SRC TR-14-002

HPV2 TEST RULE DATA ADEQUACY REVIEW (REVISED FINAL): 

REPRODUCTIVE/DEVELOPMENTAL TOXICITY

1H,3H-Benzo[1,2-c:4,5-c′]difuran-1,3,5,7-tetrone (PMDA), CASRN 89-32-7

Prepared by:

Ashlee Aldridge, Amy Nguyen, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

February 5, 2014
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

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 .......................................................................................... 3

D-1. Reproductive/Developmental Toxicity .......................................................................... 3

E. DATA ADEQUACY SUMMARY TABLE ....................................................................... 12
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

1

A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a reproductive/developmental toxicity test for 1H,3H-benzo[1,2-c:4,5-

c′]difuran-1,3,5,7-tetrone (pyromellitic dianhydride or PMDA; CASRN 89-32-7) submitted by

the PMDA HPV Consortium (members are E.I. DuPont de Nemours and Company, Inc. and 

Lonza Ltd.) of the American Chemistry Council on October 23, 2013 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 

February 5, 2014). 

The HPV2 test rule requires the following toxicological testing for CASRN 89-32-7: 

C1 (consisting of acute toxicity to fish, acute toxicity to Daphnia, and toxicity to algae testing if 

log Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 4.2), D 

(acute mammalian toxicity test), E1 (bacterial reverse mutation test), and F1 (combined repeated 

dose toxicity study with the reproduction/developmental toxicity screening test or 

reproduction/developmental toxicity screening test and repeated dose 28-day oral toxicity study 

in rodents). Since the log Kow of CASRN 89-32-7 is estimated to be 2.141, which is < 4.2, the 

C1 aquatic toxicity testing requirement is considered to include acute toxicity to fish, acute 

toxicity to Daphnia, and toxicity to algae testing. 

Given that adequate data for analogous chemicals are available for the repeated-dose toxicity 

endpoint2, EPA accepted the sponsor’s proposal to conduct reproductive/developmental toxicity 

testing according to OECD TG 421 to satisfy the remainder of the F1 testing requirement. In its 

October 23, 2013 submission (not available online as of February 5, 2014), the PMDA HPV 

Consortium supplied data from a reproduction/developmental toxicity screening test conducted 

with CASRN 89-32-7.

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

evaluated in this report.

B. EVALUATING DATA ADEQUACY

The submitted 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 current standard test guidelines to 

 

1 The estimated low Kow value of 2.14 (KOWWIN v. 1.68) for CASRN 89-32-7 was presented in a letter to EPA 

addressed to Dr. Paul Campanella, dated February 21, 2012, from Ms. Jane Bradd Andersen of E.I. DuPont de 

Nemours and Company, Inc. (Attachment 2), available at http://www.regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2007-0531-0663 as of February 5, 2014. 

2 Data from repeated-dose toxicity studies with analogous chemicals were submitted to EPA as Confidential 

Business Information (submission dated July 28, 2011 and identified by EPA as DCN 40110000061) and were 

reviewed by EPA for adequacy (EPA’s review identified as DCN 40110000066). A subset of this information is

available in a letter dated February 21, 2012 from Ms. Jane Bradd Andersen of E.I. DuPont de Nemours and 

Company, Inc. to Dr. Paul Campanella of EPA (Attachment 3), available at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0663 as of February 5, 2014.
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

2

establish whether the study met current testing requirements. If the data available for the 

endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table 

summarizing data availability/adequacy for each SIDS endpoint is included on the last page of 

this document (Section E).

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data submitted by the PMDA HPV Consortium on October 23, 2013 are adequate to satisfy the 

reproduction/developmental toxicity (component of F1) testing requirement for CASRN 89-32-7

promulgated in the final HPV2 test rule. 

A previous submission (dated July 28, 2011; classified as Confidential Business Information

[CBI]; identified by EPA as DCN 40110000061) included toxicity data for chemicals analogous 

to CASRN 89-32-7. EPA reviewed these data for adequacy (EPA’s CBI review identified as 

DCN 40110000066) and determined these data to be adequate to satisfy the following HPV2 test 

rule requirements for CASRN 89-32-7: acute toxicity to fish (component of C1), acute toxicity to 

Daphnia (component of C1), toxicity to algae (component of C1), acute mammalian toxicity (D), 

repeated-dose toxicity (component of F1), and bacterial reverse mutation (E1).

1

Therefore, all HPV test rule requirements for toxicity testing of CASRN 89-32-7 have been 

satisfied.

 

1 A subset of this information is available in a letter dated February 21, 2012 from Ms. Jane Bradd Andersen of E.I. 

DuPont de Nemours and Company, Inc. to Dr. Paul Campanella of EPA (Attachment 3), available at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0663 as of February 5, 2014.
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

3

D. DATA FOR SIDS ENDPOINTS

D-1. Reproductive/Developmental Toxicity

Summary of endpoint: Adequate

Study 1: Reproductive/developmental toxicity screening test in rats (Dunster, 2013)

Title: PMDA hydrolyzed to PMA: oral (gavage) reproduction/developmental toxicity screening 

test in the rat (OECD 421)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1H,3H-Benzo[1,2-c:4,5-c′]difuran-1,3,5,7-tetrone; CASRN 89-32-7

Remarks: Referred to as pyromellitic dianhydride or PMDA in study report; White powder; 

Batch No. NJC1207029; Purity: 99.94%. This study was designed to test the hydrolysis product

of CASRN 89-32-7, pyromellitic acid (PMA; CASRN 89-05-4). To allow hydrolysis of PMDA 

to PMA, the dosing solutions were prepared at least one day prior to dosing. 

METHOD

Method/guideline followed: OECD TG 421 (1995) “Reproduction/Developmental Toxicity 

Screening Test”

Test type: Reproductive/Developmental Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar Han:RccHan:WIST strain rats; source: Harlan Laboratories U.K. Ltd., 

Oxon, UK.; age at initiation: approximately 12 weeks old; body weights at initiation: 298–350 

grams (males), 198–222 grams (females).

Number of animals/sex/dose: 12

Route of administration: Oral (gavage)

Vehicle: Carboxymethyl cellulose (1%) and Tween 80 (0.1%)

Exposure duration: Males: 42 days; Females: 41–53 days (throughout 14 days of pre-mating, 

mating [maximum 14 days], gestation, and up to postpartum/lactation day 4)
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

4

Doses/concentrations tested: 0, 50, 250, and 750 mg/kg-bw/day

Statistical methods used: “Where considered appropriate, quantitative data was subjected to 

statistical analysis to detect the significance of intergroup differences from control; statistical 

significance was achieved at a level of p < 0.05. Statistical analysis was performed on the 

following parameters: Body Weight, Body Weight Change, Food Consumption during gestation 

and lactation, PreCoital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora 

Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, 

Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, [and]

Body Weight Relative Organ Weights.” [Dunster 2013, pp. 32–33] 

“Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module 

as detailed as follows: Where appropriate, data transformations were performed using the most 

suitable method. The homogeneity of variance from mean values was analyzed using Bartlett's 

test. Intergroup variance[s] were assessed using suitable ANOVA [analysis of variance], or if 

required, ANCOVA [analysis of covariance] with appropriate covariates. Any transformed data 

were analyzed to find the lowest treatment level that showed a significant effect using the 

Williams' test for parametric data or the Shirley test for non-parametric data. If no dose response 

was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise 

Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the 

control group. Where the data were unsuitable for these analyses, pair-wise tests... [were]

performed using the Student['s] t-test (parametric) or the Mann-Whitney U test (nonparametric).” [Dunster 2013, p. 33] 

“Data not analyzed by the Provantis data capture system were assessed separately using the 

SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene's test.

Where Levene's test was shown to be non-significant (p ≥ 0.05), parametric analysis of the data 

was applied, incorporating analysis of variance (ANOVA). If this data was shown to be 

significant, this analysis was followed by pair-wise comparisons using Dunnett's test. Where 

Levene's test was significant, non-parametric analysis of the data was analyzed incorporating the 

Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose 

response relationship was also... investigated by linear regression. Where the data... [were]

unsuitable for these analyses, then pair-wise tests were performed using the Student['s] t-test 

(parametric) or the Mann-Whitney U test (non-parametric).” [Dunster 2013, p. 33] 

“Due to the preponderance of non-normally distributed data, reproductive parameters 

(implantation losses, offspring sex ratio, and offspring surface righting) were analyzed using 

nonparametric analyses.” [Dunster 2013, p. 33] 

The standard unit of assessment for analyses of pup data was the litter.

Test method/conditions remarks: 

Test Substance Formulations

The dosing solutions were prepared as aqueous suspensions of PMDA in carboxymethyl 

cellulose (1%) and Tween 80 (0.1%) at least one day prior to dosing. Dosing formulations were 
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

5

prepared at nominal concentrations of 0, 5, 25, and 75 mg test item/mL, corresponding to doses 

of 0, 50, 250, and 750 mg/kg-bw/day, respectively. Based on stability tests showing bulk 

formulations to be stable for at least 21 days, bulk formulations were prepared every two weeks 

and stored at approximately 4 °C in the dark. During the study, samples of the dosing

formulations were collected within 3 days of preparation and analyzed using high performance 

liquid chromatography (HPLC) with UV detection (at a wavelength of 210 nm) to verify the 

concentration of the test substance. Samples were diluted with 0.01 M orthophosphoric acid 

prior to analysis. Standard solutions of test substance were prepared in 0.01 M orthophosphoric 

acid.

Dosing and Environmental Conditions

Groups of rats (12/sex/group) were orally administered the test item daily via gavage at dose 

levels of 0, 50, 250, or 750 mg/kg-bw/day1at a constant dosing volume of 10 mL/kg-bw. A 

concurrent control group was treated with the vehicle only. Treatment continued throughout the 

pre-mating (14 days), mating (maximum of 14 days), and post-mating periods. Exposure 

continued in males and females until scheduled euthanasia, for a total exposure period of 42 

consecutive days for males or through postpartum day 4 for females (41–53 consecutive days) 

with the exception of during parturition.

Prior to mating, rats were housed as groups of five/sex. Food and water were available ad 

libitum throughout the study. During the mating period, animals were paired (1 male: 1 female) 

until there was evidence of mating (i.e., the presence of sperm in a vaginal smear and/or the 

presence of a vaginal plug). Following evidence of mating (designated as day 0 post coitum), 

males were returned to their original cages. Mated females were housed individually (or with 

their litters after delivery) throughout gestation and lactation.

Males were euthanized on day 43. Females were allowed to deliver and rear their pups until 

postpartum (lactation) day 5, when all females and their pups were euthanized. Non-pregnant 

females were euthanized on day 25 post coitum.

Systemic Toxicity

Clinical Observations: All animals were observed for overt signs of toxicity, ill health, and 

behavioral changes immediately before dosing and two to three times after each daily dose.

Body Weight and Food/Water Consumption: Individual body weights of parental males and 

females were recorded on day 1 prior to dosing, at least weekly thereafter throughout the study, 

and at the time of sacrifice. Female body weights were recorded on days 0, 7, 14, and 20 of the 

postcoital period and on days 1 and 4 postpartum. Food consumption was recorded weekly on a 

per-cage basis for males during the pre-mating and post-mating periods and for females during 

the pre-mating period. Food consumption was also recorded for mated females during weekly 

intervals of the post-mating period (post coitum days 0–7, 7–14, and 14–20) and during the 

lactation period (postpartum days 1–4) for females with live litters. Water bottles were inspected 

daily for any obvious changes in water intake.

 

1

“The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project 

Number 41205014).” [Dunster 2013, p. 23] The study report for this previous toxicity work was not available for 

review.
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

6

Food efficiency (determined as the ratio of body weight change to food intake) was 

retrospectively calculated for males throughout the study (except during mating) and for females 

during the pre-mating period. “Due to offspring growth and milk production for lactation, food 

efficiency for females could not be accurately calculated for females during gestation and 

lactation.” [Dunster 2013, p. 27] 

Gross Necropsy and Histopathology: All adults and offspring were subjected to a full external 

and internal examination and any macroscopic abnormalities were recorded. Organ weights 

were recorded for all animals in all groups for the adrenals, brain (including cerebrum, 

cerebellum, and pons), epididymides, heart, kidneys, liver, ovaries, pituitary (post-fixation),

prostate, seminal vesicles, spleen, testes, thymus, and thyroid/parathyroid (post-fixation). 

Relative organ weights (calculated as the ratio of organ weight to body weight) were determined 

for each animal based on terminal body weight. Calculations of and comparisons among mean 

absolute and relative organ weights excluded values obtained from females that did not become 

pregnant.

The following tissues, which were collected from all parental animals in the control and highdose groups, any infertile males, and all females that did not give birth, were examined 

microscopically: coagulating glands, epididymides, ovaries, mammary gland, pituitary gland, 

prostate gland, seminal vesicles, testes, uterus/cervix, vagina, and gross lesions. The following 

tissues, which were collected from 5 parental rats/sex in the control and high-dose groups, were 

also examined by microscopy: adrenal glands, brain (including cerebrum, cerebellum, and 

pons), cecum, colon, duodenum, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes 

(mandibular and mesenteric), parathyroid gland, rectum, sciatic nerve, spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, and 

rectum. The testes and epididymides were “[p]reserved in Bouin’s fluid and then transferred to 

Industrial Methylated Spirits (IMS) approximately 48 hours later.” [Dunster 2013, p. 29]. All 

other tissues were preserved in buffered 10% formalin. 

Reproductive/Developmental Toxicity

Pregnant females were observed daily and the following parameters were recorded: date of 

pairing, date of mating, date and time of observed start of parturition, and date and time of 

observed completion of parturition. Parameters recorded on and after postpartum day 0 and used 

to calculate litter responses included number of offspring born, number of live offspring on 

postpartum days 1 and 4, sex of the offspring on postpartum days 1 and 4, and individual 

offspring weights on postpartum days 1 and 4. Individual offspring weights were used to 

retrospectively calculate litter weights. Calculated reproductive and offspring parameters 

included pre-coital interval, mating index, pregnancy index, gestation length, parturition index, 

pre-implantation loss, post-implantation loss, live birth index, viability index, and sex ratio.

Clinical condition of the offspring was recorded from birth to postpartum day 5. Offspring 

surface righting reflex was evaluated on postpartum day 1.

At necropsy, reproductive organs of parental animals were weighed and histological 

examinations were conducted on reproductive tissues, as described above. The numbers of 

uterine implantations and corpora lutea were recorded for each female upon examination of the 
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

7

uterus. “In addition to the routine evaluation of the testes, the seminiferous tubules of the testes 

were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the 

various cell types present within the different stages.” [Dunster 2013, pp. 296–297]

RESULTS

Endpoint value(s): 

Systemic Toxicity:

LOAEL = 750 mg/kg-bw/day (based on decreased body weight gain and reduced food efficiency 

during week 1 of the pre-mating period in males and females, increased incidence of 

inflammatory cell foci in the livers of males, increased incidence of liver congestion in females, 

increased absolute and relative liver weights in females, and increased relative and absolute 

thyroid/parathyroid weights in males1)

NOAEL = 250 mg/kg-bw/day

Reproductive/Developmental Toxicity:

NOAEL = 750 mg/kg-bw/day

Results remarks: 

Analyses of Test Substance Formulations

Measurement of test substance concentrations by HPLC in formulations prepared for dosing 

showed that formulations contained 88–111% of nominal concentrations. The chromatograms 

provided in the study report demonstrated a single peak corresponding to the test substance.

Systemic Toxicity

Clinical Observations: No parental animals died during the study. Increased salivation after 

dosing was observed in parental animals (10/sex) at the high dose. Other clinical signs included 

noisy respiration in one male at 750 mg/kg-bw/day on days 38 and 39 and in one male at 250 

mg/kg-bw/day on day 29. Scabs were also observed in one male from each of the control, lowdose, and high-dose groups during days 22 to 31, 22 to 31, and 28 to 43, respectively. 

Body Weight and Food/Water Consumption: Group mean body weight gains were statistically 

significantly (p < 0.05) reduced during week 1 of the pre-mating period (days 1–8) in parental 

males and females in the 750 mg/kg-bw/day group, with reductions of 37.9% and 97.6%, 

respectively, relative to the controls. No statistically significant effects of treatment on body 

weight gain were observed in males throughout the remainder of the study or in females during 

week 2 of the pre-mating period (days 8–15). Mean cumulative body weight gain during 

gestation (gestation days 0–20) was statistically significantly (p < 0.05) increased in low-dose

females, relative to controls; however, no significant differences were observed in mid- or high-

 

1 Although no treatment-related histological changes were observed during histopathological examination of the 

thyroid/parathyroid, the primary rationale for including increased mean absolute and relative thyroid/parathyroid 

weights in high-dose males as one of the determinants of the LOAEL for systemic toxicity is the magnitude of the 

increase (approximately 24.3% and 33.3% for absolute and relative weight thyroid/parathyroid weights, 

respectively, compared to controls).
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

8

dose females. Mean body weight gain during lactation was not clearly affected in any dose 

group. “... [F]emales treated at all dose levels showed a reduction in body weight gains during 

the lactation phase of the study in comparison to the control group, although statistical 

significance was not achieved.” [Dunster 2013, p. 34]

No statistically significant effects of treatment were observed on body weight or food 

consumption. Food efficiency was reduced by 37.2% and 100% in high-dose males and females, 

respectively, relative to controls during week 1 of the pre-mating period (statistical comparisons 

not conducted); these reductions in food efficiency corresponded to reductions in body weight 

gain. No overt treatment-related changes in water consumption were observed.

Microscopic Findings of Parental Animals at Necropsy: There were no treatment-related 

macroscopic findings in parental animals. Incidental findings, considered unrelated to treatment, 

included reddened lungs in one control male and one high-dose female, a fibrous mass on the 

median lobe of the lungs (found to be the pancreas with moderate infarction during 

histopathological examination) of a low-dose male, increased pelvic space in the kidneys of one 

low-dose female and one mid-dose male, and a fluid-filled kidney in the same mid-dose male.

Organ Weights: Mean absolute and relative thyroid/parathyroid weights were significantly

increased (p < 0.05) in males in the high-dose group, with increases of approximately 24.3% and 

33.3%, respectively, relative to the control group. Although no treatment-related histological 

changes were observed during histopathological examination of the thyroid/parathyroid, the 

increases in absolute and relative thyroid/parathyroid weights were relatively large and, 

therefore, considered to be toxicologically significant by the EPA reviewers. 

Females in the high-dose group showed statistically significant (p < 0.05) increases in absolute 

and relative liver weights, with increases of approximately 13.5% and 10.8%, respectively, 

relative to the control group. Histopathological changes in the liver, observed in both males and 

females, are discussed below.

Mean absolute and relative kidney weights were statistically significantly (p < 0.05) increased in 

males at 250 and 750 mg/kg-bw/day compared to controls. Absolute kidney weights were 

increased by 5.1% and 2.5% in mid- and high-dose males, respectively, relative to control males. 

Relative kidney weights were increased by 5.8% and 6.3% in mid- and high-dose males, 

respectively, relative to control males. “All individual values were within the normally expected 

ranges [for rats of this strain and age], and as such, the minimal increase in each case was 

considered to have arisen incidentally.” [Dunster 2013, p. 38] No corresponding 

histopathological changes were observed in the kidneys of mid- and high-dose males.

Statistically significant (p < 0.01) decreases in mean absolute and relative seminal vesicle 

weights were observed in the high-dose group, with decreases of approximately 17.5% and 

14.5%, respectively, compared to the control means. However, the mean absolute and relative 

seminal vesicle weights for the high-dose group were within the normal ranges for animals of the 

same strain and age. With the exception of one relatively low individual absolute value, the 

individual absolute and relative seminal vesicle weights for the high-dose group were within the 

normal ranges for animals of the same strain and age. In addition, three of the control animals 
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

9

had absolute and relative seminal vesicle weights that were higher than the range of normally 

expected values. Therefore, the study author dismissed the reduction in seminal vesicle weights 

in the high-dose group as unrelated to treatment. No corresponding histopathological changes 

were observed in the seminal vesicles of high-dose animals to indicate an adverse effect.

Histopathology of Parental Animals: The study author did not consider any of the 

histopathological changes to be treatment-related. However, the EPA reviewers consider some 

of the microscopic changes observed in the livers of high-dose animals to have potential 

toxicological significance. In males, an increased incidence of inflammatory cell foci (minimal 

grade) was observed at the high dose, relative to controls; incidences were 2/5 and 4/5 in the 

control and high-dose groups, respectively. In females, an increased incidence of liver 

congestion (mild grade) was observed at the high dose, relative to controls; incidences were 0/5 

and 2/5 in the control and high-dose groups, respectively. These findings, in combination with 

the significant increases in absolute and relative liver weights in high-dose females, suggest a 

treatment-related effect on the liver.

Reproductive/Developmental Toxicity

In total, 11 control females, 9 females in the 50 mg/kg-bw/day group, 12 females in the 250 

mg/kg-bw/day group, and 11 females in the 750 mg/kg-bw/day group gave birth and successfully 

reared their litters until scheduled sacrifice on postpartum day 5. 

Mating, Fertility, Gestation, and Parturition: No treatment-related effects were observed on 

mating, fertility, or gestation parameters. One control female did not become pregnant following 

evidence of mating. Three females in the low-dose group and one female in the high-dose group 

also did not become pregnant following evidence of mating. “These failures to reproduce were 

not observed in a dose-dependent manner and were therefore considered to be unrelated to 

treatment.” [Dunster 2013, p. 35] All other females successfully mated and became pregnant.

The mean pre-coital interval, mating index, gestation length, number of females with live 

offspring, and parturition index were similar among the treated and control groups. The 

pregnancy index was reduced by 18.5% in the low-dose group relative to the controls; since the 

pregnancy index in mid- and high-dose groups was similar to that in the control group, the

reduction in the low-dose group was considered to be unrelated to treatment. Gestation lengths 

ranged from 22 to 23 1⁄2 days for all animals.

The mean number of corpora lutea, number of implantation sites, sex ratio, and litter size were

similar among treated and control groups. At 50 mg/kg-bw/day, there was a statistically 

significant (p < 0.05) decrease in post-implantation loss relative to the controls; postimplantation loss of the low-dose group was reduced by 80.5% compared to controls. No other 

treatment groups showed statistically significant changes in post-implantation loss. “In the 

absence of a dose-related response, the lower implantation losses were considered to have arisen 

incidentally.” [Dunster 2013, p. 36] No statistically significant differences in pre-implantation 

loss were observed among groups.

The male partner of the female in the control group that did not become pregnant and the male 

partner of one of the females in the low-dose group that did not become pregnant were found to 
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

10

have small epididymides and small and flaccid testes. In the absence of a dose-response 

relationship, these findings were considered to be unrelated to treatment. 

Gross Necropsy and Histopathology of Reproductive Organs: Reduced seminal vesicle weights 

were observed in the high-dose group, as described above. No treatment-related

histopathological abnormalities were observed during examination of the reproductive organs. 

No abnormalities were observed in the seminiferous tubules of any animal with respect to the 

stage in the spermatogenic cycle and integrity of cell types present in the different stages of the 

cycle.

Clinical Signs, Body Weights, Survival, and Development of Offspring: No clinical signs of 

toxicity were observed in the offspring during the postnatal observation period. No statistically 

significant differences among groups were observed for mean litter weights, mean offspring 

body weights, mean offspring body weight changes, mean surface righting reflex, live birth 

index, or viability index.

Gross Necropsy of Offspring: “No treatment-related macroscopic abnormalities were detected 

for interim death or terminal kill offspring. The incidental findings observed were those 

occasionally observed in reproductive studies of this type and considered to be unrelated to 

toxicity of the test item.” [Dunster 2013, p. 37] 

CONCLUSIONS

The LOAEL for systemic toxicity is 750 mg/kg-bw/day, based on decreased body weight gain 

and reduced food efficiency during week 1 of the pre-mating period in males and females, 

increased incidence of inflammatory cell foci in the livers of males, increased incidence of liver 

congestion in females, increased absolute and relative liver weights in females, and increased 

relative and absolute thyroid/parathyroid weights in males1. The NOAEL for systemic toxicity is 

250 mg/kg-bw/day. 

The NOAEL for reproductive and developmental toxicity is 750 mg/kg-bw/day. 

STUDY RELIABILITY

[2] Reliable with restrictions. A detailed description of the methods used to prepare dosing 

formations was not provided. Insufficient evidence was provided to determine whether PMDA 

underwent complete hydrolysis to PMA prior to administration.

 

1 Although no treatment-related histological changes were observed during histopathological examination of the 

thyroid/parathyroid, the primary rationale for including increased mean absolute and relative thyroid/parathyroid 

weights in high-dose males as one of the determinants of the LOAEL for systemic toxicity is the magnitude of the 

increase (approximately 24.3% and 33.3% for absolute and relative weight thyroid/parathyroid weights, 

respectively, compared to controls).
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

11

REFERENCE

Dunster, J.S. 2013. PMDA hydrolyzed to PMA: oral (gavage) reproduction/developmental 

toxicity screening test in the rat (OECD 421). Performing Laboratory: Harlan Laboratories, Ltd., 

Derbyshire, UK. Study No. 41205015. Sponsor: The American Chemistry Council, PMDA 

Consortium, Washington, DC.

Text in quotes is taken directly from Dunster (2013).
Final HPV2 Test Rule Data Adequacy Review (February 2014) CASRN 89-32-7

12

E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No