SRC TR - 1 4 - 001

HPV2 TEST RULE: RESPONSE TO DOW AGROSCIENCES , COMMENTS DATED JANUARY 14 , 2014 (FINAL ) Phosphorochloridothioic acid, O,O - diethyl ester , CASRN 2524 - 04 - 1 Prepared by: Jennifer Rayner , Teresa Manyin , and W illiam Richards Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212 Contract No. EP - W - 09 - 027 Task FG007.4 .009.001 Submitted to: OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460 Bryan Lobar, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

January 22 , 2014HPV2 Test Rule: Response to Dow AgroSciences (Final ) CASRN 2524 - 04 - 1

1 On January 14 , 2014, Dow AgroSciences submitted an email to EPA regarding the instability of CASRN 2524 - 04 - 1 ( phosphorochloridothioic acid, O,O - diethyl ester ; O,O - diethyl phosphorochloridothioate [ DEPCT ] ) and the resulting challenges of completing the studies required in the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Che micals” (76 FR 1067, January 7, 2011; referred to herein as HPV2; available online at http://www.regulations.gov/#!documentDetail;D=EPA - HQ - OPPT - 2007 - 0531 - 0579 as of January 22 , 2014). The HPV2 test rule requires the following toxicological tests for CASRN 2524 - 04 - 1: C1 ( aquatic toxicity tests ), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus test), and F2 (reproduction/develo pmental toxicity screening test). The comments from Dow AgroSciences are addressed below: (1) Dow AgroSciences cited the results of a hydrolysis study submitted under the REACH registration for CASRN 2524 - 04 - 1 (available online at http://apps.ec ha.europa.eu/registered/data/dossiers/DISS - 9ea6e71a - f3c6 - 6f79 - e044 - 00144f67d031/AGGR - aeec32eb - 7557 - 4480 - 911a - 219171f0c3ea_DISS - 9ea6e71a - f3c6 - 6f79 - e044 - 00144f67d031.html#AGGR - aeec32eb - 7557 - 4480 - 911a - 219171f0c3ea as of January 22 , 2014). Dow AgroSciences states “ Hydrolysis half - life is 14 min. at room temp in distilled water... ” The hydrolysis test cited in the REACH registration for CASRN 2524 - 04 - 1 was not conducted according to a testing guideline or GLP , and detailed methods, test conditions, and result s were not reported. Data are insufficient to allow an independent assess ment of the reliability and adequacy of the test and its results. Therefore, the half - life results of this specific test should not be factored into decisions about the ability to c onduct , as well as the methods that may be used to conduct , toxicity tests. EPA recommends that the hydrolysis test be repeated to determine an accurate and reliable half - life of CASRN 2524 - 04 - 1. If the test confirms that the hydrolysis of the test subst ance is rapid, then precautions will need to be taken during the toxicity tests, as described below. (2) Dow AgroSciences states “ After stability testing in various media, the testing conditions may then be unrealistic for any potential route of exposure (i.e. ecotox and mammalian testing in nonaqueous media? 30 - day tox testing when the half - life is 14 minutes? etc...) .” It is recogn ized and accepted that CASRN 2524 - 04 - 1 may be unstable under testing conditions for ecological and mammalian toxicity studies and that degradation to its hydrolysis products may occur during these studies. Test solutions shall be formulated using nominal concentrations of CASRN 2524 - 04 - 1 and shall be prepared just prior to the introduction of test organisms in aquatic toxicity studies, the exposure of bacterial or mammalian cells in in vitro genotoxicity studies , and the dosing of animals in in vivo toxici ty studies . Analytical monitoring of the concentrations of CASRN 2524 - 04 - 1 and its degradation products shall be conducted during the toxicity tests, as further discussed in #3 and # 5 below.HPV2 Test Rule: Response to Dow AgroSciences (Final ) CASRN 2524 - 04 - 1

2 (3 ) Dow AgroSciences states “ Due to chemical instability of DEPCT, especially in, but not limited to aqueous media, we will not be able to follow the required testing guidelines for the following studies:... Fish tox, Daphnia tox, Algae tox. ” Although DEPCT (CASRN 2524 - 04 - 1) is a reactive chemical, there are guideli nes and methods available that will allow the chemical to be tested successfully for effects on aquatic species . The sponsor should refer to OECD Series on Testing and Assessment Number 23 , “Guidance Document on Aquatic Toxicity Test ing of Difficult Subst ances and Mixtures”. This guidance document can be used to develop test protocols that are appropriate for conducting aquatic toxicity tests for chemicals that degrade in the test system ( see s ection 3.5 ), including test substances that undergo hydrolysis under test conditions (see section 3.5.2). According to the test rule guidelines, preliminary stability tests should be conducted to assess the stability of the test substance under the test conditions of each aquatic toxicity test prior to conducting the aquatic toxicity tests . EPA recommends that toxicity tests of DEPTC for fish and Daphnia be conducted using a closed continuous flow - through system ( see OECD 23, Section s 3.4 and 3.5 ). Flow - through tests facilitate the maintenance of exposure conce ntrations especially in cases where degradation or loss of test substance may occur. The system used shall allow the undiluted test substance to mix with test dilution water immediately prior to entrance into the test system. Samples of test solution for analytical measurement shall be collected at the point of entry into the system and at the exit point of the system at least every 24 hours ; these samples shall be analyzed for concentrations of CASRN 2524 - 04 - 1 and its organic degradation products . Alternatively, toxicity tests for fish and Daphnia may be conducted using static - renewal tests. If a static renewal test is conducted, test solutions shall be prepared no more than 30 minutes prior to the introduction of test organisms to the solutions. Samples of test solution for analytical measurement of test substance concentrations ( CASRN 2524 - 04 - 1 and its organic degradation products) shall be collected at the start and end of each renewal period. For either a flow through or a static renewal test, the system must be closed (sealed to prevent exposure to air) to minimize volatilization of the test rule chemical and its degradation products from the test water to the atmosphere. Algae tests can only be conducted using static exposure technique. Due to the potential volatility of CASRN 2524 - 04 - 1 and its hydrolysis products, the following alterations to standard guideline methods must be made (as detailed in Mayer et al. [2000]):  The test vessels shall be sealed during preparation and exposure to pre vent loss of the test substance. The seal or stopper used by the testing lab must be impermeable to air.  Headspace shall be kept to a minimum to reduce loss of test substance from solution.  Test solutions (algae growth media) shall be en riched with additi onal sodium bicarbonate (e.g., 300 mg NaHCO 3 /L) to provide a source of CO 2 for algae growth. After addition of sodium bicarbonate, the pH of growth medium must be adjusted to 7.0 by the addition of HCl.  The initial cell density shall be reduced to 3 x 10 3 cells/mL to reduce demand for CO 2 .  The algae shall be added to the prepared test solutions no more than 30 minutes after preparation to maximize exposure to the parent compound .HPV2 Test Rule: Response to Dow AgroSciences (Final ) CASRN 2524 - 04 - 1

3  The duration of algae exposure shall be 72 hours. Samples for analytical measurement of the concentration of the test substance and its organic degradation products shall be collected at the beginning and end of the algae test at a minimum. ( 4 ) Dow AgroSciences states “ There are also challenges with conducting the mammalian tox studies due to instability but maybe there are other options for testing media?” With regard to in vitro geno toxicity tests, an anhydrous organic solvent that ha s no nucleophilic functional groups (e.g., dimethylformamide [DMF], acetone, dimethyl sulf oxide [DMSO]) should be used to prepare stock solutions of test substance prior to introduction into the test system. The organic solvent used should be free of water; water can be removed by adding a molecular sieve (see 40 CFR 799.9537). The use of such solvents wil l minimize degradation of CASRN 2524 - 04 - 1. In vitro genotoxicity tests shall be conducted using sealed test containers to minimize volatilization of CASRN 2524 - 04 - 1 and its degradation products. With regard to in vivo mammalian toxicity tests ( reproduct ive/developmental and micronucleus tests) , the test substance can be orally delivered undiluted or diluted in a suitable vehicle ( e.g., anhydrous corn oil). The oil should not contain fatty acids with alcohol groups. Anhydrous o il based vehicles can be u sed to minimize degradation of test substances that can react with water or other nucleophilic substances. The report must contain a justification for the use and suitability of the chosen vehicle. The dosing solutions administered should be freshly prepared prior to each dosing . According to the test rule guideline for the reproductive/developmental toxicity test, the stability of the test substance should be determined in the chosen vehicle prior to conducting the toxicity test . To maximize exposu re to the parent chemical (CASRN 2524 - 04 - 1), test solutions for mammalian toxicity studies and in vitro toxicity studies shall be used no more than 30 minutes after preparation. ( 5 ) Dow AgroSciences states “ DEPCT is highly reactive (hydrolyzes) in water ... The reactivity will have a significant influence on how testing is done - if it can be done at all. The substance will react with water, proteins, alcohols, etc. (any nucleophilic substance), thus making it unstable and difficult to verify exposure conce ntrations in ecological and mammalian toxicity tests.” Analytical monitoring of test substance concentrations used in toxicity tests must be conducted to determine actual exposure concentrations and to demonstrate repeatability of procedures used to prepare test solutions . This will include analytical determination of the concentrations of CASRN 2524 - 04 - 1 (the parent chemical) and its organic degradation products. (Analysis of the concentration of HCl is not necessary.) Concentrations of CASRN 2524 - 04 - 1 and its organic degradation products must be satisfactorily monitored throughout the duration of the test, as demonstrated by the results of a reliable, sensitive, and validated analytical metho d. The testing laboratory conducting the tests shall be familiar with the sampling, proc essing, storage , and analytical detection procedures needed for quantitation of DEPCT and its organic degradation products .HPV2 Test Rule: Response to Dow AgroSciences (Final ) CASRN 2524 - 04 - 1

4 Conclusion : EPA maintains that the requ ired aquatic and mammalian toxicity tests must be conducted to satisfy the HPV2 testing requirement s . EPA makes the following recommendations: 1. Conduct a hydrolysis test of DEPCT using OECD TG 111, which includes methods for testing unstable substances (Tiers 1 through 3). If the test confirms that the hydrolysis of the test substance is rapid, then precautions will need to be taken during the toxicity tests to minimize DEPCT hydrolysis and thereby maximize exposures to DEPCT . 2. Determine appropriate meth ods for analytical monitoring (including sampling, processing, storage, and detection procedures) during toxicity test s, based on the information obtained during the hydrolysis test and requirements of testing guidelines . 3. Submit a test plan with detailed s tudy protocols to EPA for review. To demonstrate that there is a consistent relationship between nominal and measured levels of CASRN 2524 - 04 - 1 and its degradation products, study protocols should include frequent sampling of test media and dosing solutio ns for analyses of concentrations of CASRN 2524 - 04 - 1 and its organic degradation products during the tests . REFERENCES Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M., and Tolls, J. 2000. Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental Toxicology and Chemistry 19: 2551 - 2556. OECD . 2002. “ OECD Series on Testing and Assessment Number 23: Guidance document on aquatic toxicity testing of difficult substances and mixtures”. Available at http://www.oecd ilibrary.org/environment/guidance - document - on - aquatic - toxicity - testing - of - difficult - substances and - mixtures_9789264078406 - en as of January 2 2 , 2014. OECD. 2004. “ OECD Guidelines for the Testing of Chemic als, No. 111: Hydrolysis as a function of pH”. Available at http://www.oecd - ilibrary.org/environment/test - no - 111 - hydrolysis as - a - function - of

- ph_9789264069701 - en as of January 2 2 , 2014. US EPA. 2012. “TSCA in vitro mammalian chromosome aberration test”. 40 CFR 799.9537. Available at http://www.ecfr.gov/cgi - bin/text idx?SID=b3c328624afa4723ef0e5af7b724d7dd&node=40:33.0.1.1.8.7.1.18&rgn=div8 as of January 22 , 2014.