SRC TR-15-047

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL): 

REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST

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

Prepared by:

Justine von Runnen, Amy Nguyen, William Richards, and Teresa Manyin

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

Cynthia Bowie, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

August 5, 2015
Final HPV2 Test Rule Data Adequacy Review (August 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. Reproductive/Developmental Toxicity.............................................................................. 4
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a reproduction/developmental toxicity screening test of CASRN 2524-

04-1 (phosphorochloridothioic acid, O,O-diethyl ester; O,O-diethyl phosphorochloridothioate 

[DEPCT]) submitted by Dow AgroSciences to EPA in June 2015 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/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0579 as of August 5, 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 June 2015 submission from Dow AgroSciences contained the following study report: 

Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Hukkanen, R.R. 2015. 

Phosphorochloridothioic acid, O,O-diethyl ester: Oral gavage 

reproduction/developmental toxicity screening test in Crl:CD(SD) rats. Performing 

Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and 

Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. 

These newly submitted data are evaluated for adequacy herein. No other toxicity data are 

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

The submitted toxicological 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 SIDS endpoints, study details were compared to OECD TG 421 and OPPTS 

870.35502. In addition, the study was evaluated to determine whether recommendations 

provided in EPA’s study plan reviews

3 were followed. Because CASRN 2524-04-1 has 

 

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 August 5, 2015.

2 Although the HPV2 test rule specifies that reproduction/developmental toxicity screening testing should be 

conducted according to TSCA guideline 40 CFR 799.9355, EPA previously granted a request from Dow 

AgroSciences to follow OECD TG 421 and OPPTS 870.3550 instead (request granted in EPA’s study plan review 

of CASRN 2524-04-1, “HPV2 Review of Toxicity Study Plan Submitted to EPA in August 2014: 

Reproduction/Developmental Toxicity Screening Test, Phosphorochloridothioic acid, O,O-diethyl ester, CASRN 

2524-04-1”, SRC Technical Report #14-036, dated September 3, 2014). 

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 August 5, 2015) and “HPV2 Review 

of Toxicity Study Plan Submitted to EPA in August 2014: Reproduction/Developmental Toxicity Screening Test, 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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physicochemical properties (i.e., potential for hydrolysis1and volatilization2) that necessitate 

special considerations during preparation and handling of test item formulations for the 

reproduction/developmental toxicity screening test, 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 Reproduction/Developmental Toxicity Endpoint:

Data submitted by Dow AgroSciences and reviewed in this document are considered adequate to 

satisfy the reproduction/developmental toxicity (F2) testing requirement for CASRN 2524-04-1. 

Remaining HPV2 Test Rule Toxicity Testing Requirements:

Dow AgroSciences previously submitted data from a chromosomal aberrations test of CASRN 

2524-04-1.3 These data were reviewed in a separate report4and were determined to be adequate 

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

promulgated in the HPV2 test rule.

Dow AgroSciences previously submitted data from a bacterial reverse mutation test of CASRN 

2524-04-1.5 These data were reviewed in a separate report6and were determined to be not 

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

promulgated in the HPV2 test rule.

Dow AgroSciences has submitted data from acute toxicity to fish, acute toxicity to Daphnia, and 

toxicity to algae tests of CASRN 2524-04-1, which are currently under review by EPA to 

determine whether they are adequate to satisfy the aquatic toxicity (C1) testing requirement for 

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

 

Phosphorochloridothioic acid, O,O-diethyl ester, CASRN 2524-04-1”, SRC Technical Report #14-036, dated 

September 3, 2014.

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 August 5, 2015). 

3 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 

August 5, 2015. 

4

“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 August 5, 2015.

5 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.

6

“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 August 5, 2015.
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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Summary of Data Adequacy for Toxicological Endpoints:

Available data are adequate to satisfy the testing requirements for the chromosomal 

aberrations (E2) and reproduction/developmental toxicity (F2) endpoints. Data submitted 

for the acute toxicity to fish (component of C1), acute toxicity to Daphnia (component of 

C1), and toxicity to algae (component of C1) endpoints are currently under review. The 

gene mutation (E1) endpoint is considered to be a data gap and testing is still required for 

this endpoint.
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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D. DATA FOR SIDS ENDPOINTS

D-1. Reproductive/Developmental Toxicity

Summary of endpoint: Adequate

Study 1: Reproduction/developmental oral toxicity screening test in rats (Andrus et al., 2015)

Title: Phosphorochloridothioic acid, O,O-diethyl ester: Oral gavage reproduction/developmental 

toxicity screening test in Crl:CD(SD) rats

This study is: Adequate Not Adequate

TEST SUBSTANCE 

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

Remarks: Purity: 99.3%; Lot No. C032013081501; Supplied by The Dow Chemical Company, 

Midland, MI.

METHOD

Method/guideline followed: OECD TG 421, “Reproductive/Developmental Toxicity

Screening Test” (1995); OPPTS 870.3550, “Reproductive/Developmental Toxicity Screening 

Test” (2000)

Test type: Reproduction/Developmental Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2014

Test species: Crl:CD(SD) strain rats; obtained from Charles River, Kingston, New York; Age at 

initiation: approximately 8 weeks old; body weight range at initiation (day -1): 176.6-197.4 g 

(females), 229.4-251.7 g (males)

Number of animals/sex/dose: 10/sex/dose

Route of administration: Oral (gavage)

Vehicle: Corn oil1

 

1 The study report does not fully describe the corn oil that was used as a vehicle. Given that the test item is

susceptible to hydrolysis, EPA had previously recommended (in its April 3, 2014 study plan review) that the vehicle 

be anhydrous and contain no nucleophilic functional groups to minimize hydrolysis and degradation of CASRN 

2524-04-1. The purity/composition of the corn oil vehicle used in this study is unclear from the report provided.
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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Exposure duration: Males: throughout 14 days of pre-mating, and continuing throughout

mating (up to 2 weeks), and until necropsy on test day 32; Females: throughout 14 days of premating, and continuing throughout mating (up to 2 weeks), gestation (3 weeks), and lactation (4 

days), and until necropsy on post-partum days 5-8.

Doses/concentrations tested: Nominal dose levels: 0 (vehicle control), 10, 30, and 100 mg/kgbw/day 

Statistical methods used: A Bartlett’s test for equality of variances (significance level of 0.01) 

was used to evaluate parental body weights and gestation and lactation body weight gains, litter 

mean body weights, feed consumption, and organ weights (absolute and relative). Based upon 

the findings, these parameters were evaluated by either a parametric or non-parametric analysis 

of variance (ANOVA) at a significance level of 0.05. If the results of the ANOVA were 

significant, then a Dunnett’s test or the Wilcoxon Rank-Sum test with Bonferroni’s correction 

was performed, both at a significance level of 0.05. If the feed was spilled or scratched, the feed 

consumption values were excluded from analysis.

A nonparametric ANOVA followed by the Wilcoxon Rank-Sum test with Bonferroni’s 

correction (if the ANOVA was significant) was used to analyze gestation length, average time to 

mating, and litter size. The sequential method of Grubbs (1969) was used to identify statistical 

outliers (p = 0.02). These statistical outliers were only excluded from analysis for documented, 

scientifically sound reasons. The Fisher’s exact probability test (p = 0.05) with Bonferroni’s 

correction was used to analyze the mating, conception, fertility, and gestation indices and the 

binomial distribution test (alpha = 0.05) was used to evaluate the neonatal sex ratio on postnatal 

day 1. Sex ratio calculations included the gender for pups found dead on PND 0. The censored 

Wilcoxon test (alpha = 0.05) as modified by Haseman and Hoel (1974) was used to analyze

survival indices (calculated as mortality), postimplantation loss, and other incidence data among 

neonates using the litter as the experimental unit; when incidence was greater than 5%, 

Bonferroni’s correction was applied. Non-pregnant females, females with resorptions only, or 

females found to be pregnant only after staining of their uteri were excluded from statistical

analyses. The Dunnett’s test and Bonferroni’s correction were used for multiple comparisons to 

the control, keeping an experiment-wise error rate of 0.05. 

Test method/conditions remarks: 

Test Item Preparation: 

“All dosing solutions were prepared by mixing the test material in corn oil at concentrations of 0, 

3.3, 10.0, or 33.3 mg/ml and administered at a dose volume of 3 ml/kg body weight to achieve 

the targeted dose levels. Dose volumes were adjusted daily based on individual body weights.” 

[Andrus et al. 2015, p. 15]

“Dose solutions were not corrected for purity and were prepared periodically throughout the 

study based upon stability data. Dose volumes were adjusted using the most current body 

weight. The control rats were dosed with corn oil at 3 ml/kg body weight.” [Andrus et al. 2015, 

p. 15]
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Analyses of Test Item Formulations: 

Concentration Verification and Homogeneity:

“Dose confirmation analyses of all dose solutions from the first mix were determined preexposure. A second dose confirmation analysis was performed mid way through the dosing 

period [on all dose solutions]. The homogeneity of the low-dose and the high-dose test solutions 

were determined concurrent with dose confirmation. The method used for analyzing the test 

material in corn oil was gas chromatography with electron capture detection (GC/ECD) (Perala 

and McFadden, 2014).” [Andrus et al. 2015, p. 15]

Stability:

“DEPCT was found to be stable in corn oil at concentrations ranging from 0.250 to 250 mg/mL 

for at least 24 days in a GLP stability study (Perala and McFadden, 2014). Dose solutions in the 

current study were mixed every two weeks to refresh solutions at a frequency that was well 

within the stability limits.” [Andrus et al. 2015, pp. 15-16]

“Additional stability analysis of DEPCT in corn oil was conducted throughout the duration of the 

recent range finding study (Andrus et al., 2014). In this study, only one preparation of dose 

solutions was made and the same dose containers were maintained over the course of the 14 day 

dosing period. In addition to analysis of the first mix for all dose groups, dose confirmation was 

also conducted at test days 4, 7 and 14 for the low- and high- dose solutions. Results of these 

analyses demonstrated that stability of these solutions was maintained to 14 days under the 

conditions of the range finding study, including repeated opening of containers for dosing, with 

no additional adjustments for minimizing headspace. These results indicate that there was no 

volatilization of the test compound. Therefore, in the current study, additional stability analyses 

were not conducted, and dose solutions were maintained similar to Andrus et al., 2014.” [Andrus 

et al. 2015, p. 16]

Dosing and Environmental Conditions:

Groups of sexually mature rats (10/sex/group), acclimated to the laboratory conditions for at 

least one week, were administered the test item daily via gavage at dose levels of 0 (vehicle 

only), 10, 30, or 100 mg/kg-bw/day. Food and water were available ad libitum throughout the 

study. For females, exposure continued throughout 14 days of pre-mating, and throughout

mating (up to 2 weeks), gestation (3 weeks), and lactation (4 days), and until necropsy on postpartum days 5-8. For males, exposure continued throughout 14 days of pre-mating and

throughout mating (up to 2 weeks), and until necropsy on test day 32. 

Parental animals were housed in individual solid bottom stainless steel cages during the premating period. During the mating period, females were placed with males from the same dose 

group (1:1 mating) in stainless steel cages with wire mesh floors until evidence of mating was 

observed (i.e., presence of sperm in vaginal lavage samples and/or the presence of a vaginal 

copulatory plug) or two weeks had elapsed. The day of mating was designated as gestation day 

(GD) 0. Although one section of the study report indicates that “sperm- or plug-positive 

(presumed pregnant) females were... separated from the males and returned to their home cages

[presumably meaning the individual stainless steel cages used during the pre-mating period]”

[Andrus et al. 2015, p. 20], another section of the report states “[d]uring gestation and littering, 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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dams (and their litters) were housed in plastic cages.” [Andrus et al. 2015, p. 17] Dams were 

allowed to give birth and rear their pups until lactation day (LD) 4. 

Mortality and Clinical Observations:

The animals were observed once daily throughout the study for detailed clinical signs of toxicity 

and twice daily for mortality, morbidity, the availability of feed and water, and significant 

abnormalities (decreased/increased activity, repetitive behavior, vocalization, 

incoordination/limping, injury, convulsion, fasciculation, tremor, twitches, altered respiration, 

blue/pale skin and mucous membranes, severe eye injury/rupture, alterations in fecal 

consistency, and fecal/urinary quantity). Detailed clinical observations were performed outside 

of the cage and included observations for abnormalities in the eyes, urine, feces, gastrointestinal 

tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and 

mucous membranes, as well as an assessment of general behavior, injuries, or palpable 

mass/swellings.

Body Weight and Food/Water Consumption:

Individual body weights were recorded at least once during the pre-exposure period and on the 

first day of exposure, on test day 4, and then at least weekly thereafter throughout the dosing 

period, until necropsy. Food consumption was determined twice during the first week of premating and once during the second week of pre-mating. Food consumption was not determined 

when males and females were housed together during mating. Food consumption for males was 

not measured following mating. Food consumption of mated females was measured on GD 0, 7, 

14, and 20 and on LD 1 and 4. The study report did not indicate whether water intakes were 

monitored.

Hematology and Clinical Chemistry Analyses:

No hematological or clinical chemistry analyses were conducted. 

Gross Necropsy, Organ Weights, and Histopathology:

A complete necropsy was performed on all parental animals. Adult males (fasted) were 

necropsied after a minimum of four weeks of exposure (test day 32). Females (fasted) that

produced a litter were sacrificed on LD 5-8. The fasted animals were weighed on the morning of 

the scheduled sacrifice. The brain was removed and dissected into the right and left hemispheres 

and the right hemisphere was weighed. Both hemispheres were then frozen individually and 

saved for potential assessment of brain cholinesterase (ChE) activity (no results reported). The 

eyes were examined in situ via application of a moistened microscope slide to each cornea. The 

adrenal glands, kidneys, liver, testes, and epididymides of all parental animals were weighed.

Histological examination was performed on the following tissues collected from all animals in 

the control and high-dose groups: gross lesions, kidneys, adrenals, liver, pituitary, seminal 

vesicles, coagulating glands, prostate, epididymides, testes, vagina, cervix, uterus, ovaries, 

oviducts, and mammary glands (females only). Examination of tissues from the remaining dose 

groups was limited to those tissues that demonstrated treatment-related histopathologic effects at 

the high-dose (liver) as well as all gross lesions. 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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Reproductive and Developmental Toxicity:

“Females were observed for signs of parturition beginning on or about GD 20. In so far as 

possible, parturition was observed for signs of difficulty or unusual duration. The day of 

parturition was recorded as the first day the presence of the litter was noted and was designated 

as LD 0. All litters were examined as soon as possible after delivery. The following information 

was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the 

number of live and dead pups on LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 

and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as 

they were observed during the lactation period. Any pups found dead or sacrificed in moribund 

condition were sexed and examined grossly, if possible, for external and visceral defects and 

discarded. Any pups found dead on PND 0 underwent a lung float test to determine whether 

they were born dead or alive.” [Andrus et al. 2015, p. 20] All pups surviving to PND 4 were 

sacrificed and examined for gross external alterations. Any pups found dead or which were 

sacrificed in moribund condition were sexed and examined grossly for external and visceral 

defects and discarded.

At necropsy, reproductive organs were weighed and histological examinations were conducted 

on reproductive organs, as described above. The uteri of all females were stained and the 

numbers of implantation sites were recorded. Corpora lutea were not counted. Follicular status 

was qualitatively assessed via histopathological evaluation of the ovaries.

1

Testes (sectioned and stained) from all parental males of the control and high-dose groups were 

qualitatively assessed for spermatogenesis stages. “Microscopic evaluation included a 

qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, 

and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these 

cellular associations was used to define the cycle of spermatogenesis. In addition, sections of 

both testes were examined for the presence of degenerative changes (e.g., vacuolation of the 

germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, 

mineralization, and fibrosis).” [Andrus et al. 2015, p. 22] 

RESULTS

Endpoint value(s): 

Systemic Toxicity:

LOAEL = 100 mg/kg-bw/day, based on decreased body weights of males on test days 29 and 32

(day of necropsy), increased absolute and relative liver weights of females, and a 

histopathological change in the liver of males and females characterized as tinctorial 

alteration/increased eosinophilia in the cytoplasm of centrilobular hepatocytes; NOAEL = 30

mg/kg-bw/day.

Reproductive and Developmental Toxicity:

NOAEL = 100 mg/kg-bw/day, based on no adverse effects at the highest dose tested. 

 

1

“Corpora lutea were not counted as females begin cycling within the first 24-48 hours following parturition... and, 

therefore, counts may not be representative of the current litter size. Alternatively, follicular status was qualitatively 

assessed via histopathological evaluation of the ovaries.” [Andrus et al. 2015, p. 21] 
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Results remarks: 

Analyses of Test Item Formulations:

“Analyses of all dosing solutions from the initial mix revealed mean acceptable concentrations 

ranging from 95.3 to 101.1% of targeted concentrations. Analyses of the low and high-dose dose 

solutions indicated that the test material was homogeneously distributed based on relative 

standard deviations of ≤ 3.1%. Analyses of all dosing solutions from an additional mix during 

the dosing period revealed mean acceptable concentrations ranging from 97.6 to 98.6% of 

targeted concentrations. Analyses of the low and high-dose solutions indicated that the test 

material was homogeneously distributed based on relative standard deviations of ≤ 3.6%.”1

[Andrus et al. 2015, p. 24] 

Mortality and Clinical Observations:

No mortalities or test substance-related clinical signs of toxicity occurred during the study. Two 

high-dose females had trauma to their tails; these mechanical injuries were not considered to be 

treatment-related. 

Body Weight and Food/Water Consumption:

A statistically significant (p < 0.05) decrease in mean body weight (8.6% decrease compared to 

controls) was observed in males in the high-dose group on test day 29. In addition, there was a 

dose-related decrease in final body weights of males (fasted males weighed on the morning of 

necropsy on test day 32) at all dose levels with the decrease reaching statistical significance (p < 

0.05) at the high dose (9.2% decrease compared to controls). 

“There were no treatment-related effects on body weight or body weight gain in females at any 

dose level in the pre-breeding, gestation or lactation phases. Although lactation body weight 

gains were 30.2 % lower in the 100 mg/kg/day dose group compared to controls, this effect was 

considered spurious and unrelated to treatment as these differences were not statistically 

significant as body weight gains are highly variable during lactation. Furthermore, there were no 

treatment-related effects on body weight or feed consumption during lactation.” [Andrus et al. 

2015, p. 25] There were no treatment-related effects on food consumption of parental males or 

females at any dose level tested.

Gross Necropsy, Organ Weights, and Histopathology:

No treatment-related gross abnormalities were noted at necropsy. Mechanical trauma observed 

in the tail tip of two high-dose females corresponded histopathologically to fractures of a caudal 

 

1 Given that the test item is susceptible to hydrolysis and volatilization, EPA recommended in its review (dated 

April 3, 2014) of the protocol submitted for the proposed reproduction/developmental toxicity screening test of 

CASRN 2524-04-1 that the vehicle be anhydrous and that precautions (e.g., airtight seals, minimal headspace, and 

minimal exposure to air) be taken to minimize volatilization during preparation and handling of dosing formulations. 

Although it is unclear whether the corn oil vehicle was anhydrous, and the study report does not specify whether or 

not precautions were taken to minimize volatilization, EPA considers concerns regarding hydrolysis and 

volatilization of the test substance in corn oil during the test to be negligible for the following reasons: (a) The mean 

measured concentrations of the dosing formulations in this reproduction/developmental toxicity screening test were 

within an acceptable range (mean measured concentrations were 95.3 to 101.1% of nominal concentrations); and (b) 

Two stability tests (Perala and McFadden, 2014; Andrus et al., 2014) submitted to EPA by Dow AgroSciences on 

February 23, 2015 (as attachments in an email from Dr. George Oliver of Dow AgroSciences to Mr. John Schaeffer 

of EPA) demonstrated that CASRN 2524-04-1 was stable in non-anhydrous corn oil for up to 24 days.
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

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vertebra. All other gross observations were isolated occurrences or were not dose-related and 

were therefore not considered to be related to treatment. 

Mean absolute and relative (to-body-weight) liver weights of high-dose females were increased 

compared to controls (7.7% and 9.6%, respectively) with the increase in relative liver weights 

being statistically significant (p < 0.05). Additionally, males from the mid-dose group showed a 

statistically significant (p < 0.05) increase in mean relative (to-body-weight) testes weight (8.6% 

increase compared to controls); however, this finding was not considered to be treatment-related 

as there was no dose response.

Histopathological examination of the liver revealed a higher incidence of tinctorial 

alteration/increased eosinophilia in the cytoplasm of centrilobular hepatocytes in males and 

females at the high dose, 100 mg/kg-bw/day. The incidence of this change in the control, low-, 

mid-, and high-dose groups was 1/10, 0/10, 1/10, and 6/10, respectively, in males, and 0/10, 

1/10, 1/10, and 5/10, respectively, in females (all graded as very slight). Based on the increased 

centrilobular hepatocyte eosinophilia in both sexes at the high dose, “[t]he liver was identified as 

a target organ in males and females... Remaining histological finds [sic] were interpreted to 

represent background lesions commonly seen in rats of this age and strain and were considered

[to be] unassociated with treatment.”1[Andrus et al. 2015, p. 29] 

Reproductive and Developmental Toxicity:

There were no treatment-related effects on reproductive indices, time to mating, gestation length, 

postimplantation loss, pup survival, or pup sex ratio. Percent postimplantation loss (3.89%, 

1.49%, 9.23%, and 10.44% for the control, low-, mid-, and high-dose levels, respectively) was 

statistically significantly increased in the mid- and high-dose groups (137% and 168% increases, 

respectively; p < 0.05) relative to the control group. However, the study authors considered the 

increases in postimplantation loss in the mid- and high-dose groups to be “spurious” and 

unrelated to treatment as the values (i.e., 9.23% and 10.44%) were within the laboratory’s 

historical control range (% postimplantation loss ranges in the same rat strain were 8.73-13.69% 

[n = 3 gavage studies] and 3.84-10.47% [n = 11 dietary studies], respectively).

No treatment-related observations in the offspring were noted; all recorded offspring 

observations were not considered to be related to treatment as they occurred at a low frequency. 

No treatment-related effects on litter size or pup body weights were noted at any dose level 

tested. No treatment-related histopathological findings were observed in reproductive organs.

CONCLUSIONS

The LOAEL for systemic toxicity in male and female rats was 100 mg/kg-bw/day based on 

decreased body weights of males on test days 29 and 32 (day of necropsy), increased absolute 

and relative liver weights of females, and a histopathological change in the liver of males and 

females characterized as tinctorial alteration/increased eosinophilia in the cytoplasm of 

centrilobular hepatocytes. The NOAEL for systemic toxicity was 30 mg/kg-bw/day. 

 

1 Although the study authors did not consider the changes in the liver to be adverse, EPA considers these changes to 

be toxicologically relevant due to the clear dose-response relationship. EPA also notes that these lesions were the 

basis for the study authors’ conclusion that “the liver was identified as a target organ in males and females”.
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524-04-1

11

The NOAEL for reproductive and developmental toxicity was 100 mg/kg-bw/day, based on no 

adverse effects at the highest dose tested.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Hukkanen, R.R. 2015. 

Phosphorochloridothioic acid, O,O-diethyl ester: Oral gavage reproduction/developmental 

toxicity screening test in Crl:CD(SD) rats. Performing Laboratory: The Dow Chemical 

Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: 

Dow AgroSciences LLC. 

Perala, A.W., and McFadden, J.R. 2014. Phosphorochloridothioic acid, O,O-diethyl ester: 

Method validation and stability in rodent feed, corn oil, and toluene. Performing Laboratory: 

The Dow Chemical Company (Toxicology and Environmental Research and Consulting), 

Midland, MI. Sponsor: Dow AgroSciences LLC. 

Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Sura, R. 2014. Phosphorochloridothioic acid, 

O,O-diethyl ester: 14-day oral gavage range-finding study in Crl:CD(SD) rats. Performing 

Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and 

Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

Text in quotes is taken directly from Andrus et al. (2015).