SRC TR

- 1 5

- 047

HPV 2 TEST RULE DATA ADEQU ACY REVIEW ( FINAL ) : REPRODUCTI ON/DEVELOPMENTAL TOX ICITY 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 .009.00 1

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

, 201 5Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

- 04

- 1

i 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. Repr oductive/Developmental Toxicity

................................ ................................ .............. 4Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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- 1

1 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 , 2 015).

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 K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae tests] if log K ow ≥ 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 K ow of 2.37 1 , 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: O ral g avage reproduction/developmental toxicity screening test in Crl:CD(SD) r ats. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

These newly submitted data are e valuated 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 assi gned 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 870.3550 2

. In addition, the s tudy was evaluated to determine whether recommendations provided in EPA’s study plan review s 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 gra nted 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 S creening Test, Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #14

- 0 36 , 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 HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

- 04

- 1

2 physicochemical properties (i.e., potential for hydrolysis 1 and volatilization 2 ) 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. SUMM ARY 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 (F 2) testing requirement for CASRN 2524

- 04

- 1

. Remaining HPV2 Test Rule Toxicity Testing Requirements: Dow AgroSciences previously submitted data from a chromosomal aberration s test of CASRN 2524

- 04

- 1. 3 These data were reviewed in a separate report 4 and 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 ba cterial reverse mutation test of CASRN 2524

- 04

- 1. 5 These data were reviewed in a separate report 6 and 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 Agro Sciences 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 Septe mber 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

- m 3 /mole
at 25°C (U.S. EPA. 2012. Estimation Programs Interface SuiteTM [EPI Sui te] 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 Augus t 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

- OPP T

- 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 : Salmonell a

- Escherichia coli mammalian

microsome 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,O

diethyl 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 HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

- 04

- 1

3 Summary of Data Adequacy for Toxicological Endpoints: Available data are adequate to satisfy the testing requirements for t he chromosomal aberration s (E2) and reproduction/developmental toxicity (F2) endpoints. Data submitted for t he 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 th is endpoint.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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4 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: O ral 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 ” (199 5 ); OPPTS 870.3 550 , “ 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; b ody weight range at initiation (day

- 1) : 1 76

. 6

- 1 97

. 4 g (females)

, 2 29

. 4

- 2 51

. 7 g ( males)

Number of animals/sex/dose: 1 0 /sex/dose

Route of administration: Oral (gavage)

Vehicle: Corn oil 1

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

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- 1. The purity/compositi on of the corn oil vehicle used in this study is unclear from the report provided.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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- 1

5 Exposure duration: Males: throughout 14 days of pre

- mating, and continuing through out mating (up to 2 weeks), and until necropsy on test day 32 ; Females: throughout 14 days of pre

mating, and continuing through out 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 10 0 mg/kg

bw/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 s ignificance level of 0.0 5. I f th e 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. T he 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. T he F isher ’s exact probab ility test (p = 0.05 ) with Bonferroni ’ s correction was used to analyze t he mating, conception, fertility

, and gestation indices and the binomial distribution test (alpha = 0.05 ) was used to e valuat e the neonatal sex ratio on postnatal d ay 1 . Sex ratio calculations included the g ender 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 oth er 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 o f their uteri were excluded from statistical analyses. T he 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]Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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6 Analyses of Test Item Formulations: Concentration Verification and Homogeneity: “Dose confirmat ion analyses of all dose solutions from the first mix were determined pre

exposure. 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 soluti ons 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 Mc F adden, 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 frequ ency that was well within the stability limits.” [ Andrus et al. 2015, p p. 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 d osing, 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 maint ained similar to Andrus et al., 2014.” [ Andrus et al. 2015, p. 16]

Dosing and Environmental Conditions: Groups of sexually mature rats (1 0 /sex/group)

, acclimated to the laboratory conditions for at least one week

, w ere administered the test item daily via gavage at dose levels of 0 (vehicle only), 10, 30, or 100 m g/kg

- bw/day

. Food and water were available ad libitum throughout the study. For females, exposure continued throughout 14 days of pre

- mating, and through out mating (up to 2 weeks), gestation ( 3 weeks), and lactation ( 4 days)

, and until necropsy on post

partum days 5

- 8. For males, exposure continued throughout 14 days of pre

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

Parental animals were housed in individ ual solid bottom stainless steel cages during the pre

mating 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 e vidence 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 pl ug

- 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 HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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- 1

7 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). D etailed clini cal 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 we ll 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 pre

mating 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 a nimals. Adul t 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 t he 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 tis sues collected from all animals in th e control and high

- dose groups: gross lesions, kidneys, adrenals, liver, pituitary, seminal vesicles, coagulating glands, prostate, epididymides, teste s , vagina, cervix, uterus, ovaries, oviducts, and mammary glands (fe males 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 HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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8 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 moribun d 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 w ere sexed and examined grossly for external and visceral defects and discarded

. At necropsy, r eproductive organs were weighed and histological examinations were conducted on reproductive organs , as described above. The uteri of all females were stained and t he numbers of implantation sites were recorded

. Corpora lutea were not counted . F ollicular 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 spermat ogenesis 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 spermatogene sis. 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 e t al. 2015, p. 22]

RESULTS

Endpoint value(s): Systemic Toxicity: LOAEL = 10 0 mg/kg

- bw/day, based on d ecreased body weights of males on test day s 29 and 32 (day of necropsy) , increased a bsolute 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 = 3 0 mg/kg

- bw/day.

Reproductive and Developmental Toxicity: N OAEL = 10 0 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 curren t litter size. Alternatively, follicular status was qualitatively assessed via histopathological evaluation of the ovaries.” [ Andrus et al. 2015, p. 21]Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

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9 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 dist ributed 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 a nd 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 s igns 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 s tatistically significant (p < 0.05) decrease in mean body weight ( 8.6 % decrease compared to controls ) w as observed in males in the high

- dose group on test day 29. In addition, t here 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 c ontrols).

“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 grou p 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 f ood consumption of parental males or females at any dose level tested.

Gross Necropsy, Organ Weights, and Histopathology: N o tre atment

- related gross abnormalities were noted at necropsy. Mechanical trauma observed in the tail tip of t wo 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 r eview (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 t o 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 v olatilization, 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 reproductio n/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 AgroS ciences 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 HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

- 04

- 1

10 vertebra. All other gross observations were isolated occurrences or w ere not dose

- related and were therefore not considered to be related to treatment.

Mean a bsolute 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 statisticall y 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 li ver revealed a higher i ncidence 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 g roups w as 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 group s ( 137 % and 168% increase s, respectively ; p < 0.05 ) relative to the control gr oup

. However, the study authors considered the increase s 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 lo ss 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 1 00 mg/kg

- bw/day based on decreased body weights of males on test day s 29 and 32 (day of necropsy) , increased a bsolute and relative liver weights of females, and a histopathological c hange 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 3 0 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 wa s identified as a target organ in males and females”.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CAS RN 2524

- 04

- 1

11 The N OAEL for reproductive and developmental toxicity was 10 0 mg/kg

- bw/day, based on no adverse effects at the highest dose tested.

STUDY RELIABILITY

[ 1 ] Reliable with out restrictions.

REFERENCE S

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

- diethyl ester: O ral gavage reproduction/developmental toxicity screening test in Crl:CD(SD) r ats. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Spons or: 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 E nvironmental 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).