Tydronium

Hazard statements (CLP)

H290

Code

Statements

H290

May be corrosive to metals

SECTION 2: Hazards identification

No. 1272/2008]

2.2 Label elements

2.2.1 Label Elements according GHS Classifications and Regulation (EC)

Signal word

Warning

Hazard statements

Corrosive to Metals R36/37 Irritating to eyes and skin

Precautionary statements

Minimize prolonged contact to steel, zinc, magnesium. Low Reactivity to copper, aluminum, stainless steel. S36/37/39 Wear suitable protective clothing, gloves, and eye/face protection

Hazard pictograms

Corrosive

Additional information

No fragrances added 1

2.1 Classification of the substance or mixture

B.16.1. 2.1.1 GHS Classifications and Regulation (EC) No. 1272/2008 (CLP): Corrosive to Metals 2.1.2 Directive 67/548/EEC & Directive 1999/45/EC: Corrosive to Metals 2.2 Label Elements 2.2.1 Label Elements according GHS Classifications and Regulation (EC) No. 1272/2008 (CLP) Hazard Pictogram(s) Signal Word Warning Hazard Statement Corrosive to Metals Precautionary Statements Minimize prolonged contact to steel, zinc, magnesium. Low Reactivity to copper, aluminum, stainless steel. 2.2.2 Label Elements Hazard Symbol Corrosive Risk Phrases R36/37 Irritating to eyes and skin Safety Phrases S36/37/39 Wear suitable protective clothing, gloves, and eye/face protection 2.3 Other Hazards OSHA : Non-hazardous under OSHA Hazard Communication Standard HMIS HEALTH 1 Health: 1 FLAMMABILITY 0 Flammability: 0 REACTIVITY 1 Reactivity: 1 PERSONAL PROTECTION C Personal Protection: C WHMIS (Canada): Corrosive to Metals 2.4 Additional Information: No fragrances added 1 SECTION 3: COMPOSITION/ INFORMATION ON INGREDIENTS 3.1 GHS Classification (EC Classification No. 1272/2008/EC) Ingredients Common Name CAS No. %W/W H2O Water 7732-18-5 85.0 – 86.0 % W/W H9O4: HSO4 Hydronium Sulfate 2032207-39-7 14.0 – 15.0 % W/W [Hydrogen (+1), Triaqua-u3-oxotri, sulfate (1:1)] 3.2 ADDITIONAL INFORMATION: NONE SECTION 4: FIRST AID MEASURES 4.1 Description of First Aid Measures Inhalation: Not a likely route of exposure. Remove to fresh air if irritation occurs. If symptoms develop, obtain medical attention. Skin Contact: Exposure to skin normally does not cause irritation or redness. No toxicity associated with the product being absorbed through skin. Wash exposed areas with water. In the unlikely event of irritation, seek medical advice. Eye Contact: This product may cause irritation to the eyes after direct contact with concentrate. Rinse with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Ingestion: No toxicity associated with ingestion. Ingesting large volumes may cause minor gastrointestinal distress due to separation of grease and oil in the digestive tract. Drink water to assist in digestion if swallowed. 4.2 Most Important Symptoms and Effects, Both Acute and Delayed Acute: Ingestion will cause minor distress to the gastrointestinal tract. May cause chemical and mechanical eye irritation with direct contact. Delayed and Chronic Effects: Expected to be similar to acute exposures. 4.3 Indication of the Immediate Medical Attention and Special Treatment Needed: Treat symptomatically. SECTION 5: FIREFIGHTING MEASURES Not flammable or combustible by OSHA/WHMIS criteria. Not sensitive to mechanical impact and static discharge. Flash Point Explosive Limits Temperatures > 220° C (428° F) NA Auto-Ignition NA 5.1 Extinguishing Media Suitable Extinguishing Media: Use extinguishing media appropriate to surrounding fire conditions. Unsuitable Extinguishing Media: None known 5.2 Special Hazards Arising from the Substance or Mixture: Containers may rupture from exposure to high temperatures, releasing contents that may be slippery. 5.3 Advice for Firefighters: Suitable protective clothing should be worn in fire conditions. Extinguish preferably with dry chemical, foam or water spray. 5.4 Hazardous Combustion Products: Sulfur Oxides, Calcium Oxides NFPA 704: HEALTH HAZARD–BLUE FLAMMABILITY–RED INSTABILITY- YELLOW SPECIAL HAZARD – WHITE 1 0 1 COR SECTION 6: ACCIDENTAL RELEASE MEASURES 6.1 Personal Precautions, Protective Equipment and Emergency Procedures: None 6.2 Environmental Precautions: None 2 6.3 Methods and Material for Containment and Cleaning Up: Rinse area with water. Dispose of material in accordance with local regulations. 6.4 Reference to Other Sections: See Also Section 7, 8, 13 6.5 Additional Information: None SECTION 7: HANDLING AND STORAGE 7.1 Precautions for Safe Handling: Avoid contact with eyes. Wear acid resistant personal protective gear. 7.2 Conditions for Safe Storage: Store in closed containers between 35°F and 120°F SECTION 8: EXPOSURE CONTROLS / PERSONAL PROTECTION 8.1 Control Parameters 8.1.1 Occupational Exposure Limits Substance CAS No. LTEL (8hr TWA ppm) STEL (ppm) LTEL (8hr TWA mg/m3) STEL (mg/m3) OELs are not available for non-listed components. 8.1.3 PNECs and DNELs: No PNECs or DNELs are available for this product. As with all chemical products, users are cautioned to avoid unnecessary exposures. 8.2.2 Personal Protection Equipment Respiratory Protection: Usually not needed. Eye Protection: Safety glasses are suggested as good practice. Hand Protection: Acid resistant gloves. Skin and Body Protection: Wear acid resistant personal protective gear. Engineering Controls: No special controls required. General Hygiene Considerations: Handle according to established industrial hygiene and safety practices. SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES 9.1 Information on Basic Physical and Chemical Properties Concentrate Appearance: Clear Liquid Color: Water White Liquid Odor: None to Mild Odor Threshold: Not available pH: 0 - 1.5 Melting Point NA Initial Boiling Point: Not available Boiling Point: > 112 °C (233.6° F) Flash Point: > 220 °C (428° F) Evaporation Rate: 1 (Water = 1) Flammability (solid, gas): Non-Flammable Upper/Lower Flammability Limit: Non-Flammable Auto-ignition Temperature: Non-Flammable Vapor Pressure: Not available Vapor Density: Not available Relative Density: Not available Solubilities: Infinitely Soluble in water Partition Coefficient: N-octanol/Water: Not available Decomposition Temperature: Not available Percent Volatile, wt.%: 0% Density Target: 1.035 g/mL Density Range: 1.025 – 1.045 g/ml 3 VOC Content, wt.%: 0% SECTION 10: STABILITY AND REACTIVITY 10.1 Reactivity: Chemically active on metals. 10.2 Chemical Stability: Stable under normal conditions. Avoid temperature extremes. 10.3 Possibility of Hazardous Reactions: No hazardous reactions observed. 10.4 Conditions to Avoid: Do not freeze. Do not use above ambient temperature. 10.5 Incompatible Materials: Avoid prolonged contact with metals 10.6 Hazardous Decomposition Product(s): Calcium Oxides and Sulfur Oxides. SECTION 11: TOXICOLOGICAL INFORMATION Substance: Tetraaquahydrogen+1 | CAS No. 12501-73-4 Hydrogen Sulfate | CAS No. 14996-02-2 11.1 Information on Toxicological Effects 11.1.2 Mixtures Effects of Acute Exposure Ingestion: Not Known. Product may be irritating to gastrointestinal tract. Inhalation: Not Known. Product may be irritating to nasal tissue. Skin Contact: Not Known. Prolonged contact may result in mild irritation. Eye Contact: Not Known. Contact may result in mechanical and chemical irritation. Corrosivity: None expected. Repeated Dose Toxicity: Expected to be similar to single exposures. Carcinogenicity: This product does not contain any carcinogens or potential carcinogens as listed by ACGIH, IARC, OSHA, or NTP. Mutagenicity: Not available Toxicity for Reproduction: Not available 11.2 Other Information: None SECTION 12: ECOLOGICAL INFORMATION 12.1 Toxicity: Not expected to be harmful to aquatic or soil environments. 12.2 Persistence and Degradability: Readily biodegradable organic liquid 12.3 Bio-accumulative Potential: Not available 12.4 Mobility in Soil: Not available 12.5 Results of PBT and vPvB Assessment: Not available 12.6 Additional Information on Eco-toxicity: The product does not add to the AOX-value of effluent water (DIN 38409). SECTION 13: DISPOSAL CONSIDERATIONS 13.1 Waste Treatment Methods: Disposal should be in accordance with local, state or national legislation. For disposal within the EC, the appropriate code according to the European Waste Catalogue (EWC) should be used. Containers must not be punctured or destroyed by burning, even when empty. 13.2 Additional Information: None 4 SECTION 14: TRANSPORT INFORMATION Land transport (ADR/RID) (c)(d): Not classified as dangerous for transport. U.S. Department of Transportation (DOT) (c)(d): Not classified as dangerous for transport. Canada Transportation of Dangerous Goods (TDG) (c)(d): Not classified as dangerous for transport. Sea Transport (IMDG) (c)(d): Not classified as dangerous for transport. Air Transport (ICAO/IATA) (c)(d): Not classified as dangerous for transport. (c)– Consult with transport provider. (d) – Check relevant regulations for Special Provisions. SECTION 15: REGULATORY INFORMATION 15.1 Safety, Health and Environmental Regulations and Associated Hazards for the Mixture 15.1.1 Regulations TSCA STATUS A component or components of this products is not listed on the TSCA Inventory of Existing Chemical Substances. Chemical Name 15.2 Chemical Safety Assessment: Corrosive SECTION 16: OTHER INFORMATION Hazard Statements and Precautionary Statements: Corrosive Training Advice: None Additional Information: Replaces all previous editions. References: RTECS, CAS Registry, EINECS/ESIS, Manufacturer Information Risk Phrases and Safety Phrases: R36/37 Irritating to eyes and skin S36/37/39 Wear suitable protective clothing, gloves, and eye/face protection Prepared By Tygrus Regulatory Affairs Email: info@tygrus.com Creation Date May 6, 2020 Revision Date May 6, 2020 Print Date May 6, 2020 Revision Summary This document has been updated to comply with the US OSHA HazCom 2012 Standard replacing the current legislation under 29 CFR 1910.1200 to align with the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) Disclaimer The information provided in this Safety Data Sheet is correct to the best of our knowledge, information, and belief at the date of its publication. The information given is designed only as guidance for safe handling, use, processing, storage, transportation, disposal, and release and is not to be considered a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other materials or in any process, unless specified in the text. End of Safety Data Sheet 5 SciFinder® CAS Registry Number: 2032207-39-7 H9 O4 . H O4 S Hydrogen(1+), triaqua-µ3-oxotri-, sulfate (1:1) 14996-02-2 H O4 S 12501-73-4 H9 O4 ~2 References CAS Role Patents Nonpatents Nonspecific Derivatives from Patents from Nonpatents Biological Study Properties Uses Source of Registration: CA Copyright © 2017 American Chemical Society (ACS). All Rights Reserved. cn3 lnventoryExpertService A division of the American Chemical Society Phone: 800·631-1884, 614-447-3870 Fax: 614·447•3747 E-mail: answers@cas.org Web: www.cas.orgtproduclSfother-<:as-products/cllent-servicest November 14, 2013 Millstone Properties, LLC ATTN: Mr. Lawrence R. Carlson P.O.Box 12187 Zephyr Cove, NV 89448-4187 Dear Mr. Carlson: Thank you for your Inventory Expert Service (IES) order of July 22, 2013. The processing results are attached. To ensure efficient processing of your substance(s}, it is important that these results are included with your PMN report for when reporting lo the EPA. The cost of the technical processing for your order will be applied to your American Express card. Thank you for the opportunity to provide this service. If you have any questions. please do not hesitate to contact me. Sincerely, Jennifer Moore CAS Client Services Enclosure(s) CAS 2540 Olentangy River Road P.O. Box 3343 Columbus, OH 43210-0334 USA CD NFtJ)pr\JTtAL-- Larry Carlson From: Answers <answers@cas.org > Sent: Thursday, November 14, 2013 1:21 PM To: Larry Carlson Cc: Answers Subject: IES 348687 Attachments: !ES 348687 _20131114132157.pdf Follow Up Flag: Follow up Flag Status: Flagged Dear Mr. Carlson, Please see attached for the results of your Inventory Expert Service order. Note: This is the only copy you will receive. Please retain for your records. Regards, Jennifer Jennifer Moore CAS Client Services/Inventory Expert Service CAS, a division of the American Chemical Society 2540 Olentangy River Road Columbus, OH 43202 Phone: 614-447-3870 Fax: 614-447-3747 www.cas.org/products/other-cas-products/client-services Confidentiality Notice: This electronic message transmission, including any attachment(s), may contain confidential, proprietary, or privileged information from Chemical Abstracts Service ("CAS"). a division of the American Chemical Society ("ACS"). If you have received this transmission in error, be advised that any disclosure, copying, distribution, or use of the contents of this information is strictly prohibited. Please destroy all copies of the message and contact the sender immediately by either replying to this message or calling 614-447-3600. 1 (lli' lnventoryExpertService A division of the American Chemical Society Phone: 800·631-1884, 614·447-3870 Fax: 614·447-3747 E-mail: answers@cas.org Web: www.cas.org/productsiother-cas-products/cllent-services/ INVENTORY EXPERT SERVICE REPORT IES Order Number: 348687-2 Registry Number: Not Registered CA Index Name: Hydrogen(l+), trihydrox.y-, sulfate (1:1) Please print the above CA Index Name on the appropriate page of your PMN. lf this box is checked, CAS has made correction{s) marked in red to your IES order. Please make the same corrections to your PMN before submitting It to the EPA. CAS 2540 Olentangy River Road P.O. Box 3343 Columbus, OH 43210-0334 USA lnventoryExpertService A division or lhe Ame,ican Chemical Society Phone: 800-631-1884, 614•447•3870 Fax: 614·447-3747 E-mail: answers@cas.org Web: www.cas.org/products/other-cas•products/cllent•services/ INVENTORY EXPERT SERVICE REPORT IES Order Number: 348687•1 Registry Number: Not Registered CA Index Name: Hydrogen(l+), triaqua-µ3-oxotri-, sulfate (1:1) Please print the above CA Index Name on the appropriate page of your PMN. If this box is checked, CAS has made correction(s) marked In red to your IES order. Please make the same corrections to your PMN before submitting it to the EPA. GAS 2540 Olentangy River Road P.O. Box 3343 Columbus, OH 43210·0334 USA May 12, 2020 Tygrus / EPA meeting: EPA Attendees: John Hebert ‐ AD Kimberly Nesci ‐ BEAD Demson Fuller ‐ AD Jack Hall – Risk manager, AD Cesar Cordero – Acting team lead for efficacy in AD Laura Parsons – RASSB Tina Pham – efficacy, AD Lindsey O’Dell – chem / tox Susan Lawrence – BEAD Kristin Willis – AD Diane Isbell Jenny Tao Sue Bartow Beth: Opening remarks Objective – introduce the Agency to Tygrus and the new chemistry, hydronium sulphate, initial uses, proof of concept, looking at this as a new antimicrobial. Interested in the agency thoughts on processes and idea of this as a new active ingredient. Want feedback on this as a new AI and the pathway forward. Slide 4: John Coppolino Had a meeting with EPA about 18 months ago and have been working on this material ever since. In Q419 was issued a patent. Can share the details on the patent at a later time. Build a factory in Madison Heights Michigan which can make millions of gallons per week. Are getting the facility approved. Over the past year have also been working on medical uses with Dr. Attala and especially against the SARS‐ CoV‐2 virus. Slide 5 ‐ 7: Shaun Lindsey – chemistry, the patent will be released to the EPA at an appropriate time. Is the buffering system part of the patent? Also any substances involved in stopping the reaction? Shaun – the proton from KA1 is retarding the process. There are many homologs. From organic chemistry there are 1 – 2 CAS number for 30 compounds. The process moves fast and moves around. With titration and ICP know the protons and the anions amounts. The material has a different mass spec that sulfuric acid so know it’s a new material. Lindsey – have we completed any GLP chemistry studies? Shaun – Have some chemistry studies underway, not sure if any are GLP. With the data gap analysis will make sure the complete the appropriate package. Slide 9 – Dr. Devito As an acid it is obvious it’s a new antimicrobial, though there are many acid based antimicrobials on the market. Slide 10 – Dr. Devito Hydronium sulfate can be used as a new AI for environmental surfaces Slide 11 – Dr. Devito Early anti‐viral activity, directionality towards disinfection activity Tydracide is the planned trade name 4 sprays per dish Bleach as a positive control Tydracide had a nearly 3 log reduction in FCV and a 3 log reduction in HSV1 This is a good way to profile the anti viral activity of tydracide Slide 12 – Dr. Devito Planning for the disinfection properties of the product Slide 13 – Dr. Devito Tydracide can be classified as a category IV for everything except eye and it’s not a skin sensitizer Concentrated solution is moderately irritating to the eye, sets it apart from other typical acid disinfectants Slide 14 – Beth Testing has been done for proof of concept to show there is a viable, new compound here for antimicrobial activity against a number of target organisms. Very important is the SARS‐CoV‐2. Tygrus is further defining the MP, EP and to get a better feel for the concentration of the TGAI including stability testing and what is the stable TGAI and the equilibrium. Will come in and have additional pre‐ sub meetings with the EPA as work through the data gaps and to update the agency. Hydronium sulphate is a new compound. Thoughts on the Agency on whether there is agreement this is a new AI and other ideas: Feedback on the concept for how want to address data generation overall for new data and potentially bridging to sulfuric acid and sulfates. To work on a strategy and path forward. Will then come back and speak about specifics. AI thoughts: Laura Parsons – Q1: Why do we think it’s not a new AI? Beth – when tygrus came to exponent, it wasn’t initially clear. It is a new compound, the question is wha tis the mode of action and where is the activity coming from? Tygrus said there is activity from both. Would love to be a new AI but it increased the data generation and timelines. There are many things that indicate it may not be a new AI. Also depends on how EPA thinks about this. When are looking at an inorganic complex how does the agency determine if it’s a new AI? Laura Parsons – What they hear is that we want to piggy back on the mineral acids as a bridge? Beth – yes, in some cases would want to look at bridging to the mineral acids data. There are some similarities and differences, less toxicity, less corrosive, have to do more digging into the mineral acids to argue for that piece, that would be one possible approach. Laura Parsons – For RASSB don’t have enough information to be able to say one way or another. Will need to do – give some of the bridging arguments about the basic chemistry and toxicity. Need more details before RASSB can say tthis is a new AI, think it probably is especially if has a different corrosion charactertistic than the mineral acids. Use pattern for the mineral acids and this is a broader use pattern than the mineral acids. Beth – Part of the next step will be to do what Laura said, go through the mineral acids and make the comparisons and look at the arguments. We have to do more work to be able to come back more definitively on the new AI piece. Inclined that this is new. For the uses / labels we do recognize that are looking for a broader label and more claims than registered for the other products. John Hebert – From a regulatory pov, this has a unique CAS# then from a regulatory pov that it is a new AI. If it is included in other classes for data bridging has to be determined. Believes it is a new AI based on the CAS# and it’s a new compound. Beth – John / Laura seems to be amenable to looking at the approaches for the other actives and chemistries and with simomilary to come back with a new plan. Then can fulfill the data gaps and if there is opportunity for not having to test or to make sure the agency agrees with the approach. Demson – If want to come back in later, what studies do we want to bridge. There are forms that we can fill out and if we believe there are data to use for bridging we can site the information to help RASSB. Beth – starting with 40CFR 158 (W). Starting with what is required, other areas to fulfill the data requirements. Have a template for the requirements and go through each one and determine if data or bridge. Can the agency share the template they use? Demson – Yes, they can share the template. Beth – other questions / thoughts, etc. John Hebert – Food uses? Will we have an exemption from tolerance or tolerance limits? Beth – Yes, we will likely be looking for an exemption from tolerance. There are things that may be measurable and ubiquitous so a tolerance can be challenging. It is on our mind. We want to be able to move quickly. It’s an exciting chemistry so want to be efficient and it’s a difficult time right now. Especially to combat the SARS‐CoV‐2. Also this could be a low risk compound so anything we can do to move this through to registration would be an objective. John – Are coming out with expedited process for COVID‐19 but does not include new AIs but they would consider this. Beth – not often get a new AI sot that adds antoher dimension. Will take the Agency guidance into consideration. John – when would we have a draft label? Beth – we would love to have one really fast but that may be premature. Perhaps 4 – 6 weeks we will have the strategy and we are moving fast on testing. In about 2 months to talk with the agency again. We would have a better idea of use patterns then. Diane will be working with Tygrus on the microbioloty and a better sense of the testing and labeling. In the next few weeks we will be able to solidify thoughts on timing and further information on a label. Beth – thank you for yoru time and attention. We hope you are as excited as we are. We are moving quidkly and will prepare the minutes and send those over. Sue – Send the minutes to Sue. Sue will type up the attendee list from the EPA. John – Sue will be sending out minutes of the meeting and we can work off of those, the EPA minutes are the official minutes. Suggest we stick to using the EPA minutes as official. Jenny – from the agency perspective, the agency follows and promotes use of in vitro testing for animal testing for eye and sensitization. Acute dermal / oral waiver. If have any questions let them know through Sue. The agency appreciates the reduction in animal testing. Beth – when we prepare the data gap analysis we will take that into account. We appreciate the reminder. We will also look at bridging for other options for bridging from the mineral acids. If at any time there are questions please reach out to Exponent. We can use Sue as the conduit. Meeting closed: 3:58 PM Environmental Impact of Tydracide™ for disinfecting COVID-19 & Comparison to Bleach Summary Due to the COVID-19 pandemic there is an extreme level of attention on disinfecting surfaces to reduce the spread of new infections. Tydracide is a novel low toxicity acid that is proven to be more effective at killing COVID-19 viruses than the most widely used solution, sodium hypochlorite or bleach with each is at its recommended disinfection use concentrations. Additionally, Tydracide offers further benefits by reducing toxicity to individuals and the environment, which may allow simplified disinfectant protocols and create new disinfectant applications such as possibly disinfecting N95 respirators for health care personnel. This whitepaper will discuss Tydracide environmental test results using U.S. EPA (Environmental Protection Agency) Test Procedures focused on Daphnia Magna, a water flea, as a measure of environmental toxicity. The Tydracide LC50 (Lethal Concentration) for daphnia is 1.2667mg/l, which is the concentration which was statistically measured to kill 50% of a daphnia population under specific laboratory conditions. The recommended usage concentration for disinfecting with Tydracide is 5%. In order to meet the LC50 concentration from 1 gallon of Tydracide-5 it would need to be diluted with 81.7 gallons of water. Sodium hypochlorite is significantly more lethal to daphnia with a LC50 concentration of 0.033- 0.048mg/l. At 500ppm recommended use concentration for disinfecting, 1 gallon of sodium hypochlorite would need to be diluted with 12,346 gallons of water to meet the LC50 concentration. This comparison is summarized in Table 1. Table 1. Tydracide and Sodium Hypochlorite LC50 for Daphnia and Dilution Factors Recommended Daphnia Dilution to Chemical Name Disinfection LC50 Meet LC50 Concentration Sodium Hypochlorite 500ppm or mg/l 0.033-0.048 mg/l 12,346 (bleach) Tydracide 5% 1.2667 mg/l 81.7 Using the dilution rates from recommended disinfection use concentration to LC50 for daphnia as representative model for the environmental impact of these two chemistries, it can be concluded that sodium hypochlorite is more than 150 time more toxic to the environment than Tydracide. Introduction The U.S. EPA utilizes acute toxicity tests for the National Pollutant Discharge Elimination System (NPDES) to identify effluents containing toxic materials in acutely toxic concentrations1. LC50 is the testing protocol for acute toxicity used in this process. LC50 is the medial lethal concentration or the concentration of a material in water that is estimated to be lethal to 50% of the test organisms. The LC50 can also be used to estimate acute and chronic toxicity in the receiving water based on appropriate dilution and other factors. The LC50 provides a concentration measurement result based on statistical testing of lethality to specific organisms in a controlled and reproducible environment. One way to use the LC50 information when applied to pesticides, viracides and other chemicals that may be introduced to the environment, is to consider the dilution rates with water required from shipping or recommended use concentrations of these chemicals in order to meet the LC50 concentration. The higher the dilution rate to meet the LC50 concentration, the more toxic the chemical is to the specific species tested. Using these species as representatives to the overall environmental impact may allow a more general comparison of the environmental impact between specific chemicals. The LC50 tests can be performed on multiple organisms to provide representative viewpoint of the environmental impact of a particular material when released as an effluent to waters. The most widely used organisms for LC50 testing are Daphnia magma and Daphnia pulex, and the EPA has established a specific test protocol for these organisms under Test Method 2021.0. No single test method or test organism can be expected to satisfy a comprehensive approach to environmental protection2. The Fathead minnow is another commonly used species for LC50 tests and the EPA protocol for this species is Test Method 2020.0. Daphnid is a freshwater microcrustacean invertebrate, commonly known as the water flea, which are a major component of the freshwater zooplankton throughout the world and may be the dominant herbivore in lakes. Species of daphnids include Daphnia magna and Daphnia pulex. Daphnia magma is a lake and pond dweller in waters of western and northern North America2. These organisms are an important link in many aquatic food chains and are a significant source of food for juvenile fish species. Daphnids are sensitive to a board range of aquatic contaminants and are widely used for evaluating the toxicity of chemicals. The small size and short life cycles of daphnids simplifies the test protocol with this organism. As result the LC50 concentration for daphnia is one of the most common metrics to determining the environmental impact for a potential chemical release into the environment. Tydracide LC50 Results and Dilutions LC50 testing for Tydracide™ was performed by Paragon Laboratories in Livonia, MI. Paragon Laboratories, Inc. is a privately held provider of chemical, physical, and biological testing services. In operation since 1996, Paragon is capable of performing over 225 unique analytical procedures. With significant experience in the application of ASTM, EPA, EN, and Standard Methods, Paragon has developed a reputation as a proven leader across a range of disciplines – including fuels and lubricants, chemicals, and waters – for various industries to meet quality and regulatory standards. Paragon Laboratories performed LC50 on Tydracide-50 for Daphnia magna per EPA method 2021.0 and for Fathead minnows per EPA method 2020.0. Paragon’s results are summarized in Table 2. Table 2. Tydracide-50 LC50 Concentrations for Daphnia and Minnows LC50 value Test Species Test Duration (grams Tydracide-50/liter) Daphnia Magna 48 hours 1.2667 Fathead Minnows 96 hours 1.0946 Tydracide-50 is a concentrated version of Tydracide with a specific gravity of 1.035 grams/ml or 1,035grams/liter. To reach the LC50 concentration of 1.2667 grams of Tydracide-50/liter Tydracide-50 would need to be diluted with 817 parts water to 1 part Tydracide-50 (=1035/1.2667). Similar calculations are shown in Table 3 for 10%, 5%, 2% and 1% concentrations of Tydracide for both Daphnia Magna and Fathead Minnow. Table 3. Dilution to Meet LC50 for various Tydracide Concentrations Tydracide 50 Tydracide Specific Gravity Daphnia LC50 Minnow LC50 Content Concentrations (grams/liter) Dilution Tydracide-50 1,035 817 945 (50% concentration) Tydracide-10 (10% concentration; 1,007 207 163 189 4:1 dilution) Tydracide-5 (5% concentration; 1,003.5 103.5 81.7 94.5 9:1 dilution) Tydracide-2 (2% concentration; 1,001.4 41.4 32.6 37.8 24:1 dilution) Tydracide-1 (1% concentration; 1,000.7 20.7 16.3 18.9 49:1 dilution) The recommended usage concentration for Tydracide for disinfecting surfaces of COVID-19 is (expected) 5%, which is a 9:1 dilution of the more concentrated Tydacide-50. The LC50 dilution factor for daphnia magna with a 5% concentration of Tydracide is 81.7X. One potential measure of environmental toxicity is a comparison of these LC dilution rates to other COVID-19 viracides such as sodium hypochlorite. Sodium Hypochlorite LC50 Results and Dilutions The most widely used viracide for COVID-19 is sodium hypochlorite or bleach. The daphnia LC50 concentration for sodium hypochlorite is 0.033-.048mg/l3 or an average of 0.0405mg/l. Household bleach is normally shipped as a 5,000ppm or mg/l product. Recommended usage is at 500mg/l as a disinfectant (9:1 dilution of concentrate), and at 200mg/l as a sanitizer. The dilution rates to meet the daphnia LC50 is shown in Table 4. Table 4. Dilution to Meet Daphnia LC50 for various Bleach Concentrations Sodium Hypochlorite Concentration Daphnia LC50 (Bleach) (mg/l or ppm) Dilution Concentrations Shipping Concentration 5,000 123,457 Disinfecting Concentration 500 12,346 Sanitizing Concentration 200 4,938 References 1 – Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organism Fifth Edition, US EPA, October 2002 2- Biological Test Method: Acute Lethality Test Using Daphnia, Canada Environmental Technology Centre, Report EPA 1/RM/11, July 1990 with 1996 amendments 3- Sodium Hypochlorite/Calcium Hypochlorite (Clorox, Bleach) Chemical Fact Sheet 2/86 http://pmep.cce.cornell.edu/profiles/miscpesticides/methylchloride- xanthangum/hypochlorite/hypochl_prf_0286.html Better Everything EPA-mandated Animal Toxicity Tests Confirm the Safety of Tydracide-50 This report summarizes the performance of Tydracide-50 in a battery of toxicology tests required for EPA registration of chemicals, disinfectants and other antimicrobial products. The toxicology tests (known in the vernacular as the standard “Six-Pack”) are performed on small laboratory animals to simulate the human health impact of chemical substances. First published in 1982 and periodically updated, the current EPA guidelines (870.1000) require acute or short-term toxicity testing be performed on all registered chemicals according to their probable routes of human exposure. Tydracide-50 is a novel product based on the naturally occurring hydronium ion (H3O+), which is the defining particle of all acids. Using the proprietary manufacturing processes developed at Tygrus, the hydronium ion can now be produced in its stabilized form (H9O4) on a commercial scale. This chemical substance, called Tydronium™, is manufactured as a pure product concentrate and can be formulated for application in various markets. Given the acidic nature of Tydronium™, it is important to conclusively demonstrate that technical grade active ingredient derivative products, like Tydracide-50, behave differently than traditional acids in regards to their overall safety profile. The following six tests were conducted on Tydracide-50 according to the EPA guidelines: Acute Dermal Toxicity, Acute Oral Toxicity (Up & Down Method), Acute Inhalation Toxicity, Primary Eye Irritation, Primary Skin Irritation, Dermal Sensitization (Local Lymph Node Assay). Testing was performed by Product Safety Laboratories (PDL) of Dayton, New Jersey. PDL is now part of Eurofins Scientific, an international group of laboratories headquartered in Luxembourg that provides certified testing and support services to the pharmaceutical, food, environmental and consumer products industries and to government organizations. Eurofins Scientific is a multinational contract research organization that offers its customers documented, top- of-the-line laboratory services to determine product effectiveness, safety and to support regulatory approval. All six toxicity tests of Tydracide-50 were performed during the second quarter of 2018 using the same lot/batch. The following paragraphs summarize the test results obtained: An Acute Dermal Toxicity Test was performed by applying a patch containing Tydracide-50 directly to the skin of ten healthy rats (5 male and 5 female). Each patch contained 5000 milligrams (mg) of Tydracide-50 per kilogram (kg) of body weight for each rat. The animals were observed for mortality, signs of gross toxicity, and behavioral changes during the first several hours after application, after patch removal, and then at least once daily thereafter for 14 days. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma. Individual body weights of the animals were recorded prior to Tydracide-50 application and again on Days 7 and 14, followed by gross necropsy of euthanized animals where tissues and organs of the thoracic and abdominal cavities were examined. All animals remained healthy during the study, gained weight, and there were no adverse findings recorded on any animals except for slight redness of the skin at the dose site of a single male rat that resolved within 24 hours. The data show that for acute dermal toxicity Tydracide-50 has a LD50 (dose at which 50% of the animals show negative effects) above 5000 mg/kg. As such, according to the Global Harmonized System for chemical classification (GHS), Tydracide-50 is a Category 5 substance…i.e. practically non-toxic. The US EPA applies a slightly more restricted four-category rating and Tydracide-50 would be considered a Category 4 product requiring no hazard statements to be present on the product label. Confidential Information ©2019 Tygrus, LLC October 17, 2019 Using related methodologies, an Acute Oral Toxicity study was performed to determine the potential for Tydracide-50 to produce toxicity from a single dose via oral administration. Three healthy female rats (typically more sensitive to chemical substances than males) were dosed at 5000 mg/kg by oral gavage and observed for 14 days as described in the previous paragraph. Despite hypoactivity and irregular respiration following dosing, all animals appeared normal and no gross abnormalities were observed upon necropsy. These data are consistent with assigning Tydracide-50 a GHS Oral Toxicity classification of Category 5 and an EPA classification of Category 4….essentially non-toxic. Identical results were collected in an Acute Inhalation Toxicity study where five healthy male and five healthy female rats were continuously exposed to Tydracide-50 aerosol (1-4 micron particle size) over a four- hour period at a 5.13 mg/liter concentration. Following exposure, the animals were monitored for signs of gross toxicity and observed for 14 days as described above. All animals survived exposure to the test atmosphere saturated with Tydracide-50 and gained body weight during the study. Immediately following exposure, all rats exhibited irregular respiration, however, all animals recovered and appeared active and healthy for the remainder of the 14-day observation period with no gross abnormalities seen during necropsy. Tydracide-50 was also evaluated in a Primary Skin Irritation study using the more sensitive rabbit species as a test subject. Methods used were similar to those previously described for the rat. Results showed no skin corrosion from Tydracide-50 exposure and only slight skin irritation following a four-hour exposure that resolved by the study termination at day seven. These data assign Tydracide-50 to the lowest toxicity categories for skin irritation on both GHS and EPA scales (Category 3 and 4, respectively). Again using a rabbit model, Primary Eye Irritation was measured by instillation of a 100 microliter drop of Tydracide-50 into one eye each of three healthy, naive animals without pre-existing ocular irritation. Ocular irritation was scored by a widely accepted method (Draize scoring) where (at 1, 24, 48, 72 hours and at 4, 7, 10, 14, 17, and 21 days post installation) lesions and variations in the appearance of the cornea, iris and conjunctivae were recorded. Over the first 24 hours following test substance instillation, two treated eyes exhibited corneal opacity and ‘positive’ conjunctivitis. There was no iritis observed in any treated eye during this study. The overall incidence and severity of irritation decreased gradually with time. Positive irritation cleared from the two treated eyes by Day 21. Minimal conjunctivitis persisted in one treated eye through Day 21 (study termination), all resulting in a total numerical score of 19.7 and classification of Tydracide-50 as moderately irritating to the eye (EPA Category 3, GHS Category 2B). The final study in the EPA six-pack suite was performed in mice to determine if Tydracide-50 has the capacity to sensitize rodent skin. This test procedure is called the Local Lymph Node Assay (LLNA) and is quite sophisticated. It involves large numbers of mice and directly measures immune cell proliferation in the lymph nodes caused by a chemical applied to the skin. The amount of cell proliferation is assessed by radioactive isotope incorporation into newly synthesized DNA. For the test occasion where Tydracide-50 performance was assessed, a cell stimulation index (SI) of 4.5 was calculated for the positive control substance alpha- Hexylcinnamaldehyde, a moderate dermal sensitizer. The average SI of three Tydracide-50 concentrations (25, 50, and 100%) was 0.98, far below the 3.0 cutoff established to designate a chemical as a contact sensitizer. As such, Tydracide-50 is not considered to be a contact dermal sensitizer and would require no classification by GHS or EPA. The results from these pivotal studies demonstrate that Tydracide-50 is considered safe according to the “Six- Pack” EPA toxicity testing paradigm. Unlike traditional acid products that are highly corrosive and highly toxic, Tydracide-50 can be assigned the lowest chemical toxicity rating in most categories, and is only moderately irritating to the eyes. Clearly, Tydracide-50 has the potential to be used safely and effectively as a technical grade active ingredient in economically important markets and where environmentally friendly, green chemistries are preferred. Confidential Information ©2019 Tygrus, LLC October 17, 2019 2017 Hydronium / Citric Acid Trials for Crescendo Stabilization 2/17/2017 trials begin. 500ml of Crescendo with pH reduced to 3.8 pH in 500ml flasks with cap hand tightened. Kept in incubator @ 78 degrees for 12 weeks. 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