SRC TR-15-023

HPV2 TEST RULE DATA ADEQUACY REVIEW (REVISED FINAL):

BACTERIAL REVERSE MUTATION TEST 

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

Prepared by:

Ashlee Aldridge, Amy Nguyen, Teresa Manyin, and William L. 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

March 4, 2015
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 4

D-1. Genetic Toxicity – Gene Mutation..................................................................................... 4
Final HPV2 Test Rule Data Adequacy Review (March 2015) 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 March 4, 2015). 

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.

The submission from Dow AgroSciences contained the following study report: 

 Tendulkar, K.E. 2014. Phosphorochloridothioic acid, O,O-diethyl ester: SalmonellaEscherichia coli mammalian-microsome reverse mutation assay pre-incubation method with 

a confirmatory assay. Performing Laboratory: JAI Research Foundation, Gujarat, India. 

Sponsor: Dow AgroSciences LLC.

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 4712. In addition, the study was evaluated to determine whether recommendations provided 

in EPA’s study plan review3 were followed. Because CASRN 2524-04-1 has physicochemical

 

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/opptintr/

exposure/pubs/episuitedl.htm as of March 4, 2015. 

2 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 March 4, 2015). 

3

“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-HQ-OPPT-2007-0531-0805 as of March 4, 2015.
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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properties (i.e., potential for hydrolysis1and volatilization2) 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 Data Submitted for the Gene Mutation Endpoint:

Data submitted by Dow AgroSciences for the bacterial reverse mutation (E1) endpoint are not 

considered adequate because no precautions were taken to minimize losses of test material by 

volatilization during the study. Due to the potential volatility of CASRN 2524-04-1, EPA 

previously commented in its review3of the study plan submitted by Dow AgroSciences for the 

gene mutation study that “all test containers (including vessels used for the preparation of test 

item formulations, tubes used for pre-incubation of bacteria, and agar plates onto which bacteria 

are transferred) shall have minimal headspace and minimal exposure to air and shall be covered 

with airtight seals.” However, the study report for the gene mutation study submitted by Dow 

AgroSciences provides no indication that any precautions were taken to minimize losses of test 

material by volatilization during the study. Of particular concern is test substance loss by 

volatilization during the 20-minute pre-incubation period and the 48-hour plate incubation 

period. Although measurement of CASRN 2524-04-1 in stock formulations in acetone (lowest 

and highest concentration) performed by gas chromatography/electron capture detection 

(GC/ECD) after 4 hours in an ice bath showed minimal degradation and/or loss of the test 

substance, these results are not representative of the extent of degradation and/or loss by 

volatilization anticipated during the mutagenicity test due to the differences in solvent used 

(acetone in stability test vs. aqueous medium in mutagenicity test) and temperature (near 0 °C in 

stability test vs. 37 °C in mutagenicity test). 

Although a positive correlation between nominal test substance concentration and cytotoxicity

was observed, it is unknown whether the observed cytotoxicity was due to CASRN 2524-04-1, 

its degradation products, or a combination thereof. Therefore, the observation of cytotoxicity 

cannot be used as an indirect indicator that the test methods were acceptable. The lack of 

precautions taken to minimize volatilization suggests that undegraded CASRN 2524-04-1 was 

free to volatilize to the atmosphere throughout the study; consequently, it is unknown whether or 

not the test results are representative of those that would be obtained had precautions been taken 

to minimize volatilization of CASRN 2524-04-1. 

This study needs to be repeated under conditions that minimize the loss of the test material by 

volatilization during the 20-minute pre-incubation period and during the 48-hour plate incubation 

 

1 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. 

2 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/opptintr/

exposure/pubs/episuitedl.htm as of March 4, 2015). 

3

“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-HQ-OPPT-2007-0531-0805 as of March 4, 2015.
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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period. EPA requests that all test containers (including vessels used for the preparation of test 

item formulations, tubes used for pre-incubation of bacteria, and agar plates onto which bacteria 

are transferred) have minimal headspace and minimal exposure to air and be covered with 

airtight seals. In addition, EPA recommends that the mutagenicity assay be performed using the 

plate incorporation method (also described in OECD TG 471) instead of the pre-incubation 

method to minimize losses of test substance by volatilization. EPA also recommends that any 

additional gene mutation testing be performed using triplicate plating for all definitive

mutagenicity tests. 

EPA recommends that the sponsor submit a revised study plan prior to conducting any 

additional testing for the gene mutation endpoint.

Remaining HPV2 Test Rule Toxicity Testing Requirements:

Dow AgroSciences submitted a study report of a micronucleus test of CASRN 2524-04-1

1

, 

which was reviewed by EPA in a separate report2and determined to be adequate to satisfy the 

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

HPV2 test rule.

Data have not yet been submitted to EPA for the following toxicity testing requirements for 

CASRN 2524-04-1: acute toxicity to fish (component of C1), acute toxicity to Daphnia

(component of C1), toxicity to algae (component of C1), reproductive toxicity (component of 

F2), and developmental toxicity (component of F2).

Summary of Data Adequacy for Toxicological Endpoints:

Adequate data have been submitted for the chromosomal aberrations endpoint. The acute 

toxicity to fish, acute toxicity to Daphnia, toxicity to algae, gene mutation, reproductive 

toxicity, and developmental toxicity endpoints are considered data gaps and testing is still 

required for these endpoints.

 

1 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 

March 4, 2015.

2

“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.
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D. DATA FOR SIDS ENDPOINTS

D-1. Genetic Toxicity – Gene Mutation

Summary of endpoint: Not adequate 

Study 1: Reverse mutation assay in Salmonella typhimurium and Escherichia coli

(Tendulkar, 2014)

Title: Phosphorochloridothioic acid, O,O-diethyl ester: Salmonella-Escherichia coli

mammalian-microsome reverse mutation assay pre-incubation method with a confirmatory assay

This study is: Adequate Not Adequate

This study is not considered adequate because no precautions were taken to minimize losses of 

test material by volatilization during the study. Due to the potential volatility of CASRN 2524-

04-1, EPA previously requested in its study plan review of the proposed gene mutation study that 

all test containers (including vessels used for the preparation of test item formulations, tubes 

used for pre-incubation of bacteria, and agar plates onto which bacteria are transferred) have 

minimal headspace and minimal exposure to air and be covered with airtight seals. However, the 

study report for the gene mutation study provides no indication that any precautions were taken 

to minimize the loss of test material by volatilization during the study. 

TEST SUBSTANCE 

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

Remarks: Purity: 99.3%; Colorless to light yellow liquid; Lot No. C032013081501

METHOD

Method/guideline followed: OECD TG 471 (1997) “Bacterial Reverse Mutation Test”, OPPTS 

870.5100 (1998) “Bacterial Reverse Mutation Test”, and Method B.13/14 of Commission 

Regulation (EC) No. 440/2008 “Mutagenicity: Reverse Mutation Test using Bacteria”

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

Year study performed: 2014

Test species: Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 and 

Escherichia coli strain WP2 uvrA (pKM101); obtained from Molecular Toxicology, Inc., Boone, 

NC.

Metabolic activation: With (S9 fraction of liver from rats induced with Aroclor 1254) and 

without
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Concentrations tested: Initial toxicity/mutagenicity assay: 0 (solvent control), 1.5, 5, 15, 50, 

150, 500, 1500, and 5000 μg/plate; “Confirmatory” mutagenicity assay: 0 (negative control), 0 

(solvent control), 0.15, 0.5, 1.5, 5, 15, 50, and 150 μg/plate

Vehicle: Acetone

Statistical methods used: No statistical analyses provided

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 item.” [Tendulkar 2014, p. 7]1

“Solubility and precipitation tests of the test item were performed prior to the cytotoxicity test.

The test item was [considered by the study author to be] insoluble in sterile distilled water while 

found to be soluble in DMSO [dimethyl sulfoxide]. Release of gas was smelled and heating of 

the tube containing [the] dose formulation was observed in the dose solution prepared in 

DMSO.” [Tendulkar 2014, p. 14]

“To overcome the problems like heating of [the] tube and gas release while using DMSO as the 

solvent, the test item was further tested for its solubility in acetone, as recommended by the 

sponsor and found to be soluble in acetone, though with slight release of gas. Precipitation was 

not observed at the concentration of 5000 μg/plate. Therefore, 5000 μg/plate was selected as the 

highest concentration to be used in the initial toxicity test both in the absence and presence (5% 

v/v S9 mix) of metabolic activation.” [Tendulkar 2014, p. 15]

Preparation of Bacterial Cultures and Cell Viability Test: 

“Fresh cultures for the test were prepared by inoculating frozen permanent cultures into a flask 

containing 10 mL of sterile nutrient broth N° 2 (Oxoid). The flasks were then incubated at 

37 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to early stationary or late 

exponential phase.” [Tendulkar 2014, p. 15]

“After the incubation period, the culture flasks were removed from the orbital shaking incubator. 

Aseptically, the cultures were diluted with Oxoid nutrient broth (ONB) and the optical density 

was measured at 660 nm using a Spectrophotometer... Oxoid nutrient broth was used as the 

control blank. Cell viability of the tester strains was determined prior to treatment. The optical 

density of the cultures was found to be in the acceptable range and they were used for the study.”

[Tendulkar 2014, p. 15]

 

1 Although the study report referred to the second test as a “confirmatory mutagenicity assay”, EPA does not agree 

that this was a confirmatory test. The “initial toxicity-mutation assay” was conducted using duplicate plating, rather 

than triplicate plating as recommended by OECD TG 471, and did not provide a sufficient range of concentrations to 

allow for five analyzable concentrations, as required by OECD TG 471, due to cytotoxicity and, therefore, the initial 

assay should not be considered a definitive test for mutagenicity evaluation. Consequently, EPA would characterize 

the second test as a “definitive mutagenicity test”, rather than a “confirmatory mutagenicity test”. 
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“After plating for [the] initial toxicity assay and [the] confirmatory mutation assay, the bacterial 

suspensions of each tester strain were further diluted up to 10-6in PBS (Phosphate buffered 

saline) and 100 μL of each diluted suspension were plated on nutrient agar plates in triplicate. 

These plates were incubated at 37 ± 1 °C for 24 hrs. After incubation, colonies in each plate 

were counted and expressed as number of colony forming unit[s] [(CFU)] per mL of bacterial 

suspensions. Bacterial cell count was within the required range of 1-2 x 109 CFU/mL” 

[Tendulkar 2014, p. 15]

Initial Toxicity/Mutagenicity Assay:

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

WP2 uvrA (pKM101) were exposed to test item concentrations ranging from 1.5 to 

5000 μg/plate, with and without S9 mix (5% v/v). All test item concentrations were plated in 

duplicate. Concurrent positive and solvent (acetone) controls were also plated in duplicate. The 

protocol used was the same as in the “confirmatory” mutagenicity assay; however, only two 

plates per concentration were used in the initial assay, while three were used in the 

“confirmatory” assay. 

Stock solutions of the test item were prepared in acetone by serial dilution. Concentrations of 

stock solutions were 30, 100, 300, 1000, 3000, 10,000, 30,000, and 100,000 μg/mL for treatment 

concentrations of 1.5, 5, 15, 50, 150, 500, 1500, and 5000 μg/plate, respectively.

“All [of] the stock solutions were maintained in [an] ice bath throughout the experiment. Tubes 

containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. 

Volumes of 50 μL of the relevant stock solution of test item and acetone were used for [the] 

treatment and negative control, respectively.” [Tendulkar 2014, pp. 20-21]

“A volume of 500 μL of 5% v/v S9 mix was added in the presence of metabolic activation and 

500 μL of 0.2 M phosphate buffer was added in the absence of metabolic activation. Finally 

100 μL of bacterial culture was added to the tubes and mixed... This treatment mixture was 

incubated in [a] shaking water bath for 20 ± 2 minutes at 37 ± 1 °C.1 After incubation, 2 mL 

molten top agar was added to this treatment mixture and poured on MGA [minimal glucose agar] 

plates and allowed to solidify. Duplicate sets were maintained for each concentration of... [the 

test item], negative control and positive control. The petri plates were incubated at 37 ± 1 °C for 

48 hours and then examined to assess the background bacterial lawn pattern and reduction in 

number of colonies.” [Tendulkar 2014, p. 21] The method that was used for scoring bacteria on 

the culture plates was not described in the study report.

“Confirmatory” Mutagenicity Assay:

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

WP2 uvrA (pKM101) were exposed to test item concentrations ranging from 0.15 to 

150 μg/plate, with and without S9 mix (5% v/v). Exposure to the test item was conducted using 

 

1 Based on the protocol submitted in Appendix 6 of the study report, it appears that the test substance (i.e., 50 μL of 

stock solution) was added to the 500 μL of phosphate buffer (for exposures without metabolic activation) or S9 mix 

(for exposures with metabolic activation) plus 100 μL of bacterial culture prior to the 20-minute pre-incubation 

period. At the end of the pre-incubation period, 2 mL of molten top agar was added and the mixture was poured 

onto plates.
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the pre-incubation method as described above for the initial toxicity/mutagenicity assay. All test 

item concentrations were plated in triplicate. Concurrent positive, solvent (acetone), and 

negative (distilled water) controls were also plated in triplicate. 

Stock solutions of the test item were prepared in acetone by serial dilution. Concentrations of 

stock solutions were 3, 10, 30, 100, 300, 1000, 3000, and 10,000 μg/mL for treatment 

concentrations of 0.15, 0.5, 1.5, 5, 15, 50, and 150 μg/plate, respectively. 

“All [of] the stock solutions were maintained in [an] ice bath throughout the experiment. 

Volumes of 50 μL of relevant stock solutions [or acetone or distilled water for the solvent and 

negative controls, respectively]... were used to obtain the required test concentrations for 

treatment both in the absence and presence (5% v/v S9 mix) of the metabolic activation 

system... The numbers of revertant colonies were recorded approximately after the 48h

incubation period.” [Tendulkar 2014, p. 22] The method that was used for scoring bacteria on 

the culture plates was not described in the study report.

Composition of Top Agar and Agar Plates:

The top agar contained the following components: Bacto agar (0.6 g), sodium chloride (0.6 g), 

0.5 mM histidine/biotin solution (10 mL), and distilled water (90 mL). The minimal glucose 

agar plates contained the following: Bacto agar (7.5 g), distilled water (450 mL), 50x Vogal 

Bonner medium (10 mL), and 20% glucose solution (25 mL). 

Metabolic Activation System:

Male Sprague-Dawley rats (5-6 weeks) were induced with Aroclor 1254 (500 mg/kg-bw) 

administered by intraperitoneal injection. The S9 post-mitochondrial supernatant fraction was 

collected, buffered (with 0.15 M potassium chloride), and stored at -70oC. The S9 mix was 

prepared fresh from thawed S9 fraction and co-factor mix and was maintained in an ice bath 

prior to use. Each 10 mL of the 5% v/v S9 mix contained 9.5 mL of co-factor mix and 0.5 mL of 

the S9 fraction. The cofactor mix contained 0.80 g D-glucose 6-phosphate; 1.75 g βnicotinamide adenine dinucleotide phosphate; 0.90 g magnesium chloride; 1.35 g potassium 

chloride; 6.40 g sodium phosphate, dibasic; 1.40 g sodium phosphate, monobasic; and 450 mL 

distilled water and was stored below 0 °C. 

“The ability of the [S9] sample to activate ethidium (EtBr) and cyclophosphamide (CPA) to 

intermediates mutagenic to TA98 and TA1535, respectively, was determined according to Lesca 

et al...” [Tendulkar 2014, p. 68] “Dilutions of the sample S9, ranging from 0.2-10% in S9 mix, 

were [also] tested for their ability to activate benzo(a)pyrene (BP) and 2-aminoanthracene (2-

AA) to intermediates mutagenic to TA100. Assays were conducted as described by Maron and 

Ames.” [Tendulkar 2014, p. 68]

“Treatment with 2-aminoanthracene in the absence of metabolic activation was performed for 

tester strain TA100 in both [of] the [initial toxicity/mutagenicity and “confirmatory”

mutagenicity] trials [and compared with results of TA100 treated with 2-aminoanthrocene in the 

presence of metabolic activation] to demonstrate the efficiency of the S9 fraction used in the 

study.” [Tendulkar 2014, p. 22] Results without metabolic activation were expected to be 
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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negative for mutagenic activity, while results with metabolic activation were expected to be 

positive. 

Positive Controls:

Without S9 activation:

TA98: 2-Nitrofluorene (7.5 μg/plate)

TA100: Sodium azide (5 μg/plate) 

TA1535: Sodium azide (0.5 μg/plate)

TA1537: 9-Aminoacridine hydrochloride hydrate (75 μg/plate)

WP2 uvrA (pKM101): 4-Nitroquinoline 1-oxide (0.5 μg/plate)

With S9 activation:

TA98 and TA100: 2-Aminoanthracene (5 μg/plate)

TA1535, TA1537, and WP2 uvrA (pKM101): 2-Aminoanthracene (10 μg/plate)

DMSO was the solvent for 9-aminoacridine hydrochloride hydrate, 2-nitrofluorene, and 2-

aminoanthracene. Distilled water was the solvent for sodium azide and 4-nitroquinoline 1-oxide.

Analysis of Test Concentrations and Stability of the Test Item in Stock Solutions (in Acetone) 

Maintained in an Ice Bath:

During the “confirmatory” mutagenicity assay, samples from the stock solutions maintained in 

an ice bath were collected for analysis of concentrations of the test item in the stock solutions at 

0 hours (for all stock concentrations) and after an additional 4 hours in the ice bath (for the 

lowest and highest stock concentrations) for stability analysis. Analyses were performed using a 

gas chromatography/electron capture detection (GC/ECD) method. Samples were not collected 

for test concentration or stability analysis from the bacterial incubation samples. 

For concentration analysis, “[t]wo sets of a single replicate of a 1 mL aliquot... of each 

concentration (3, 10, 30, 100, 300, 1000 and 3000 μg/mL) and vehicle (acetone) were taken from 

the middle portion [of stock solutions maintained in an ice bath] for active ingredient [CASRN 

2524-04-1] concentration analysis at 0 hour. Two sets of a single replicate of a 1 mL aliquot of 

the highest concentration (3000 μg/mL) and the lowest concentration (3 μg/mL) were taken for 

stability analysis after [an additional] 4 hours [in the ice bath], i.e., at the end of plate exposure

[sic]1. Stock solutions were maintained in [an] ice bath throughout the experimental phase. All 

[of] the aliquots were stabilized by immediate freezing (-80 ± 5 °C) to quench any degradation of 

the test item. The first sets of replicates were sent to Department of Chemistry (JRF [JAI

Research Foundation]) for analysis. Second set of replicates... [was] stored in [a] deep freezer 

(-80 ± 5 oC) as backup... These unused aliquots can be used for reanalysis, as necessary, or will 

be discarded following issuing of the final report.” [Tendulkar 2014, p. 22] 

“The following criteria for acceptable specification for the concentration of the test item in the 

vehicle were used to determine a valid assay: Accuracy of 85 to 115% of nominal with < 10% 

 

1 Although the study report states that samples for stability analysis were taken “after 4 hours, i.e., at the end of plate 

exposure” [Tendulkar 2014, p. 22], the duration of plate exposure appeared to be 48 hours, rather than 4 hours, for 

the initial toxicity/mutation assay and the “confirmatory” mutagenicity assay. Therefore, it is unclear why the study 

author indicates that plate exposure was complete after only 4 hours.
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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relative standard deviation (% RSD) at each concentration. For the test item to be considered 

stable in the vehicle for this test system, the stability calculation result must be ≤ 15% of the time 

0 hour concentration.” [Tendulkar 2014, p. 23]

Criteria for Determining a Mutagenic Response and Cytotoxicity: 

The criteria for determining a positive (mutagenic) response were as follows:

 “The conditions necessary for determining a positive result were that there should be a doserelated increase in the mean revertants per plate of at least one tester strain over a minimum

of two increasing doses of the test item either in the absence or presence of the metabolic 

activation system.” [Tendulkar 2014, p. 25] 

 For Salmonella typhimurium strains TA98, TA1535, and TA1537: “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 negative control value.” [Tendulkar 2014, p. 25]

 For Salmonella typhimurium strains TA100 and Escherichia coli WP2 uvrA (pKM101):

“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 negative control value.” [Tendulkar 

2014, p. 25]

“A response that did not meet all three of the above criteria (magnitude, concentrationresponsiveness, reproducibility1

) was determined to be non-mutagenic.” [Tendulkar 2014, p. 25]

“Cytotoxicity is detectable as more than [a] 50% reduction in mean revertants per concentration 

relative to the mean negative control value (Teo et al., 2003) and by thinning of the bacterial 

background lawn.” [Tendulkar 2014, p. 25]

RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable)2: ≥ 15 μg/plate in the absence of metabolic activation; 

≥ 50 μg/plate in the presence of metabolic activation

Results remarks: 

Initial Toxicity/Mutagenicity Assay:

“No positive mutagenic responses were observed with any of the tester strains in either the 

presence or absence of S9 activation. Precipitate was not observed with any of the tester strains 

in either the presence or absence of S9 activation. Toxicity was observed beginning at 15 μg per 

plate in the absence of S9 activation and at 50 μg per plate in the presence of S9 activation

[based on thinning of the bacterial lawn in all strains at these concentrations]. [The] Percent 

reduction [in mean revertants] was 43.75 % in TA1537, 40.91% in TA1535, 61.90% in TA98, 

27.69% in TA100 and 46.34% in E. coli WP2uvrA (pKM101) at [a] test concentration of 50 

μg/plate in [the] absence of [the] metabolic activation system. In the presence of [the] metabolic 

activation system [the] percent reduction [in mean revertants] observed was 47.37% in TA1537, 

50.00% in TA98, [and] 27.20% in TA100 at [a] test concentration of 50 μg/plate... [and] 47.73% 

 

1 The “above criteria” described in the study report did not include reproducibility. It is unclear how reproducibility 

factored into the criteria for a positive response. 

2 Cytotoxic concentrations are based on thinning of the bacterial lawn.
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

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in TA1535 at 150 μg/plate and 41.77% in E. coli WP2uvrA (pKM101) at 500 μg/plate.” 

[Tendulkar 2014, p. 26]

“Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the 

confirmatory mutagenicity assay was 150 μg per plate.” [Tendulkar 2014, p. 26]

“Confirmatory” Mutagenicity Assay:

“In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with 

any of the tester strains in either the presence or absence of S9 activation... Precipitate was not 

observed with any of the tester strains in either the presence or absence of S9 activation. 

Toxicity was observed beginning at 15 μg per plate in the absence of S9 activation and at 50 μg 

per plate in the presence of S9 activation [based on thinning of the bacterial lawn in all strains at 

these concentrations]. [The] Percent reduction [in mean revertants] was 44.44% in TA1537, 

44.08% in TA1535, 46.57% in TA98, 22.17% in TA100 and 35.33% in E. coli WP2uvrA 

(pKM101) at [a] test concentration of 50 μg/plate in [the] absence of [the] metabolic activation 

system. In the presence of [the] metabolic activation system [the] percent reduction [in mean 

revertants] observed was 48.29% in TA1537, 45.57% in TA98, [and] 23.44% in TA100 at [a] 

test concentration of 50 μg/plate... [and] 39.35% in TA1535 [at 150 μg/plate] and 2.16% in E. 

coli WP2uvrA (pKM101) at 50 and 150 μg/plate.” [Tendulkar 2014, p. 26]

“In both the initial toxicity-mutation assay and the confirmatory mutagenicity assay, the values 

[for numbers of revertants] for the negative controls (acetone and sterile distilled water) in all 

strains were within [the] limits of historical range of distilled water. Results of the concurrent 

negative control (acetone) were comparable with the results of [the] concurrently run additional 

negative control (distilled water). Positive controls (both in the absence and presence of 

metabolic activation during both the trials) exhibited a clear increase in the number of revertants 

when compared with the concurrent negative control and were within the historical ranges. This 

demonstrated the efficiency of the test system and suitability of the procedures employed in the 

assay. An increase in the mean number of revertants was not observed in Salmonella 

typhimurium tester strain TA100 (initial toxicity test and confirmatory mutation test) treated with 

2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in 

the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in 

this assay.” [Tendulkar 2014, p. 27]

Dose Formulation Analysis:

The measured concentration of test substance “was found to be within [an] acceptable range of 

± 15% of nominal (% RSD < 10%) in acetone at all the tested concentrations [of stock solutions 

maintained in an ice bath] during [the] confirmatory mutation assay. The doses complied with 

the presence of test item for its claimed concentration (± 15% of nominal) of active ingredient.

Average recovery [at 0 hours] was 101%, 98.7%, 99.2%, 98.0%, 98.4%, 96.7% and 99.7% at test 

[stock solution] concentrations of 3, 10, 30, 100, 300, 1000 and 3000 μg/mL, respectively. 

Average recovery for stability samples... [analyzed for stock solutions that had been stored for 4 

hours in an ice bath] was 100% and 99.1% at test concentrations of 3 and 3000 μg/mL, 

respectively. % RSD ranged from 0.034% to 0.613% across the dosing range.” [Tendulkar 2014, 

p. 27] 
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

11

It should be noted that the results for the stability analysis performed on formulations of CASRN 

2524-04-1 in acetone are not representative of the degree to which hydrolysis

1

and volatilization2

of CASRN 2524-04-1 may have occurred under the exposure conditions used in this study for 

the following reasons: (1) the stability test was performed in acetone rather than the aqueous 

medium used during the pre-incubation or incubation period and, therefore, would not 

demonstrate hydrolysis of the test substance; (2) the stability test was conducted for 4 hours, as 

compared to the 48 hours and 20 minutes for the sum of the plate incubation and pre-incubation 

periods, allowing less time for degradation and volatilization; and (3) the stability test was 

conducted under a much colder temperature (i.e., in an ice bath) than the bacterial incubation 

temperature (i.e., 37 ± 1 °C) and, therefore, the test substance would be less likely to volatilize. 

In addition, it should be noted that the test containers were not covered with airtight seals during 

each phase of the test (e.g., preparation of stock solutions, 20-minute pre-incubation period, and 

48-hour plate incubation period). 

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

[3] Not reliable. This study is considered unreliable because no precautions were taken to 

minimize losses of test material by volatilization during the study. Due to the potential volatility 

of CASRN 2524-04-1, EPA previously commented in its review3of the study plan submitted by 

Dow AgroSciences for the gene mutation study that “all test containers (including vessels used 

for the preparation of test item formulations, tubes used for pre-incubation of bacteria, and agar 

plates onto which bacteria are transferred) shall have minimal headspace and minimal exposure 

to air and shall be covered with airtight seals.” However, the study report for the gene mutation 

study submitted by Dow AgroSciences provides no indication that any precautions were taken to 

minimize losses of test material by volatilization during the study. Of particular concern is test 

substance loss by volatilization during the 20-minute pre-incubation period and the 48-hour plate 

incubation period. Although measurement of CASRN 2524-04-1 in stock formulations in 

acetone (lowest and highest concentration) performed by GC/ECD after 4 hours in an ice bath

showed minimal degradation and/or loss of the test substance, these results are not representative 

of the extent of degradation and/or loss by volatilization anticipated during the mutagenicity test 

 

1 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. 

2 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/opptintr/

exposure/pubs/episuitedl.htm as of March 4, 2015). 

3

“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-HQ-OPPT-2007-0531-0805 as of March 4, 2015.
Final HPV2 Test Rule Data Adequacy Review (March 2015) CASRN 2524-04-1

12

due to the differences in solvent used (acetone in stability test vs. aqueous medium in 

mutagenicity test) and temperature (near 0 °C in stability test vs. 37 °C in mutagenicity test). 

Although a positive correlation between nominal test substance concentration and cytotoxicity

was observed, it is unknown whether the observed cytotoxicity was due to CASRN 2524-04-1, 

its degradation products, or a combination thereof. Therefore, the observation of cytotoxicity 

cannot be used as an indirect indicator that the test methods were acceptable. The lack of 

precautions taken to minimize volatilization suggests that undegraded CASRN 2524-04-1 was 

free to volatilize to the atmosphere throughout the study; consequently, it is unknown whether or 

not the test results are representative of those that would be obtained had precautions been taken 

to minimize volatilization of CASRN 2524-04-1. 

This study needs to be repeated under conditions that minimize the loss of the test material by 

volatilization during the 20-minute pre-incubation period and during the 48-hour plate incubation 

period. EPA requests that all test containers (including vessels used for the preparation of test 

item formulations, tubes used for pre-incubation of bacteria, and agar plates onto which bacteria 

are transferred) have minimal headspace and minimal exposure to air and be covered with 

airtight seals. In addition, EPA recommends that the mutagenicity assay be performed using the

plate incorporation method (also described in OECD TG 471) instead of the pre-incubation 

method to minimize losses of test substance by volatilization. EPA also recommends that any 

additional gene mutation testing be performed using triplicate plating for all definitive

mutagenicity tests. 

REFERENCE

Tendulkar, K.E. 2014. Phosphorochloridothioic acid, O,O-diethyl ester: SalmonellaEscherichia coli mammalian-microsome reverse mutation assay pre-incubation method with a 

confirmatory assay. Performing Laboratory: JAI Research Foundation, Gujarat, India. Sponsor: 

Dow AgroSciences LLC.