SRC TR

- 1 4

- 0 02

HPV2 T EST RULE DATA ADEQUACY REVIEW ( REVISED FINAL ) :

REPRODUCTI VE /DEVELOPMENTAL TOXIC ITY

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 Willi am 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 Pennsylv ania Avenue Washington, D.C. 20460

Bryan Lobar, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

February 5

, 201 4Final 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

................................ ................................ ....... 12Final 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

- b enzo[1,2

- c:4,5

- c′]difuran

- 1,3,5,7

- tetrone ( pyromellitic di anhydride 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/#!docum entDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of February 5 , 201 4 ).

The HPV2 test rule requires the following toxicological testing for CASRN 89

- 32

- 7 : C 1 (consisting of acute toxicity to fish, acute toxicity to Daphnia

, and toxicity to algae testing if l og K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log K ow ≥ 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 d ose 28

- day oral toxicity study in rodents ). Since the log K ow of CASRN 89

- 32

- 7 is estimated to be 2.14 1 , which is < 4.2, the C1 aquatic toxicity testing requirement is considered to include acute toxicity to fish, acute toxicity to Daphnia , and toxicity t o algae testing.

Given that adequate data for analogous chemical s are available for the repeated

- dose toxicity endpoint 2

, EPA accepted the sponsor’s proposal to conduct reproductive/developmental toxicity testing according to OECD TG 421 to satisfy the remain der of the F1 testing requirement. In its October 23 , 2013 submission ( not available online as of February 5

, 201 4 ), the PMDA HPV Consortium supplied data from a reproduction/developmental toxicity screening test conducted with CASRN 89

- 32

- 7.

The se 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 for mat 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 K ow 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 , fro m Ms. Jane Bradd Andersen of E.I. DuPont de Nemours and Company, Inc. (Attachment 2), available at http://www.regulations.gov/#!documentDetail;D=EPA

HQ

- OPPT

- 2007

- 0531

- 0 663 as of February 5

, 201 4

. 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 esta blish 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 Ju ly 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 (comp onent 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 to xicity 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/

d evelopmental t

oxicity s creening t est in r ats ( Dunster, 2013 )

Title : PMD A hydrolyzed to PMA: oral (gavage) reproduction/developmental toxicity screening test in the rat (OECD 421)

Th is 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 p yromellitic 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 , pyromel litic 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”

Tes t type: Reproductive/Developmental Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar Han :RccH an:WIST strain rats ; s ource: Harlan Laboratories U.K. Ltd., Oxon, UK.; a ge at initiation: approximately 1 2 weeks o ld; body weights at initiation: 298

– 350
g rams ( males ) , 198

– 222 g rams ( females )

.

Number of animals/sex/dose: 12

Route of administration: Oral (g avage )

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

Exposure duration: Males: 42 days; Femal es: 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, quantita tive 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, B ody 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 Bod y Weight Change, Offspring Surface Righting, Absolute Organ Weights

, [and] Body Weight Relative Organ Weights.” [Dunster 2013, p p . 32

– 33 ]

“Data were analyzed using the decision tree from the Provantis TM 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 ' t est for parametric data or the Shirley t est for non

- para metric 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 (non

parametric).” [Dunster 2013, p. 33]

“Data not analyzed by the Provantis data capture system were assessed sep arately 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 va riance (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 analy z ed incorporating the Kruskal

- Wallis tes t 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

- tes t (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 analy z ed 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 Formulation s T he 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 wereFinal 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, respec tively

. 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 collecte d within 3 days of preparation and analyzed using high performance liquid chromatography (HPLC) with UV detect ion ( 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 m g/kg

- bw/day 1 at 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 m ales 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 group s 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 vag inal plug). Following evidence of mating ( designated as day 0 post coitum ), males were returned to the ir original cage s . Mated females were housed individually (or with their litters after delivery) throughout gestation and lactation.

Males were euthani zed on day 43. Females were allowed to deliver and rear their pups until post partum (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 femal es 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

- ma ting period ( post coi tum 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

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- 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 accura tely 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 an d 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 t hyroid/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 weig hts excluded values obtained from females that did not become pregnant.

The following tissues , which were collected fr om all parental animals in the control and high

dose group s, any infertile males, and all females that did not give birth

, were examined microscopically: coagulating gland s , 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 con trol and high

- dose group s , were also examined by microscopy: adrenal glands , brain (including cerebrum, cerebellum

, and pons), cecum, colon, duodenum, heart, ileum, jej u num, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), parathyroid gland

, rectum, sciatic nerve, spinal cord (cervical, mid

thoracic

, 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 Meth ylated 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: da te 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 b orn, 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 . I ndividual offspring weights were used to retrospectively calculate litter weights. Ca lculated 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 cond ition of the offspring was recorded from birth to postpartum day 5. Offspring surface righting reflex was evaluated on postpartum day 1.

At necropsy, reproductive organ s of parental animals were weighed and histological examinations were conducted on rep roductive tissues, as described above . The numbers of uterine implantations and corpora lutea were recorded for each female upon examination of theFinal 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, p p . 29 6

– 297 ]

RESULTS

Endpoint value(s):

Systemic Toxicity: LOAEL = 750 mg/kg

- bw/day ( base d 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 abs olute and relative liver weights in females, and increased relative and absolute thyroid/parathyroid weights in males 1 ) 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 correspon ding to the test substance

. Systemic Toxicity Clinical Observations : No parental animals died during the study. Increased salivation after dos ing was observed in parental animals (10/sex) at the high dose. Other clinical signs included noisy respiratio n in one male at 750 mg/kg

- bw/day on d ays 38 and 39 and in one male at 250 mg/kg

- bw/day on d ay 29. Scabs were also observed in one male from each of the control, low

dose, and high

- dose groups during d ays 22 to 31 , 22 to 31, and 28 to 43, respectively

. Body Weight and Food/Water Consumption: Group mean body weight gain s 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). Mea n cumulative body weight gain during gestation ( gestation days 0

– 20) was statistically significant ly (p < 0.05) increased in low

- dose females , relative to controls ; h owever, 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 signi fican ce was not achieved.” [Dunster 2013, p. 34]

No statistically significant effects of treatment were observed on body weight or food consumption. F ood efficiency was reduced by 37.2% and 100% in high

- dose males and females, respectively, relative to c ontrols 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 r eddened lungs in one control male and one high

- dose female

, a fibrous m ass on the median lobe of the lungs (found to be the pancreas with moderate infarction during histopathological examination) o f 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 i n 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 in creas es of approximately 24.3 % and 33.3 %
, respectively , relative to the contr ol group

. Although no treatment

- related histological changes were observed during histopathological examination of the thyroid/parathyroid, the increase s 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) increase s in absolute and relative liver weights , with increases of approximately 1 3

. 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 c ompared 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 histo pathological changes were observed in the kidneys of mid

- and high

- dose males.

S tatistically 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, t he 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 lo w 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 animalsFinal HPV2 Test Rule Data Adequacy Review ( February 2014) CASRN 89

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- 7

9 had absolute and relative sem inal 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. N o 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 cons ider some of the microscopic changes observed in the liver s of high

- dose animals to have potential toxicological significance. In males, a n 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

- dos e 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 f

emales, 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 the ir litter s until scheduled sacrifice on postpartum day 5.

Mating, Fertility, Ges tation, 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

- do se 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 successfull y 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
p regnancy 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, th e 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 corpor a lutea, number of implantation sites, sex ratio, and litter size were similar among treated and control groups. At 50 mg/k g

- bw/day, there was a statistically significant (p < 0.05) decrease in post

- implan tatio n loss relative to the controls ; post

implantation 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 female s in the low

- dose group that did not become pregnant were found toFinal HPV2 Test Rule Data Adequacy Review ( February 2014) CASRN 89

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- 7

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

- response relationship, these findings w ere 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 h istopathological 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 Weight s , 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 lit ter 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

co ngestion in females, increased absolute and relative liver weights in females, and increased relative and absolute thyroid/parathyroid weights in males 1

. 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 restriction s

. 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 absolu te 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,
respe ctively, compared to controls).Final HPV2 Test Rule Data Adequacy Review ( February 2014) CASRN 89

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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

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12 E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Ef

fects 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 Aberrat ions Yes Yes No Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No