SRC TR-16-007

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

BACTERIAL REVERSE MUTATION ASSAY

Phosphorochloridothioic acid, O,O-diethyl ester, CASRN 2524-04-1

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, 2016
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a bacterial reverse mutation test for CASRN 2524-04-1 

(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 2524-04-1: 

C1 (aquatic toxicity, consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia, 

and toxicity to algae tests] if log Kow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and 

toxicity to algae tests] if log Kow ≥ 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 Kow of 2.371, Test Group 1 testing is required to determine the toxicity 

of CASRN 2524-04-1 to aquatic organisms.

Dow AgroSciences previously submitted a study report of a bacterial reverse mutation test of 

CASRN 2524-04-1.2 This study was reviewed in a separate report3and was determined to be 

inadequate to satisfy the gene mutation (E1) testing requirement for CASRN 2524-04-1 

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 AgroSciences submitted a study plan to EPA for a second bacterial 

reverse mutation test of CASRN 2524-04-1.

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 2016, Dow AgroSciences submitted the following study to EPA: 

 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.

 

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-screeningtools/epi-suitetm-estimation-program-interface#download as of February 10, 2016. 

2 Tendulkar, K.E. 2014. Phosphorochloridothioic acid, O,O-diethyl ester: Salmonella-Escherichia coli mammalianmicrosome 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,Odiethyl ester, CASRN 2524-04-1”, SRC Technical Report #15-023, dated March 4, 2015. Available online at 

http://www.regulations.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,Odiethyl ester. Test Facility: BioReliance Corporation, Rockville, 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 2524-04-1”, SRC Technical Report #15-055, dated 

September 22, 2015. 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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 2524-04-1, study details 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 2524-04-1 has physicochemical 

properties (i.e., potential for hydrolysis2and volatilization3) 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 REQUIRED 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 2524-04-1. 

Other HPV2 Test Rule Toxicity Testing Requirements for CASRN 2524-04-1:

Dow AgroSciences previously submitted a study report of a micronucleus test4of CASRN 2524-

04-1. This study was reviewed in a separate report5and was determined to be adequate to satisfy 

the chromosomal aberrations (E2) testing requirement for CASRN 2524-04-1 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

instead (request granted in EPA’s study plan review of CASRN 2524-04-1, “HPV2 Review of Toxicity Study Plan 

Dated February 25, 2014, Phosphorochloridothioic acid, O,O-diethyl ester, CASRN 2524-04-1”, SRC Technical 

Report #14-008, dated April 3, 2014; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-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 Dow Chemical Company 

(Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. 

3 CASRN 2524-04-1 is considered volatile based on an estimated Henry’s Law Constant of 6.72 x 10-4

atm-m3/mole 

at 25 °C (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-screeningtools/epi-suitetm-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 Laboratory: 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 HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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Dow AgroSciences previously submitted a study report of a reproduction/developmental toxicity 

screening test1of CASRN 2524-04-1. This study was reviewed in a separate report2and was 

determined to be adequate to satisfy the reproduction/developmental toxicity (F2) testing 

requirement for CASRN 2524-04-1 promulgated in the HPV2 test rule.

Dow AgroSciences previously submitted study reports of acute toxicity to fish3, acute toxicity to 

Daphnia4, and toxicity to algae5tests of CASRN 2524-04-1. These studies were reviewed in a 

separate report6and were determined to be adequate to satisfy the aquatic toxicity (C1) testing 

requirement for CASRN 2524-04-1 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-

04-1.

 

1 Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Hukkanen, R.R. 2015. Phosphorochloridothioic acid, O,Odiethyl 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 #15-047, 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, MI. 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 acid, O,Odiethyl 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 LLC.

6

“HPV2 Test Rule Data Adequacy Review: Acute Toxicity to Fish, Acute Toxicity to Daphnia, 

and Toxicity To Algae, Phosphorochloridothioic acid, O,O-diethyl ester, CASRN 2524-04-1”, SRC Technical 

Report #15-051, dated August 27, 2015.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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 2524-04-1

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 with 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, and 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: Acetone
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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 initial 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 mutagenic 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 test 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 prepared 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 plated 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 

0.375 mL aliquot of appropriate positive control. Each plate was sealed with Petri-SealTM

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 condition 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 enumerated either 

by hand or by machine.” [Wagner and Jayakumar 2016, p. 12] (If extreme toxicity was observed, 

revertant colonies were counted by hand.)
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Confirmatory Mutagenicity Assay:

Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain 

WP2 uvrA were exposed to test item concentrations ranging from 0.6 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 toxicity/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, containing 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 at 

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 MgCl2 and 33 mM KCl 

in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham 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 activate 

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 HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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. Distilled 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.” [Wagner 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 by 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 and 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 volatility 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 for 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 concentrations 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 toxicity/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 the study 

protocol, the measured test substance concentrations must be within 90-110% of the initial 

concentrations in order for the test substance formulations to be considered stable.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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 vehicle 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 Jayakumar 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 the presence of 

metabolic activation; ≥ 500 μg/plate in TA98 and TA1535 in the presence of metabolic 

activation

Results remarks: 

Initial Toxicity/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. 

 

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 HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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 TA1535 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 concentrations ≥ 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 uvrA in the presence of 

S9 activation) and 2000 μg per plate (with tester strains TA1537 and WP2 uvrA 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 WP2 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 the 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 confirmatory 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 revertants 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 2524-04-1

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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; measured 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 values 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 

acceptable 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 protocolspecified acceptance criteria for a solution (85.0 to 115.0% of target concentration), 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 formulations (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 concentrations 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.