SRC TR-14-008

HPV2 REVIEW OF TOXICITY STUDY PLAN DATED FEBRUARY 25, 2014

(FINAL)

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

Prepared by:

Teresa Manyin, William L. Richards, and Laura Morlacci

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

April 3, 2014
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

1

HPV2 Toxicity Study Plan Review: 

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

In response to the 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/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0579 as of 

April 3, 2014), Dow AgroSciences submitted a study plan on February 25, 2014 for several 

toxicity tests with phosphorochloridothioic acid, O,O-diethyl ester (CASRN 2524-04-1; O,Odiethyl phosphorochloridothioate [DEPCT]).

The HPV2 test rule requires the following toxicological tests for CASRN 2524-04-1: 

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

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

testing] if log Kow ≥ 4.2), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or 

micronucleus test), and F2 (reproduction/developmental toxicity screening test). 

The February 25, 2014 study plan from Dow AgroSciences included protocols for the following 

toxicity tests and preliminary tests for the reproduction/developmental toxicity screening test:

 Bacterial Reverse Mutation Assay (to be conducted by Jai Research Foundation)

 Mammalian Erythrocyte Micronucleus Test (to be conducted by The Dow Chemical 

Company)

 Test for Stability in Rodent Feed and Corn Oil (preliminary test, to be conducted by The 

Dow Chemical Company)

 14-Day Oral Gavage Range-Finding Study in Rats (preliminary test, to be conducted by 

The Dow Chemical Company)

General Comments:

Due to the potentially rapid hydrolysis rate of CASRN 2524-04-1, EPA recommends that Dow 

AgroSciences conduct a hydrolysis test of CASRN 2524-04-1 prior to initiating toxicity testing. 

If the test confirms that the hydrolysis of the test substance is rapid, then precautions will need to 

be taken during the toxicity tests to minimize hydrolysis of CASRN 2524-04-1 and thereby 

maximize exposure to CASRN 2524-04-1. Additionally, the degradation products of CASRN 

2524-04-1 should be identified by analytical determination. Since CASRN 2524-04-1 and its 

hydrolysis products are potentially volatile, precautions may be needed to minimize the loss of 

these chemicals by volatilization during the hydrolysis study.

Bacterial Reverse Mutation Assay:

1) The protocol states that the bacterial reverse mutation assay will be conducted according to 

the following guidelines: OECD TG 471, OPPTS 870.5100, EEC B.13/14, and JMAFF 2-1-

19-1.

Although the test rule specifies that the required bacterial reverse mutation testing be 

conducted according to TSCA guideline 40 CFR 799.9510, EPA considers the use of 

OECD TG 471 acceptable to satisfy the test rule requirement.
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

2

2) The protocol states that analytical methods will be used to verify concentrations of the test 

item in formulations and to determine the stability of the test item in the vehicle. 

CASRN 2524-04-1 is likely to undergo hydrolysis if exposed to water or to react with 

other molecules with nucleophilic functional groups if such molecules are present in the 

test item formulation. If preliminary stability tests demonstrate that the concentration of 

CASRN 2524-04-1 declines over time (i.e., degrades) in the dosing formulation, EPA 

suggests that analytical measurements include analytical determination of the 

concentrations of not only CASRN 2524-04-1 (the parent chemical) but also its organic 

degradation products. Analysis of the concentration of inorganic degradation products 

(such as HCl) is not necessary. The testing laboratory conducting the tests should be 

familiar with the sampling, processing, storage, and analytical detection procedures 

needed for quantitation of CASRN 2524-04-1 and its organic degradation products. 

Furthermore, due to the potential volatility of CASRN 2524-04-1 and its degradation 

products, measures must be taken to minimize volatilization during sample collection, 

transfer, storage, processing, and analytical detection (e.g., no headspace in sample vials, 

minimal exposure to air). The final report should provide data from the analytical 

measurement of test item concentrations and determination of the stability of the test item 

in the vehicle. 

3) The protocol states that the vehicle will be chosen on the basis of solubility of the test item 

and lists the following possible vehicles, in order of preference: water, dimethyl sulfoxide 

[DMSO], ethanol, and acetone. 

Water and ethanol are not acceptable vehicles. The chosen vehicle must be an anhydrous 

organic solvent that has no nucleophilic functional groups (e.g., DMSO, acetone,

dimethylformamide [DMF]). The organic solvent used shall be free of water; water can 

be removed by the use of drying agents such as molecular sieves (see 40 CFR 799.9537). 

The use of such solvents will minimize hydrolysis of CASRN 2524-04-1. 

4) The protocol states that an initial toxicity-mutation assay will be conducted in which each test 

item concentration, each positive control, and the vehicle control will be plated in duplicate.

If the toxicity assay (described in section E, p. 7 of the protocol) is to be considered as an 

initial mutation test and the second assay (described in section F, p. 8 of the protocol) is 

to be considered a confirmatory mutation test, then each concentration of the test item 

and each control shall be plated in triplicate, rather than duplicate, during the initial assay 

as specified in OECD TG 471. If duplicate plating is used in the initial test, then any 

equivocal or negative results from the second test will need to be confirmed or clarified 

by conducting further mutation tests (as indicated in OECD TG 471).

5) The protocol does not specify that test containers will be sealed to minimize volatization.

Due to the potential volatility of CASRN 2524-04-1 and its hydrolysis products, 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. 
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

3

6) The protocol lists the criteria for a positive response, followed by the statement “A response 

that does not meet all three of the above criteria (magnitude, concentration-responsiveness, 

reproducibility) will not be evaluated as positive.”

The “above criteria” specified in the protocol do not mention reproducibility. The 

protocol should be revised to clarify how reproducibility will affect the determination of 

a positive result. For example, OECD TG 471 states that “a reproducible increase at one 

or more concentrations in the number of revertant colonies per plate in at least one strain 

with or without metabolic activation system” can be used as a possible criterion for a 

positive result.

7) In addition to the information specified in the protocol, the following items must be included 

in the final study report:

 Composition of the test substance, including identities and concentrations of major 

ingredients and of impurities (e.g., certificate of analysis)

 Justification for the choice of vehicle

 A description of measures that were taken to minimize volatilization and degradation 

of the test substance prior to and during the test

8) Item II.10 of the “Protocol Annexure Form” at the end of the protocol for the bacterial 

reverse mutation assay indicates that the product is expected to be stable under standard 

conditions for the duration of the study.

EPA suggests that this statement be modified since CASRN 2524-04-1 is an electrophile 

that will react not only with water but also with other components of this biological test 

system that have nucleophilic functional groups (e.g., amines, alcohols, thiols).

Mammalian Erythrocyte Micronucleus Test:

1) Dow AgroSciences submitted a protocol for a mammalian erythrocyte micronucleus test in 

response to the E2 testing requirement in the HPV2 test rule.

According to the test rule, the E2 testing requirement may be satisfied by conducting any 

one of the following three tests for chromosomal damage:

 In vitro Mammalian Chromosome Aberration Test according to 40 CFR 799.9537

 In vivo Mammalian Chromosome Aberration Test according to 40 CFR 799.9538

 In vivo Mammalian Erythrocyte Micronucleus Test according to 40 CFR 799.9539

As stated in the test rule, “Persons required to conduct testing for chromosomal damage 

are encouraged to use the in vitro Mammalian Chromosome Aberration Test (40 CFR 

799.9537) to generate the needed data unless known chemical properties (e.g., 

physical/chemical properties, chemical class characteristics) preclude its use. A subject 

person who uses one of the in vivo methods instead of the in vitro method to address a 

chromosomal damage test requirement must submit to EPA a rationale for conducting 

that alternate test in the final study report.” Therefore, if Dow AgroSciences conducts an

in vivo mammalian erythrocyte micronucleus test, rather than an in vitro mammalian 

chromosome aberration test, to satisfy the E2 testing requirement, the final study report 

must include a rationale for this decision.
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

4

2) The protocol states that the mammalian erythrocyte micronucleus test will be conducted 

according to the following guidelines: OECD TG 474, OPPTS 870.5395, and EC B.12.

Although the test rule specifies that in vivo mammalian erythrocyte micronucleus testing 

be conducted according to TSCA guideline 40 CFR 799.9539, EPA considers the use of 

OECD TG 474 acceptable to satisfy the test rule requirement with the caveat that the 

study report must include all information listed in item 32 of OECD TG 474.

3) The protocol does not specify the matrix that will be used for preparation of dosing solutions.

EPA recommends that the test substance be diluted and orally delivered in a suitable 

vehicle. The vehicle must be anhydrous and contain no nucleophilic functional groups to 

minimize hydrolysis and degradation of CASRN 2524-04-1. For example, anhydrous 

oil-based vehicles (e.g., anhydrous corn oil) can be used. The oil should not contain fatty 

acids with alcohol groups. 

4) The protocol states that concentrations of the test item in dosing formulations will be verified 

analytically. 

CASRN 2524-04-1 is likely to undergo hydrolysis if exposed to water or to react with 

other molecules with nucleophilic functional groups if such molecules are present in the 

test item formulation. If preliminary stability tests demonstrate that the concentration of 

CASRN 2524-04-1 declines over time (i.e., degrades) in the dosing formulation, EPA 

suggests that analytical measurements include analytical determination of the 

concentrations of not only CASRN 2524-04-1 (the parent chemical) but also its organic 

degradation products. Analysis of the concentration of inorganic degradation products 

(such as HCl) is not necessary. The testing laboratory conducting the tests should be 

familiar with the sampling, processing, storage, and analytical detection procedures 

needed for quantitation of CASRN 2524-04-1 and its organic degradation products. 

Furthermore, due to the potential volatility of CASRN 2524-04-1 and its degradation 

products, measures must be taken to minimize volatilization during sample collection, 

transfer, storage, processing, and analytical detection (e.g., no headspace in sample vials, 

minimal exposure to air). The final report should provide data from the analytical 

measurement of test item concentrations and determination of the stability of the test item 

in the vehicle. 

5) The protocol states that groups of six mice per dose (6/dose) will be administered the test 

substance.

This statement should be revised to indicate that six mice per sex per dose (6/sex/dose) 

will be administered the test substance. As indicated in OECD TG 474, each treated and 

control group must include at least 5 analyzable animals per sex. Both sexes must be 

tested unless the results of the range-finding test demonstrate that there are no substantial 

differences in toxicity between sexes, in which case testing of a single sex is sufficient. 
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

5

6) The protocol does not describe measures that will be taken to minimize volatization during 

preparation and handling of test item formulations.

Due to the potential volatility of CASRN 2524-04-1 and its degradation products, all 

containers used for preparation of test item formulations shall have minimal headspace 

and minimal exposure to air and shall be covered with airtight seals. 

7) In addition to the information specified in the protocol, the following items must be included 

in the final study report:

 Composition of the test substance, including identities and concentrations of major 

ingredients and of impurities (e.g., certificate of analysis)

 Justification for the choice of vehicle

 A description of measures that were taken to minimize volatilization and degradation 

of the test substance prior to and during the test.

8) The protocol indicates that the stability of the test item under the conditions of administration 

will be provided in the final study report.

The protocol did not describe how or when the stability of the test item will be 

determined. The stability of the test item in the vehicle should be determined before the 

range-finding test is conducted.

9) Appendix A of the protocol is entitled “Dose Selection Criteria”.

The title of Appendix A should be revised to “Dose Selection Criteria for the RangeFinding Test”, so that it will not be confused with dose selection criteria for the main 

(micronucleus) test.

Test for Stability in Rodent Feed and Corn Oil:

1) The protocol includes testing to determine the stability of the test substance in rodent feed.

The reason for testing stability of CASRN 2524-04-1 in rodent feed is unclear. The 

protocol submitted for the 14-day range-finding study (preliminary test for the 

reproduction/developmental screening test) specifies that gavage will be the means of 

exposure and states that “Dietary administration of the test material is not feasible due to 

instability in rodent feed.” EPA agrees that CASRN 2524-04-1 would not be stable in 

feed due to its expected reactivity with water and other nucleophilic components of feed. 

Additionally, there is concern that CASRN 2524-04-1 (and its degradation products) 

would volatilize from feed. Since exposure of rats in the reproduction/developmental 

screening test will be conducted using gavage, testing of stability of CASRN 2524-04-1 

in rodent feed is unnecessary.

2) The protocol does not specify whether the corn oil used for stability testing will be anhydrous.

Since CASRN 2524-04-1 is likely to undergo hydrolysis if exposed to water, the corn oil 

used for stability testing and oral administration of the test substance to rats must be 

anhydrous.
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

6

3) The protocol includes testing to determine the stability of the test substance in toluene.

The reason for testing stability of CASRN 2524-04-1 in toluene is not provided. Since 

toluene is not an appropriate vehicle for use in administration of the test substance to 

animals, the relevance of stability in toluene is unclear. EPA speculates that toluene may

be used in the analytical methods used to measure test substance concentration in dosing 

formulations. A clarification of the relevance of stability testing in toluene is requested.

4) The protocol states that concentrations of the test item in the matrix of interest will be 

measured analytically. 

Due to the potential volatility of CASRN 2524-04-1 and its degradation products, 

measures must be taken to minimize volatilization during sample collection, transfer, 

storage, processing, and analytical detection (e.g., no headspace in sample vials, minimal

exposure to air). 

5) The protocol does not describe measures that will be taken to minimize volatization during 

preparation and handling of test item formulations.

Due to the potential volatility of CASRN 2524-04-1 and its degradation products, all 

containers used for preparation of test item formulations shall have minimal headspace 

and minimal exposure to air and shall be covered with airtight seals. 

14-Day Oral Gavage Range-Finding Study in Rats:

1) The dose levels proposed for the range-finding study were selected based on inhibition of 

cholinesterase (ChE) observed in previous studies of CASRN 2524-04-1 and a “structurally 

similar” chemical (chlorpyrifos) in rats. Furthermore, ChE activity in the red blood cells and 

brain will be included in the endpoints measured during the range-finding study. 

Caution must be used in the interpretation of data showing inhibition of ChE activity. 

Although inhibition of ChE may be used as an indicator of exposure to organophosphorus 

compounds, it does not necessarily correlate with nervous system symptomatology (Lotti, 

1995) or with changes in other endpoints that need to be examined to fully evaluate 

systemic toxicity. Inhibition of ChE by itself is not sufficient evidence of toxicity for use 

in dose selection. Therefore, the selection of doses for the range-finding study may need 

to be revisited to consider other available data describing the oral toxicity of CASRN 

2524-04-1. If the sponsor proceeds with the range-finding study at the dose levels 

proposed in the protocol (5, 25, 50, and 100 mg/kg-bw/day) and the only effect observed 

is ChE inhibition, then an additional range-finding study will need to be conducted at 

higher dose levels and/or with measurement of additional endpoints (e.g., histopathology, 

functional observational battery, etc.) to demonstrate clear physiological evidence of 

toxicity in order to provide useful results for the selection of doses for the 

reproduction/developmental toxicity screening test. Doses chosen for the 

reproduction/developmental toxicity screening test must follow the guidance provided in 

TSCA guideline 799.9355: “The highest dose level should be chosen with the aim of 

inducing toxic effects but not death or severe suffering. Thereafter, a descending 

sequence of dose levels should be selected in order to demonstrate any dose response 

relationships and no adverse effects at the lowest dose level.” Inhibition of ChE should 

not be used as the sole basis for measuring toxicity; additional evidence of toxicity (e.g., 
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

7

adverse effects on histology or functional observational battery measurements) must be 

demonstrated at the dose levels used for the reproduction/developmental screening test.

2) The protocol does not specify that the corn oil used for dosing will be anhydrous.

To minimize hydrolysis and degradation of CASRN 2524-04-1, the corn oil vehicle must 

be anhydrous and contain no nucleophilic functional groups. 

3) The protocol does not specify whether the dosing formulations will be prepared immediately 

prior to use or stored for an unspecified length of time. The protocol states that CASRN 

2524-04-1 has been shown to be stable in corn oil at concentrations ranging from 0.25 to 250 

mg/L for at least 16 days, citing Perala (in progress).

The citation provided (Perala) appears to be a reference to the stability test for which a 

protocol was submitted in Dow AgroSciences’ February 25, 2014 study plan. It is 

unclear if these are preliminary data, or if the data were obtained from a definitive test 

(conducted prior to the receipt of EPA’s above comments on the submitted protocol). 

Regardless, if definitive data show that CASRN 2524-04-1 is not stable in corn oil, 

dosing solutions must be prepared fresh daily prior to dosing.

4) The protocol does not describe measures that will be taken to minimize volatization during 

preparation and handling of test item formulations.

Due to the potential volatility of CASRN 2524-04-1 and its degradation products, all 

containers used for preparation of test item formulations shall have minimal headspace 

and minimal exposure to air and shall be covered with airtight seals. 

5) In addition to the information specified in the protocol, the following items should be 

included in the final study report:

 Composition of the test substance, including identities and concentrations of major 

ingredients and of impurities (e.g., certificate of analysis)

 A description of measures that were taken to minimize volatilization and degradation 

of the test substance prior to and during the study.

 Mean and individual animal data for all measured parameters
Final HPV2 Study Plan Review (April 2014) CASRN 2524-04-1

8

REFERENCES

Lotti, M. 1995. Cholinesterase inhibition: Complexities in interpretation. Clinical Chemistry 

41: 1814-1818. Available at http://www.clinchem.org/content/41/12/1814.full.pdf as of April 2, 

2014.

OECD. 1997a. Bacterial reverse mutation test. Test Guideline 471. Available at 

http://www.oecd.org/chemicalsafety/risk-assessment/1948418.pdf as of April 2, 2014

OECD. 1997b. Mammalian erythrocyte micronucleus test. Test Guideline 474. Available at

http://www.oecd.org/chemicalsafety/risk-assessment/1948442.pdf as of April 2, 2014.

US EPA. 2012a. TSCA bacterial reverse mutation test. 40 CFR 799.9510. Available at 

http://www.ecfr.gov/cgi-bin/text-idx?SID=fcbcf7e639c49659136e088ea6cc5387

&node=40:33.0.1.1.8.7.1.16&rgn=div8 as of April 2, 2014.

US EPA. 2012b. TSCA in vitro mammalian chromosome aberration test. 40 CFR 799.9537. 

Available at http://www.ecfr.gov/cgi-bin/text-idx?SID=b3c328624afa4723ef0e5af7b724d7dd&

node=40:33.0.1.1.8.7.1.18&rgn=div8 as of April 2, 2014.

US EPA. 2012c. TSCA mammalian bone marrow chromosome aberration test. 40 CFR 

799.9538. Available at http://www.ecfr.gov/cgi-bin/retrieveECFR?gp=&SID=f1b9034d1e2

dfc59e8f96f6d6d31f52b&r=SECTION&n=40y33.0.1.1.8.7.1.19 as of April 2, 2014.

US EPA. 2012d. TSCA mammalian erythrocyte micronucleus test. 40 CFR 799.9539. 

Available at http://www.ecfr.gov/cgi-bin/retrieveECFR?gp=&SID=f1b9034d1e2dfc59e

8f96f6d6d31f52b&r=SECTION&n=40y33.0.1.1.8.7.1.20 as of April 2, 2014.

US EPA. 2012e. TSCA reproduction/developmental toxicity screening test. 40 CFR 799.9355. 

Available at http://www.ecfr.gov/cgi-bin/text-idx?SID=fcbcf7e639c49659136e088ea6cc5387

&node=40:33.0.1.1.8.7.1.9&rgn=div8 as of April 2, 2014.