SRC TR-10-062 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

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

Reproductive/Developmental Toxicity 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 Work Assignment Manager 

March 5, 2010 
HPV1 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 22202 
Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 78-11-5

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity 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 guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline 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 assigned a Klimisch reliability code. If the study 

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review 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 Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual 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. 

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

REPRODUCTIVE/DEVELOPMENTAL TOXICITY 

Title: Reproductive and Developmental 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-implantation losses, and pup survival to day 4; individual 

animal data are not provided for most endpoints, 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, described by the study authors as incidental, is not 

adequately explained or documented. 

TEST SUBSTANCE 

 

Identity: Pentaerythritol tetranitrate (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 toxicity screening test 

GLP compliant?: Yes 

Year study performed: 2007 

Test species: Sprague-Dawley rats; supplier: Charles River Laboratories, Wilmington, MA; age: 

approximately 10 weeks; mean initial weight: 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), during 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 including day 3 post-partum for reproductive females or a total of 

52-54 days for non-reproductive females; maximum duration = 56 days 

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

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Draft 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 check 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 compare treatment groups using treatment and sex as 

factors. If data did not meet the ANOVA 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 significance 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 corn 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 doses 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 mating, 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. Females 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 pregnancy 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, general clinical observations were made at least once a day and more 

frequently when signs of toxicity were observed. Pertinent behavioral changes, signs of difficult 

or prolonged parturition, and signs of toxicity, including mortality, were recorded. The duration 

of gestation was recorded and calculated from day 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 parturition (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 special attention being paid to the reproductive organs. The numbers 

of implantation sites and corpora lutea were recorded. The ovaries, testes, and epididymides of 

adults were weighed. Dead pups and pups euthanized at day 4 post-partum, or shortly thereafter, 

were carefully examined for gross abnormalities. The ovaries, testes, epididymides, and all 

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Draft 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 performed on the ovaries, testes, and epididymides of 

the animals of the highest dose group 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 hematoxylin) was conducted with special emphasis on 

stages of spermatogenesis and histopathology interstitial testicular cell structure. Examination of 

the intact epididymis included the caput, corpus, and cauda, which can be accomplished by 

evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, 

change in prevalence of cell types, aberrant 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 PETN toxicity and that the deaths of four rats 

prior to the study's completion were from esophageal perforations caused by oral gavage. 

However, five deaths at the highest dose are evident (one on day 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 gestation 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 was born from a control female which died at 2 

days due to lack of being fed. No gross lesions were observed for any of these four pups. 

No differences in mean male body weight were observed among treatment levels at any stage of 

the study. Adult females that received 100 and 500 mg/kg-bw/day PETN weighed more than 

control females and those of the 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 other 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 

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

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

No differences in testes and epididymides weights, expressed as organ/body weight ratios, were 

observed among treatments. No gross lesions were observed in adult males upon necropsy. No 

differences in ovary weights (expressed as organ/body weight ratios), number of implantation 

sites, and number of corpora lutea were observed among treatments. No gross lesions were 

observed in adult females upon necropsy. 

No abnormal behaviors were observed in pups prior to euthanasia. Upon necropsy, testicular 

hematomas were observed from four control pups, three pups from the 100 mg/kg-bw/day dose 

group, one pup from the 500 mg/kg-bw/day dose 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 group. No other gross abnormalities were observed. 

The study authors and pathology report authors 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 considered by the authors of the pathology report 

to be “a background finding with no relationship to the test article treatment.” The overall 

microscopic incidence table in the pathology report 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 testicular atrophy, autolysis, 

minimal focal degeneration, and spermatic granuloma 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/day. 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 intervals, fertility rates, pre-and post-implantation 

losses, and pup survival to day 4; individual animal data are not provided for most endpoints, 

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, described 

by the study authors as incidental, is not adequately explained or documented. 

REFERENCE

Quinn, M.J., et. al. 2008. Reproductive and Developmental 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. 

5 
Review 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 Oral Exposure to PETN (Pentaerythritol 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) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures 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 summary 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 provided for each dose level. Therefore, individual data are missing for many 

parameters, including body weight (for both adults and pups), food consumption, 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/dose, 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 discrepancy 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. Therefore, at least five deaths occurred during the study, in 

contrast to the four deaths in the statement 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 dose; 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 intervals, fertility rates, pre- and 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/Branch/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 met, 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).