SRC TR-14-001

HPV2 TEST RULE:

RESPONSE TO DOW AGROSCIENCES, COMMENTS DATED JANUARY 14, 2014

(FINAL)

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

Prepared by:

Jennifer Rayner, Teresa Manyin, and William Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

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

1

On January 14, 2014, Dow AgroSciences submitted an email to EPA regarding the instability of 

CASRN 2524-04-1 (phosphorochloridothioic acid, O,O-diethyl ester; O,O-diethyl 

phosphorochloridothioate [DEPCT]) and the resulting challenges of completing the studies 

required in the final test rule, “Testing of Certain High Production Volume Chemicals; Second 

Group of 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 

January 22, 2014). 

The HPV2 test rule requires the following toxicological tests for CASRN 2524-04-1: C1 (aquatic 

toxicity tests), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus 

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

The comments from Dow AgroSciences are addressed below:

(1) Dow AgroSciences cited the results of a hydrolysis study submitted under the REACH 

registration for CASRN 2524-04-1 (available online at 

http://apps.echa.europa.eu/registered/data/dossiers/DISS-9ea6e71a-f3c6-6f79-e044-

00144f67d031/AGGR-aeec32eb-7557-4480-911a-219171f0c3ea_DISS-9ea6e71a-f3c6-6f79-

e044-00144f67d031.html#AGGR-aeec32eb-7557-4480-911a-219171f0c3ea as of January 22, 

2014). Dow AgroSciences states “Hydrolysis half-life is 14 min. at room temp in distilled 

water...”

The hydrolysis test cited in the REACH registration for CASRN 2524-04-1 was not conducted 

according to a testing guideline or GLP, and detailed methods, test conditions, and results were 

not reported. Data are insufficient to allow an independent assessment of the reliability and 

adequacy of the test and its results. Therefore, the half-life results of this specific test should not 

be factored into decisions about the ability to conduct, as well as the methods that may be used to 

conduct, toxicity tests. EPA recommends that the hydrolysis test be repeated to determine an 

accurate and reliable half-life of CASRN 2524-04-1. If the test confirms that the hydrolysis of 

the test substance is rapid, then precautions will need to be taken during the toxicity tests, as 

described below.

(2) Dow AgroSciences states “After stability testing in various media, the testing conditions may 

then be unrealistic for any potential route of exposure (i.e. ecotox and mammalian testing in 

nonaqueous media? 30-day tox testing when the half-life is 14 minutes? etc...).”

It is recognized and accepted that CASRN 2524-04-1 may be unstable under testing conditions 

for ecological and mammalian toxicity studies and that degradation to its hydrolysis products 

may occur during these studies. Test solutions shall be formulated using nominal concentrations 

of CASRN 2524-04-1 and shall be prepared just prior to the introduction of test organisms in 

aquatic toxicity studies, the exposure of bacterial or mammalian cells in in vitro genotoxicity 

studies, and the dosing of animals in in vivo toxicity studies. Analytical monitoring of the 

concentrations of CASRN 2524-04-1 and its degradation products shall be conducted during the 

toxicity tests, as further discussed in #3 and #5 below.
HPV2 Test Rule: Response to Dow AgroSciences (Final) CASRN 2524-04-1

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(3) Dow AgroSciences states “Due to chemical instability of DEPCT, especially in, but not 

limited to aqueous media, we will not be able to follow the required testing guidelines for the 

following studies:... Fish tox, Daphnia tox, Algae tox.”

Although DEPCT (CASRN 2524-04-1) is a reactive chemical, there are guidelines and methods 

available that will allow the chemical to be tested successfully for effects on aquatic species. 

The sponsor should refer to OECD Series on Testing and Assessment Number 23, “Guidance 

Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”. This guidance 

document can be used to develop test protocols that are appropriate for conducting aquatic 

toxicity tests for chemicals that degrade in the test system (see section 3.5), including test 

substances that undergo hydrolysis under test conditions (see section 3.5.2). 

According to the test rule guidelines, preliminary stability tests should be conducted to assess the 

stability of the test substance under the test conditions of each aquatic toxicity test prior to 

conducting the aquatic toxicity tests. 

EPA recommends that toxicity tests of DEPTC for fish and Daphnia be conducted using a closed 

continuous flow-through system (see OECD 23, Sections 3.4 and 3.5). Flow-through tests 

facilitate the maintenance of exposure concentrations especially in cases where degradation or 

loss of test substance may occur. The system used shall allow the undiluted test substance to mix 

with test dilution water immediately prior to entrance into the test system. Samples of test 

solution for analytical measurement shall be collected at the point of entry into the system and at 

the exit point of the system at least every 24 hours; these samples shall be analyzed for 

concentrations of CASRN 2524-04-1 and its organic degradation products. Alternatively, 

toxicity tests for fish and Daphnia may be conducted using static-renewal tests. If a staticrenewal test is conducted, test solutions shall be prepared no more than 30 minutes prior to the 

introduction of test organisms to the solutions. Samples of test solution for analytical 

measurement of test substance concentrations (CASRN 2524-04-1 and its organic degradation 

products) shall be collected at the start and end of each renewal period. For either a flowthrough or a static renewal test, the system must be closed (sealed to prevent exposure to air) to 

minimize volatilization of the test rule chemical and its degradation products from the test water 

to the atmosphere.

Algae tests can only be conducted using static exposure technique. Due to the potential volatility 

of CASRN 2524-04-1 and its hydrolysis products, the following alterations to standard guideline 

methods must be made (as detailed in Mayer et al. [2000]):

 The test vessels shall be sealed during preparation and exposure to prevent loss of the test 

substance. The seal or stopper used by the testing lab must be impermeable to air.

 Headspace shall be kept to a minimum to reduce loss of test substance from solution.

 Test solutions (algae growth media) shall be enriched with additional sodium bicarbonate 

(e.g., 300 mg NaHCO3/L) to provide a source of CO2 for algae growth. After addition of 

sodium bicarbonate, the pH of growth medium must be adjusted to 7.0 by the addition of 

HCl.

 The initial cell density shall be reduced to 3 x 103cells/mL to reduce demand for CO2. 

 The algae shall be added to the prepared test solutions no more than 30 minutes after 

preparation to maximize exposure to the parent compound.
HPV2 Test Rule: Response to Dow AgroSciences (Final) CASRN 2524-04-1

3

 The duration of algae exposure shall be 72 hours.

Samples for analytical measurement of the concentration of the test substance and its organic 

degradation products shall be collected at the beginning and end of the algae test at a minimum. 

(4) Dow AgroSciences states “There are also challenges with conducting the mammalian tox 

studies due to instability but maybe there are other options for testing media?”

With regard to in vitro genotoxicity tests, an anhydrous organic solvent that has no nucleophilic 

functional groups (e.g., dimethylformamide [DMF], acetone, dimethyl sulfoxide [DMSO])

should be used to prepare stock solutions of test substance prior to introduction into the test 

system. The organic solvent used should be free of water; water can be removed by adding a 

molecular sieve (see 40 CFR 799.9537). The use of such solvents will minimize degradation of 

CASRN 2524-04-1. In vitro genotoxicity tests shall be conducted using sealed test containers to 

minimize volatilization of CASRN 2524-04-1 and its degradation products.

With regard to in vivo mammalian toxicity tests (reproductive/developmental and micronucleus 

tests), the test substance can be orally delivered undiluted or diluted in a suitable vehicle (e.g., 

anhydrous corn oil). The oil should not contain fatty acids with alcohol groups. Anhydrous oilbased vehicles can be used to minimize degradation of test substances that can react with water 

or other nucleophilic substances. The report must contain a justification for the use and 

suitability of the chosen vehicle. The dosing solutions administered should be freshly prepared

prior to each dosing. According to the test rule guideline for the reproductive/developmental 

toxicity test, the stability of the test substance should be determined in the chosen vehicle prior to 

conducting the toxicity test.

To maximize exposure to the parent chemical (CASRN 2524-04-1), test solutions for 

mammalian toxicity studies and in vitro toxicity studies shall be used no more than 30 minutes

after preparation.

(5) Dow AgroSciences states “DEPCT is highly reactive (hydrolyzes) in water... The reactivity 

will have a significant influence on how testing is done - if it can be done at all. The substance 

will react with water, proteins, alcohols, etc. (any nucleophilic substance), thus making it 

unstable and difficult to verify exposure concentrations in ecological and mammalian toxicity 

tests.”

Analytical monitoring of test substance concentrations used in toxicity tests must be conducted

to determine actual exposure concentrations and to demonstrate repeatability of procedures used 

to prepare test solutions. This will include analytical determination of the concentrations of 

CASRN 2524-04-1 (the parent chemical) and its organic degradation products. (Analysis of the 

concentration of HCl is not necessary.) Concentrations of CASRN 2524-04-1 and its organic

degradation products must be satisfactorily monitored throughout the duration of the test, as 

demonstrated by the results of a reliable, sensitive, and validated analytical method. The testing 

laboratory conducting the tests shall be familiar with the sampling, processing, storage, and 

analytical detection procedures needed for quantitation of DEPCT and its organic degradation 

products. 
HPV2 Test Rule: Response to Dow AgroSciences (Final) CASRN 2524-04-1

4

Conclusion: EPA maintains that the required aquatic and mammalian toxicity tests must be 

conducted to satisfy the HPV2 testing requirements. EPA makes the following 

recommendations: 

1. Conduct a hydrolysis test of DEPCT using OECD TG 111, which includes methods for 

testing unstable substances (Tiers 1 through 3). If the test confirms that the hydrolysis of 

the test substance is rapid, then precautions will need to be taken during the toxicity tests

to minimize DEPCT hydrolysis and thereby maximize exposures to DEPCT.

2. Determine appropriate methods for analytical monitoring (including sampling, 

processing, storage, and detection procedures) during toxicity tests, based on the 

information obtained during the hydrolysis test and requirements of testing guidelines.

3. Submit a test plan with detailed study protocols to EPA for review. To demonstrate that 

there is a consistent relationship between nominal and measured levels of CASRN 2524-

04-1 and its degradation products, study protocols should include frequent sampling of 

test media and dosing solutions for analyses of concentrations of CASRN 2524-04-1 and 

its organic degradation products during the tests. 

REFERENCES

Mayer, P., Nyholm, N., Verbruggen, E.M.J., Hermens, J.L.M., and Tolls, J. 2000. Algal growth 

inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental 

Toxicology and Chemistry 19: 2551-2556. 

OECD. 2002. “OECD Series on Testing and Assessment Number 23: Guidance document on 

aquatic toxicity testing of difficult substances and mixtures”. Available at http://www.oecdilibrary.org/environment/guidance-document-on-aquatic-toxicity-testing-of-difficult-substancesand-mixtures_9789264078406-en as of January 22, 2014.

OECD. 2004. “OECD Guidelines for the Testing of Chemicals, No. 111: Hydrolysis as a 

function of pH”. Available at http://www.oecd-ilibrary.org/environment/test-no-111-hydrolysisas-a-function-of-ph_9789264069701-en as of January 22, 2014.

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

Available at http://www.ecfr.gov/cgi-bin/textidx?SID=b3c328624afa4723ef0e5af7b724d7dd&node=40:33.0.1.1.8.7.1.18&rgn=div8 as of 

January 22, 2014.