constituents 4.1 Acute toxicity Human evidence based on casuistic reports clearly demonstrates that TTO may cause severe acute toxicity following oral exposure to neat TTO. Temporary depression of the central nervous system has been reported in children drinking no more than a few teaspoons of 100% TTO. Based on the published cases, intoxicated children have not experienced prolonged and severe sequela. However, more serious and potentially fatal effects following unintentional oral intake of neat TTO can not be excluded, and such risks should be minimized. There are no reports available on human intoxications due to oral intake or dermal use of diluted/formulated TTO products. In experimental animals, the oral as well as dermal LD50 values are generally in the range of 1000-5000 mg/kg b.w. for TTO constituents, which is in accordance with the LD50 value above 5000 mg/kg b.w. reported for TTO. However, in experimental studies intraperitoneal administration of high doses (100 or 200 mg/kg b.w.) of myrcene or limonene to mice caused sedative as well as motor relaxant effects (Gurgel do Vale, Couto Furtado et al. 2002). Further, TTO administered orally at doses greater than 1500 mg/kg b.w. to female rats appeared to induce persistent neurotoxic lesions in pathways controlling limb movements (Kim, Cerven et al. 2002). The implication of this observation is limited due to the high dose needed. Should new studies demonstrate this kind of neurotoxicity at significantly lower doses, this may have implications for the safety assessment of TTO. A human LD50 value between 500 and 5000 mg/kg b.w. has been suggested for limonene (Gosselin, Hodge et al. 1976). As limonene only constitutes approximately 2.5% of TTO, this value does not change the overall conclusion regarding the acute toxicity of TTO products/formulations. Thus, TTO products and formulations have generally, except for the neat products, been reported to be without significant risk for acute human toxicity. 4.2 Skin and eye irritation 4.2.1 In vitro and animal data ρ-Cymene and γ-terpinene were not irritating when assessed in vitro using the HET-CAM assay (Demirci, Paper et al. 2004). Irritation was not evident when 1,8-cineole (Opdyke 1975) and terpinolene (Opdyke 1976) were applied to intact or abraded rabbit skin for 24 h with occlusion. α-Pinene applied neat to the skin of mice and swine was not irritating (Urbach and Forbes cited in (Opdyke 1978)). 9 Moderate irritation was seen when terpinen-4-ol (100%) (Opdyke 1982), ρ-cymene (100%) (Opdyke 1974), γ-terpinene (100%) (Opdyke 1976), terpineol (Opdyke 1974), d-limonene (Opdyke 1975), α-phellandrene (100%) (Opdyke 1978), α- pinene (Opdyke 1978) and myrcene (Opdyke 1976) were applied to intact or abraded rabbit skin for 24 h with occlusion. Evaluation of skin damage and cytotoxicity of a range of terpenes on rat abdominal skin showed no irritation for 1,8-cineole and α-terpineol, whereas significant histopathological changes and cytotoxicity against human keratinocytes were evident for terpinolene, α-terpinene and limonene at very low concentrations (Kitahara, Ishiguro et al. 1993). The irritancy of α-terpinene, terpinolene and limonene to rabbits was further evaluated by the Draize test, and terpinolene was more irritating than limonene, which was in turn more irritating than α-terpinene (Okabe, Obata et al. 1990). The interpretation of the in vitro observations in relation to irritation of human skin is complicated, as evidence from the studies in rabbits and clinical studies in human do not appear to demonstrate the same degree of toxicity to the skin. Investigation of the irritant capacity of several terpenes by transepidermal water loss (TEWL) and histological observations suggested that α-terpineol is potentially irritating (Fang, Hung et al. 2003). Based on the information that no eye irritation in rabbits was observed at 1% sabinene (Yao and Chiou 1993) and that sabinene constitutes below 2% of TTO, it can be anticipated that an irritant response due to sabinene in a TTO product is unlikely. In a report on acute dermal irritation in the rabbit of TTO, the skin irritation index was determined by the Draize method using NZ White rabbits exposed to undiluted TTO (batch 88/375). The Draize irritation index for undiluted TTO was found to be 5.0, indicating a severe irritant (Bolt 1989). This result has been observed in several studies with neat TTO. In a study in rabbits from 1996 (Pharmatox) following OECD guideline 404, TTO was applied for 4 hours with a semi- occlusive patch application followed by a 14 days observation period. The study demonstrated that: TTO (75%) was found to be a mild to moderate irritant, TTO (50%) was found to be a minimal irritant, TTO (25%) was found to be a non- irritant, and TTO (12.5%) was found to be a non irritant. Thus, a clear and expected dose relationship between concentration of TTO and irritancy was observed. Primary eye irritation of TTO was studied in the rabbit (female, Japanese White) under GLP conditions (Oyama 2000). Two groups of three rabbits were given a single ocular dose (0.1 mL) of TTO (1% or 5% in liquid paraffin). After instillation of the test substance, no abnormal signs in the clinical conditions were observed among the rabbits. Ocular responses using Draize’s criteria demonstrated a conjunctival discharge lasting for up to six hours following instillation of 1% TTO and conjunctival redness and discharge for up to 24 hours following instillation of 5% TTO. In both groups, the maximal response was observed after one hour. Based on these observations, the author concludes, that both TTO solutions can be classified as “minimally irritating” (Oyama 2000). 4.2.2 Human data When Patch testing human volunteers, the following TTO constituents were non- irritating: terpinen-4-ol (5-10%) (Opdyke 1982; Knight and Hausen 1994), γ- 10 terpinene (5%) (Opdyke 1976; Southwell, Freeman et al. 1997), α-terpinene (5%) (Opdyke 1976; Knight and Hausen 1994), 1,8-cineole (4-28%) (Opdyke 1975; Knight and Hausen 1994; Southwell, Freeman et al. 1997), ρ-cymene (4%) (Opdyke 1974; Knight and Hausen 1994), terpinolene (20%) (Opdyke 1976; Knight and Hausen 1994), terpineol (12%) (Opdyke 1974; Knight and Hausen 1994), α-pinene (10%) (Opdyke 1978; Knight and Hausen 1994), cadinene (10%) (Opdyke 1973), myrcene (4%) (Opdyke 1976; Knight and Hausen 1994), α- phellandrene (4-8%) (Opdyke 1978; Knight and Hausen 1994) aromadendrene (1%) (Knight and Hausen 1994) and limonene (1%) (Knight and Hausen 1994). In a larger multicenter study, a set of 5 to 10 fragrances at 2 concentrations was patch tested. Besides scores for allergic response, the researchers indicated the frequency of doubtful or irritant reaction that was not seen as allergy. A total of 1323 patients were patch tested in 11 centres and none of them demonstrated irritancy to α-terpineol (Frosch, Pilz et al. 1995). A later study by six of the same dermatological departments demonstrated that among 18 fragrances tested in 1606 consecutive patients, the lowest reactivity was observed with α-terpineol, yelding only 1 positive (<0.1%) allergic respons and 11 (0.7 %) doubtful/irritant reactions in a patch test with 5% α-terpineol (Frosch, Johansen et al. 2002). Eye irritation thresholds between 100ppm and 1000 ppm for ρ-cymene, 1,8- cineole, α- and γ- terpinene, α-pinene, limonene have also been determined (Cometto-Muñiz, Cain et al. 1998; Cometto-Muñiz, Cain et al. 1998). Limonene was not a respiratory irritant when tested in humans at concentrations of 10, 225, and 450 mg/m3. At the highest exposure level a temporary decrease in lung capacity was observed (Falk-Filipsson, Lof et al. 1993). Using a protocol based on the original Draize method, the potential of six TTO products to induce skin irritancy and/or allergenicity in humans was tested (Skin&CancerFoundationAustralia 1997). A total of 311 persons were included in the study and exposed to 100% TTO, 25% TTO in cream, 25% TTO in ointment, 25% TTO in gel, 5% TTO in cream and 5% TTO + 5% synergist in cream. No information as to the synergist was given. The test substances were applied to the skin a minimum of seven times during a three-week induction period. These observations indicate that products with concentrations of TTO below or equal to 25% are not causing irritancy to the participants. Likewise, undiluted TTO is not an irritant for the vast majority of the participants, but a small fraction of the population (in this study 5.5%) seems to be more susceptible to TTO and demonstrates positive skin reactions towards undiluted TTO. The small fraction of participants with an increased susceptibility to TTO was not further characterised regarding previous incidensies of skin irritation. All data indicate that the irritative effects of TTO and TTO constituents depend on the dose. A range of individual TTO constituents as well as TTO has been demonstrated to be irritants when applied undiluted. Experimental studies in rabbits demonstrate that 75-100% TTO is a strong irritant, 50% TTO a mild irritant, and that 25% TTO and lower concentrations are non-irritative. Studies in humans are limited to studies on neat TTO (or constituents) and concentrations from 25% and below. The human data support the experimental data in so far as the neat oil is a significant irritant, whereas irritative effects are not observed when the concentration of TTO is below 25%. Thus, the no-observed-effect-level for irritative effects of TTO is expeted to be at least 25% based on human studies and considering the experimental studies 11 probably below 50%. Moreover, the assumption that cineole should be a main culprit is not supported by the published evidence of irritative effects following exposure to TTO. 4.3 Skin sensitization A skin sensitizer is an agent that is able to cause an allergic response in susceptible individuals. The consequence of this is that following subsequent exposure via the skin, the characteristic adverse health effects of allergic contact dermatitis may be provoked. As yet, there is not a validated in vitro test method accepted for skin sensitisation. Two validated in vivo laboratory animal tests to evaluate the potential of a substance to cause skin sensitisation exist: The local lymph node assay (LLNA, OECD 429) and the guinea pig maximisation test (GPMT, OECD 406). 4.3.1 In vitro and animal data α-Pinene (10%) was sensitising using an open epicutaneous test in guinea pigs whereas l-carvone (1%), ρ-cymene (4%), d-limonene (8%), terpinene-4-ol (5%), 1,8-cineole (16%) and 1-terpineol (12%) were not (Klecak 1985). Likewise, no sensitizing capacity of 1,8-cineole was observed in guinea pigs (Hausen, Reichling et al. 1999). The amount of α-pinene in TTO is 3.5%, and a 25% TTO product will therefore have around 0.9% α-pinene. d-Limonene did not produce sensitisation reactions when applied to guinea pigs whereas oxidised d-limonene did (Karlberg, Boman et al. 1991). A more recent study supporting the initial observation demonstrated that only the oxidation products of d-limonene, (R)-(-)-carvone, (+)-limonene oxide, along with air oxidized d-limonene, were potent sensitizers in the Freund complete adjuvant test and in the guinea pig maximization test (Haneke 2002). Limonene at 25 and 50% did not produce a response in the local lymph node assay, but 100% did (Warbrick, Dearman et al. 2001). This was regarded as being a weak response. The concentration of limonene in TTO is 2-3%, and the amount of limonene in a 25% TTO product would be reduced to 0.6%. In a report on skin sensitisation in the guinea pig following exposure to TTO (Bolt 1989), groups of 20 albino guinea pigs (HA strain) were tested according to the Magnusson & Kligman method. The induction procedure consisted of two intradermal injections (5% TTO in paraffin or 5% TTO with Freund´s complete adjuvant) or an epidermal induction application (undiluted TTO). The dose used for challenge was 30% TTO. The erythemal reactions were measured 24 hours after removal of the patch of the challenge test. There were no responses in either group. The experimental methodology stated in the report appears to follow OECD 406 guidelines. These in vitro and animal data do not suggest that any of the TTO constituents tested (except for oxidation products) are sensitizers. 4.3.2 Human data Using a maximisation test and 25 human volunteers, the following components did not produce sensitisation reactions; terpinen-4-ol (5%) (Opdyke 1982; Klecak 1985), γ-terpinene (5%) (Opdyke 1976), α-terpinene (5%) (Opdyke 1976), 1,8- cineole (16%) (Opdyke 1975; Klecak 1985), terpinolene (20%) (Opdyke 1976; Klecak 1985), ρ-cymene (4%) (Opdyke 1974; Klecak 1985), d-limonene (8%) (Opdyke 1975; Klecak 1985), cadinene (10%) (Opdyke 1973), l-carvone (1%) 12 (Klecak 1985) and myrcene (4%) (Opdyke 1976), whereas α-pinene (10%) did (Klecak 1985). An interesting observation in relation to the pinenes was that β- pinene did not cause sensitisation reactions (Klecak 1985). A maximization test on 25 volunteers with α-phellandrene at a concentration of 4% in petrolatum produced one sensitization reaction (Opdyke 1978). In view of the autoxidation problems, it was decided that the maximization procedure should be repeated on α-phellandrene using a freshly distilled sample processed under a blanket of nitrogen and containing an antioxidant. The same maximization test was carried out on another 25 volunteers using 8% in petrolatum of this freshly processed sample, and no sensitization reactions were observed (Opdyke 1978). However, phellandrene was identified as a sensitizer in another study on the sensitizing potential of some essential oils and their constituents (Woeber and Krombach 1969), and α-phellandrene induced a positive patch test in four of the eleven patients included in a study on patients from a dermatological department (Hausen, Reichling et al. 1999). To what extent the positive findings in the two latter studies are caused by oxidative degradation products of phellandrene is not clear. 4.3.2.1 Contact dermatitis The results of patch testing of TTO-sensitised individuals with TTO components in three large studies (Knight and Hausen 1994; Southwell, Freeman et al. 1997; Hausen, Reichling et al. 1999) are summarised below (Table 3). A high fraction of TTO-sensitised patients demonstrated positive patch tests against 5% ascaridol (9 out of 11), 5% α-Terpinene (15 out of 21), and terpinolene when tested with 10% oil in ethanol (17 out of 18) (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Positive patch test results were also recorded for aromadendrene (5 out of 18), limonene (6 out of 18), α-Phellandrene (5 out of 18), and 1,2,4- Trihydroxymenthane (4 out of 11) (Knight and Hausen 1994; Hausen, Reichling et al. 1999). It is, however, noteworthy that clear differences occur between skin- reactions recorded in different studies. Thus, α-Terpinene tested at comparable concentrations cause significantly different results in the studies from the group around Hausen (Knight and Hausen 1994; Hausen, Reichling et al. 1999) and Southwells study published in 1997 (Southwell, Freeman et al. 1997). Likewise, limonene and aromadendrene caused skin reactions in five or six out of seven participants in the Knight and Hausen study from 1994 when applied in 1% as compared to zero or one in eleven subjects exposed to 5% aromadendrene or limonene in the study from 1999 (Hausen, Reichling et al. 1999). Differences do occur between dermal reactions recorded in different studies with limited number of participants. However, these differences are often equally well explained by presense of impurities or oxidative product in test oils. The present data from these studies do not allow a closer evaluation on the potential presence of oxidation products. 13 Table 3. Number of presensitized dermatological patients reacting to TTO components (% of component tested) Component Hausen et al., 1999 Southwell et al., 1997 Knight & Hausen, n = 11 n = 3 1994 n = 7 Aromadendrene 0 (5) 5 (1) Ascaridol 9 (5) d-Carvone 0 (5) l-Carvone 0 (5) 1,8-Cineole 0 (5) 0 (1.4) 0 (5) ρ-Cymene 0 (5) 0 (1.5) 1 (1) Limonene 1 (5) 0 (0.7) 6 (1) Myrcene 2 (5) 0 (1,5) α-Phellandrene 4 (5) 1 (1) α-Pinene 0 (10) 0 (0.7) β-Pinene 0 (0.9) Sesquiterpene hydrocarbons 3 (1.5) α-Terpinene 7 (5) 1 (5.9) 7 (5) γ-Terpinene 0 (5.2) Terpinen-4-ol 0 (10) 0 (9.5) 2 (10) α-Terpineol 0 (1.3) 0 (1,10) Terpinolene 11 (10) 0 (1.1) 0 (1) 6 (10) 1,2,4-Trihydroxymenthane 4 (5) Viridiflorene 1 (5) Limonene cause skin reactions in six of seven participants in the Knight and Hausen study from 1994 when applied in 1% as compared to only one in eleven subjects exposed to 5% limonene (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Other studies have, however, not supported the high fraction of positive reactons recorded in the study from 1994 (Knight and Hausen 1994). Thus, patch testing with limonene (1%) produced 1 irritant or doubtful positive reaction in 192 participants, whereas 0.1% limonene produced no reactions (Frosch, Pilz et al. 1995). Further, patch testing with 3% limonene produced only 7 positive in 1606 dermatology patients (Frosch, Johansen et al. 2002). Whether the positive reactions observed in the 1994 study on limonene were caused by impurities or oxidative products is not to say, but positive patch test reactions to oxidised limonene are common amongst dermatology patients (Karlberg, Dooms-Goossens et al. 1997; Matura, Goossens et al. 2002; Matura, Karlberg et al. 2003). In contrast to the study in guinea pigs, α-pinene produced no dermal sensitization when tested at concentration of 10% and 12% in petroleum, respectively in a dermal human sensitization study (EPA 2005). In experiments with oil of turpentine and α-pinene, it was shown that only the autoxidation products of oil of turpentine and not the terpenes themselves were eczematogenic. Autoxidation of a- pinene in the presence of air and light was sufficient to produce the eczematogenic agent, but its formation could be prevented by addition of inhibitors such as hydroquinone and pyrogallol (Opdyke 1978). Patch testing of 100 dermatological patients with 1% and 5% terpineol produced no irritant reactions (Frosch, Pilz et al. 1995). Consecutive testing of 1606 patients attending the patch test clinic of 6 European departments of dermatology demonstrated that the standard fragrance mix produced the highest reactivity in all centres (mean 11.4%; range 9.3–17.9%), whereas caryophyllene caused positive reactions in 0.6% and α-terpineol in less than 0.1% of the patients (Frosch, Johansen et al. 2002). In a more recent study, 1511 consecutive dermatitis patients in 6 European dermatology centres were patch tested with oxidized fragrance terpenes and some oxidation fractions and compounds. About 0.5% of the patients reacted to oxidized caryophyllene (Matura, Sköld et al. 2005). 14 There have been a number of human contact dermatitis cases due to topical application of TTO with well over a dozen published cases within the last ten years (Apted 1991; De Groot and Weyland 1992; Selvaag, Eriksen et al. 1994; Van Der Valk, De Groot et al. 1994; De Groot 1996; Bhushan and Beck 1997). The applications included 100% TTO as well as lower concentrations of TTO in different formulated products. In an older study on occupational skin disorders, terpinolene was found not to be a sensitizer for human skin (Woeber and Krombach 1969) and a high fraction of TTO-sensitised patients with existing skin disease demonstrated positive patch tests against terpinolene when tested with 10% oil in ethanol (17 out of 18), whereas patch testing with terpinolene (1%) did not show any positive respons (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Using a protocol based on the original Draize method, the potential of six TTO products to induce skin irritancy and/or allergenicity in humans was tested (Skin&CancerFoundationAustralia 1997). A total of 311 persons were included in the study and exposed to 100% TTO, 25% TTO in cream, 25% TTO in ointment, 25% TTO in gel, 5% TTO in cream and 5% TTO + 5% synergist in cream. The report concludes that TTO is a mild allergen as only 1% of the participants (3/308) were sensitised, ie. made allergic, to TTO by means of the Draize test (Skin&CancerFoundationAustralia 1997). Based on an Italian study in 725 persons patch tested according to GIRDCA guidelines, the authors conclude that the sensitization potential of Melaleuca oil is poor, and that the response in patch tests appears to be dose dependent, and primarily observed after exposure to undiluted TTO. Positive responses to patch tests were more frequent in subjects with existing allergic contact dermatitis or atopic dermatitis (Lisi, Meligeni et al. 2000). The prevalence of hypersensitivity to a number of allergens was tested in a group of 219 volunteers (Greig, Carson et al. 2000). The findings were slightly higher than in other studies. The prevalence for hypersensitivity to TTO was found to be 2.3%. The authors argue that the prevalence found might be too high due to selection bias as the population studied were self-selected (Greig, Carson et al. 2000). In 1997, 1216 patients were patch tested at a dermatologic clinic (Fritz, Burg et al. 2001). Products containing Melaleuca alternifolia oils were tested concentrated or diluted. Seven patients with an allergic contact dermatitis due to TTO were identified. Two of them also exhibited delayed type IV hypersensitivity towards fragrance-mix or colophony suggesting the possibility of cross reaction or an allergic group reaction. The allergic potential of low concentrations of freshly produced Melaleuca alternifolia oil is presumed to be low on healthy skin, whereas photoaged Melaleuca alternifolia oil must be considered to be a stronger sensitizer due to formation of oxidative degradation products (Fritz, Burg et al. 2001). By 2003 close to 7000 patients at German dermatological clinics had been tested epicutaneously with a 5% dilution of oxidised TTO containing the original constituents as well as oxidation products (Hausen 2004). Seventy patients (1%) had a positive reaction to TTO (Hausen 2004). The most important allergens of TTO appears to be terpinolene, ascaridol, α-terpinene, and 1,2,4-trihydroxy menthane for which the prevalence of allergic respons among patients visiting dermatological clinics vary between 0.4% and 0.6% (Hausen 2004). Ascaridol and 1,2,4-trihydroxy menthane have repeatedly been found as oxidation products in aged TTO products. 15 The more recent appreciation of the potential presence of oxidative degradation products in TTO and TTO formulations is important as most of the earlier studies do not describe the age or storage condition for the TTO, TTO products or individual constituents applied. Thus earlier studies may have been conducted with potentially partly oxidised oils, which may explain some of the apparently contradicting results obtained between studies and the observation that a low concentration may induce a positive response in one study, whereas a repetition with a higher dose does not. This is the case with α-pinene, α-terpinene and terpinolene. Further, the test concentrations applied are considerable higher than what would be expected from the use of TTO products. To what extent aging of test formulation has been a problem in the larger clinical studies is equally uncertain, but several studies clearly demonstrate that replacement of old test samples with fresh TTO reduces the occurrence of positive findings. The prevalence of positive findings following exposure of pre-sensitized dermatological patients in the clinical studies is generally around 0.4-0.6%. Thus TTO has probably a weak sensitizing potential, though the present known numbers may be an overestimate due to problems with aged test material. Further surveillance of skin sensitization due to exposure to TTO should therefore be encouraged with due focus on the test material used. On the other hand, oxidative degradation products from TTO appear to possess a clear sensitizing potency. The formation of oxidation products in TTO and TTO products need therefore to be controlled. Whether these degradation products are formed during distillation, product formulation or during storage at retailers or consumers is not clear. Whether this apparently technical problem can be dealt with during production, through addition of anti-oxidants, or through documented shelf-lives for the products is an issue that needs consideration. 4.4 Dermal/percutaneous absorption Several terpenes (thymol, menthone and 1,8-cineole) do not cause toxicity themself, but enhance the percutaneous penetration of other substances (e.g. propranolol, piroxicam, zidovudine, insulin, haloperidol) (Doliwa, Santoyo et al. 2001; Vaddi, Ho et al. 2002; Pillai and Panchagnula 2003; Narishetty, Panchagnula et al. 2004; Amnuaikit, Ikeuchi et al. 2005).The degree of enhancement depends on the lipophilicity of the terpene as well as the lipophilicity of the drug in question (El-Kattan, Asbill et al. 2001). The levels of terpenes absorbed or deposited in the skin are seldom reported. The effect of three cyclic terpenes (carveol, terpinene-4-ol, α-terpineole) on the transdermal penetration of water was studied in vitro. The maximum increase in permeability coefficients of carveol, terpinen-4-ol and α-terpineol was 10.6, 8.7 and 10.9, respectively (Magnusson, Runn et al. 1997), thus demonstrating clear effects on skin integrity. Likewise, treatment of human epidermis with terpene penetration enhancers has been shown to increase electrical conductivity. The increase in ion transport suggests that terpenes open new polar pathways across the stratum corneum. A correlation between increases in ion transport and previously reported increases in 5-fluorouracil penetration suggests that terpene enhancers may create micro-pores in the intercellular lipids through which both ions and polar drugs may pass (Cornwell and Barry 1993). A quantitative study in mice and rabbits demonstrated that p-cymene is well absorbed through the skin (Wepierre 1963; Wepierre 1963) . Following absorption, 16 the ring substituent is oxidized to yield aromatic alcohol and carboxylic acid metabolites that are excreted free or conjugated in the urine. The presently available data on penetration through human skin demonstrates that in experimental studies on dermal penetration of different ingredients of TTO, the first component to penetrate the skin and reach the subcutaneous fat layer (within 1 hour) was terpinen-4-ol. After two hours exposure α-terpineol was also found in the subcutaneous fat layer (Hayes, Leach et al. 1997). As exposure time was increased, more ingredients were detected (1,8-cineole, α-terpinene, p-cymene, α- terpinolene), but all in considerably lower amounts (Hayes, Leach et al. 1997). A more recent study revealed that among seven major constituents of TTO (terpinene-4-ol, 1,8-cineole, p-cymene, terpinolene, α-terpineol, α-terpinene, γ- terpinene) present on the upper side of the skin, only three (terpinen-4-ol, α- terpineol, eucalyptol) could positively be identified as being absorbed through the skin (Nielsen and Nielsen 2006). γ–Terpinene which was found to appear in higher amounts than α-terpineol and 1,8-cineole in the TTO applied to the skin was not detected as absorbed. The three constituents absorbed were those compounds among the seven constituents with the lowest log Pow values – the least lipophilic (Nielsen and Nielsen 2006). Thus, the relative occurrences of individual constituents of TTO differ between what is applied to the skin and what eventually get absorbed (Nielsen and Nielsen 2006). The penetration rates for the TTO constituents eventually penetrating the skin were low, and a the penetration coefficient (Kp) around 20µm/h for terpinene-4-ol was reported as was a lag-time from 4-6 hours for terpinene-4-ol (Nielsen and Nielsen 2006). The penetration of TTO through human epidermal membranes was also evaluated experimentally by use of Franz cells (static diffusion cells) (Edwards-Jones, Buck et al. 2004). TTO was applied topically as the pure oil and as a 20 % formulation in ethanol. Following the 24 hr experimental period, terpinen-4-ol, α-terpineol, and 1,8-cineole were detected in the receptor phase. None of the other TTO constituents could be detected in the receptor phase, but a fraction of sesqui- terpinene compounds together with terpinen-4-ol and α-terpineol was seen in epidermis (Cross and Roberts 2006). These recent observations are in agreement with studies using a matrix-type transdermal system describing the levels of terpenes and their effects on the stratum corneum after dermal application (Cal, Janicki et al. 2001). In this study, dermis did not present a barrier for penetration of terpenes. For all terpenes the penetration was, however, slower in the presence of epidermis, and large amounts of terpenes were found in epidermis indicating that affinity of these compounds to the stratum corneum is very high (Cal, Janicki et al. 2001). When the difference in thickness of epidermis and dermis is taken into consideration, the higher affinity of terpenes to epidermis than dermis can be demonstrated. The dry mass of epidermis is approximately 2–3 mg/cm2. Thus, the amounts of terpenes found in epidermis, most probably to stratum corneum, correspond to over 50% of the total mass (Cal, Janicki et al. 2001). Penetration of limonene, terpinolene, and cineole had lag-times close to two hours and an absorption through the matrix-type barrier between 8% and 13% of the applied amount (Cal, Janicki et al. 2001) 17 4.5 Repeat dose toxicity Based on the toxicokinetic evidence, accumulation of TTO, its constituents, or metabolites is not expected. Metabolism occurs primarily in the liver followed by renal excretion. Relevant target organs for non-genotoxic effects following repeated and intended use of TTO products is therefore the liver and the kidneys. After acute high dose exposure, effects on the gastrointestinal tract (intestinal atony) and the central nervous system have been observed (see section on acute toxicity). The mutagenic/carcinogenic potential of TTO and constituents is discussed in section 4.6. Terpinen-4-ol did not induce changes in the morphology or function of the kidneys of male Sprague-Dawley rats following 28 days of repeated oral exposure to 400 mg/kg b.w., and was considered to be non-toxic (Schilcher and Leuschner 1997). The available literature on systemic effects of terpinen-4-ol is very limited. Based on the 28-days study on kidney toxicity in rats, the NOAEL after oral exposure may be estimated to be 400 mg/kg. As terpinen-4-ol on average constitutes 40% of TTO, this NOAEL for terpinen-4-ol corresponds to an oral NOAEL for TTO (based on renal toxicity of terpinen-4-ol) of 1000 mg/kg. Cineole given to B6C3F1 mice by gavage for 28 days at doses up to 1200 mg/kg/day did not result in any changes. When given encapsulated at doses corresponding to 600 – 5607 mg/kg/day, some hypertrophy of hepatocytes was seen, but was not considered significant (National Toxicology Program, cited in (De Vincenzi, Silano et al. 2002)). Cineole (8 or 32 mg/kg/body weight) was given by gavage to male SPF CFLP mice 6 days per week for 80 weeks. No changes were evident in mice given cineole when compared to control mice (Roe, Palmer et al. 1979). Based on the studies on hepatic and renal toxicity evaluated by BIBRA, a NOAEL might be estimated as 300 mg/kg body weight, which is in agreement with the evaluation from the Norwegian Food Control Authorities in 1999. As 1,8- cineole on average constitutes 5% of TTO, this NOAEL for 1,8-cineole corresponds to an oral NOAEL for TTO (based on liver and kidney toxicity of 1,8- cineole) of 6000 mg/kg. Exposure to α-terpinene (125 or 250 mg/kg b.w.) for nine consecutive days caused decreased body weight gain in pregnant Wistar rats (Araujo, Souza et al. 1996). No maternal toxicity was observed at 60 mg/kg b.w., and a NOAEL of 60 mg/kg b.w. for systemic effects following repeated exposure to α-terpinene is suggested. Based on the amount of α-terpinene present in TTO, this corresponds to a NOAEL of 660 mg/kg b.w. for TTO. The effects of ρ-cymene on the brain chemistry of rats was studied by exposing male Long-Evans rats to 0, 50 or 250 ppm p-cymene by inhalation (Lam, Ladefoged et al. 1996). Rats were exposed for 6 hours per day, 5 days per week for four weeks and then had an 8 week wash-out period. No obvious toxicity was seen during the exposure period and body weights did not differ after the 12 week trial period. Levels of synaptosomal protein were significantly reduced in treated rats, whereas relative amounts of noradrenaline and dopamine were increased. A limited number of relevant repeat-dose studies are available and the inhalation route is often used for cumene. A NOAEL of 488 ppm based on inhalation might be suggested as might also a LOAEL of 769 mg based on the only study with oral exposure. Based on the oral study and using an uncertainty factor of 10, a NOAEL for cumene/p-cymene of 75 mg/kg body weight is suggested. As p-cymene on average constitutes 6% of TTO, this NOAEL for p-cymene corresponds to an oral 18 NOAEL for TTO (based on possible renal effects of p-cymene) of 1200 mg/kg body weight. In a 3-month oral toxicity study, rats were fed an alpha-pinene resin or pinene polymer made predominantly from alpha-pinene. (The ratio of alpha-and beta- pinene was 10:1.) The dose levels were 0, 1, 3 or 5% in the diet. Effects seen at 5% (3967 mg/kg/day) included an increase in relative liver weight in both sexes, and absolute liver weight in females only. Increased relative thyroid weights in males were noted at the 3 and 5% dose levels. In the absence of histopathological alterations, these changes were not considered treatment related. No effects were noted at 1%, which corresponds to roughly 800 mg/kg/day (EPA 2005). Based on the amount of pinene present in TTO, this corresponds to a NOAEL for TTO above 5000 mg/kg b.w. Based on the study using dietary exposure of rats to concentrations of α-terpineol corresponding to 500 mg/kg b.w. (Hagan, Hansen et al. 1967), a NOAEL for α- terpineol of 500 mg/kg bw can be suggested as the study did not demonstrate any toxicity. As α-terpineol on average constitutes 5% of TTO, this NOAEL for α- terpineol corresponds to an oral NOAEL for TTO (based on the only available study on systemic toxicity for α-terpineol) of 10.000 mg/kg body weight might be suggested. Adult beagle dogs were gavaged twice daily for 6 months with 100 or 1000 mg d- limonene/kg body weight per day. Limonene ingestion did not affect feed consumption or body weight. Increased kidney weight was seen but no histopathological kidney changes were seen. No nephropathy was evident (Webb, Kanerva et al. 1990). An activated immune response from alveolar macrophages has been observed in rats following oral exposure to limonene at doses from and above 250 mg /kg b.w. It is unclear how these observations would add to a potential risk following dermal exposure to a TTO product containing around 2.5% limonene. However, if the data was used to estimate a NOAEL for TTO, this NOAEL would probably be above 2000 mg/kg b.w. Methyleugenol administered by gavage at a maximum dosage of 1000 mg/kg body weight to F344/N rats and B6C3F1 mice for 14 weeks resulted in erythrocyte microcytosis and thrombocytosis in rats (Abdo, Cunningham et al. 2001). Other results were suggestive of impaired liver function and protein digestion. Adverse effects seen in the salivary glands, adrenal glands, testis and uterus were considered to be secondary to the liver and stomach effects. The no-observed-effect level (NOEL) was estimated to be 10 mg/kg for both species (Abdo, Cunningham et al. 2001). Methyl eugenol is present in trace amounts in TTO (below 0.1%) and the estimated NOAEL for TTO based on the repeated dose toxicity of this minor constituent (methyl eugenol) would exceed 1000 mg/kg b.w. A range of toxic effects have been reported after repeated exposure to TTO or TTO constituents and used to estimate NOAEL values. For α-terpinene the estimated NOAEL is based on weight loss in pregnant rats, and given the presence of 9% α- terpinene in TTO, this would equal a NOAEL for TTO of 660 mg/kg. The direct extrapolation from a NOAEL for a constituent to a NOAEL for TTO is only acceptable when no other constituent is reported to affect the same target. In case of TTO, three constituents have been reported to affect the kidneys. 19 TTO constituent Target toxicity Estimated NOAEL Conc. in TTO Terpinen-4-ol renal 400 mg/kg 40% Cineole renal 300 mg/kg 4.5% Cumene renal 75 mg/kg 6% To estimate a NOAEL for TTO based on the renal toxicity data, information on the estimated constituent-specific NOAEL as well as relative presence in TTO needs to be considered. When available data from terpinen-4-ol, cineole, and cumene is used, a NOAEL may be estimated using the formula: (40%/400mg/kg + 4.5%/300mg/kg + 6%/75mg/kg) x NOAEL = 100% This formula gives an estimated NOAEL for TTO of 510 mg/kg Lack of data on possible renal effects of the remaining constituents may decrease the NOAEL further. A worst case scenario would be that the remaining 49.5% of TTO had a constituent-specific NOAEL equal to cumene. Incorporating this estimate in the calculation of a NOAEL for TTO gives an adjusted formula: (40%/400mg/kg + 4.5%/300mg/kg + 6%/75mg/kg + 49.5%/75mg/kg) x NOAEL = 100% The worst case scenario estimate for a NOAEL for TTO would be 117 mg/kg Based on the available information on the repeat dose toxicity, the renal effects would have the lowest estimated NOAEL of 117 mg/kg b.w.. A margin of safety estimate for dermal use of TTO products based on this value would need to incorporate the fraction of an applied dose absorbed and the actual concentration of TTO in the product besides an estimate of the amount of TTO applied on the skin. 4.6 Mutagenicity/carcinogenicity 4.6.1 Bacterial assays The mutagenic potential of tea tree oil (Melaleuca alternifolia) was examined using the Ames Test. One of the major components, the monoterpenoid terpinen-4-ol, was also examined to determine if it demonstrated any mutagenic potential. Salmonella typhimurium (TA102, TA100 and TA98) was utilised in the Ames test. Commercially available tea tree oils were tested. No mutagenic effect was determined in any of the brands of tea tree oil on any of the strains of Salmonella examined with or without metabolic activation (Fletcher, Cassella et al. 2005). The same negative results were obtained for the terpinen-4-ol component examined. There was a clear evidence of toxicity of tea tree oil on all Salmonella strains and also by terpinen-4-ol at higher dose levels. It is suggested that terpinen-4-ol may contribute significantly to the widely reported antibacterial activity of tea tree oil (Fletcher, Cassella et al. 2005). Further, the following TTO constituents were found to be non-mutagenic using bacterial assays such as the Ames test: α-terpinene (Gomes-Carneiro, Viana et al. 2005), 1,8-cineole (Yoo 1985; Gomes-Carneiro, Felzenszwalb et al. 1998), α- terpineol (Florin, Rutberg et al. 1980), limonene (Florin, Rutberg et al. 1980; Watabe, Hiratsuka et al. 1981; Connor, Theiss et al. 1985), α-pinene (Rockwell and Raw 1979; Florin, Rutberg et al. 1980; Connor, Theiss et al. 1985; Gomes- Carneiro, Viana et al. 2005), cymene (Rockwell and Raw 1979), and β-myrcene (Gomes-Carneiro, Viana et al. 2005). 20 Though no mutagenicity was observed when tested directly, weak mutagenic activity toward TA100, but not TA98, was observed in an older study with ether extracts of urine from rats fed β-terpineol (Rockwell and Raw 1979). Repetition of this finding has not been published and it is difficult to evaluate the implications of this observation given that the effect is observed in β-terpineol and it is the α-form that occurs in TTO. Terpineol was negative using the Bacillus subtilis rec- assay (Oda, Hamano et al. 1978), but caused a slight increase in the number of revertants for one of four test strains (Gomes-Carneiro, Felzenszwalb et al. 1998). α-Terpineol was negative in 5 out of six salmonella strains. However, the result from the last strain (TA102) can not be ignored as a false positive finding because of dose-related toxicity. However, in support of a lack of genotoxic potential, α- terpineol did not induce lung tumors in mice following repeated intraperitoneal administrations. 4.6.2 Tests with mammalian cells γ-Terpinene increased DNA strand breakage in human lymphocytes at high doses (0.2 mM) when tested in the Comet assay, but significantly reduced chemically- induced DNA damage at lower doses (Aydin, Basaran et al. 2005). Cineole, d-(+)-limonene, l-phellandrene and β-pinene at concentrations ranging from 10 – 1000 μM did not increase the frequency of spontaneous sister-chromatid exchanges in Chinese hamster ovary cells (Sasaki, Imanishi et al. 1989). β-Myrcene is non-mutagenic in mammalian cells (Kauderer, Zamith et al. 1991) and is not genotoxic in bone marrow cells of rats administered β-myrcene orally (Zamith, Vidal et al. 1993). Limonene produced renal tumors in male F344 rats (Turner, Tinwell et al. 2001; Sekihashi, Yamamoto et al. 2002). No tumors are found in female F344 rats, other rats or mice. It is a non-genotoxic carcinogen in male F344 rats, but is considered to be non-mutagenic and of no cancer risk to humans (Flamm and Lehman- McKeeman 1991; Whysner and Williams 1996; Rivedal, Mikalsen et al. 2000). Cineole, d/l-carvone, d-limonene, terpineol, and thymol did not induce primary lung tumors in male or female A/He mice following 24 ip injections during an eight week period with 24 weeks follow-up. The doses used were either MTD (maximal tolerated dose) or 20% of MTD (Stoner, Shimkin et al. 1973). In conclusion, two of the TTO constituents (1,8-cineole and phellandrene) may act as weak promoters. There is no strong evidence that any of the TTO constituents are mutagenic. The carcinogenic mechanism explaining the gender and species specific renal tumors induced by limonene in male F344 rats is not seen in humans. Based on the available information, neither TTO nor its constituents are expected to pose any carcinogenic risk to humans. 4.7 Reproductive toxicity The available literature on reproductive toxicity of TTO and constituents is limited. Therefore, results from studies on myrcene, linalool, and cumene which are terpenes/terpenoids with some structural and chemical resemblancies with the major components of TTO, are included. 21 α-Terpinene was given to female Wistar rats at 30, 60, 125 and 250 mg/kg body weight on days six to 15 of pregnancy. The two highest doses were maternally toxic, and the highest dose also caused a reduction in the proportion of pregnant females. Foetuses from rats given 250 mg/kg had reduced body weights and increased kidney weights. Abnormal ossification of bones and minor skeletal abnormalities were evident in foetuses from females given 60 mg/kg or more. Thus the NOAEL for embryofoetotoxicity was set at 30 mg/kg body weight (oral route) (Araujo, Souza et al. 1996). β-Myrcene was given to female Wistar rats at 250, 500, 1000 and 1500 mg/kg by gavage from day 15 of pregnancy until postnatal day 21. Offspring from rats given 250 mg/kg did not show adverse effects but those given 500 mg/kg or more showed decreased birth weight, increased perinatal mortality and delayed postnatal development. Fertility of female offspring of rats given 1000 or 1500 mg/kg was impaired. The data suggest a NOAEL for peri- and postnatal developmental of 250 mg β-myrcene/kg body weight (Delgado, De Almeida Nogueira et al. 1993). In a similar study, β-Myrcene (0, 100, 300 and 500 mg/kg) was given by gavage to male and female Wistar rats prior to mating, during mating and pregnancy, and up to postnatal day 21. Male and female rats showed increased liver and kidney weights but no other signs of toxicity. β-Myrcene did not affect the proportion of females impregnated nor the pregnancy index. There was no evidence of maternal toxicity or external malformations at any dose. At 500 mg/kg there was an increased resorption rate and more skeletal abnormalities in fetuses. Myrcene did not affect postnatal weight gain but developmental milestones were slightly delayed. These data suggested a NOAEL for toxic effects on fertility and general reproductive performance of 300 mg β-myrcene/kg body weight (Paumgartten, De- Carvalho et al. 1998). Studies with cumene (which is closely related to ρ-cymene) have indicated a low potential for reproductive toxicity (EPA, cited in (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002)). The effects of cumene vapour on development in CD rats and New Zealand white rabbits has been examined (Darmer Jr, Neeper-Bradley et al. 1997). The effects of coriander oil (72.9% linalool, 22.3% other terpenoids, balance unknown) on reproduction and development has been studied in rats (United National Environmental Program 2002). The NOAEL for linalool based on foetotoxicity is suggested at a level of 365 mg linalool/kg bw (United National Environmental Program 2002). Among constituents of TTO for which evidence of potential foetotoxicity is available, α-terpinene has the lowest estimate of a NOAEL (30 mg/kg bw) and the highest relative occurrence (9% on average) in TTO. Based on reproductive toxicity, a NOAEL for TTO can tentatively be set at 330 mg TTO/kg bw following oral exposure. 4.8 Toxicokinetics A discussion of the toxicokinetics of TTO is essentially meaningless since TTO is a mixture of some 14+ individual substances. However, data do exist on some of the constituents and parallels exist between different constituents belonging to the same chemical groups. Thus, the structural formulas of TTO constituents illustrated in chapter 7 demonstrate the striking structural resemblances among the main constituents of TTO. 22 Most dat on the toxicokinetics of TTO is based on oral exposures. Chemicals absorbed via the gastrointestinal tract may undergo metabolism in the liver before reaching the general circulation, whereas chemicals absorbed through the skin avoid this first pass metabolism. However, in relation to elimination kinetics, biotransformation, and organ deposition, data from oral exposures are also relevant to the dermal exposure situation. Major biotransformation is expected to take place in the liver and to a lesser extent in other organs by the cytochrome P-450 dependent monooxigenases. These phase I reactions play a key role in converting more lipophilic terpenes into more hydrophilic compounds, which may then conjugate with glucoronic acid (or other phase II reactions) to generate even more hydrophilic metabolites that eventually are excreted. Excretion is expected to be dominated by renal elimination, but bile excretion followed by faecal elimination will also occur. Conjugates will be expected to be excreted within 2-3 days post exposure. An important notion is that intestinally absorbed chemicals will be transported directly to the liver before entering the systemic circulation. In this way the body has an evolutionary developed defence against oral exposure to toxicants. Following dermal absorption, however, this first pass metabolism is circumvented with the consequence that absorbed chemicals may enter critical organs for toxicity before having passed the liver and thus before being metabolised. A simple toxicokinetic model for metabolic pathways for the terpenes 1,8-cineole, p-cymene and terpinen-4-ol (major terpenes found in melaleuca oil) has been described by Villar et al. (Villar, Knight et al. 1994) In this model, less than 10% of the absorbed oil is expected to be eliminated through the faeces, and 60-80% of an oral dose is expected to be eliminated through the urine within 48-72 hours (Villar, Knight et al. 1994). A number of terpenes (α-pinene, d-limonene, α-terpinene, β-myrcene, terpineol, and 1,8-cineole) have been demonstrated in vitro (liver microsomes prepared from phenobarbital-treated rats) to dose-dependently inhibit the liver enzyme CYP2B1 (De-Oliveira, Ribeiro-Pinto et al. 1997; De-Oliveira, Fidalgo-Neto et al. 1999). An inhibited isoenzyme will affect the metabolism of those chemicals.that depend on CYP2B1, though IC50-values between 0.1µM and 15µM in microsomal preparations will be expected to require a substantial in vivo dose to reach significant target organ concentrations. Based on observations from in vivo intoxications in man and animals, the expected target organs for toxicity during the acute phase will be the gastrointestinal tract and the central nervous system. Effects on these targets will be expected to be clearly dose dependent with existence of non-toxic exposure levels. At lower doses no acute systemic toxicity is expected. The known toxicokinetics indicate transport to the liver, hepatic biotransformation followed by renal elimination. The relatively short elimination half-lives expected on the basis of the presently known information on TTO constituents does not indicate significant accumulation of either parent compound or metabolites. Therefore, toxicity following low-dose repeated exposure has focussed on targets like hepatic toxicity, renal toxicity, mutagenicity of parent constituent as well as metabolites, and foetotoxicity. 23 4.9 Degradation/oxidation products Like most natural oils several constituents of TTO may undergo oxidative degradation during storage as well as metabolism after being absorbed. Photooxidation may also be an issue for some chemicals. A few descriptions of degradation of TTO with time are described in the literature. Section 6.15 includes a discussion of the most prominent degradation/oxidation products. In section 6.15 the known toxic effects of these compounds have been included along with the parent compounds and stratified according to toxicity target. 4.10 Phototoxicity At a concentration of 100%, TTO did not produce phototoxic effects when applied to the skin of hairless mice, but some irritation was noted (Forbes and Davies 1982). The potential phototoxic effects of TTO and its constituents are expected to be covered by the inclusion of degradative and oxidative products in the previous sections. All methods have their strengths and weaknesses and this study from 1982 is not outstanding. But if 100 % TTO does not produce phototoxic reactions, those products that may be used on the skin and exposed to sun light, which may contain 5-10 % TTO can generally be regarded as safe. However, as with several other questions on toxicity of TTO, the question that needs focus is the degradation and oxidation products formed in aged TTO products. 24 5 Toxicity profile for TTO Tea tree oil (TTO) is a mixture of many individual constituents. From a clinical point of view, toxicity testing of that specific mixture of constituents that makes TTO is the most relevant, but for scientific and preventive purposes the toxicity profiles of the individual constituents are equally important. Thus, knowledge on individual profiles will allow the focus to be directed against the constituents that are most problematic (lowest margin of safety) and allow a discussion how these constituents may be eliminated, reduced, or controlled during processing and storage of TTO products. The present chapter will focus on the literature that has tested TTO either as neat oil, as mixtures with different carriers or as sales products, whereas chapter 6 will focus on the individual constituents and their known degradation/oxidation products. Acute toxicity Oral exposure TTO can be toxic if ingested, as evidenced by experimental studies in rats and from cases of human poisoning. The oral LD50 for TTO in a rat model is 1.9 – 2.6 ml/kg (Russell 1999). Rats dosed with 1500 mg TTO/kg body weight appeared lethargic and ataxic and showed depressed activity levels 72 h post dosing (Kim, Cerven et al. 2002). By day 4, however, all but one animal given this dose had regained all locomotor functions. Although values determined in animal models are not necessarily directly related to human toxicity, the experimental data do indicate that TTO dose-dependently is orally toxic. Published cases of oral poisoning in humans tend to be more dramatic in children because of their low body weight compared to an adult. One such case report involved a 23-month-old child who drank less than 10 ml of 100% pure TTO (Jacobs and Hornfeldt 1994). After a nap of approximately 30 minutes, he was unsteady on his feet and appeared as if ‘drunk’. The child was taken to a hospital and treated with activated charcoal and sorbitol via a naso-gastric tube, and approximately 5 h later he appeared to be asymptomatic. All other signs (such as respiratory rate, oxygen saturation, pupil reactivity, electrolytes and blood glucose) were normal throughout (Jacobs and Hornfeldt 1994). The authors attributed the clinical symptoms to a central nervous system depression caused by the ingested TTO. Similar symptoms were reported in a 17-month-old boy beginning 10 minutes after the ingestion of an unknown but less than 10ml volume of 100% pure TTO (Del Beccaro 1995). Under observation in hospital, complete resolution of symptoms occurred after approximately 5 h. In a third case, the ingestion of 2 teaspoons of 100% pure TTO by a 4-year-old boy led to symptoms of ataxia within 30 minutes followed by unconsciousness and unresponsiveness requiring intubation (Morris, Donoghue et al. 2003). The boy’s neurologic status improved gradually over 10 h and he was discharged from hospital 24 h after admission without respiratory or neurologic sequelae. A case of poisoning in an adult occurred when a patient drank approximately half a tea cup of TTO corresponding to a dose of approximately 0.5-1.0 ml/kg body weight (Seawright 1993). The patient was comatose for 12 h, and semi-conscious and hallucinatory for the following 36 h. Symptoms of abdominal pain and diarrhoea continued for approximately 6 weeks after this. In another incident, a 60- year-old man who swallowed one and a half teaspoonfuls of TTO as a preventative for a cold presented with a red rash which covered his feet, knees, upper body and 25 arms including his palms and elbows (Elliott 1993). His hands, feet and face were also swollen. The rash and other symptoms gradually disappeared and approximately one week later he had more or less recovered. Dermal exposure Toxicity following dermal application of inappropriately high doses of melaleuca oil to cats or dogs treated for fleas has been described. Animals had typical signs of depression, weakness, uncoordination and muscle tremors. However, the treatment of clinical signs has been sufficient to achieve recovery without sequelae within 2-3 days (Villar, Knight et al. 1994). For the same reason (fleas) three cats each had 120 ml of 100 % pure TTO applied to their shaved but intact skin (Bischoff and Guale 1998). All three cats experienced severe symptoms (hypothermia, uncoordination, dehydration, trembling), and one died after three days. The other two cats recovered within 24 and 48 h, respectively. The authors noted that the cat that died had elevated blood urea and persistent dehydration, which suggests that the animal may have had pre-existing renal damage unrelated to the TTO poisoning (Bischoff and Guale 1998). The toxicity observed in cats and dogs are parallel to the effects observed in orally intoxicated humans (i.e. effects on the central nervous system). Dose comparisons are not possible between the dermal exposure of cats or dogs and the oral human exposure given that the skin area with dermal exposure and the absorption of TTO are not known. However, a dose of 120 mL on a cat appear to be a dose resembling the estimated dose from the study by Seawright from 1993. In a study following OECD guideline 402 – limit test for acute dermal toxicity a group of 5 male and 5 female rabbits (NZ whites) were treated with TTO dermally (2000 mg/kg bw, undiluted sample, batch 88/375, skin area appr. 175 cm2, 24 hours exposure) (Bolt 1989). Slight diarrhoea was observed on day three in one animal. No weight loss or other signs of toxicity was recorded during the two weeks observation period. The author concludes that the test sample is essentially non-toxic at a dose level of 2000 mg/kg bw. In another study, the acute dermal LD50 in rabbits was recorded as in excess of 5000 mg/kg bw since this dose caused 2/10 deaths in rabbits (FragranceRawMaterialsMonograph 1988). Dermal penetration Experimental studies on dermal penetration of different ingredients of TTO demonstrated that the first component to penetrate the skin and reach the subcutaneous fat layer (within 1 hour) was terpinen-4-ol. After two hours exposure α-terpineol was also found in the subcutaneous fat layer (Hayes, Leach et al. 1997). As exposure time was increased, more ingredients were detected (1,8-cineole, α- terpinene, p-cymene, α-terpinolene), but all in considerably lower amounts (Hayes, Leach et al. 1997). The penetration of TTO through human epidermal membranes was also evaluated experimentally by use of Franz cells (static diffusion cells) (Edwards-Jones, Buck et al. 2004). TTO was applied topically as the pure oil and as a 20 % formulation in ethanol. Following the 24 hr experimental period, terpinen-4-ol, α-terpineol, and 1,8-cineole were detected in the receptor phase. None of the other TTO constituents could be detected in the receptor phase, but a fraction of sesqui- terpinene compounds together with terpinen-4-ol and α-terpineol was seen in epidermis (Cross and Roberts 2006). Close to 15 % of the applied amounts of terpinen-4-ol and α-terpineol were recovered in the receptor phase, but over all less than 3% of the applied TTO penetrated the skin within the 24 hr experimental 26 period (Cross and Roberts 2006). There was a general experimental problem relating to recovery in these studies, which the authors explain by evaporation of volatile constituents during the experimental period. The results on fractional dermal penetration of the TTO are, however, in good agreement with earlier studies described below (Hayes, Leach et al. 1997). Another experimental study using Franz cells evaluated the influence of topical application of TTO on the penetration of benzoic acid and methiocarb through human skin and identified the same three TTO constituents (terpinen-4-ol, α- terpineol, 1,8-cineole) as the only TTO constituents to quantifiable penetrate the skin (Nielsen and Nielsen 2006). Further, this study demonstrated that TTO significantly decreased the penetration rate as well as the total amount of benzoic acid and the pesticide methiocarb penetrating the skin (Nielsen and Nielsen 2006). An important observation from these studies is that apparently only the least lipophilic constituents of TTO penetrate the human skin (Nielsen and Nielsen 2006). Thus, despite a low overall dermal penetration of TTO, the constituents that do penetrate the skin, penetrate in higher amounts. The low (lack of) penetration of the more lipophilic constituents of TTO may also have implications for the risk assessment related dermal exposure to these constituents. An experimental study on human skin discs with 12 hours exposure to 200 µL (180 mg) TTO demonstrated a skin penetration of 4 mg TTO. This number was used to estimate risk in a worst case scenario based on topical application of 10 mL (9 g) neat TTO, which would correspond to a skin penetration of 200 mg TTO. This would equate some 2.8 mg/kg bw for an adult (70 kg) and 20 mg/kg for a child (10 kg). When this calculation is repeated on a 4% TTO product, assuming identical dermal penetration and exposure to 10 mL, an expected exposure would be 0.11 mg/kg for an adult and 0.8 mg/kg for a child. Histopathological assessment of skin discs exposed to 4% TTO indicated no major cellular damage apart from a few sporadic vacuolated cells (Hayes, Leach et al. 1997). Skin irritation Human data In an assessment of the skin sensitivity and irritant potential of TTO, twenty-eight volunteers received applications of 1, 2.5, 5 or 10% TTO in sorbolene cream in a double blind placebo controlled pattern in occlusive patch testing for 21 days (5 days a week for three weeks) (Altman 1991). Irritancy was rated on a scale from 0 (no irritation) to 4 (erythema with oedema and blistering). Four persons exhibited slight irritation on one or two days out of 15 observations (concentrations of TTO used in four persons experiencing one day with slight irritation were 1% TTO, 2.5% TTO, 5% TTO, 5% TTO, respectively). One person reported slight irritation on 11 out of 15 days using the 10% formulation. No volunteers treated with placebo (sorbolene) reported any skin irritation. Using a protocol based on the original Draize method, the potential of six TTO products to induce skin irritancy and/or allergenicity in humans was tested (Skin&CancerFoundationAustralia 1997). A total of 311 persons were included in the study and exposed to 100% TTO, 25% TTO in cream, 25% TTO in ointment, 25% TTO in gel, 5% TTO in cream and 5% TTO + 5% synergist in cream. No information as to the synergist was given. The test substances were applied to the skin a minimum of seven times during a three-week induction period. Skin reactions were scored on a scale ranging from no reaction (grade 0), erythema (grade 1), erythema and oedema (grade 2), vesiculation (grade 3) to bulla formation (grade 4). Irritancy was only observed after exposure to the undiluted TTO. The report argues that based on this study and the use of average values for irritancy, 27 TTO should be considered to be a low-irritant substance (Skin&CancerFoundationAustralia 1997). However, use of average values in inhomogeneous populations may not be correct. Thus, more than one third (118/306) of the participants had a positive reading for TTO on one of nine days in contrast to below 1% following exposure to the other products tested and in controls. Further, 17 persons (5.5%) had a stronger reaction than erythema on at least one day during the induction phase. Based on these observations, the conclusion might look different: The products with concentrations of TTO below or equal to 25% are not causing irritancy to the participants. Likewise, undiluted TTO is not an irritant for the vast majority of the participants, but a small fraction of the population (in this study 5.5%) seems to be more susceptible to TTO and demonstrates positive skin reactions towards undiluted TTO. Unfortunately, the report does not give information as to the distribution of skin reactions between observation days for the individuals. This information could have been valuable for a discussion of the lengths and severity of the adverse reactions in the susceptible individuals given undiluted TTO. Experimental data The effect of TTO on wound healing was evaluated in a study on rabbits with or without surgically produced wounds. The rate of wound closure was not affected by exposure to undiluted TTO during a seven days observation period (Bolt 1989). In a report on acute dermal irritation after exposure to TTO, the skin irritation index was determined by the Draize method using NZ White rabbits exposed to undiluted TTO (batch 88/375). The Draize irritation index for undiluted TTO was found to be 5.0, indicating a severe irritant (Bolt 1989). To what extent this finding is affected by occlusion causing an overestimate of the Draize index is not clear. In a report on dermal irritation in the rabbit due to TTO exposure, the test sample of TTO was applied to the dorsal skin region of six rabbits (NZ Whites) at a rate of 0.5 mL of initially undiluted TTO, but from day 2 a 25% solution in paraffin oil over an area of 15 cm2 during 30 days (Bolt 1989). Assessment of irritation was made on days 2, 7, 14, 21 and 30. Terminal skin biopsies were carried out and histological analysis performed. The undiluted TTO caused severe irritation after 24 hours. Hence the concentration was reduced to 25% (It is surprising that the authors initiate the study with undiluted TTO given that they 2 months before in an earlier report conclude that undiluted TTO is a severe irritant). The 25% solution of TTO in paraffin oil was not a visible irritant to the skin, but did cause minor (grade 1+) pathological changes consistent with mild irritation. The changes were seen as consistent with a non-specific dermatitis due to topical application of an irritant preparation. The authors conclude that the observed lesions following exposure to 25% TTO in paraffin were superficial and reversible. Two studies on acute dermal irritation/corrosion following OECD guidelines have been reported in 1996: 1 Pharmatox. Acute dermal irritation/corrosion of TTO in the rabbit (T1836.A): Guideline: OECD 404 Species/strain: Rabbit / New Zealand White Group size: 3 female rabbits Test substance: Tea tree oil (TTO) Batch No.: 28220296 Dose: 500 µL undiluted applied on 4x4 cm patches GLP: in compliance 28 TTO (100%) was found to be a mild irritant at 60 minutes post exposure, a severe irritant at 24 and 48 hours, a moderate irritant at 72 hours and a mild irritant 7 and 14 days following a 4 hour semi-occlusive patch application on intact skin. At 21 days the skin had returned to normal. 2. Pharmatox. Acute dermal irritation/corrosion of 75, 50, 25 and 12.5% TTO solutions in the rabbit (T1836.B): Guideline: OECD 404 Species/strain: Rabbit / New Zealand White Group size: 3 female rabbits Test substance: Tea tree oil (TTO) Batch No.: 28220296 Dose: 500 µL diluted to with peanut oil and applied on 4x4 cm patches GLP: in compliance TTO was applied for 4 hours with a semi-occlusive patch application followed by a 14 days observation period. The study demonstrated that: TTO (75%) was found to be a mild to moderate irritant, TTO (50%) was found to be a minimal irritant, TTO (25%) was found to be a non-irritant, TTO (12.5%) was found to be a non irritant Based on the experimental studies with TTO, it is concluded that irritant reactions will not be expected to occur at TTO concentrations below 25 %. Mucous membrane irritation The guidelines describe that tests for mucous membrane irritation should follow either OECD 405 or the alternative bovine cornea opacity-permeability test. However, the HET-CAM test (Hen’s Egg Test – ChorioAllantoic Membrane) (Gilleron, Coecke et al. 1997) is an alternative method often used in screening studies for finished cosmetic products. It is presently being validated, and may be taken up in the legislation of some EU member states (e.g. France). Using the HET-CAM test TTO products were screened for eye irritation potential under GLP conditions (Schilcher and Leuschner 1997). TTO powder and TTO ground leaf were both evaluated as non-irritant. Undiluted TTO, water-soluble TTO, 25% TTO with 5% surfactant and 10% TTO with 10% surfactant were all rated as severe irritants, whereas 5% TTO with 8% surfactant was rated as a slight irritant. However, the placebo group (0% TTO and 10% surfactant) was also rated as a severe irritant. As the surfactant by it self caused a high irritation index, the results obtained with diluted TTO cannot be used for evaluating the irritancy of TTO. It is not clear from the report whether the water-soluble TTO was tested as undiluted or as a 10% solution. Further, no information is available as to the composition of the surfactant used. These data demonstrates the importance of differentiation between testing of TTO and testing of a TTO product. Thus, the irritancy of a TTO-product need not be due to TTO, and the absence of irritancy of TTO does not assure the safety of a TTO- product. The primary eye irritation of TTO was also studied in the rabbit (female, Japanese White) under GLP conditions (Oyama 2000). Two groups of three rabbits were given a single ocular dose (0.1 mL) of TTO (1% or 5% in liquid paraffin). After instillation of the test substance, no abnormal signs in the clinical conditions were observed among the rabbits. Ocular responses using Draize’s criteria demonstrated 29 a conjunctival discharge lasting for up to six hours following instillation of 1% TTO and conjunctival redness and discharge for up to 24 hours following instillation of 5% TTO. In both groups, the maximal response was observed after one hour. Based on these observations, the author concludes, that both TTO solutions can be classified as “minimally irritating” (Oyama 2000). Skin sensitisation Human data The human data on contact dermatitis was recently reviewed in a PhD-thesis (Hayes, Leach et al. 1997). The thesis states, that there has been an increase in the number of human contact dermatitis cases due to topical application of TTO with well over a dozen published cases within the last ten years (Apted 1991; De Groot and Weyland 1992; Selvaag, Eriksen et al. 1994; Van Der Valk, De Groot et al. 1994; De Groot 1996; Bhushan and Beck 1997). The applications included 100% TTO as well as lower concentrations of TTO in different formulated products. In 1997, 1216 patients were patch tested at a dermatologic clinic (Fritz, Burg et al. 2001). Fourteen of them used products containing TTO. The patients used creams, hair products and essential oils containing Melaleuca alternifolia oil for cosmetic reasons and to treat skin infections. They were patch tested for a standard panel of allergens, topical emulgators, perfumes, plants, topical medications, metal, gloves, topical disinfectants and preservatives, dental products and rubber derivatives. Products containing Melaleuca alternifolia were tested concentrated or diluted. Seven patients with an allergic contact dermatitis due to tea tree oil were identified. Two of them also exhibited delayed type IV hypersensitivity towards fragrance- mix or colophony suggesting the possibility of cross reaction or an allergic group reaction caused by contamination of the colophony with the volatile fraction of turpentines. The allergic potential of low concentrations of Melaleuca alternifolia oil is presumed to be low on healthy skin. Photoaged Melaleuca alternifolia oil must be considered to be a stronger sensitizer (Fritz, Burg et al. 2001). The prevalence of hypersensitivity to a number of allergens was tested in a group of volunteers (Greig, Carson et al. 2000). The findings were slightly higher than in other studies. The prevalence for hypersensitivity to TTO was found to be 2.3%. The authors argue that the prevalence found might be too high due to selection bias as the population studied were self-selected (Greig, Carson et al. 2000). 219 volunteers took part in the study. Close to 50% of the volunteers demonstrated hypersensitivity to dust mites and rye grass. This is a high number compared to the around 30% fraction of people expected to react to prick tests for dust mites or rye grass. 2.4% - 4.3% demonstrated marked irritancy to 100% TTO, whereas 7.2% - 10.1% demonstrated mild irritancy to 100% TTO (Greig, Carson et al. 2000). No participants demonstrated irritancy of any kind to 10% TTO. The bias could, however, go both ways and the prevalence for hypersensitivity to TTO is close to the study by (Leach 2000). Leach concluded that TTO must be considered a mild allergen as only 2% of the 150 panellists showed an allergic reaction (Leach 2000). Based on an Italian study in 725 persons patch tested according to GIRDCA guidelines, the authors conclude that the sensitization potential of Melaleuca oil is poor, and that the response in patch tests appears to be dose dependent, and primarily observed after exposure to undiluted TTO. Positive responses to patch tests were more frequent in subjects with existing allergic contact dermatitis or atopic dermatitis (Lisi, Meligeni et al. 2000). 30 Using a protocol based on the original Draize method, the potential of six TTO products to induce skin irritancy and/or allergenicity in humans was tested (Skin&CancerFoundationAustralia 1997). A total of 311 persons were included in the study and exposed to 100% TTO, 25% TTO in cream, 25% TTO in ointment, 25% TTO in gel, 5% TTO in cream and 5% TTO + 5% synergist in cream. No information as to the synergist was given. The test substances were applied to the skin a minimum of seven times during a three-week induction period. After a two- week period without skin exposure to TTO, a single 48-hour challenge for each product was applied on a new area of skin. The report concludes that TTO is a mild allergen as only 1% of the participants (3/308) were sensitised, ie. made allergic, to TTO by means of the Draize test (Skin&CancerFoundationAustralia 1997). Experimental data In a report on skin sensitisation in the guinea pig of TTO (Bolt 1989), groups of 20 albino guinea pigs (HA strain) were tested according to the Magnusson & Kligman method. The induction procedure consisted of two intradermal injections (5% TTO in paraffin or 5% TTO with Freund´s complete adjuvant) or an epidermal induction application (undiluted TTO). The dose used for challenge was 30% TTO. The erythemal reactions were measured 24 hours after removal of the patch of the challenge test. There were no irritant responses in either group. The experimental methodology stated in the report appears to follow OECD 406 guidelines. Mutagenicity Several in vitro genotoxicity tests are available. The SCCNFP for the in vitro base level testing of cosmetic ingredients recommend three assays (adopted by SCCNFP in December 2003): 1. Gene mutation tests Bacterial reverse mutation test (Ames test, OECD 471) 2. Test for clastrogenicity In vitro mammalian cell chromosome aberration test (OECD 473) 3. Test for aneugenicity and non-disjunction In vitro micronucleus test (OECD 474) The first two tests are usually considered to provide sufficient evidence of mutagenic and/or genotoxic potential. There are, however, situations in which mutagenicity testing beyond the base level (two tests) may be required. Normally, if there is clear structural alert for mutagenicity or when some concern is raised by positive results from in vitro tests, further testing may be justified, e.g. micronucleus test in mammalian cells (OECD 474) In vitro data The mutagenicity of complete TTO was evaluated by the Salmonella/microsome assay (TA98, TA100, TA102) with and without metabolic activation (Bolt 1989). The sample of TTO was markedly antibacterial and doses above 50 µg were toxic to S. Typhimurium. At doses of 50 µg or less TTO did not demonstrate toxicity against the indicator strains. At these dose levels no reversion-inducing activity towards either of the indicator strains was observed. This study therefore indicates that TTO at doses below 50 µg is not mutagenic in this assay (Bolt 1989). A more recent study gave supporting evidence of the absence of a mutagenic potential of TTO when tested by the bacterial reverse mutation assay on the TA98 and TA100 Salmonella strains (Evandri, Battinelli et al. 2005). A study following OECD guideline 474 and testing the potential of TTO to induce micronucleis in bone marrow demonstrated absence of any chromosomal damage at 48 hours following in vivo oral exposure of mice to TTO at doses ranging from 31 6 Toxicity profiles for individual TTO constituents 6.1 Terpinen-4-ol The acute toxicity of terpinen-4-ol has been studied in mice, rats and rabbits with different administration routes (oral, subcutaneous, intraperitoneal, intramuscular, and dermal) (National Toxicology Program 2005). The dermal LD50 value in rabbits was described as above 2500 mg/kg (Opdyke 1982; National Toxicology Program 2005). Terpinen-4-ol at 400 mg/kg was administered orally to male Sprague-Dawley rats for 28 days to assess nephrotoxicity. Terpinen-4-ol did not induce changes in the morphology or function of the kidneys, and was considered to be non-toxic at this dose level (Schilcher and Leuschner 1997). The effect of terpinen-4-ol on intestinal relaxation was studied in vitro in rabbit duodenum. The intestinal relaxation induced by terpinen-4-ol is consistent with previous studies undertaken in the guinea-pig and rat ileum. The effect was dose related and was achieved at relatively low concentrations (200 µM). The relaxation of the rabbit duodenum and the decrease in spontaneous mechanical activity induced by terpinen-4-ol were promptly reversed by washing out the compound from the bath, showing functionally that terpinen-4-ol did not cause damage to the tissue contractile apparatus (Nascimento, Leal-Cardoso et al. 2005). Terpinen-4-ol (2% in gel) significantly enhanced the percutaneous permeation of hydrocortisone formulated in HPMC (hydroxypropylmethyl Cellulose) gel systems (El-Kattan, Asbill et al. 2000). Moderate irritation was seen when terpinen-4-ol (100%) was applied to intact or abraded rabbit skin for 24 h with occlusion (Opdyke 1982). Using 48 h closed patch tests and human volunteers, terpinen-4-ol (5%) in petrolatum was found to be non-irritating (Opdyke 1982). Patch testing of 10 volunteers with terpinen-4-ol (5- 10%) did not show any irritant reactions (Knight and Hausen 1994). Terpinen-4-ol (5%) was not sensitising using an open epicutaneous test in guinea pigs (Klecak 1985). Using a maximisation test and 25 human volunteers, terpinen- 4-ol (5%) did not produce sensitisation reactions (Opdyke 1982). In a patch test of seven dermatological patients with positive patch tests for TTO and seven control persons, one pre-sensitised patient gave a positive reaction to terpinen-4-ol at 1% and an additional patient when the concentration of terpinen-4-ol was increased to 10%. None of the non-sensitized controls gave positive reactions to 10% terpinen- 4-ol (Knight and Hausen 1994). Likewise, 10% terpinen-4-ol gave no response when tested in 10 guinea pigs (Knight and Hausen 1994). In a more recent study by the same group, terpinen-4-ol (10%) was applied on 11 dermatological patients, and none of them gave positive reactions to terpinen-4-ol (Hausen, Reichling et al. 1999). 33 The mutagenic potential of terpinen-4-ol was examined in the Ames test using Salmonella typhimurium (TA102, TA100 and TA98). No mutagenic effect was determined for the terpinen-4-ol component of TTO in any of the strains of Salmonella examined with or without metabolic activation. There was a clear evidence of toxicity against all Salmonella strains by terpinen-4-ol at higher dose levels. It is suggested that terpinen-4-ol may contribute significantly to the widely reported antibacterial activity of tea tree oil (Fletcher, Cassella et al. 2005). Evaluation: Irritancy: neat terpinen-4-ol induce irritancy, but not at 5-10%. Sensitisation: possibly a weak sensitiser at 10% in pre-sensitised patients. Mutagenicity: not mutagenic. Systemic toxicity: The available literature on systemic effects of terpinen-4-ol is limited. Based on the 28-days study on kidney toxicity in rats, the NOAEL after oral exposure may be estimated to be 400 mg/kg. As terpinen-4-ol on average constitutes 40% of TTO, this NOAEL for terpinen-4-ol corresponds to an oral NOAEL for TTO (based on renal toxicity of terpinen-4-ol) of 1000 mg/kg. 34 6.2 γ-Terpinene Most of the literature on γ-terpinene is more than 30 years old and several reports were not published in international journals. For regulatory purposes this literature was reviewed by Opdyke in 1976 (Opdyke 1976). The acute oral LD50 in rats was reported as 3.7 g/kg body weight and the dermal LD50 in rabbits exceeded 5 g/kg body weight (Opdyke 1976). Neat γ-terpinene was moderately irritating to intact and abraded rabbit skin when applied for 24 hours under occlusion, whereas 48 hours closed-patch test of human exposed to 5% γ-terpinene in petrolatum produced no irritation (Opdyke 1976). γ- Terpinene did not demonstrate any irritative effects or toxicity in the CAM (Chorioallantoic Membrane) assay (Demirci, Paper et al. 2004). An investigation on skin reactions to γ-terpinene (5% in soft white paraffin) using an occlusive patch test on 25 human subjects for 21 days demonstrated neither irritation nor allegic response (Southwell, Freeman et al. 1997). A maximization test carried out in 1975 by Kligman et al. on 25 volunteers using 5% γ-terpinene in petrolatum revealed no sensitization reactions. A study published in german including 20 pre-sensitized persons found one positive reaction to γ- terpinene (Opdyke 1976). γ-terpinene induced DNA damage in human lymphocytes in the comet assay at concentrations from and above 0.2mM (Aydin, Basaran et al. 2005). In contrast, it was found that below DNA damaging concentrations γ-terpinene protected lymphocytes against DNA damage induced by other chemicals (Aydin, Basaran et al. 2005). The interpretation of these data in relation to human risk is difficult as dose-comparisons between these in vitro studies and the human exposure situation with dermal exposure is complicated. Evaluation: Irritancy: neat γ-terpinene was moderately irritating in rabbits, whereas more relevant concentrations of γ-terpinene did not induce irritation in humans. Sensitisation: possibly a weak sensitiser in patients pre-sensitised to terpenes. In volunteers without a past history of allergic reactions to cosmetic products, 5% γ-terpinene did not induce any allergic response during 21 days observation Mutagenicity: No data from Ames test available. γ-terpinene induce DNA damage at high doses when tested in the Comet assay. Systemic toxicity: The available literature on systemic effects of γ-terpinene is not sufficient to reach conclusions on chronic toxicity or estimate a NOAEL. The dermal LD50 value above 5 mg/kg indicates low toxicity. 35 6.3 α-Terpinene The oral LD50 value of α-terpinene was reported to be 1680 mg/kg body weight in rats (Opdyke 1976). α-terpinene has been demonstrated in vitro (liver microsomes prepared from phenobarbital-treated rats) to dose-dependently inhibit the liver enzyme CYP2B1 (De-Oliveira, Ribeiro-Pinto et al. 1997; De-Oliveira, Fidalgo-Neto et al. 1999). An inhibited isoenzyme will affect the metabolism of those chemicals,.that depend on CYP2B1, though IC50-values between 0.1µM and 15µM in microsomal preparations will be expected to require a substantial in vivo dose to reach significant target organ concentrations. α-Terpinene (30, 60, 125 and 250 mg/kg body weight) in corn oil was given by gavage to female Wistar rats from day 6 to 15 of pregnancy. Caesarean sections were performed on day 21 of pregnancy. The number of implantation sites, living and dead foetuses, resorptions and corpora lutea was recorded. All foetuses were weighed, examined for externally visible malformations. A reduction in body weight minus uterine weight at term indicated that the two highest oral doses tested (125 and 250 mg α-terpinene/kg body weight) were maternally toxic. Signs of delayed ossification (poorly ossified and not ossified bones as well as irregular spongy bones) and a higher incidence of minor skeletal malformations were observed at doses of 60 mg/kg body weight or more. These findings indicate that the no-observed-adverse-effect level for α-terpinene-induced embryofoetotoxicity can be set at 30 mg/kg body weight by the oral route (Araujo, Souza et al. 1996). Ten hours following application of 0.1% α-terpinene on rat abdominal skin histopathology demonstrated effects on epidermis (liquefaction, desquamation) as well as dermis/hypodermis (collagen fiber swelling) (Kitahara, Ishiguro et al. 1993). The same authors also demonstrated that α-terpinene at a concentration in cell cultures of 0.1% caused significant toxicity by affecting the cell survival of human keratinocytes (Kitahara, Ishiguro et al. 1993). The irritancy of α-terpinene, terpinolene and limonene to rabbits was evaluated by the Draize test (Okabe, Obata et al. 1990). Terpinolene was more irritating than limonene, which was in turn more irritating than α-terpinene. The interpretation of the in vitro observations in relation to irritation of human skin is complicated, as evidence from the study in rabbits and human evidence does not appear to demonstrate the same degree of toxicity to the skin. Thus, using 48 h closed patch tests and human volunteers α- terpinene (5%) in petrolatum was non-irritating (Opdyke 1976). Using a maximisation test and 25 human volunteers, α-terpinene (5% in petrolatum) did not produce sensitisation reactions (Opdyke 1976). However, in a more recent study α-terpinene appears to be among the most important allergens of TTO for which the prevalence of allergic response among patients visiting dermatological clinics vary between 0.4% and 0.6% (Hausen 2004). The mutagenicity of α-terpinene was evaluated by the Salmonella/microsome assay (TA100, TA98, TA97a and TA1535 tester strains), without and with addition of an extrinsic metabolic activation system (rat liver S9 fraction induced by Aroclor 1254). Results from the present study indicated that α-terpinene is not mutagenic in the Ames test (Gomes-Carneiro, Viana et al. 2005). 36 Evaluation: Irritancy: based on human evidence, α-terpinene will not be expected to induce irritancy at a concentration of 5%. Sensitisation: equivocal evidence. α-terpinene is probably a weak sensitiser in pre-sensitised patients. Mutagenicity: not mutagenic. Systemic toxicity: There is limited evidence of systemic toxicity following exposure to α-terpinene. However, the study on α-terpinene- induced embryofoetotoxicity suggests a NOAEL for α- terpinene of 30 mg/kg body weight. As α-terpinene on average constitutes 9% of TTO, this NOAEL for α-terpinene corresponds to an oral NOAEL for TTO (based on α-terpinene embryotoxicity) of 330 mg/kg. 37 6.4 1,8-Cineole – eucalyptol The acute oral LD50 in rats 2480 mg/kg bw (Jenner, Hagan et al. 1964). The European Commission (European Commission 2002) states that eucalyptol undergoes oxidation in vivo with the formation of hydroxycineole which is excreted as glucuronide. In rats, 2-hydroxycineole, 3-hydroxycineole and 1,8- dihydroxycineol-9-oic acid were identified as main urinary metabolites (Madyastha and Chadha 1986). Single subcutaneous doses of 250 or 500 mg/kg bw increased the activity of drug- metabolizing enzymes and stimulated bile flow (Jori, Di Salle et al. 1972). Liver microsomal-enzyme activity was greatly enhanced in adult rats treated with eucalyptol both during and after pregnancy and was also increased in the foetal and newborn offspring of such rats. In these offspring, a more marked stimulation of the generally poor drug-metabolizing capacity was demonstrated in connection with the O- demethylation of p-nitroanisole than with the p-hydroxylation of aniline. Suckling rats treated directly with eucalyptol also showed an increase in liver-enzyme activity, but administration of the oil to lactating mothers did not lead to any enzyme induction in the suckling rats. It thus appears that while eucalyptol is able to penetrate the placental barrier and reach a concentration in the foetal blood high enough to stimulate hepatic enzyme activity, it is unable to cross the blood-milk barrier to any effective extent. Its placental mobility is compatible with its high lipid solubility, a property reported to have a direct bearing on placental penetration (Jori and Briatico 1973). In relation to another member of the CYP-family of liver enzymes, 1,8-cineole has been found in vitro (liver microsomes prepared from phenobarbital-treated rats) to dose- dependently inhibit the liver enzyme CYP2B1 (De-Oliveira, Ribeiro-Pinto et al. 1997; De-Oliveira, Fidalgo-Neto et al. 1999). Apparently, 1,8-cineole induces or inhibite different liver enzymes, thus potentially affecting hepatic metabolism. Cineole in a concentration of 5% enhance the skin permeation of propranolol in polymer films significantly (Amnuaikit, Ikeuchi et al. 2005). However, in another study on the enhancing effect of naturally occurring terpenes, 1,8-cineole was demonstrated to be a poor enhancer of the in vitro percutaneous absorption of diclofenac sodium from carbopol gels containing propylene glycol (Arellano, Santoyo et al. 1996). 1-8-cineole acts to reduce the intensity of lipid based reflections. Decreases in reflection intensities may be linked to a disruption of lipid packing within the bilayers and/or to a disturbance in the stacking of the bilayers (Cornwell, Barry et al. 1996). Ten hours following application of 0.1% cineole on rat abdominal skin histopathology demonstrated no effects on epidermis (liquefaction, desquamation) as well as dermis/hypodermis (collagen fiber swelling) (Kitahara, Ishiguro et al. 1993). The same authors also demonstrated that cineole at a concentration in cell cultures of 0.1% did not cause cell toxicity by affecting the cell survival of human keratinocytes (Kitahara, Ishiguro et al. 1993). Groups of 6 male and 6 female B6C3F1 mice were fed eucalyptol for 28 days either by stomach tube on 5 days/wk at doses of 150, 300, 600 and 1200 mg/kg bw or in encapsulated form at concentrations of 3750, 7500, 15000 and 30000 mg/kg, equivalent to 600 – 5607 mg/kg bw/day for male and 705-6777 mg/kg bw/day for female mice. The liver weight/body weight ratio in males was increased at all but the lowest dose given in encapsulated form as was the brain weight/body weight ratio in females at the top dose level. Microscopic examination revealed a minimal hypertrophy of centrilobular hepatocytes in animals of both sexes fed the encapsulated compound, especially at the two highest dose levels (Wolff et al, 1987b). A parallel study with groups of 6 male and 6 female Fischer 344 rats exposed the animals to eucalyptol for 28 days either by stomach tube on 5 days/wk 38 at doses of 150, 300, 600 and 1200 mg/kg bw or in encapsulated form with the diet at concentrations of 3750, 7500, 15000 and 30000 mg/kg, equivalent to 381 – 3342 mg/kg bw/day for the male rats and to 353 – 3516 mg/kg bw/day for the female rats (Wolff et al., 1987a cited in (European Commission 2002)). At dose levels of 600 mg/kg bw and higher, dose-related decrease of body weight gain and absence of a normal degree of hepatic centrilobular cytoplasmic vacuolization was observed in male rats. In addition, other dose-related lesions in the liver, kidneys and parotid salivary glands were found at all dose levels in male rats fed encapsulated eucalyptol (Wolff et al., 1987a cited in (European Commission 2002)). Groups of 10 male Wistar rats were given 0, 500, or 1000 mg eucalyptol/kg bw/day by gavage for 28 days. Statistically significant decreases in the terminal body weight and increased relative liver and kidney weights were found in both dose groups, whereas the relative brain weight was increased only in the highest dose group. No macroscopical changes were seen. Only brain, liver and kidneys were examined histopathologically, showing no changes in the brain and minor focal infiltration of mononuclear cells in the liver among all groups. In kidneys, a dose- related accumulation of eosinophilic protein droplets containing α2u-globulin in the cytoplasma of proximal tubular epithelial cells was induced (Kristiansen and Madsen 1995). Eucalyptol was tested as constituent of toothpaste in an oral long-term study with specific pathogen-free CFLP mice. Groups of 52 male mice were given 0, 8 and 32 mg eucalyptol/kg bw/day in 1 ml toothpaste base/kg bw/day by gavage 6 days/week for 80 weeks followed by an observation period between 16 and 24 weeks according to the number of survivors. No treatment-related effects on body weight, food consumption, survival, weight of adrenals, kidneys, liver, lungs or spleen, on the microscopic appearance of brain, lungs, liver and kidneys and on the tumour incidence were observed (Roe, Palmer et al. 1979). Evaluation of skin damage and cytotoxicity of 1,8-cineole on rat abdominal skin showed no irritation (Kitahara, Ishiguro et al. 1993). Using 48 h closed patch tests and human volunteers, 1,8-cineole (16%) in petrolatum was non-irritating (Opdyke 1975). Patch testing of 7 volunteers with 1,8-cineole did not show any irritant reactions (Knight and Hausen 1994).Skin irritancy following occlusive patch testing for 21 days was not detected in 28 humans exposed to any of eight preparations of pure cineole in concentrations ranging from 3.8% to 28.1% in soft paraffin (Southwell, Freeman et al. 1997). Among a group of 25 human subjects without prior allergic reactions to cosmetic products none gave a positive response when tested with up to 28.8% 1,8-cineole in a TTO mixture with terpinen-4-ol in the occlusive patch test (daily readings and replacement for 21 days) (Southwell, Freeman et al. 1997). Three participants in the same study initially gave an allergic reaction to TTO, but when retested with new and substantially pure 1,8-cineole (1.4%) no reactions were found. The authors argue that impurities or oxidation products might have influenced their first trial and that 1,8-cineole is not an allergen (Southwell, Freeman et al. 1997). Likewise, in a study in 11 human subjects sensitized to TTO none demonstrated allergic reaction to the 1,8-cineole constituent (5%) nor could its sensitizing capacity be shown in experimentally sensitized guinea pigs exposed to 5% 1,8- cineole (Hausen, Reichling et al. 1999). 1,8-cineole was not mutagenic when evaluated by the Salmonella/microsome assay (TA97a, TA98, TA100 and TA102 tester strains), without and with addition of an extrinsic metabolic activation system (lyophilized rat liver S9 fraction induced by Aroclor 1254) (Gomes-Carneiro, Felzenszwalb et al. 1998). Eucalyptol did not 39 show mutagenic effects in the following strains of Salmonella typhimurium with or without metabolic activation: TA 98, TA 100, TA 1535 and TA 1537 (Haworth, Lawlor et al. 1983). In CHO cells, eucalyptol did not induce chromosome aberrations with or without metabolic activation. Sister chromatid exchanges were induced in CHO cells only in the absence of metabolic activation at doses that induced cell cycle delay (Galloway, Armstrong et al. 1987). Sister chromatid exchanges induced by mitomycin C in CHO K-1 cells were not increased by posttreatment with eucalyptol (Sasaki, Imanishi et al. 1989). Cineole at concentrations ranging from 10 – 1000 μM did not increase the frequency of spontaneous sister-chromatid exchanges in Chinese hamster ovary cells (Sasaki, Imanishi et al. 1989). A number of in vitro studies have come to different conclusions in relation to mutagenicity, chromosomal damage. Taken together, it is the impression that 1,8-cineole is possibly a weak promoter. If 1,8-cineole acts as a promoter only and not by itself damages DNA, it may be defendable to calculate no-effect-levels. Cineole did not induce primary lung tumors in male or female A/He mice following 24 ip injections during an eight week period with 24 weeks follow-up. The doses used were either MTD (maximal Tolerated Dose) or 20% of MTD (Stoner, Shimkin et al. 1973). BIBRA in 1991 suggested a NOAEL for eucalyptol of 300 mg/kg. Using an uncertainty-factor of 100, this would give an estimated ADI of 3 mg/kg anticipating 100% dermal absorption in the absence of specific data on dermal absorption. Based on these estimates, the Norwegian Food Control Authorities in 1999 calculated, that exposure to eucalyptol using a 1 gram facial cosmetic TTO cream daily holding 3% TTO of which 15% was eucalyptol would cause an exposure of approximately 2% of the ADI (Acceptable Daily Intake, Norwegian Food Control Authorities, December 1999). Other exposure scenarios with other TTO products were presented, none of them causing an exceeded ADI for eucalyptol. Currently eucalyptol is regarded as GRAS (generally recognised as safe) by FEMA (1965) and is approved by the US Food and Drug Administration (FDA) for food use. The FDA advisory review panels on over-the-counter drugs have concluded that eucalyptol is safe for a variety of products, such as lozenges taken every 0.5 - 1 hr at 0.2 – 15 mg or taken every 2 hrs at 1 –30 mg of eucalyptol (US Food and Drug Administration, 1976 – 1990). Maximum concentrations of eucalyptol in cosmetic products have been reported to be 0.4% in soap, 0.04% in detergents, 0.1% in creams and lotions and 1.6% in perfume (Opdyke 1975). Evaluation: Irritancy: Neither animal nor human evidence indicate that 1,8-cineole should have any significant potential as irritant. Sensitisation: Available information from animal as well as human exposure indicate that pure 1,8-cineole is not a sensitiser. Mutagenicity: 1,8-cineole is not mutagenic in Ames test. 1,8-cineole is possibly a weak promoter, but not carcinogenic in mice tested at MTD. Systemic toxicity: Based on the studies on hepatic and renal toxicity a NOAEL might be estimated as 300 mg/kg body weight, which is in agreement with the BIBRA evaluation from 1991 and used by the Norwegian Food Control Authorities in 1999. As 1,8- cineole on average constitutes 5% of TTO, this NOAEL for 40 6.5 Terpinolene Terpinolene has low acute toxicity. Oral and dermal LD50s are 3800 mg/kg in rats and mice, and >5000 mg/kg in rabbits (Opdyke 1988). A study on the skin penetration from matrix-type transdermal systems demonstrate that close to 12% of the dose penetrate the epidermis within 8 hour, corresponding to 0.7 mg/cm2 in this experimental setup (Cal, Janicki et al. 2001). Terpinolene applied full strength to intact or abraded rabbit skin for 24 hr under occlusion was not irritating (Opdyke 1976). Tested at 20% in petrolatum it produced no irritation after a 48-hr closed-patch test on human subjects (Opdyke 1976). Ten hours following application of 0.1% terpinolene on rat abdominal skin histopathology demonstrated effects on epidermis (liquefaction, desquamation) as well as dermis/hypodermis (collagen fiber swelling) (Kitahara, Ishiguro et al. 1993). The same authors also demonstrated that terpinolene at a concentration in cell cultures of 0.1% caused significant toxicity by affecting the cell survival of human keratinocytes (Kitahara, Ishiguro et al. 1993). The interpretation of this study is difficult considering the effects observed at very low concentrations of terpinolene compared to the concentrations applied to human skin in vivo with no irritant effects. To gain credibility, these observations on cell cultures need to be repeated. In a maximization test on 24 volunteers terpinolene was tested at a concentration of 20% in petrolatum and produced no sensitization reactions (Opdyke 1976). In an older study on occupational skin disorders, terpinolene was found not to be a sensitizer for human skin (Woeber and Krombach 1969). A high fraction of TTO- sensitised patients with existing skin disease demonstrated positive patch tests against terpinolene when tested with 10% oil in ethanol (17 out of 18), whereas patch testing with terpinolene (1%) did not show any erythema (Knight and Hausen 1994; Hausen, Reichling et al. 1999). A 52-year-old man developed an acute contact dermatitis after application of undiluted TTO to his scalp. Patch tests revealed a specific hypersensitivity to TTO and to 6 of its constituents including terpinolene (Reindl, Gall et al. 2000). Terpinolene was given the status of a “generally regarded as safe” (GRAS) direct food additive by the Flavor Extract Manufacturers Association (FEMA No. 3046) in 1965 and is approved by the FDA for use in foods (Opdyke, 1988). The Council of Europe included terpinolene in the list of artificial food flavouring substances that may be added to food without risk to human health in 1974 (Opdyke, 1988). Significant dietary exposure to terpinolene occurs through ingestion of such foods as ice cream and ices (64 mg/kg), candies (0.12 - 48 mg/kg), non-alcoholic beverages (16 mg/kg), and baked goods (49 mg/kg). Dermal exposure can occur from such products as soaps (200 – 4000 mg/kg), lotions (100 –1000 mg/kg), perfumes (1200 – 5000 mg/kg), and detergents (20 – 400 mg/kg). Evaluation: Irritancy: Human evidence indicates that terpinolene does not cause irritant reactions at exposures below 20%. Sensitisation: Available information from human exposure indicate that terpinolene is a weak sensitiser in pre-sensitised individuals (no effect at 1%, significant response at 10%). 42 Mutagenicity: No mutagenicity, genotoxicity, or carcinogenicity studies were identified for terpinolene Systemic toxicity: No subchronic, chronic, or foeto- toxicity studies were identified for terpinolene, and a NOAEL for systemic toxicity can not be estimated. Based on irritancy and sensitization a NOAEL of 1% may be suggested. As terpinolene constitutes approximately 3.3% of TTO, this would equal a NOAEL of 30% for TTO regarding irritancy and sensitization. 43 6.6 p-Cymene p-Cymene is formed through oxidation of α-terpinene or γ-terpinene (McGraw, Hemingway et al. 1999), and oxidised tea tree oil contains increased levels of p- cymene and decreased levels of α-terpinene, γ-terpinene (Brophy, Davies et al. 1989; Hausen, Reichling et al. 1999; Hausen 2004). Cumene is a different chemical than cymene, as cumene is lacking the methyl-group residing in the para-position on p-cymene. Studies on the metabolism of p-cymene in rabbits do not indicate that cumene is among the primary metabolites of p-cymene as metabolism appears to affect the isopropyl group (Matsumoto, Ishida et al. 1992). However, as studies on the further metabolism of cumene and cymene demonstrate considerable similarities, they are expected to have comparable toxicological profiles. Thus, several recent toxicological evaluations, including one by for US EPA (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002), have due to insufficient data on p-cymene used data on cumene to develop NOAEL values for cymene. This approach will also be used in this report with the exceptions of evaluations of sensitising and mutagenic effects where even minor chemical differences may have implications for the outcome. The acute oral LD50 of p-cymene in rats was reported as 4750 mg/kg (Jenner, Hagan et al. 1964). The lethal dose by ip administration was 2162 mg/kg in the guinea-pigs and the acute dermal LD50 of p-cymene in rabbits was reported as > 5000 mg/kg (Opdyke 1974). p-Cymene is well absorbed through the skin. In studies with 14C-labelled p-cymene on mice and rats, the penetration observed was around 250 µg/cm2 in 60 min (Wepierre 1963; Wepierre 1963). Likewise, cumene is rapidly absorbed by oral administration or inhalation exposure (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002). Following absorption, the ring substituent is oxidized to yield aromatic alcohol and carboxylic acid metabolites that are excreted free or conjugated in the urine. There is no evidence that cumene accumulates in the body even following high dose or repeat dose exposure (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002). The effects of p-cymene on the brain chemistry of rats was studied by exposing male Long-Evans rats to 0, 50 or 250 ppm p-cymene by inhalation (Lam, Ladefoged et al. 1996). Rats were exposed for 6 hours per day, 5 days per week for four weeks and then had an 8 week wash-out period. No obvious toxicity was seen during the exposure period and body weights did not differ after the 12 week trial period. Levels of synaptosomal protein were significantly reduced in treated rats, whereas relative amounts of noradrenaline and dopamine were increased (Lam, Ladefoged et al. 1996). The doses used were, however, in excess of the occupational TLV’s (Threshold Limit Values), and the relevance of the study is probably limited in relation to topical use of TTO oil with minor amounts of cymene. Repeat dose toxicity studies have been performed with cumene (Wolf, Rowe et al. 1956; Cushman, Norris et al. 1995). In the only oral toxicity study on cumene, rats were gavaged with cumene up to 769 mg/kg bw/day, 5 days/week for a period of 6 months (Wolf, Rowe et al. 1956). Following necropsy and hematological examination, the only effect reported was an increase in average kidney weight (not specified if absolute or relative weight) in the 2 highest dose groups (no statistical analysis). This finding was not accompanied by histopathological renal changes. In all probability the kidney weight changes may be early indications of 44 species and sex specific alpha-2μ-globulin-induced nephrotoxicity. Other terpene hydrocarbons including limonene and camphene have been reported to produce alpha-2μ-globulin-induced nephrotoxicity in male Fisher 344 rats. This phenomenon is specific to Fisher 344 male rats and has neither been observed in other sexes or strains of rats, other rodents, nor in humans. A recent well-conducted developmental toxicity study was conducted with cumene in CD rats and New Zealand white rabbits. Rats and rabbits were used to assess the potential developmental toxicity of cumene (Darmer Jr, Neeper-Bradley et al. 1997). Pregnant rats were exposed to atmospheres containing up to 1,200 ppm of cumene inhalation, 6 hours/day during gestation days 6-15 and pregnant rabbits were exposed at up to 2,300 ppm of cumene 6 hours/day during gestation days 6- 18. In rats, reported effects included reduced food consumption, reduced body weight gain, perioral wetness, encrustation, and increased relative maternal liver weight. No statistically significant effects were reported in the fetuses. In rabbits, the reported effects included, death of 2 does at the highest concentration, reduced body weight gain, reduced food consumption, increased incidence of perioral wetness, lung color changes in 33% of high-dose does, and increased relative maternal liver weight. No statistically significant effects were reported in the fetuses. There was a significant increase in the incidence of skeletal and visceral variations; however, they were not exposure related. In reviewing this study, EPA (EPA 1997) set the maternal NOAEL at 488 ppm in rats based on the significant decrease in body weight gain during exposure and increased relative liver weight. Even at maternally toxic concentrations, exposure to cumene vapor did not produce developmental toxicity in rats. In further review of this study, EPA determined that the changes in gestational parameters of the rabbits, though not significant, were consistent in indicating possible developmental effects and therefore set the NOAEL in rabbits for both developmental and maternal effects at 1,206 ppm and the LOAEL at 2,297 ppm, respectively (EPA 1997). p-cymene was not irritating when assessed in vitro using the HET-CAM assay (Demirci, Paper et al. 2004). Moderate irritation was seen when neat p-cymene was applied to intact or abraded rabbit skin for 24 h with occlusion (Opdyke 1974). Using 48 h closed patch tests and human volunteers p-cymene (4%) in petrolatum applied daily for 10 days to the same spot on the backs of volunteers did not produce irritation (Opdyke 1974). Patch testing of 10 volunteers with 1% p-cymene did not show any irritant reactions (Knight and Hausen 1994). Eye irritation thresholds in humans between 100 ppm and 1000 ppm has been determined for p- cymene (Cometto-Muñiz, Cain et al. 1998; Cometto-Muñiz, Cain et al. 1998). p-Cymene (4%) was not sensitising using an open epicutaneous test in guinea pigs (Klecak 1985). Using a maximisation test and 25 human volunteers p-cymene (4%) did not cause positive responses (Opdyke 1974). The results of the patch testing of 21 TTO-sensitised individuals with TTO components (Knight and Hausen 1994; Southwell, Freeman et al. 1997 Knight, 1994 #362; Hausen, Reichling et al. 1999) demonstrated that one patient gave a positive response to p-cymene at 1%. An extensive review on the toxicity of cymene and cumene has recently (2002) been submitted to the US EPA under the HPV (high production volume) Challenge Program by The Flavor and Fragrance High Production Volume Chemical Consortia (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002). This review includes detailed descriptions of the available literature including data on metabolism, accumulation, elimination, and potential for systemic toxicity as well as mutagenicity/genotoxicity. Regarding possible mutagenic/genotoxic effects, they conclude: that p-Cymene produced no increase in the frequency of mutations when tested in Sd-4-73 Escherichia coli. 45 Concentrations up to 2,000 μg/plate of cumene did not increase the number of revertants in Salmonella typhimurium strains (TA97, TA98, TA100, TA1535, and TA1537) in the Ames preincubation assay with or without metabolic activation (NTP unpublished results). In cultured mammalian cells, cumene showed no consistent evidence of mutagenicity or genotoxicity at non-cytotoxic concentrations. Cumene did not increase mutations in the CHO/HGPRT test with or without metabolic activation at concentrations of up to 175 μg cumene/plate. Cultured rat hepatocytes treated with cumene up to 5,000 μg/ml showed cytotoxicity at concentrations of 128 μg/ml and higher and unscheduled DNA synthesis was reported at 16 μg/ml. However, the authors note that the results between triplicates were highly variable and inconsistent (The Flavour and Fragrance High Production Volume Consortia - the Terpene Consortium 2002). Cymene was found to be non-mutagenic using bacterial assays such as the Ames test (Rockwell and Raw 1979). The US EPA has concluded that cumene does not appear to metabolize to highly reactive chemical species and in terms of metabolism, cumene is analogous to methyl benzene for which a 2-year inhalation study was conducted by NTP in 1990, and no evidence of carcinogenic activity was reported in either rats or mice (EPA 1997). Overall, the EPA concluded “there is not much suspicion that cumene would pose a significant carcinogenic hazard.” Evaluation: Irritancy: p-cymene is a moderate irritant to rabbits at high concentrations. Tested up to 4%, p-cymene was not an irritant to humans. Sensitisation: Based on the available information from human exposure, p- cymene is not expected to be a sensitiser. Mutagenicity: Neither p-cymene nor cumene appear to be mutagenic or genotoxic at non-cytotoxic concentrations. There is not much suspicion that cumene would pose a significant carcinogenic hazard to humans. Systemic toxicity: A limited number of relevant repeat-dose studies are available and the inhalation route is often used for cumene. A NOAEL of 488 ppm based on inhalation might be suggested as might also a LOAEL of 769 mg based on the only study with oral exposure. Based on the oral study and using an uncertainty factor of 10, a NOAEL for cumene/p-cymene of 75 mg/kg body weight is suggested. As p-cymene on average constitutes 6% of TTO, this NOAEL for p-cymene corresponds to an oral NOAEL for TTO (based on possible renal effects of p- cymene) of 1200 mg/kg body weight. 46 6.7 α-Pinene In a memorandum from April 2005 (EPA 2005), the US EPA (registration division, office of prevention, pesticides and toxic substances) has assessed the toxicity of α- and β-pinene and conclude: α-and β-pinene are the major components of turpentine. The two chemicals are closely related, having the same empirical formula of C10H16 and the same basic ring structure. The predominant uses of the pure forms of α- and β-pinene are as fragrances. Dermal LD50 (rabbit) for α- as well as β-pinene was larger than 5000 mg/kg bw (Opdyke 1978; Opdyke 1978). A patient attempting suicide ingested 400-500 ml pine oil and was admitted to the clinic. Since more than the lethal dose had been ingested hemoperfusions with activated charcoal and amberlite and hemodialysis were performed. The composition of the ingested pine oil was determined by gas chromatography/mass spectrometry. Four monoterpenes were identified: 57% α-pinene, 8% β-pinene, 26% carene, 6% limonene and 3% other hydrocarbons. The blood and urine monoterpene concentrations were continuously monitored. The data suggest that monoterpenes are poorly resorbed in the gastrointestinal tract. The resorbed portion of the hydrocarbons cumulates in the lipophilic body compartments and is slowly metabolized and then excreted by the kidneys. The main metabolic pathways are hydration, hydroxylation, and rearrangement, and acetylation. Five metabolites were identified (Koppel, Tenczer et al. 1981). α-Pinene is well-absorbed via the skin, lungs, and gastro-intestinal tract (EPA 2005). In a 3-month oral toxicity study, rats were fed an α-pinene resin or pinene polymer made predominantly from α-pinene. (The ratio of α-and β-pinene was 10:1.) The dose levels were 0, 1, 3 or 5% in the diet. Effects seen at 5% (3967 mg/kg/day) included an increase in relative liver weight in both sexes, and absolute liver weight in females only. Increased relative thyroid weights in males were noted at the 3 and 5% dose levels. In the absence of histopathological alterations, these changes were not considered treatment related. No effects were noted at 1%, which corresponds to roughly 800 mg/kg/day (EPA 2005). The effect of oral pretreatment with α-pinene on the hexobarbital sleeping time was examined in healthy female rats and rats rendered cirrhotic with thioacetamide. After pretreatment with α-pinene the sleeping time of both healthy and cirrhotic rats was significantly shortened. This is attributed to microsomal enzyme induction (Marosi, Pap et al. 1973). A mixture of α- and β-pinene (and other terpene hydrocarbons) was tested in three developmental toxicity studies. Summaries of the results of these studies report that no maternal or developmental effects were noted in mice, hamsters, or rats at the highest dose levels, 560, 600, or 260 mg/kg/day, respectively. α- and β-pinene are not structurally related to any known developmental or reproductive toxicants (EPA 2005). Undiluted α-pinene applied to the backs of hairless mice and swine was not irritating. However, once applied to intact or abraded rabbit skin for 24 hr under occlusion it was a moderate irritant. When tested at 10% in petroleum it produced 47 no irritation after a 48 hr closed patch test on two different panels of human subjects. Beta pinene applied full strength to intact or abraded rabbit skin for 24 hr under occlusion was a moderate irritant. When tested at 12% in petroleum it produced no irritation after a 48 hr closed patch test on human subjects (EPA 2005). Too few human subjects in an in vivo exposure chamber reported eye- irritation for α-pinene and α-terpineol to allow estimates of thresholds of these compounds which therefore have much less irritative potency than n-butanol, 3- carene, and limonene (Mølhave, Kjaergaard et al. 2000). In a dermal human sensitization study, α- and β-pinene produced no dermal sensitization when tested at concentration of 10% and 12% in petroleum, respectively (EPA 2005). In experiments with oil of turpentine and α-pinene it was shown that only the autoxidation products of oil of turpentine and not the terpenes themselves were eczematogenic. Autoxidation of a-pinene in the presence of air and light was sufficient to produce the eczematogenic agent, but its formation could be prevented by addition of inhibitors such as hydroquinone and pyrogallol (Opdyke 1978). No phototoxic effects were reported for undiluted α-pinene on hairless mice and swine (Opdyke 1978). The mutagenicity of (+) and (-)-α-pinene was evaluated by the Salmonella/microsome assay (TA100, TA98, TA97a, TA1535, and TA1537 tester strains), without and with addition of an extrinsic metabolic activation system (rat liver S9 fraction induced by Aroclor 1254). Results indicated that (+) and (-)-α- pinene are not mutagenic in the Ames test (Florin, Rutberg et al. 1980; Gomes- Carneiro, Viana et al. 2005). β-pinene at concentrations ranging from 10 – 1000 μM did not increase the frequency of spontaneous sister-chromatid exchanges in Chinese hamster ovary cells (Sasaki, Imanishi et al. 1989). No chronic or carcinogenicity studies were identified; however, α- and β-pinene are not structurally related to any known carcinogens (EPA 2005). Evaluation: Irritancy: Neat α- and β-pinene are moderate irritants to rabbits but not to mice and swine. Tested at 10-12% in petroleum neither α- nor β-pinene were irritants to humans. Sensitisation: Based on the available information from human exposure, the pinenes are not expected to be sensitisers. The oxidation product may be eczematogenic. Mutagenicity: Neither α- nor β-pinene appear to be mutagenic or genotoxic. Systemic toxicity: Given the low acute toxicity by the oral, dermal and inhalation routes, the low subchronic toxicity, the lack of reproductive or developmental effects at high dose levels, and the extensive naturally-occurring (primarily inhalation and oral) exposures, the US EPA concluded that a quantitative approach was not needed. If a NOAEL had to be developed it could be based on the study on developmental toxicity in the most susceptible animal species and result in a NOAEL for pinene of 260 mg/kg/day. As α-pinene on average constitutes 3.5% of TTO, this NOAEL for α-pinene corresponds to an oral NOAEL for TTO (based on possible developmental effects of α-pinene) above 7000 mg/kg body weight. 48 6.8 α-Terpineol The acute toxicity of α-terpineol is limited with an oral LD50 value in rats above 4000 mg/kg and a dermal LD50 value in rabbits above 3000 mg/kg (Opdyke 1974). However, human cases of intoxication include several cases of accidental ingestion of large amounts of α-terpineol (400-500 mL) with fatal as well as non-fatal outcome and including young as well as older individuals (Hill, Barer et al. 1975; Welker and Zaloga 1999; Cording, Vallaro et al. 2000). In his review on terpineol Opdyke (Opdyke 1974) describe that terpineol is rapidly absorbed through the intact shaved abdominal skin of the mouse. Ten (10) male and 10 female weanling Osborne-Mendel rats were fed alpha- terpineol acetate in the diet for 20 weeks at concentrations of 0, 1000, 2500 or 10,000 ppm (Hagan, Hansen et al. 1967). These dietary levels were calculated by the US FDA to result in daily intakes of 0, 50, 125 and 500 mg/kg bw, respectively. All animals were examined for growth, hematology, and macroscopic changes in the tissues. Microscopic examination was performed on 6-8 male and female animals in the high dose and control groups. No statistically significant adverse effects were reported (Hagan, Hansen et al. 1967). Terpineol has been demonstrated in vitro (liver microsomes prepared from phenobarbital-treated rats) to dose-dependently inhibit the liver enzyme CYP2B1 (De-Oliveira, Ribeiro-Pinto et al. 1997; De-Oliveira, Fidalgo-Neto et al. 1999). An inhibited isoenzyme will affect the metabolism of those chemicals.that depend on CYP2B1, though IC50-values between 0.1µM and 15µM in microsomal preparations will be expected to require a substantial in vivo dose to reach significant target organ concentrations. Ten hours following in vitro application, 0.1% α-terpineol caused significant cytotoxicity by affecting the cell survival of human keratinocytes and fibroblasts in vitro (Kitahara, Ishiguro et al. 1993). Moderate irritation was seen when terpineol was applied to intact or abraded rabbit skin for 24 h with occlusion (Opdyke 1974). Investigation of the irritant capacity of several terpenes by transepidermal water loss (TEWL) and histological observations suggested that α-terpineol is potentially irritating (Fang, Hung et al. 2003). Evaluation of skin damage and cytotoxicity of a range of terpenes on rat abdominal skin showed no irritation for α-terpineol (Kitahara, Ishiguro et al. 1993). Patch testing of 10 volunteers with 1% α-terpineol did not show any irritant reactions (Knight and Hausen 1994). Using 48 h closed patch tests and human volunteers, terpineol (12%) in petrolatum was non-irritating (Opdyke 1974). In a larger multicenter study, a set of 5 to 10 fragrances at 2 concentrations was patch tested in each centre on a minimum of 100 consecutive patients seen in the patch test clinic. These patients were also patch tested to a standard series with the 8% fragrance mix and its 8 constituents. In patients with a positive reaction to any of 48 food fragrancies, a careful history with regard to past or present reactions to perfumed products was taken. A total of 1323 patients were patch tested in 11 centres and none of them demonstrated irritancy or allergic response to α-terpineol (Frosch, Pilz et al. 1995). An earlier study by six of the same dermatological departments demonstrated that among 18 fragrances tested in 1606 consecutive patients from these dermatological clinics, the lowest reactivity was observed with α-terpineol, yelding only 1 positive (<0.1%) and 11 doubtful reactions in a patch 49 test with 5% α-terpineol (Frosch, Johansen et al. 2002) The results of the patch testing of 10 TTO-sensitised individuals with α-terpineol (1.1-1.3%) did not demonstrate any positive response (Knight and Hausen 1994; Southwell, Freeman et al. 1997). Terpineol was negative using the Bacillus subtilis rec- assay (Oda, Hamano et al. 1978). α-Terpineol was not mutagenic when assayed for mutagenicity towards four Salmonella-strains (TA 98, TA 100, TA 1535, TA 1537) with and without metabolic activation (Florin, Rutberg et al. 1980). Terpineol caused a slight but dose-related increase in the number of hisq revertants with TA102 tester strain both without and with addition of S9 mixture, but results were negative with the TA97a, TA98, and TA100 tester strains (Gomes-Carneiro, Felzenszwalb et al. 1998). Terpineol did not induce primary lung tumors in male or female A/He mice following 24 ip injections during an eight week period with 24 weeks follow-up. The doses used were either MTD or 20% of MTD (Stoner, Shimkin et al. 1973). α-Terpineol was included in a study on the male rat specific renal toxicity/carcinogeniciy mediated through formation of hyaline droplets (this species-specific toxic effect is also discussed in relation to limonene). The authors evaluated a new ligand displacement model, and α-terpineol as well as its metabolites demonstrated minimal ligand displacement (Lehman-McKeeman and Caudill 1999). Thus, α-terpineol had a binding affinity for the ligand that was 175 times lower than the positive control and seven times lower than limonene, and the binding affinity for the metabolites were lower (Lehman-McKeeman and Caudill 1999). The data demonstrates that α-terpineol does not induce this kind of renal toxicity. Evaluation: Irritancy: An in vitro study and a study on rabbits indicate that α- terpineol is an irritant at high concentrations. Human dermal exposure to up to 12% α-terpineol in petroleum does not appear to induce irritancy Sensitisation: Based on the available information from human exposure, α- terpineol is not expected to be a sensitiser. Mutagenicity: α-Terpineol was negative in 5 out of six salmonella strains. However, the result from the last strain (TA102) can not be ignored as a false positive finding because of the dose-related toxicity. To exclude mutagenicity, another study based on the TA102 tester strain is needed. However, in support of a lack of genotoxic potential, α-terpineol did not induce lung tumors in mice following repeated intraperitoneal administrations. Systemic toxicity: Based on the study using dietary exposure of rats, a NOAEL for α-terpineol of 500 mg/kg bw can be suggested. As α- terpineol on average constitutes 5% of TTO, this NOAEL for α-terpineol corresponds to an oral NOAEL for TTO (based on the only available study on systemic toxicity for α-terpineol) of 10,000 mg/kg body weight might be suggested. 50 6.9 Aromadendrene Aromadendrene is one in a row of minor constituents of TTO for which published scientific literature on potential toxicity is absent or limited. No data on acute, sub-chronic, or chronic toxicity is available. No information on mutagenicity or potential genotoxicity is available. Following topical application, patch testing of 10 volunteers with 1% aromadendrene did not show any irritant reactions (Knight and Hausen 1994). A high fraction of TTO-sensitised patients (5 out of 7) demonstrated positive patch tests against 1% aromadendrene (Knight and Hausen 1994). On the other hand, none of the 11 patients tested in the Hausen et al. study from 1999 demonstrated any positive reaction to aromadendrene when tested at 5% (Hausen, Reichling et al. 1999). Differences do occur between dermal reactions recorded in different studies, but these differences are often explained by presense of impurities or oxidative product in test oils. Evaluation: Irritancy: One study in humans is available in which aromadendrene did not demonstrate irritancy at a concentration of 1%. Sensitisation: Two studies in humans by the same group of authors are published. The most recent, with the highest concentration of aromadendrene (5%) including 11 patients did not demonstrate a sensitisation potential. Mutagenicity: No published data are available. Systemic toxicity: No published data are available, and no NOAEL can be suggested. Aromadendrene is a minor constituent of TTO (3.5%). Comparison with other chemicals of close chemical resemblance does not indicate that aromadendrene exposure following topical use of TTO products should pose a significant risk for systemic toxicity. 51 6.10 δ-Cadinene Cadinene is the principle component of Juniperus oxycedrus tar, and some of the available information on the toxicity of cadinene is limited to studies using oils derived from the various varieties of juniper. The acute oral LD50 of cadinene was reported to be higher than 5 g/kg in the rat and the acute dermal LD50 in the rabbit was likewise above 5 g/kg (Opdyke 1973). The activities of testosterone hydroxylation and the levels of P4502B1 and 3A2 were increased following experimental exposure to cadinene. The P450 isoform induced by cadinene is similar to that induced by phenobarbital. However, the magnitude of induction by cadinene was less than that by phenobarbital at the dose levels studied (Hiroi, Miyazaki et al. 1995). When cadinene was tested at a concentration of 10 % in petroleum it produced no irritation in a 48-hr closed-patch test in 25 human subjects (Opdyke 1973). The oil from Juniperus communis was not phototoxic in animal tests (Anonymous 2001). Juniperus oxycedrus Tar was genotoxic in several assays (Anonymous 2001). However, no genotoxicity data were available for any of the extracts which means that cadinene was not tested alone but only as part of the tar. Evaluation: Irritancy: One study in humans is available in which cadinene did not demonstrate irritancy at a concentration of 10%. Sensitisation: A single study on 25 volunteers has been published. Cadinene at a concentration of 10% inpetroleum did not demonstrate a sensitisation potential. Mutagenicity: No published data are available on cadinene. Systemic toxicity: No published data are available; except for a study demonstrating that cadinene induce liver enzymes in animal experiments, which is insufficient for suggesting a NOAEL. Cadinene is a minor constituent of TTO (4%). Comparison with other chemicals of close chemical resemblance together with the low acute toxicity does not indicate that cadinene exposure following topical use of TTO products should pose a significant risk for systemic toxicity. 52 6.11 Limonene Acute toxicity d-Limonene is rated as moderately toxic (with a probable lethal dose in humans of 0.5-5.0 g/kg (between 40 and 400 gram for a 80-kg adult) (Gosselin, Hodge et al. 1976). No toxicity was reported after humans were given a single dose of 20 g d- limonene in an attempt to dissolve gallstones (Igimi 1976). Both the acute oral LD50 in rats and the acute dermal LD50 in rabbits exceeded 5 g/kg (Opdyke 1975). Toxicokinetics Lemonade prepared with whole lemon (Mediterranean-style lemonade) contains high levels of d-limonene. In humans drinking 800-1200 mL of lemonade (containing 447-596 mg limonene), no toxicity was observed and maximal concentration of the primary metabolite, perillic acid, was reached after one hour and declined rapidly with a terminal elimination half-life ranging from 1-2 hours (Chow, Salazar et al. 2002). Limonene was well absorbed on to the skin of rats (Opdyke 1975). The toxicokinetics of d-limonene was studied in Sprague-Dawley rats following intravenous and oral administration at 200 mg/kg each. Blood concentration–time profiles after intravenous administration showed a biphasic decline with a mean initial t½ of 12.4 min and a terminal t½ of 280 min. The plasma/red blood cell partition was found to be 0.84. The plasma protein binding of d-limonene was found to be 55.3% at 20 mg/ml. The mean total clearance was 49.6 ml/min/kg, the volume of distribution at steady-state was 11.7 l/kg, and median residence time was 263 min. The blood concentration–time decline following oral administration also showed a biphasic decline with a mean initial t½ of 34 min and terminal t½ of 337 min. The oral bioavailability of d-limonene was 43.0 % (Chen, Chan et al. 1998). Carvone and carveol are oxidation/degradation products of limonene (Anandaraman and Reineccius 1986) Systemic toxicity After intraperitoneal administration of high doses (100 or 200 mg/kg bw) of limonene to mice, sedative as well as motor relaxant effects were observed (Gurgel do Vale, Couto Furtado et al. 2002). d-Limonene given orally to rats (250, 500, 1000 mg/kg/d) for 8 consecutive days resulted in a marked increase in both the number and the phagocytic activity of alveolar macrophages compared to the controls. These results suggest that d- limonene taken up from the thoracic duct lymph moves to the lung and directly activates the immune response of alveolar macrophages there, or indirectly activates it through activated lymphocytes (Hamada, Uezu et al. 2002). In vitro studies using the L929 cell line demonstrated cytotoxicity at concentrations as low as 0.25% in the tissue medium (Vajrabhaya and Suwannawong 2004). The susceptibility in specified cell lines and general problems related to dose transferal between in vitro studies and the in vivo situation do, however, complicate quantitative use of in vitro data for the risk evaluations. Renal toxicity Renal toxicity following exposure to limonene has received special focus as this is one of the cases where one gender of a specific strain of a specified species (male rats of the Fisher 344 strain) develops a characteristic toxic response. Thus, 53 limonene produces renal tumors in male F344 rats (Turner, Tinwell et al. 2001; Sekihashi, Yamamoto et al. 2002). Under the conditions of 2-year gavage studies, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. However, there was no evidence of carcinogenic activity of d-limonene for female F344/N rats that received 300 or 600 mg/kg. There was no evidence of carcinogenic activity of d-limonene for male B6C3F1 mice that received 250 or 500 mg/kg. There was no evidence of carcinogenic activity of d-limonene for female B6C3F1-mice that received 500 or 1,000 mg/kg. A range of studies using different strains and species have been published in order to support the hazard evaluation regarding renal toxicity following exposure to limonene. d-Limonene given to male Fischer 344 rats at 75, 150 or 300 mg/kg body weight 5 days per week for up to 4 weeks resulted in hyaline droplet formation by the 6th day (Kanerva, Ridder et al. 1987). In another study by the same group of researchers limonene administered by oral gavage at 150-2400 mg/kg/day in a subchronic (91- day) study induced renal alterations in male rats at all dose levels, whereas kidneys of male mice, female rats and female mice were unaffected (Kanerva and Alden 1987). In a separate subchronic study, groups of 5-wk-old male Fischer 344 rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30 or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of α2µ-globulin , that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans (Webb, Ridder et al. 1989). In a study to assess the presence or absence of this response in a non-rodent species, adult beagle dogs were gavaged twice daily for 6 months with 100 or 1000 mg d-limonene/kg body weight per day. Limonene ingestion did not affect feed consumption or body weight and there were no evidence of hyaline droplet accumulation nor of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene. Thus, dogs are refractory to the hyaline droplet nephropathy observed in male rats, thereby providing additional evidence that the male rat kidney is uniquely sensitive to hydrocarbons like d- limonene, and that this specific male rat nephropathic response may be inappropriate for interspecies extrapolation and human risk assessment (Webb, Kanerva et al. 1990). d-Limonene administered to 10-wk-old Wistar rats for 4 weeks (125, 500 and 4000 ppm) caused damage to the epithelial cells of the proximal tubes. The dosage of 4000 ppm reduced growth slightly in males whereas 500 ppm did not. Other changes in males included slightly increased kidney weights, and/or slight histopathological changes in the kidneys and epithelial cells in the urine (Jonker, Woutersen et al. 1993). d-Limonene produces tumors only in the kidneys of male rats in association with hyaline droplet nephropathy, which is due to the accumulation of the rat-specific, low molecular weight protein α2µ-globulin in the P2 segment cells of renal proximal tubules. Human urine contains no α2µ-globulin and, compared with the male rat, much less protein and almost no low molecular weight protein. Genotoxicity tests for d-limonene are negative, and the mechanism of 54 tumorigenesis involves tumor promotion and enhanced cell proliferation. There is no risk of cancer for humans from d-limonene, since the binding of d-limonene to α2µ-globulin would not occur (Whysner and Williams 1996). In line with this argument, Flamm and Lehman-McKeeman states: The three major lines of evidence supporting the human safety of d-limonene are (1) the male rat specificity of the nephrotoxicity and carcinogenicity; (2) the pivotal role that α2µ- globulin plays in the toxicity, as evidenced by the complete lack of toxicity in other species despite the presence of structurally similar proteins; and (3) the lack of genotoxicity of both d-limonene and d-limonene-1,2-oxide, supporting the concept of a nongenotoxic mechanism, namely, sustained renal cell proliferation. Collectively, the evidence that the renal effects of d-limonene are confined to male rats because of the unique presence of α2µ-globulin is quite compelling. In this regard, d-limonene is readily distinguished from classical renal carcinogens and should, therefore, not be subjected to traditional interspecies extrapolation and quantitative risk assessment. As d-limonene shows no toxicity or carcinogenicity in female rats or male and female mice when administered over a lifetime, it is considered safe for human consumption (Flamm and Lehman-McKeeman 1991). Dermal toxicity In an in vivo study in rats on penetration enhancing effects and skin irritation, 1% d-limonene was demonstrated to significantly enhance the percutaneous penetration of the test substance ketoprofen (Okabe, Obata et al. 1990). The same study also demonstrated that limonene at a 5-10% concentration on the skin did not induce skin irritation (edema or erthema) during 72 hours observation period following application (Okabe, Obata et al. 1990). Moderate irritation was seen when neat d-limonene was applied to intact or abraded rabbit skin for 24 h with occlusion (Opdyke 1975). Evaluation of skin damage and cytotoxicity on rat abdominal skin showed histopathological changes and cytotoxicity against human keratinocytes after exposure to limonene (Kitahara, Ishiguro et al. 1993). Using 48 h closed patch tests and human volunteers, d,l-limonene (dipentene) (20%) in petrolatum was non-irritating (Opdyke 1974). Patch testing of 10 volunteers with 1% d-limonene did not show any irritant reactions (Knight and Hausen 1994). Limonene was not a respiratory irritant when tested in humans at concentrations of 10, 225, and 450 mg/m3. At the highest exposure level a temporary decrease in lung capacity was observed (Falk-Filipsson, Lof et al. 1993). d-Limonene (8%) was not sensitising using an open epicutaneous test in guinea pigs (Klecak 1985). Limonene at 25% and 50% did not produce a response in the local lymph node assay, but 100% did (Warbrick, Dearman et al. 2001). This was regarded as being a weak response. d-Limonene did not produce sensitisation reactions when applied to guinea pigs whereas oxidised d-limonene did (Karlberg, Boman et al. 1991). Using a maximisation test and 25 human volunteers, d- limonene (8%) did not produce sensitisation reactions (Opdyke 1975). Limonene cause skin reactions in six out of seven participants in the Knight and Hausen study from 1994 when applied in 1% as compared to only one in eleven subjects exposed to 5% limonene (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Whether the positive reactions observed in the 1994 study on limonene were caused by impurities or oxidative products is not to say, but positive patch test reactions to oxidised limonene are common amongst dermatology patients (Karlberg, Dooms-Goossens et al. 1997; Matura, Goossens et al. 2002; Matura, Karlberg et al. 2003). Patch testing with limonene (1%) produced one irritant or doubtful positive reaction in 192 participants, whereas 0.1% limonene produced no reactions (Frosch, Pilz et al. 1995). Further, patch testing with 3% limonene 55 produced only 7 positive in 1606 dermatology patients (Frosch, Johansen et al. 2002). Autoxidation of d-limonene readily occurs to give a variety of oxygenated monocyclic terpenes that are strong contact allergens (Karlberg, Dooms-Goossens et al. 1997). Thus, patch testing with oxidized R-(+)-limonene was performed on 2273 patients at 4 dermatology clinics in Europe, and a total of 63 patients (2.8%) showed positive reactions (Matura, Goossens et al. 2002). Oxidation products of d- limonene, (R)-(-)-carvone, (+)-limonene oxide, along with air oxidized d-limonene, were found to be potent sensitizers in the Freund complete adjuvant test and in the guinea pig maximization test (Haneke 2002). Reproductive toxicity Pregnant rabbit were administered oral doses of 250 or 1,000 mg/kg d-limonene (Kodama, Okubo et al. 1977). Decrements in feed intake and body weight gain and deaths in 6/21 animals were observed in the high dose group. These effects were not seen at 250 mg/kg d-limonene; no teratogenic effects were observed. Pregnant rats were given 2,869 mg/kg d-limonene orally from day 9 to 15 of gestation (Tsuji, Fujisaki et al. 1975). Body weight gain of the dams was decreased, and a prolongation of the ossification of metacarpals and proximal phalanges was observed in the foetuses. Oral administration of 2,363 mg/kg d-limonene to mice between days 7 and 12 of gestation also caused maternal body weight decrements and increased incidences of abnormal bone formation in the foetuses (Kodama, Okubo et al. 1977). Mutagenicity and genotoxicity d-(+)-Limonene at concentrations ranging from 10 – 1000 μM did not increase the frequency of spontaneous sister-chromatid exchanges in Chinese hamster ovary cells (Sasaki, Imanishi et al. 1989). d-Limonene exposures failed to result in observable mutations either in vitro or in vivo (Haneke 2002). Limonene was not mutagenic when assayed for mutagenicity towards four salmonella-strains (TA 98, TA 100, TA 1535, TA 1537) with and without metabolic activation (Florin, Rutberg et al. 1980). Limonene was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 when tested in a pre-incubation protocol in both the presence and absence of Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver S9 (Haworth, Lawlor et al. 1983). Watabe et al. investigated the mutagenicity, with and without induced S9, of d-limonene and two presumed intermediate metabolites (the 1,2- and 8,9-epoxides, which are in turn converted to the corresponding glycols) in Salmonella typhimurium, and they also observed no increase in revertants (Watabe, Hiratsuka et al. 1980). Carcinogenicity d-Limonene is classified as a group 3 carcinogen by the IARC (evidence of carcinogenicity is inadequate in humans or limited in experimental animals. Limonene did not induce primary lung tumors in male or female A/He mice following 24 ip injections during an eight week period with 24 weeks follow-up. The doses used were either MTD or 20% of MTD (Stoner, Shimkin et al. 1973). Elegbede et al. (1986) compared orange peel oil and DMBA in a two-stage skin carcinogenesis model with female CD-I mice and confirmed that topically applied orange peel oil was a very weak promoter of both skin papillomas and carcinomas but that minor terpene components, and not topically applied d-limonene, possessed the promoter activity (Elegbede, Maltzman et al. 1986). 56 Evaluation: Irritancy: Neat limonene is a moderate irritant to rabbits. Limonene does not induce irritancy in humans when tested up to a concentration of 20% Sensitisation: Neat limonene induces a response in the LNNA, but concentrations of 50% and below did not. In the absence of oxidation/degradation products the published literature describes limonene as a non-sensitiser. However, autooxidation of limonene has repeatedly been demonstrated to generate potent sensitisers. Mutagenicity: There is no support in the literature that limonene is mutagenic. Limonene is potentially a very weak promoter and the evidence for carcinogenicity is rated as limited in experimental animals by IARC. Systemic toxicity: Limonene is generally of limited acute toxicity and a natural ingredient in many soft drinks and lemon juice products. Renal carcinogenicity and toxicity in humans following topical application of limonene is not seen as relevant. Based on the study on reproduction, 250 mg/kg orally is suggested as a NOAEL value for limonene. As limonene on average constitutes 2,5% of TTO, this NOAEL for limonene corresponds to an oral NOAEL for TTO (based on the study on reproductive toxicity for limonene) of 10,000 mg/kg body weight. 57 6.12 Sabinene Sabinene constitutes on average below 2% of TTO. The only published literature on sabinene describe that sabinene (1%) has an anti- inflammatory effect when tested against experimentally induced eye inflammation in rabbits. No sign of eye irritation due to sabinene at this concentration is reported (Yao and Chiou 1993). Based on the information that no eye irritation was observed at 1% sabinene and that sabinene constitutes below 2% of TTO, it can be anticipated that an irritant response due to sabinene in a TTO product is unlikely. There is no available published literature describing mutagenic, genotoxic, or systemic effects in humans, in experimental animals, or in vitro. 58 6.13 Globulol Globulol constitutes on average 1.5% of TTO. Globulol has been evaluated and topical treatment with globulol is found useful in controlling Th2-type inflammatory cutaneous disorders. These disorders may include atopic dermatitis (Hori, Nonomura et al. 2001). No published literature on toxicity of globulol is available and a toxicological profile can not be developed. The chemical nature of this Sesquiterpene alcohol and the amounts present in TTO product does, however, not indicate that acute or systemic toxicity can be expected. 59 6.14 Viridiflorol Viridiflorol constitutes on average below 1% of TTO. The only available information on viridiflorol is that it inhibits acetylcholinesterase (Miyazawa, Watanabe et al. 1998). Thus, there is no information available for a judgement of the potential toxicity of this very minor ingredient in TTO. 60 6.15 Degradation products Like most natural oils several constituents of TTO may undergo oxidation. This is a natural process that occurs over time and primary depends on storage conditions related to temperature, access to oxygen, and presence of antioxidants. Photooxidation may also be an issue for some chemicals. A few descriptions of degradation of TTO with time are described in the literature. It has been stated that the oxidation products formed may increase the irritancy capacity of the TTO. In one experiment TTO was stored on a window sill to study the influence of light, oxygen and warmth. GC analysis demonstrated an increase in peroxides within 4 days from 50 ppm to more than 500 ppm. Peroxides, epoxides and endoperoxides were formed (Hausen et al. 1999). Chemicals of this type are well recognized as demonstrating a range of toxic effects including skin irritation, irritation of mucous membranes, formation of lipid peroxides (membrane damage), mutagenicity, and adduct formation with DNA. Besides these peroxides, degradation products from the original constituents of TTO may be formed, which may potentially exert a different degree of toxicity than their parent TTO constituent. Oxidised tea tree oil contains increased levels of ρ-cymene and decreased levels of α-terpinene, γ-terpinene and terpinolene (Brophy, Davies et al. 1989; Hausen, Reichling et al. 1999; Hausen 2004). The amount and rate of transformation may be illustrated by the observation, that during a 4 day period the p-cymene content in a TTO sample increased from 2% to 11.5%, while the contents of α-terpinene, γ-terpinene as well as terpinolene were reduced to one half of their original concentrations during the same period (Hausen et al. 1999). Not all constituents do, however undergo degradation to the same extent. Thus, a detailed study on the autoxidation of terpenes in cell-free nutrient medium demonstrated that alpha-pinene and beta-pinene were both autoxidized to a certain extent, while limonene remained unaffected (Lindmark-Henriksson, Isaksson et al. 2004). These natural processes related to the ageing of a product have led regulatory bodies to focus not only on parent constituents of a product, but also on degradation products formed during storage. Thus, Directive 2003/15/EC from February 2003 states that limitations and restrictions as to the use of certain ingredients in cosmetics will be implemented besides a general requirement for information on minimum durability (‘best used before the end of ‘ on the label followed by a date or details of where it appears on the packaging). Among ingredients present in TTO, d-limonene is mentioned specifically: The presence of d-limonene in the product must be indicated in the list of ingredients when the concentration exceeds 0.001 % in leave-on products and 0.01 % in rinse-off products. Further to d-limonene; limolene and natural products containing substantial (??) amounts of it, should only be used when the level of peroxides is kept to the lowest practical level, for instance by adding antioxidants at the time of production. Such products should have a peroxide value of less than 20 millimoles peroxide per liter (IFRA guidelines) (SCCNFP/0392/00, final adopted September 2001). Degradation products derived from individual components found in tea tree oil are listed below (Table 4). These have been derived by autoxidation from exposure to heat, air or oxygen, rather than by biotransformation or metabolic processes. An important notion was that the rate of the autoxidation was more than one order of magnitude slower than that of the biotransformation. Moreover, different products 61 were formed by autoxidation than by biotransformation (Lindmark-Henriksson, Isaksson et al. 2004). Some of the degradation products occur already in newly distilled TTO and are part of the ISO list of the 14 main constituents of TTO. The toxicological profiles of these compounds have already been described in the above section, but the most important of the remaining known degradation products will be described in the detail that the available literature allows. Table 4. Oxidation products of tea tree oil components Component Oxidation/degradation product(s) References(s) α-Terpinene ρ-cymene (McGraw, Hemingway et al. 1999) Thymol (McGraw, Hemingway et al. 1999) Carvacrol (McGraw, Hemingway et al. 1999) 1,8-cineole (McGraw, Hemingway et al. 1999) Ascaridol (Karapire, Kus et al. 2005) 1,2,4-trihydroxymenthane (Hausen, Reichling et al. 1999) γ-Terpinene ρ-cymene (Foti, Sortino et al. 2005) Limonene (+)-limonene oxide (Haneke 2002; Marine and Clemons 2003) (R)-(-)-carvone (Anandaraman and Reineccius 1986; Haneke 2002) Carveol (Anandaraman and Reineccius 1986) Limonene-(1,2)-epoxide (Anandaraman and Reineccius 1986) α-Pinene Sobrerol (Haneke 2002) Verbenone (Lindmark-Henriksson, Isaksson et al. 2004) β-Pinene α-terpineol (Lindmark-Henriksson, Isaksson et al. 2004) Pinocarvone (Lindmark-Henriksson, Isaksson et al. 2004) 1,8-cineole (Lindmark-Henriksson, Isaksson et al. 2004) Degradation of α-terpinene caused formation of mainly p-cymene, but degradation products also included 1,8-cineole (already described above), thymol, ascaridol, iso-ascaridol, and 1,2,4-trihydroxymethane (Hausen, Reichling et al. 1999). Especially peroxides, epoxides (e.g. iso-ascaridol) and endoperoxides (e.g. ascaridol) generated through photooxidation seems to be toxicologically important products (Hausen, Reichling et al. 1999). 62 6.15.1 Thymol and carvone The oral LD50 of carvone and thymol in rats were found to be 1640 mg/kg bw and 980 mg/kg bw, respectively (Jenner, Hagan et al. 1964). The metabolism of thymol and carvacrol in rats was studied using gas chromatographic-mass spectrometric methods. The urinary excretion of metabolites was rapid. Only very small amounts were excreted after 24 hrs. Although large quantities of carvacrol and, especially, thymol were excreted unchanged (or as their glucuronide and sulphate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred (Austgulen, Solheim et al. 1987). Thymol and carvone did not induce any subacute or chronic toxicity following dietary exposure of rats to 2500 ppm in the feed (Hagan, Hansen et al. 1967). l-Carvone (1%) was not sensitising using an open epicutaneous test in guinea pigs (Klecak 1985). In accordance with this observation in guinea pigs, carvone (5%) was not positive in two separate studies with patch tests of 18 humans presensitized to TTO (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Neither carvone nor thymol were mutagenic when assayed for mutagenicity towards four salmonella-strains (TA 98, TA 100, TA 1535, TA 1537) with and without metabolic activation (Florin, Rutberg et al. 1980; Stammati, Bonsi et al. 1999). Likewise, DNA-repair tests of thymol and carvone were negative at exposure-relevant concentrations, though inhibition of DNA-repair was observed at high doses of carvone (Stammati, Bonsi et al. 1999). The genotoxic potential of major compounds of thyme oil, i.e. thymol and carvacrol, were investigated in human lymphocytes by single-cell gel electrophoresis. Also, the effects of these substances on the induction of DNA damage by 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and mitomycin C (MMC) were evaluated. No increase in DNA strand breakage was observed at thymol concentrations below 0.1 mM, but at the higher concentration of 0.2 mM significant increases in DNA damage were seen. Thymol significantly reduced the DNA strand breakage induced by IQ and MMC at the lower concentrations studied. Carvacrol, which is an isomer of thymol, seemed to protect lymphocytes from the genotoxic effects of IQ and MMC at non-toxic concentrations below 0.05 mM, but at the higher concentration of 0.1 mM carvacrol itself induced DNA damage (Aydin, Basaran et al. 2005). Thus, these data indicate that thymol and carvacrol protect against DNA damage at concentrations below 0.1 mM, but cause DNA damage themselves at higher concentrations. Interpreting these findings in a human risk assessment is complicated, but as the DNA damage only emerges at the millimolar range, this kind of toxicity will not be expected to occur during topical use of TTO products containing thymol and/or carvacrol as degradation products. Thymol and d/l-carvone did not induce primary lung tumors in male or female A/He mice following 24 ip injections during an eight week period with 24 weeks follow-up. The doses used were either MTD or 20% of MTD (Stoner, Shimkin et al. 1973). Evaluation: Based on the available published literature and the fact that thymol as well and carvone are minor degradative constituents of TTO, acute or systemic toxicity from these compounds will not be expected. Likewise, none of the two compounds appears to pose and mutagenic or carcinogenic risk to humans. 63 6.15.2 Ascaridol / isoascaridol and 1,2,4-trihydroxymenthane A high fraction of TTO-sensitised patients demonstrated positive patch tests against 5% ascaridol (9 out of 11) (Knight and Hausen 1994; Hausen, Reichling et al. 1999). Positive patch test results were also recorded for 1,2,4- Trihydroxymenthane at a concentration of 5% (4 out of 11 patients) (Knight and Hausen 1994; Hausen, Reichling et al. 1999). By 2003 close to 7000 patients at German dermatological clinics had been tested epicutaneously with a 5% dilution of oxidised TTO containing the original constituents as well as oxidation products (Hausen 2004). Seventy patients (1%) had a positive reaction to TTO (Hausen 2004). The most important allergens of TTO appear to be terpinolene, ascaridol, α-terpinene, and 1,2,4-trihydroxy menthane for which the prevalence of allergic responses among patients visiting dermatological clinics varies between 0.4% and 0.6% (Hausen 2004). Evaluation: Sensitisation: These degradation products of TTO have clear allergic potencies demonstrated repeatedly in independent studies in humans. 64 6.16 Other components of interest Besides the known major and minor constituents of TTO and known degradation products, for which the toxicological profiles have been described above, a few other terpenes of close familiarity to the TTO constituents have been evaluated because toxicological information on these structurally comparable terpenes/terpenoids were available that could supplement the available information on the more important constituents of TTO. These compounds include myrcene, phellandrene, and caryophyllene. 6.16.1 Myrcene Myrcene can be found in TTO in concentrations up to 0.5%, and is the only TTO constituent without a ring structure. Myrcene has a low acute toxicity (Opdyke 1976) with oral as well as dermal LD50 values above 5000 mg/kg bw. Myrcene is not mutagenic in Ames test (Gomes-Carneiro, Viana et al. 2005) and systemic toxicity related to this constituent is not expected. Myrcene is included in this section because three relevant studies on reproductive toxicity are available for this minor constituent of TTO. Therefore, only the reproductive data are described to any length in this section. Data on acute toxicity, induction of hepatic enzymes, allergy, and mutagenicity are available, but not considered to add to the review of the toxicity of TTO and constituents. In a study on the embryo-foetotoxic potential of beta-myrcene in the rat beta- myrcene (250, 500 and 1200 mg/kg) in corn oil was given orally to Wistar rats from day 6 to 15 of pregnancy. From the data presented the NOAEL for embryo- foetotoxicity could be set at 500 mg beta-myrcene/kg body weight (Delgado, Carvalho et al. 1993). Another study by the same authors with the aim to provide data on the peri- and postnatal developmental toxicity of beta-myrcene used doses of beta-myrcene (250, 500, 1000 and 1500 mg/kg) in corn oil and given by gavage to female Wistar rats from day 15 of pregnancy, parturition and throughout the period of lactation up to weaning (postnatal day 21). From the data presented in this paper the NOAEL for peri- and postnatal developmental toxicity was set at 250 mg beta-myrcene/kg body weight (Delgado, De Almeida Nogueira et al. 1993). The effects of myrcene on fertility and general reproductive performance were studied in the rat (Paumgartten, De-Carvalho et al. 1998). Myrcene (0, 100, 300 and 500 mg/kg) in peanut oil was given by gavage to male Wistar rats (15 per dose group) for 91 days prior to mating and during the mating period, as well as to females (45 per dose group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactation up to postnatal day 21. Myrcene did not affect the mating index (proportion of females impregnated by males) or the pregnancy index (ratio of pregnant to sperm-positive females). No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg) did myrcene induce an increase in the resorption rate and a higher frequency of fetal skeleton anomalies. No adverse effect of myrcene on postnatal weight gain was noted but time of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring. On the basis of the data presented in this paper the NOAEL for toxic effects on fertility and general reproductive performance was set at 300 mg of ß-myrcene/kg body weight by the oral route (Paumgartten, De-Carvalho et al. 1998). 65 Reproductive toxicity from myrcene is not expected to be relevant for exposures related to TTO due to the low amount of myrcene present in TTO. However, the data may serve as a supplement to the limited data on reproductive toxicity available on more dominant constituents of TTO. 6.16.2 Phellandrene The available newer literature on the toxicological profile of phellandrene is limited. The older literature is covered in a review by Opdyke (Opdyke 1978). The acute oral LD50 in rats was reported as 5.7 g/kg (4.7-6.7 g/kg) and the acute dermal LD50 in rabbits exceeded 5 g/kg (Opdyke 1978). Phellandrene was readily absorbed through the skin of rats (Opdyke 1978). In sheep, α-phellandrene apparently undergoes reduction of one double bond and oxidation of the methyl group to give phellandral, which is further oxidized to phellandric acid; conjugation with glycine gives rise to phellanduric acid, which is then excreted in the urine (Opdyke 1978). α-Phellandrene applied full strength to intact or abraded rabbit skin for 24 hr under occlusion was moderately irritating (Opdyke 1978). Tested at 4% and 8% in petrolatum, it produced no irritation after a 48-hr closed-patch test on two separate panels of human subjects. An older study by Valette and Cavier from 1954 cited by Opdyke (Opdyke 1978) states that α-Phellandrene is readily absorbed through the skin of rats. A maximization test was carried out on 25 volunteers. The material (RIFM no. 71- 68) was tested at a concentration of 4% in petrolatum and produced one sensitization reaction (Opdyke 1978). In view of the autoxidation problems, it was decided that the maximization procedure should be repeated on α-phellandrene using a freshly distilled sample processed under a blanket of nitrogen and containing butylated hydroxyanisole as an antioxidant. The same maximization test was carried out on another 25 volunteers using this freshly processed sample (RIFM no. 72-76). The material was tested at a concentration of 8% in petrolatum and produced no sensitization reactions (Opdyke 1978). However, phellandrene was identified as a sensitizer in another study on the sensitizing potential of some essential oils and their constituents (Woeber and Krombach 1969), and α- phellandrene induced a positive patch test in four of the eleven patients included in a study on patients from a dermatological department (Hausen, Reichling et al. 1999). To what extent the positive findings in the two latter studies are caused by oxidative degradation products of phellandrene is not clear. l-Phellandrene at concentrations ranging from 10 – 1000 μM did not increase the frequency of spontaneous sister-chromatid exchanges in Chinese hamster ovary cells (Sasaki, Imanishi et al. 1989). Phellandrene has in a study by Roe and Field in 1965 (reviewed by Opdyke in 1978) been reported to promote tumour formation on the skin of mice treated with the primary carcinogen 7,12-dimethylbenz[a]anthracene (Opdyke 1978). Evaluation: Irritancy: Neat phellandrene is a moderate irritant to rabbits. α- Phellandrene does not induce irritancy in humans when tested up to a concentration of 8% 66 Sensitisation: In the absence of oxidation/degradation products the published literature describes α-phellandrene as a non-sensitiser. However, autooxidation of α-phellandrene has been demonstrated to generate sensitisers and two studies on humans have demonstrated positive patch tests after exposure to products based on α-phellandrene. Mutagenicity: The only study available does not support that α-phellandrene is mutagenic. α-Phellandrene is potentially a weak promoter. Systemic toxicity: There are no studies available on systemic toxicity of α- phellandrene. However, the high LD50 value, the chemical familiarity with other terpenes, and the expected quantitative occurrence in TTO products do not indicate that systemic toxicity caused by α-phellandrene would be likely. 6.16.3 Caryophyllene Consecutive testing of 1606 patients attending the patch test clinic of 6 European departments of dermatology was performed. The standard fragrance mix produced the highest reactivity in all centres (mean 11.4%; range 9.3–17.9%), whereas caryophyllene caused positive reactions in 0.6% of the patients (Frosch, Johansen et al. 2002). In a more recent study, 1511 consecutive dermatitis patients in 6 European dermatology centres were patch tested with oxidized fragrance terpenes and some oxidation fractions and compounds. About 0.5% of the patients reacted to oxidized caryophyllene (Matura, Sköld et al. 2005). Evaluation: Caryophyllene induced positive allergic response in 0.5% of approximately 3000 dermatological patients participating in two independent European studies. 67 7 Structural formulas This section includes the structural formulas and chemical constituents of the most important TTO constituents and their metabolites. CH3 H3 C CH3 α-Terpinene (C10H16) CH3 H O CH3 H3 C Terpinen-4-ol (C10H18O) CH3 H2 C CH3 H3 C CH3 d-Limonene (C10H16) α-phellandrene (C10H16) CH3 H3 C CH3 H3 C CH3 p-Cymene (C10H14) Terpinolene (C10H16) 68 CH3 H3 C CH3 γ-Terpinene (C10H16) CH3 OH H3 C CH3 α-Terpineol (C10H18O) CH3 O H3 C CH2 Carvone (C10H14O) H3 C CH3 H3 C CH3 Thymol (C10H14O) 1,8-Cineole (C10H18O) CH3 H3 C CH3 H3 C CH3 Cumene (C9H12) Cadinene (C15H24) H2 C CH2 CH3 H3 C H3 C CH3 α-Pinene (C10H16) H3 C Myrcene (C10H16) 69 CH2 H3 C OH H3 C CH3 H3 C CH3 Aromadendrene (C15H24) Viridiflorol (C15H26O) CH2 H3 C H3 C OH H3 C Sabinene (C10H16) Linalool (C10H18O) CH3 O CH3 O H3 C CH3 CH2 Methyleugenol (C11H14O2) Ascaridol (C10H16O2) 70 8 References Abdo, K., M. Cunningham, et al. (2001). "14-Week toxicity and cell proliferation of methyleugenol administered by gavage to F344 rats and B6C3F1 mice." Food and Chemical Toxicology 39(4): 303-316. Altman, P. (1991). 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Brazilian Journal of Medical & Biological Research 26(1): 93-98. 79 From: Tony Larkman [mailto:tlarkman@attia.org.au] Sent: Tuesday, 8 December 2020 4:14 PM To: 'Bart Heldreth' <heldrethb@cir-safety.org>; 'Monice Fiume' <fiumem@cir-safety.org> Cc: Phillip Prather <phil@downunderenterprises.com> Subject: CIR Expert Review for tea Tree Oil Dear Bart & Monice, First my apologies for being unable to attend the meeting last night; Phillip Prather, a Director of ATTIA Ltd, stood in for me and has provided me with a report on the discussion and his input. 1. Phil has advised that the Panel wanted a copy of the ATTIA White Paper on the stability of TTO – I have attached a copy of this; please note that this is available, along with other information from this URL: https://teatree.org.au/teatree_about_packaging.php . I have also attached two reports commissioned by ATTIA and RIRDC in 2012 from which some of the Stability White Paper was derived. 2. Phil also advised that the Panel wants to know what oxidation rates might occur in a formulated product. I am unable to respond to this substantively as all of ATTIA’s work focuses on the TTO itself rather than formulated products. There are an infinite number of formulations available and the rate of oxidation of TTO or its components will be governed both by the other ingredients present and of course the level of exposure to the atmosphere. As part of a comprehensive dossier prepared for the EU’s SCCS (formerly SCCP) a report was prepared in 2006 titled “Literature review on tea tree oil: Toxicity profiles for tea tree oil, constituents of tea tree oil and known oxidation products” by JB Nielsen; I have attached a copy of this and while this is somewhat dated now it may assist you particularly in the areas raised (and noted by Phil) of Tox and genal, dermal tox & skin sensitisation. Other comments: · I am pleased that it was noted by the Panel that TTO has GRAS status; this is an important consideration. · I was delighted that the Panel clearly noted the dichotomy between oxidised and fresh oil in the literature relating to dermal irritation and draw your attention to my specific comments on this in my submission to the Panel and ask you to note that the levels of reaction are close to non-existent when fresh, unoxidised TTO is deployed. · Adulteration, which I raised repeatedly in my submission, was not addressed substantively. Again please refer to my submission comments on this area and the fact that oxidative products are often detected in these fraudulent samples along with a long list of extraneous products sourced from incomplete fractionation of other essential oils, principally pine, Eucalyptus and White Camphor oils. Some of these products have adverse effects when deployed on humans and animals. · The Panel called for more toxicity data: these data exist as part of a REACH dossier submitted to the EU’s ECHA in 2018; they are not available publically but it may be possible for the Panel to request summary evidence from the ECHA for specific data required. · Concentrations of TTO have, as noted by the panel “…come down from 15% to 3-4% over last 15+ years”. This is, in my opinion, largely driven by fears of dermatitis or other skin conditions and is almost wholly driven by the deliberate deployment of oxidised TTO in patch testing for TTO and strangely, turpentine. As you will have read in my emails on the subject researchers claim this is being done to obtain higher response rates because ‘patients may therefore be exposed to oxidised fragrance chemicals and develop an allergy’ per an email I shared with you earlier from Dr Sophie Rolls of Dermatology, ST4 - University Hospital Wales, Cardiff who stated on 30 Apr 2020: Thank you for your interest in our paper. We can confirm that it is oxidised tea tree that we are patch testing with as has been recommended on the British Society for Cutaneous Allergy facial series. We agree that there seem to be many fewer problems of allergy to non-oxidised chemicals such as limonene, linalool and tea tree, compared to oxidised samples. In everyday practice we see patients who do not always follow advice labels with respect to correct storage of their products and who often ignore sell-by-dates. Patients may therefore be exposed to oxidised fragrance chemicals and develop allergy. As our aim is to identify the underlying cause of a patient's dermatitis it is the oxidised TTO which is tested. We will ensure in future if we write further papers that it is made clear that it is oxidised TTO which is being tested. It remains beyond my comprehension why this is being recommended and done for TTO alone of all essential oils in the series and ask you to note that when challenged to explain this anomaly (cc to all others in the research group) no response whatsoever was forthcoming. Please do not hesitate to contact me if you have any further questions. Regards, Tony Larkman CEO - ATTIA Ltd ': 02 4017 1336 È: 0434 263 664