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HPV2 TEST RULE DATA ADEQU ACY REVIEW ( FINAL ): BACTERIAL REVERSE MU TATION ASSAY Phosphorochloridothioic acid, O,O

- diethyl ester , CASRN 2524

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Prepared by: Christopher Janson, Teresa Manyin, Amy Nguyen, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 12

- 003 Task FA552.4 .011.001

Submitted to:
OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460 Cynthia Bowie, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

February 10 , 2016Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a bacterial reverse mutat ion test for CASRN 2524

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- 1 (phosphorochloridothioic acid, O,O

- diethyl ester; O,O

- diethyl phosphorochloridothioate [DEPCT]) submitted by Dow AgroSciences to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2; available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of February 10 , 2016 ). The HPV2 test rule requires the following toxicological testing for CASRN 2524

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- 1: C1 (aquatic toxicity, consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae tests] if log K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae tests] if log K ow ≥ 4.2), E1 (bacterial reverse mutation test), E2 (chromosomal aberrations or micronucleus test), and F2 (reproduction/developmental toxicity screening test). Based on an estimated log K ow of 2.37 1 , Test Group 1 testing is required to determine the toxici ty of CASRN 2524

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- 1 to aquatic organisms. Dow AgroSciences previously submitted a study report of a bacterial reverse mutation test of CASRN 2524

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- 1. 2 This study was reviewed in a separate report 3 and w as determined to be in adequate to satisfy the gene mutation (E1) testing requirement for CASRN 2524

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- 1 promulgated in the HPV2 test rule because no precautions were taken to minimize losses of test material by volatilization during the study. On August 25, 2015 , Dow AgroScience s submitted a study p lan to EPA for a second bacterial reverse mutation test of CASRN 2524

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. 4 EPA reviewed the study plan and provided comments and recommendations for conducting an adequate study to satisfy the gene mutation (E1) testing requirement

. 5

In January of 201 6 , Dow AgroSciences submitted the following stud y to EPA :

 Wagner, V.O., and Jayakumar, G. 2016

. Bacterial reverse mutation assay with p hosphorochloridothioic acid, O,O

- diethyl ester. Performing Laboratory: BioReliance, Rockville, MD . Sponsor: The Dow Chemical Company

.

1 U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft ® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/tsca

- screening

tools/epi

- sui tetm

- estimation

- program

- interface#download as of February 10 , 2016. 2 Tendulkar, K.E. 2014. Phosphorochloridothioic acid, O,O

- diethyl ester : Salmonella

- Escherichia coli mammalian

microsome reverse mutation assay pre

- incubation method with a confirmatory assay. Performing Laboratory: JAI Research Foundation, Gujarat, India. Sponsor: Dow AgroSciences LLC. 3 “HPV2 Test Rule Data Adequacy Review: Bacterial Reverse Mutation Test, Phosphorochloridothioic acid, O,O

diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 023, dated March 4, 2015. Available online at http://www.regul ations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0847 as of February 10 , 2016

. 4 Dakoulas, E.W. (Study Director)

. 2015. Bacterial reverse mutation assay with phosphorochloridothioic acid, O,O

diethyl ester. Test Facility: BioReliance Corporation, Roc kville, MD. Study No. AE23VX.503001.BTL. Sponsor: The Dow Chemical Company. 5 “HPV2 Review of Toxicity Study Plan Dated August 25, 2015: Bacterial Reverse Mutation Assay , Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

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- 1” , SRC Technical Report # 15

- 055 , dated S eptember 22, 2015.Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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2 These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.
B. EVALUATING DATA ADEQUACY The submitted data are evaluated for adequacy in Section D. The study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy the E1 testing requirement for CASRN 2524

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- 1, study detail s were compared to OECD TG 471

. 1 In addition, the study was evaluated to determine whether recommendations provided in EPA’s study plan review were followed. Because CASRN 2524

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- 1 has physicochemical properties (i.e., potential for hydrolysis 2 and vola tilization 3 ) that necessitate special considerations in the gene mutation testing process, EPA had previously provided recommendations aimed to minimize loss of the test substance during toxicity testing.
C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRE D BY TEST RULE Evaluation of Study Submitted for the Bacterial Reverse Mutation (E1) Testing Requirement : The study submitted by Dow AgroSciences and reviewed in this document is considered adequate to satisfy the gene mutation (E1) testing requirement for CASRN 2524

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- 1. Other HPV2 Test Rule Toxicity Testing Requirements for CASRN 2524

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- 1: Dow AgroSciences previously submitted a study report of a micronucleus test 4 of CASRN 2524

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- 1. This study was reviewed in a separate report 5 and was determined to be adequate to satisfy the chromosomal aberrations (E2) testing requirement for CASRN 2524

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- 1 promulgated in the HPV2 test rule.

1 Although the HPV2 test rule specifies that gene mutation testing should be conducted according to TSCA guideline 40 CFR 799.9510, EPA previously granted a request from Dow AgroSciences to follow OECD TG 471 ins tead (request granted in EPA’s study plan review of CASRN 2524

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- 1, “HPV2 Review of Toxicity Study Plan Dated February 25, 2014 , Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

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- 1”, SRC Technical Report #14

- 008, dated April 3, 2014; availabl e online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OPPT

- 2007

- 0531

- 0805 as of February 10 , 2016 ). 2 West, R.J., and Hu, J. 2014. Phosphorochloridothioic acid, O,O

- diethyl ester: Determination of hydrolysis as a function of pH according to OECD Guideline 111. Performing Laboratory: The D ow Chemical Company (Toxicology and Environmental Research and Con sulting), Midland, MI. Sponsor: Dow AgroSciences LLC. 3 CASRN 2524

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- 1 is considered volatile based on an estimated Henry’s Law Constant of 6.72 x 10

- 4 atm

- m 3 /mole at 25 °C (U.S. EPA. 2012. Estimation Programs Interface SuiteTM [EPI Suite] for Microso ft ® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/tsca

- screening

tools/epi

- su itetm

- estimation

- program

- interface#download as of February 10 , 2016 ). 4 Schisler, M.R., and McFadden, L.G. 2014. Evaluation of phosphorochloridothioic acid, O,O

- diethyl ester (DEPCT) in the mouse peripheral blood micronucleus test. Performing Laborato ry: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0819 as of February 10 , 2016

. 5 “HPV2 Test Rule Data Adequacy Review: Micronucleus Test, Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 024, dated March 4, 2015. Available online at http://www. regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0848 as of February 10 , 2016

.Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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3 Dow AgroSciences previously submitted a study report of a reproduction/developmental toxicity screening test 1 of CASRN 2524

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- 1. This study was reviewed in a separate report 2 and was determined to be adequate to satisfy the reproduction/developmental toxicity (F2) testing requirement for CASRN 2524

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- 1 promulgated in the HPV2 test rule. Dow Agr oSciences previously submitted study reports of acute toxicity to fish 3 , acute toxicity to Daphnia 4
, and toxicity to algae 5 tests of CASRN 2524

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- 1. These studies were reviewed in a separate report 6 and were determined to be adequate to satisfy the aquatic toxicity (C1) testing requirement for CASRN 2524

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- 1 promulgated in the HPV2 test rule. Summary of Data Adequacy for Toxicological Endpoints: Adequate data have been submitted by Dow AgroSciences to satisfy the testing requirements for the acute toxicity to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to algae (component of C1), gene mutation (E1), chromosomal aberrations (E2), reproductive toxicity (component of F2 ), and developmental toxicity (component of F2) endpoints

. There are no outstanding HPV2 test rule requirements for toxicity testing of CASRN 2524

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.

1 Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Hukkanen, R.R. 2015. Phosphorochloridothioic acid, O,O

diethyl ester: Oral gavage reproduction/developmental toxicity screening test in Crl:CD(SD) rats. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. 2 “HPV2 Test Rule Data Adequacy Review: Reproduction/Developmental Toxicity Screening Test, Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 04 7 , dated August 5 , 2015. 3 Marino, T.A., Hutchinson, K.L., Louch, D.W., Holzheuer, W.B., and Nelson, E.J. 2015a. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute toxicity study with the rainbow trout, Oncorhynchus mykiss

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, M I. Sponsor: Dow AgroSciences LLC. 4 Marino, T.A., Hutchinson, K.L., Louch, D.W., Holzheuer, W.B., and Nelson, E.J. 2015b. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute toxicity study with the freshwater Cladoceran, Daphnia magna
. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. 5 Marino, T.A., Hutchinson, K.L., Holzheuer, W.B., and Nelson, E.J. 2015c. Phosphorochloridothioic aci d, O,O

diethyl ester: Growth inhibition test with the freshwater green alga, Pseudokirchneriella subcapitata

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences L LC. 6 “HPV2 Test Rule Data Adequacy Review : Acute To xicity to Fish, Acute Toxicity t o Daphnia , a nd Toxicity To Algae, Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

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- 1”, SRC Technical Report #15

- 0 51 , dated August 27 , 2015.Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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4 D. DATA FOR SIDS ENDPOINTS

D

- 1. Genetic Toxicity

– Gene Mutation Summary of endpoint: Adequate Study 1: Reverse mutation assay in Salmonella typhimurium and Escherichia coli (Wagner and Jayakumar, 2016

)

Title: Bacterial reverse mutation assay with phosphorochloridothioic acid, O,O

- diethyl ester This study is: Adequate Not Adequate

TEST SUBSTANCE Identity: Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

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Remarks : Purity: 99.3%; Pale whitish yellow clear liquid; Lot No. C032013081501

METHOD Method/guideline followed: OECD TG 471 (1997) “Bacterial Reverse Mutation Test”
Test type: Bacterial Reverse Mutation Test GLP compliant?: Yes, with the exception that concentration checks were not performed for the positive control substances Year study performed: 2015 Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2 uvrA ; Salmonella tester strains were derived from Dr. Bruce Ames’ cultures and E. coli tester strains were obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland

. Metabolic activation: With (S9 fraction of liver from rats induced wit h Aroclor 1254) and without

Concentrations tested: Initial toxicity/mutagenicity assay: 0 (solvent control), 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 , and 5000 μg/plate (all strains in the presence and absence of metabolic activation); Confirmatory mutagenicity assay: 0 (solvent control), 0.6, 2, 6, 20, 60, 200 , and 600 μg/plate (TA98, TA100, and TA1535 in the absence of metabolic activation and TA100, TA1537, a nd WP2 uvrA in the presence of metabolic activation) and 0 (solvent control), 2, 6, 20, 60, 200, 600 , and 2000 μg/plate (TA1537 and WP2 uvrA in the absence of metabolic activation and TA98 and TA1535 in the presence of metabolic activation)

Vehicle: AcetoneFinal HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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5 Statistical methods used: The mean and standard deviation of the number of revertants per plate were calculated.

Test method/conditions remarks: “The assay was performed in two phases using the preincubation method. The first phase, the init ial toxicity

- mutation assay, was used to establish the dose

- range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mut agenic potential of the test substance.” [ Wagner and Jayakumar 2016 , p. 7] “Acetone [purity: 99.6%] was selected as the solvent of choice based on information provided by the Sponsor, and compatibility with the target cells. The Sponsor indicated the tes t substance formed clear solutions in acetone at a concentration of approximately 500 g/L.” [ Wagner and Jayakumar 2016, p. 14 ] The highest formulation concentration used in the mutagenicity assays was 100 g/L. Test substance dilutions [in acetone] were p repared immediately before use and delivered to the test system at room temperature under yellow light.” [Wagner and Jayakumar 2016, p. 8] Initial Toxicity/Mutagenicity Assay: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2 uvrA were exposed to test item concentrations ranging from 0.15 to 5000 μg/plate, with and without S9 mix (10% v/v). All test item concentrations were plated in triplicate. Concurrent positive and solvent (acetone) controls were also pla ted in triplicate. “The standard treatment volumes were increased [by] 7.5X [compared to the standard protocol] to minimize the headspace of the treatment vessel... [As a result,] 3.75 mL S9 mix or sham mix, 0.75 mL of tester strain (cells seeded) and 0.375 mL of vehicle or test substance dilution were added to 4 mL air tight sealed, capped glass culture tubes pre

- heated to 37±2 °C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2 °C. Following the preincubation, 0.65 mL of treatment mixture was added to 2.0 mL of selective top agar for each plate and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 0.3 75 mL aliquot of appropriate positive control. Each plate was sealed with Petri

- Seal TM adhesive sealing film (Sigma

- Aldrich Cat. No. Z376922). After the overlay had solidified, the plates were inverted and incubated in an appropriate number of ≥ 9

- liter desiccators by dose level and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2

- 8 °C until colony counting could be conducted.” [Wagner and Jayakumar 2016, p. 11]

The conditi on of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification... As appropriate, colonies were e numerated either by hand or by machine.” [Wagner and Jayakumar 2016, p. 12] (If extreme toxicity was observed, revertant colonies were counted by hand.)Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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6 Confirmatory Mutagenicity Assay: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Es cherichia coli strain WP2 uvrA were exposed to test item concentrations ranging from 0.6 to 2000 μg/plate, with and without S9 mix (10% v/v). Exposure to the test item was conducted using the pre

- incubation method as described above for the initial toxici ty/mutagenicity assay. All test item concentrations were plated in triplicate. Concurrent positive and solvent (acetone) controls were also plated in triplicate. Composition of Top Agar and Agar Plates:

“ On the day of its use, minimal top agar, containi ng 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L

- histidine, D

- biotin and L

- tryptophan solution to a final concentration of 50 μM each... Bottom agar was Vogel

- Bonner minimal medium E... containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel

- Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).” [Wagner and Jayakumar 2016, p. 11] Metabolic Activation System: Male Sprague

- Dawley rats (5

- 6 weeks) were induced with Aroclor 1254 (500 mg/kg

- bw) administered by intraperitoneal injection five days prior to sacrifice. The S9 fraction was prepared by Moltox (Boone, NC) and stored at temperatures ≤

- 60°C upon arrival a t BioReliance.

“ The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose

- 6

- phosphate, 4 mM ß

- nicotinamide

- adenine dinucleotide phosphate, 8 mM MgCl 2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (S ham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.” [Wagner and Jayakumar 2016, p. 10] “The ability of the [S9] sample to activate ethidium (EtBr) and cyclophosphamide (CPA) to intermediates mutagenic to TA98 and TA1535, respectively, was determined... Dilutions of the sample S9, ranging from 0.2

- 10% in S9 mix, were [also] tested for their ability to activa te benzo(α)pyrene (BP) and 2

- aminoanthracene (2

- AA) to intermediates mutagenic to TA100.” [ Wagner and Jayakumar 2016, p. 67] These tests confirmed the ability of the S9 batch to activate the tested compounds.

Concurrent Positive Controls: Without S9 activation: TA98: 2

- Nitrofluorene (1.0 μg/plate ) TA100: Sodium azide (1.0 μg/plate ) TA1535: Sodium azide (1.0 μg/plate) TA1537: 9

- Aminoacridine (75 μg/plate ) WP2 uvrA : Methyl methanesulfonate (1000 μg/plate )Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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7 With S9 activation: TA98 and TA1535: 2

- Aminoanthracene (1.0 μg/plate) TA100 and TA1537: 2

- Aminoanthracene (2.0 μg/plate) WP2 uvrA : 2

- Aminoanthracene (15 μg/plate)

DMSO was the solvent for 2

- nitrofluorene, 9

- aminoacridine, methyl methanesulfonate, and 2

- aminoanthracene. Dis tilled water was the solvent for sodium azide. To meet the criteria for a valid test, “[t]he mean of each positive control must exhibit at least a 3.0

- fold increase in the number of revertants over the mean value of the respective vehicle control.” [ Wagne r and Jayakumar 2016, p. 13] Analysis of Test Concentrations and Stability of the Test Item (in Acetone) Maintained at Room Temperature: During the initial toxicity and confirmatory mutagenicity assays, test substance formulations were analyzed in

- house b y gas chromatography with flame ionization detection (GC/FID) using a method that had previously been validated. For each assay, two samples (one for analysis, one for backup) were collected from the highest test substance formulation concentration (100 a nd 40 mg/mL, corresponding to 5000 and 2000 μg/plate for the initial toxicity/mutagenicity assay and confirmatory mutagenicity assay, respectively), lowest analyzable test substance formulation concentration (0.03 and 0.04 mg/mL corresponding to 1.5 and 2. 0 μg/plate for the initial toxicity/mutagenicity assay and confirmatory mutagenicity assay, respectively), and vehicle control solutions. “Samples were brimmed to minimize headspace in the vials.” [Wagner and Jayakumar 2016, p. 10] “Due to potential vo latility concerns of the Test Substance, all of the samples and standards were prepared and analyzed on the same day. Additional efforts were made to the preparation of the samples and standards to minimize headspace where appropriate and diluted aliquots were transferred to autosampler vials in excess then capped and sealed to eliminate as much headspace as possible.” [Wagner and Jayakumar 2016, p. 59] Each sample of test substance formulation was analyzed in duplicate; a single measurement was made fo r each sample of vehicle control solution. To meet the acceptance criteria, as specified in the study protocol, the measured test substance concentrations must be within 85.0 to 115.0% of nominal concentrations and the ratio of the small/large measured co ncentrations for the duplicate dilutions of each sample must be > 0.925. To determine stability over 3 hours, samples derived from the initial toxicity/mutagenicity assay formulations (nominal concentrations of 0.03 and 100 mg/mL) were analyzed once and then reanalyzed following storage for 3 hours at room temperature. To determine stability over 5 hours (intended to cover the amount of time required to complete dosing), independent formulations of the low and high concentrations used for the initial to xicity/mutagenicity assay (nominal concentrations of 0.03 and 100 mg/mL) were measured immediately following preparation and again following 5 hours of storage at room temperature. According to t he study protocol, the measured test substance concentration s must be within 90

- 110% of the initial concentrations in order for the test substance formulations to be considered stable.Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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8 Criteria for Determining a Mutagenic Response and Cytotoxicity: The criteria for determining a positive (mutagenic) response were as follows:

 “ For the test substance to be evaluated [as] positive, it must cause a concentration

- related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below... ” [Wagner and Jayakumar 2016, p. 13]

 For Salmonella typhimurium strains TA98, TA1535, TA1537 , and WP2 uvrA : “ Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0

- times the mean vehicle control value. ” [Wagner and Jayakumar, p. 13]

 For Salmonella typhimurium strain TA100: “Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0

- times the
mean vehi cle control value.” [Wagner and Jayakumar 2016, p. 13]

“ An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a concentration

- responsive increase that does not achieve the respective threshold cited above or a non

- concentration responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.” [Wagner and J ayakumar 2016, p. 13]

“ A concentration is considered toxic if one or both of the following criteria are met: (1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt concentration

- dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn...” [Wagner and Jayakumar 2016, p. 13]

RESULTS Genotoxicity result: Negative in the presence and absence of metabolic activation Cytotoxic concentration (if applicable) 1 : ≥ 150 μg/plate in TA98, TA100, and TA1535 in the absence of metabolic activation; ≥ 500 μg/plate in TA1537 and WP2 uvrA in the absence of metabolic activation; ≥ 150 μg/plate in TA100, TA1537, and WP2 uvrA in th e presence of metabolic activation; ≥ 500 μg/plate in TA98 and TA1535 in the presence of metabolic activation

Results remarks: Initial Toxicity/Mutagenicity Assay: N o positive mutagenic responses were observed in the presence or absence of metabolic act ivation. No evidence of test substance precipitation was observed in the presence or absence of metabolic activation.

1 Cytotoxic concentrations shown here are based on reduction of bacterial lawn (degree of lawn reduction classified as moderate or greater) and/or > 50% reduction in the mean number of revertants per plate as compared to the vehicle control.Final HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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9 In the absence of metabolic activation, the lowest concentrations to cause a significant reduction in background lawn (degree of lawn reduction classified as moderate or greater) were 150 μg/plate in TA98, TA100, and TA1535 and 500 μg/plate in TA1537 and WP2 uvrA . In the presence of metabolic activation, the lowest concentrations to cause a significant reduction in background lawn were 150 μg/plate in TA100, TA1537, and WP2 uvrA and 500 μg/plate in TA98 and TA1535. In the absence of metabolic activation, a > 50% reduction in the mean number of revertants per plate was observed at concentrations ≥ 500 μg/plate in TA98, TA100 , and TA153 5 and at concentrations ≥ 1500 μg/plate in TA1537 and WP2 uvrA . In the presence of metabolic activation, a > 50% reduction in the mean number of revertants per plate was observed at concentrations ≥ 500 μg/plate in TA100, TA1537 , and WP2 uvrA and at conce ntrations ≥ 1500 μg/plate in TA98 and TA1535.

“ Based on the findings of the initial toxicity

- mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 600 μg per plate (with tester strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100, TA1537 and WP2 uvr A in the presence of S9 activation) and 2000 μg per plate (with tester strains TA1537 and WP2 uvr A in the absence of S9 activation and TA98 and TA1535 in the presence of S9 activation).” [Wagner and Jayakumar, p. 15] Confirmatory Mutagenicity Assay: No positive mutagenic responses were observed in the presence or absence of metabolic activation. No evidence of test substance precipitation was observed in the presence or absence of metabolic activation. In the absence of metabolic activation, the lowest concentrations to cause a significant reduction in background lawn (degree of lawn reduction classified as moderate or greater) were 200 μg/plate in TA100 and TA1535 and 600 μg/plate in TA98, TA1537, and W P2 uvrA . In the presence of metabolic activation, the lowest concentrations to cause a significant reduction in background lawn were 200 μg/plate in TA100, TA1537, and WP2 uvrA and 600 μg/plate in TA98 and TA1535.
In the absence of metabolic activation, a > 50% reduction in the mean number of revertants per plate was observed at concentrations ≥ 200 μg/plate in TA100 and at concentrations ≥ 600 μg/plate in TA98, TA1535, TA1537, and WP2 uvrA . In the presence of metabolic activation, a > 50% reduction in t he mean number of revertants per plate was observed at concentrations ≥ 200 μg/plate in TA100 and WP2 uvrA and at concentrations ≥ 600 μg/plate in TA98, TA1535, and TA1537. Response of Controls: In both the initial toxicity

- mutation assay and the confirma tory mutagenicity assay, the values for numbers of revertants for the solvent control in all strains were within the limits of the historical range of negative controls (including but not limited to deionized water, dimethyl sulfoxide, ethanol, and acetone ). Positive controls (both in the absence and presence of metabolic activation during both assays) exhibited a clear (> 3

- fold) increase in the number of revertantsFinal HPV 2 Test Rule Data Adequacy Review (February 2016) CAS RN 2524

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10 when compared with the concurrent negative control and results were within the historical ranges. Test Substance Formulation Analysis: For the initial toxicity/mutagenicity assay, the mean measured concentrations of the test substance formulations were 105 and 0.0300 mg/mL for nominal concentrations of 100 and 0.03 mg/mL, respectively; measure d values were 99.9

- 105% of nominal values. For the confirmatory mutagenicity assay, the mean measured concentrations of the test substance formulations were 42.5 and 0.0296 mg/mL for nominal concentrations of 40 and 0.04 mg/mL, respectively; measured valu es were 74.1

- 106.3% of nominal values. The ratios of the small/large (S/L) measured concentrations for the duplicate dilutions of each sample were all > 0.925.

“ The 0.0400 mg/mL formulation for... [the confirmatory assay] was found to be below the acceptab le range for concentration (85.0 to 115.0% of target) but met the S/L ratio of > 0.925. Although the actual concentration of the low dose formulation was lower than the protocol

specified acceptance criteria for a solution (85.0 to 115.0% of target concen tration), the critical top dose formulation met the acceptance criteria indicating that the test guideline recommended limit dose was achieved. Therefore, the Study Director has concluded that the difference in nominal concentration at the low dose had no adverse impact on the study results or validity of the study conclusion. No test article was detected in the vehicle control sample.” [Wagner and Jayakumar 2016, p. 16] In the first stability test, mean measured concentrations of test substance formu lations (nominal concentrations of 0.03 and 100 mg/mL) were 95.8

- 99.1% of initial measured concentrations after 3 hours of storage at room temperature. In the second stability test, mean measured concentrations of test substance formulations (nominal conc entrations of 0.03 and 100 mg/mL) were 97.0

- 100% of initial measured concentrations after 5 hours of storage at room temperature. The criterion for stability (90

- 110% of initial concentration) was met by the results of both stability tests.

CONCLUSIONS Under the conditions of this test, none of the bacterial strains exhibited a positive mutagenic response either in the presence or absence of metabolic activation.

STUDY RELIABILITY
[1] Reliable without restriction.

REFERENCE Wagner, V.O., and Jayakumar, G. 2016. Bacterial reverse mutation assay with phosphorochloridothioic acid, O,O

- diethyl ester. Performing Laboratory: BioReliance, Rockville, MD. Sponsor: The Dow Chemical Company.