SRC TR-10-062

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

1,3-Propanediol, 2,2-bis[(nitr ooxy)methyl]-, dinitrate (e ster), CAS No. 78-11-5

Reproductive/Developmental To xicity Screening Test

Prepared by:

Ashlee Aldridge, Teresa Manyin, and Joan D. Garey

Environmental Science Center

SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Wo rk Assignment Manager

March 5, 2010HPV1 Test Rule – Data Adequacy Assessment

1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester), CAS No. 78-11-5

Reproductive/Developmental Toxicity Screening Test

March 2010

Submitted to: High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460-0001

Prepared By:

SRC, Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city study conducted in response to the first

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test guideline and professional judgment as guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline is not always the test guideline required by the test rule, because some test sponsors requested and received approval from EPA to use a different guideline .) The study was summarized according to a standard format, assessed for adequacy, and assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w of TSCA Section 4 Final Study Report”. Any deviations from the applicable test guideline are described. The study is also evaluated for compliance with Standard Operating Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual animal data, calculations, and statistical analyses are described, and internal inconsistencies within the study report are identified. An overall assessment is provided for each of the following: study conduct, data reliability, and data utility.

1Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

REPRODUCTIVE/DEVELOPMENTAL TOXICITY

Title: Reproductive and Developmenta l Effects of Oral Exposure to PETN (Pentaerythritol Tetranitrate) in the Rat

The study is: Adequate Not Adequate

The test report does not provide sufficient information; missing results include mating/precoital intervals, fertility rates, pre-and post-implantati on losses, and pup survival to day 4; individual animal data are not provided for most endpoint s, including body weights (for adults and pups), food consumption, organ weights, and breeding, reproductive, and developmental parameters; the mortality of animals at the high dose, desc ribed by the study authors as incidental, is not adequately explained or documented.

TEST SUBSTANCE

Identity: Pentaerythritol tetranit rate (1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester); PETN), CAS No. 78-11-5

Remarks: 98.43% purity

METHOD

Method/guideline followed: EPA OPPTS 870.3550

Test type: Reproductive/developmental toxi city screening test

GLP compliant?: Yes

Year study performed: 2007

Test species: Sprague-Dawley rats; supplier: Charles Ri ver Laboratories, Wilmington, MA; age: approximately 10 weeks; mean initial we ight: 360 g for males, 222 g for females

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

Route of administration: Oral gavage

Vehicle: Corn oil

Exposure duration: Males: prior to mating (14 days), dur ing mating (up to 14 days), and postmating for a total of 28 days; females: prior to mating (14 days), during mating (up to 14 days), during gestation, and up to and incl uding day 3 post-partum for reproductive females or a total of 52-54 days for non-reproductive fema les; maximum duration = 56 days

Doses/concentrations tested: 0, 100, 500, or 1000 mg/kg-bw/day

2Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

Statistical methods used: Bartlett's Test for homogeneity of variance and a KolmogorovSmirnoff test for normality were used as a ch eck of the analysis of variance (ANOVA) assumptions. When assumptions were met, a two-way ANOVA followed by a Newman Kuels multiple comparisons test was used to compar e treatment groups using treatment and sex as

factors. If data did not meet the AN OVA assumptions, a Kruskal-Wallis nonparametric ANOVA for each sex at each time point followed by the Dunn's Method were used to compare treatment groups. Statistical si gnificance was defined as p < 0.05.

Test method/conditions remarks: Male and female Sprague-Dawley rats were used: 10 male and 10 female rats were assigned to each group. Rats were exposed daily to co rn oil (control), or 100, 500, or 1000 mg/kg-bw PETN, which was determined to be 98.43% pure, via gavage for a maximum of 56 days. The highest treatment level was determined based on weight loss experienced in mice and rats receiving estimated daily dos es of 3170 mg/kg –bw for 14 days and 2025 mg/kg-bw for 14 weeks, respectively. Rats were weighed on day -1 and weekly thereafter. The daily dose for each treatment was based on the most recent body weight for each individual for each dose group. The dosing solution was administered based on volume using a single PETN/diluent

concentration for all treatments. All rats had continuous access to water and feed.

Dosing of both sexes began 2 weeks prior to mati ng, after a 7-day acclimation period. Dams with offspring were euthanized on day 4 postpartum. Females that showed no evidence of copulation were euthanized 24 to 26 days after the last day of the mating period. Dosing was continued daily in both sexes during the mating period. Fema les remained with the same male they were paired with until a sperm plug was observed or until 2 weeks had elapsed. Daily dosing of the parental females continued throughout pregnanc y and up to and including day 3 postpartum. Males were dosed after the mating period until a total dosing period of 28 days had been completed, and were then euthanized. Pups received PETN only through lactation.

Throughout the test period, ge neral clinical ob servations were made at least once a day and more frequently when signs of toxicity were observed. Pertinent behavioral ch anges, signs of difficult or prolonged parturition, and signs of toxicity, including mortality, were recorded. The duration of gestation was recorded and calculated from da y 0 of pregnancy as indicated by presence of a sperm plug. Each litter was examined as soon as possible after delivery to establish the numbers of total pups, still births, live births, and runts (pups that are significantly smaller than corresponding control pups), sex of pups, and the presence of gross abnormalities. Litters were weighed within 24 hours of parturitio n (day 1) and on day 4 post-partum.

Males and females were individually weighed the day before and on the first day of dosing, weekly thereafter, and at termination of the study. During pre-mating, pregnancy and lactation, food consumption was measured at least weekly.

At the time of euthanasia or death, the adult animals were examined for any abnormalities or pathological changes, with spec ial attention being paid to the reproductive organs . The numbers of implantation sites and corpora lutea were reco rded. The ovaries, testes, and epididymides of adults were weighed. Dead pups and pups euthani zed at day 4 post-partum, or shortly thereafter, were carefully examined for gross abnormalities. The ovaries, testes, epididymides, and all

3Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

organs showing macroscopic lesions of all adult animals were preserved.

Detailed histological examinations were performe d on the ovaries, testes, and epididymides of the animals of the highest dose gr oup and the control group. Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 μm thickness, and staining with PAS and hematoxy lin) was conducted with special emphasis on stages of spermatogenesis and hist opathology interstitial te sticular cell structure. Examination of the intact epididymis included the caput, co rpus, and cauda, which can be accomplished by evaluation of a longitudinal sec tion. The epididymis was eval uated for leukocyte infiltration, change in prevalence of cell types, aberra nt cell types, and phagocytosis of sperm.

RESULTS

Endpoint value(s): Based on the limited data provided in the study report, the NOAEL for systemic, reproductive, and developmental toxicity is 1000 mg/kg-bw/day. No adverse effects were reported at the highest dose tested.

Results remarks The study authors state that no rats died due to P ETN toxicity and that th e deaths of four rats prior to the study's completion were from esopha geal perforations caused by oral gavage. However, five deaths at the hi ghest dose are evident (one on da y 5 and four on day 17) from the histopathological report, and no evidence of esophageal perforations is provided. No outward signs of toxicity (e.g., behavior) were reported at any treatment level.

No differences were observed for mean gestati on duration, mean number of pups produced (live or dead not stated), and mean pup sex ratios. One stillborn pup was observed from a female from the 100 mg/kg-bw/day dose group and one pup from a different female from the same group was found dead at 2 days. Only one runt wa s born from a control female which died at 2 days due to lack of being fed. No gross lesi ons were observed for any of these four pups.

No differences in mean male body weight were observed among treatment le vels at any stage of the study. Adult females that received 100 a nd 500 mg/kg-bw/day PETN weighed more than

control females and those of th e 1000 mg/kg-bw/day treatment level at day 20 of exposure. Mean body weights of females from the 100 and 500 mg/kg-bw/day treatment levels were higher than controls at day of necropsy.

No differences in mean body weight were observed in female pups at day of birth; however, the average body mass of male pups from the 500 mg /kg-bw/day treatment group was greater than those from the control and 1000 mg/kg-bw/day groups. No treatment-related differences in mean body mass were observed in males or females at day 4.

Mean feed consumption was greater in males from the 100 and 500 mg/kg-bw/day treatment groups than the 1000 mg/kg-bw/day group during the pre-mating period; feed consumption by the control males was not different than any ot her treatment level for this time period. Feed consumption in females from the 100 and 500 mg/kg-bw/day treatment groups was greater than in females from the control and the 1000 mg/ kg-bw/day treatment group during the pre-mating

4Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

and pregnancy periods; feed consumption in females did not diffe r during lactation.

No differences in testes and epididymides weight s, expressed as organ/body weight ratios, were observed among treatments. No gross lesions we re observed in adult males upon necropsy. No differences in ovary weights (expressed as or gan/body weight ratios), number of implantation sites, and number of corpora lutea were obser ved among treatments. No gross lesions were observed in adult females upon necropsy.

No abnormal behaviors were observed in pups prio r to euthanasia. Upon necropsy, testicular hematomas were observed from four control pup s, three pups from the 100 mg/kg-bw/day dose group, one pup from the 500 mg/kg-bw/day dos e group, and zero from the 1000 mg/kg-bw/day treatment group. Mottled livers were observed in only four pups from the 100 mg/kg-bw/day group and three from the 500 mg/kg-bw/day grou p. No other gross abnormalities were observed.

The study authors and pathology report aut hors report that no treatment-induced histopathological effects in any tissues from any treatment level were observed. Minimal or mild

ovarian interstitial cell hyperplasia was observed in 1 of 10 and 3 of 10 females given 0 and 1000 mg/kg-bw/day PETN, respectively. This was co nsidered by the authors of the pathology report to be “a background finding with no relationship to the test article trea tment.” The overall microscopic incidence table in the pathology repo rt also shows minimal unilateral testicular atrophy in 2 of 10 control males, with 4 of 10 high dose males having varying testicular effects considered outside of normal limits (including minimal unilateral testic ular atrophy, autolysis, minimal focal degeneration, and spermatic granul oma as findings distributed among the four males). These observations in the males were not specifically discussed in the pathology report.

CONCLUSIONS

Based on the limited data provided in the study report, the NOAEL for systemic, reproductive, and developmental toxicity is 1000 mg/kg-bw/da y. No adverse effects were reported at the highest dose tested.

STUDY RELIABILITY

Klimisch code = [4]. Not assignable; the test report does not provide sufficient information; missing results include mating/precoital interval s, fertility rates, pre-and post-implantation losses, and pup survival to day 4; individual an imal data are not provi ded for most endpoints, including body weights (for adults and pups), food consumption, organ weights, and breeding, reproductive, and developmental parameters; the mo rtality of animals at the high dose, described by the study authors as incidental, is not adequately explained or documented.

REFERENCE

Quinn, M.J., et. al. 2008. Reproductive and Developmenta l Effects of Oral Exposure to PETN (Pentaerythritol Tetranitrate) in the Rat. U.S. Army Center for Health Promotion and Preventive Medicine, Toxicology Study No. 87-XC-07TY-08, Laboratory Project ID 07TY-52-07-07-01. Study completed December 2008.

5Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester)

CAS Registry Number: 78-11-5

Report Title: Reproductive and Developmental Effects of Or al Exposure to PETN (Pent aerythritol Tetranitrate) in

the Rat

Test Sponsor: Dyno Nobel Inc., Salt Lake City, UT

Testing Lab: U.S. Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD

Test Standard: 40 CFR 799.9355

Date Study Received by EPA: N/A, posted to http://www.regulations.gov on 5/19/08

Date Study Due to EPA: 5/17/07

Special Instructions (e.g., regarding approved protocol modifications, etc.):

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [ A ], Disagree [ D ], Not Applicable [ NA ], or Cannot Determine [ C ], and add comment, below, as needed

[A] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from the EPA test standard requirements, including “shall,” “must,” or “will” violations. List significant test standard deviations and “shall,” “must,” or “will” violations in comments below.

[A] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol that may have a bearing on the study conclusions. List these in comments below.

[D] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented.

[D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed.

[D] 5. The Statement of Quality Assurance (QA) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures may be inadequate.

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed.

[D] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or

Enforceable Consent Agreement (ECA).

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations from temperature, lighting, dosing) are apparent.

[D] 9. The final study report inadequately describes the study method.

[D] 10. The final study report inadequately describes the test system.[D] 11. The final study report inadequately describes the dosage information.

[A] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete observations pertinent to the type of study conducted), or individual data were not provided.

[D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used.

Part II. Conclusions

Place a check mark beside the summar y statement(s) that apply to this review and add comments below as appropriate

Part A. To be filled out by Reviewer

Study Conduct 1. The study was well-conducted. [ ] 2. The study was generally well-conducted, with some minor [ ] deficiencies (list in notes below). 3. The study conduct was significantly deficient or questionable, (give [X] reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [ ] 5. Appropriate methods were used to determine [X] statistical significance. 6. If repeated, the test results would be expected to be the [ ]

same.

Data Utility 7. The data are useful for risk assessment for the endpoint tested. [ ] 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 9. The data are not useful for any purpose (describe in notes below). [X]

Reviewer Comments (continue on separate page, if needed): Part I (1): “Must” violations: The test report does not provide mating/precoital intervals, fertility rates, and calculations of pre-and post-implantation losses (although the mean numbers of implantations and corpora lutea are provided for each dose). Additionally, the test report does not provide pup survival to day 4 for each dose.

Part I (2): “Should” violations: Individual animal data are not provided, with the exception of histopathological data. Instead, only mean values are provid ed for each dose level. Therefore, individual data are missing for many parameters, including body weight (for both adults and pups), food consum ption, organ weights, mating success, pregnancy rate, numbers of implantations and corpora lutea per female, numbers of pre- and post-implantation losses, time to mating, gestation length, number of litters/d ose, number of live pups per litter, number of dead pups per litter, pup sex ratio for each litter, number of runts for each litter, pup clinical observations, pup gross abnormalities, and survival of individual pups.

Part I (12): Individual data were provided only for histopathology, and not for any other parameter measured.

Part II, Study Conduct: Although the test methods appear to be relatively sound, the reporting of results was deficient. In addition to the missing results noted above, a di screpancy occurred in the description of mortality. The authors state that, “No rats died due to PETN toxicity; the death of four rats prior to the study’s completion were from esophageal perforations caused by oral gavage.” The authors do not state the doses at which these deaths occurred. However, it is evident from the histopathology report (which contains only data for the controls and high dose) that 3 animals at the high dose were found dead during the study and an additional 2 animals at the high dose were killed moribund. The histopathology report shows that no deaths occurred in control animals, but the numbers of deaths at the low and mid-doses are not provided. Theref ore, at least five deaths o ccurred during the study, in

contrast to the four deaths in the st atement above. This discrepancy does not reflect well on the accuracy of the results reported.In addition, there is no evidence to substantiate the claim of esophageal perforations in the animals that were found dead or killed moribund; full necropsy details are not provided. Therefore, the death of five animals at the high dose cannot be verified as non-related to toxicity of the test substance, but rather, may be evidence of a toxicological

effect.

Part II, Data Reliability: The authors provided data only in the form of means for each do se; in the absence of data for individual animals, it is impossible to verify the mean values provided by the study authors. Therefore, the precision and accuracy of the data cannot be judged without data on individual animals.

Part II, Data Utility: The study authors do not provide data for all of the required reproductive and developmental parameters (i.e., data is missing for mating/precoital interv als, fertility rates, pre- an d post-implantation losses, and pup survival to day 4). Therefore, the study is not adequate for risk assessment of the reproductive and developmental endpoints.

Reviewer Name/Title/Bran ch/Division/Office: Ashlee Aldridge/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was me t, additional testing is indicated. [ ] (when applicable) 2. The trigger for additional (tiered) testing was not met. [ ]

Study Acceptability 3. The study is acceptable. [ ] 4. The study may not be acceptable [ ] (provide rationale in memo to CITB chief).