Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
TESTING ON VERTEBRATE ANIMALS

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Pre-existing data:
There are no pre-existing data available that would address the study endpoint (GLP or non-GLP). There are no Historical human data that could be used in place of this study.
- (Q)SAR
There are no accepted QSAR approaches for predicting the outcome of this endpoint
- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint
- Weight of evidence
There are no data from which to generate a weight of evidence
- Grouping and read-across
We have not identified any suitable analogues that could be used to provide data for this endpoint.
- Substance-tailored exposure driven testing
Exposure-based waiving is not relevant for this substance.


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Column 2 considerations are not relevant for this substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(1,4-dimethylpentyl)-N'-phenylbenzene-1,4-diamine
EC Number:
221-374-3
EC Name:
N-(1,4-dimethylpentyl)-N'-phenylbenzene-1,4-diamine
Cas Number:
3081-01-4
Molecular formula:
C19H26N2
IUPAC Name:
N1-(5-methylhexan-2-yl)-N4-phenylbenzene-1,4-diamine
Details on test material:
Santoflex 14
Specific details on test material used for the study:
Identification: N-(1,4 Dimethylpentyl)-N'-Phenyl-p-Phenylenediamine (also referred to as 7PPD)
Lot No.: W9C26UD001
Receipt Date: 29 Mar 2019
Physical Description: Dark pink, translucent liquid
Purity: 95.5 %
Storage Conditions: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, protected from light, under nitrogen
Supplier: Eastman Chemical Company

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System

Receipt
On 30 May and 11 Jun 2019, female and male Crl:CD(SD) rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 10 weeks old and weighed between 218 and 374 g at the initiation of dosing.
Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE) during the pretest period were excluded from the study, therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter loss, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 per group at termination.

Animal Identification
Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition.

Environmental Acclimation
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

Selection, Assignment, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals with findings during the palpation of the testes, at extremes of body weight range, or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital and discarded.

Husbandry
Housing
On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68 °F to 78 °F (20 °C to 26 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study.
The feed was analyzed by the supplier for nutritional components and environmental contaminants.Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test substance and vehicle were administered as a single daily oral gavage dose.Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia (a total of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (a total of 49 to 60 doses). All animals were dosed at approximately the same time each day.

The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.
Details on mating procedure:
After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12 hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by high performance liquid chromatography using ultraviolet absorbance detection using a validated analytical procedure
Duration of treatment / exposure:
The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia (a total of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (a total of 49 to 60 doses). All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.
Frequency of treatment:
The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia (a total of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (a total of 49 to 60 doses). All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
10/sex/group except in the high dose group whic had 15/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (a total of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (a total of 49–60 doses).
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 3 hours postdose
Oestrous cyclicity (parental animals):
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
Sperm parameters (parental animals):
not conducted
Litter observations:
Viability
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled euthanasia due to reasons unrelated to test substance administration (e.g., death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 10, and 13.

Sex Determination
Pups were individually sexed on PND 0, 4, and 13.

Body Weights
Pups were weighed individually on PND 1, 4, 7, 10, and 13.

Preweaning Developmental Landmarks
Anogenital Distance
The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

Assessment of Areolas/Nipple Anlagen
On PND 13, all male pups were evaluated for the presence of nipples/areolae The number of nipples was recorded.

Thyroid Hormone Analysis
Sample Collection
Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants.

Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
Postmortem examinations (parental animals):
Unscheduled Deaths
If necessary, for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained as noted in Text Table 8.
For females euthanized for humane reasons during gestation, the number and location of viable fetuses, corpora lutea, and implantation sites were recorded. For females euthanized during lactation, the number of corpora lutea and former implantation sites were recorded. Recognizable fetuses were examined externally and preserved in 10 % neutral buffered formalin. All pups were euthanized by an intraperitoneal injection of sodium pentobarbital in the scapular region and necropsied.

Scheduled Euthanasia
All surviving animals, including females with total litter loss, were euthanized by carbon dioxide inhalation.

Necropsy
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed.

Organ Weights
The organs identified in Text Table 9 were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative samples of the tissues identified in Text Table 10 were collected from all animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated

Histology
Tissue trimming was performed at the Testing Facility. Tissues identified in Text Table 10 from all animals in the control and high-dose groups and from all animals dying spontaneously, as well as gross lesions from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Text Table 10 for microscopic examination were evaluated from all animals in the control and high dose groups and from all animals euthanized in extremis. The liver (in males and females), mammary gland in males, and adrenal glands (in females) were considered potential target tissues. The liver (in males and females) and mammary glands (males) were microscopically examined in all dose groups. The adrenal glands in the low- and mid dose groups were not collected for microscopic evaluation per protocol, and therefore the adrenal glands were not examined microscopically in all dose group. The adrenal glands were only considered a potential target tissue based on the organ weight data and gross lesions correlations. After reviewing the mammary glands in all dose groups including the control group, the male mammary gland was no longer considered a target tissue.
Postmortem examinations (offspring):
Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary, for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained as noted.
Intact offspring that were found dead or euthanized in extremis during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Findings were recorded as developmental variations or malformations, as appropriate.

Scheduled Euthanasia
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.

Necropsy
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

Organ Weights
The organs identified were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ weights were not recorded for animals found dead or euthanized in extremis.

Statistics:
Presented in "other methods" box due to character limit here.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related higher incidences of clear material around the mouth and/or on the ventral neck were noted for males and females at 100 and 200 mg/kg/day at approximately 3 hours postdose sporadically throughout the study. These findings generally did not persist to the daily examinations and were not considered adverse. Other clinical observations noted at the daily examinations or approximately 3 hours following dose administration, including hair loss and yellow and/or brown material on various body surfaces, occurred infrequently, similarly in the control group, and/or in a manner that was not dose-related
Mortality:
mortality observed, treatment-related
Description (incidence):
One female each in the 100 and 200 mg/kg/day groups was euthanized in extremis, and 1 and 4 females in the 50 and 200 mg/kg/day group, respectively, were euthanized due to total litter loss.

In the 200 mg/kg/day group, Female No. 4553 was euthanized in extremis on Gestation Day 23 following prolonged labor (dystocia); clinical observations noted for this female included unkempt appearance, partial closure of the eyes, and red and brown material on various body surfaces at the daily examinations and/or approximately 3 hours postdose during Gestation Days 21–23. Macroscopic observations of pale kidneys and microscopic observations of vacuolation of the liver and kidneys and centrilobular hypertrophy of the liver were noted for this female. In addition, Female No. 4544 in the 100 mg/kg/day group was euthanized in extremis on Lactation Day 0 following observations of an unkempt appearance, and pale and cool extremities and body at the daily examination on the day of euthanasia and prolonged labor and dystocia. At necropsy, macroscopic findings included a pale brain, kidney, liver, and pituitary gland and microscopically was noted with centrilobular hepatocellular necrosis in the liver. The moribund condition of both females was considered test substance-related.
Additionally, 1 and 4 females in the 50 and 200 mg/kg/day groups were euthanized due to total litter losses early in lactation, often preceded by dystocia.

Female No. 4528 in the 200 mg/kg/day group was euthanized due to dystocia and a total litter loss on Lactation Day 2. Prior to euthanasia, this female was noted with unkempt appearance, hunched posture, and red, yellow, and/or brown material on various body surfaces at the daily examinations and/or approximately 3 hours postdose primarily from Gestation Day 21 through the day of euthanasia. Macroscopic examination of this female revealed enlarged adrenal glands and a small thymus, and microscopic findings of vacuolation of the kidneys and centrilobular hypertrophy of the liver were noted.

Female No. 4558 in the 200 mg/kg/day group was euthanized due to dystocia and a total litter loss on Lactation Day 0. This female was noted with clinical observations of unkempt appearance and red and/or material on various body surfaces during Gestation Day 22 through euthanasia. At necropsy, this female was noted with enlarged adrenal glands and a small thymus macroscopically and with vacuolation and hypertrophy of the adrenal cortex and vacuolation of the kidney microscopically.

Female No. 4571 in the 200 mg/kg/day group was euthanized due to a total litter loss on Lactation Day 2. This female was noted with red material around the nose on Gestation Day 21 and Lactation Day 0. There were no remarkable macroscopic findings noted for this female, but vacuolation of the kidney and centrilobular hypertrophy of the liver were noted microscopically.

Female No. 4547 in the 200 mg/kg/day group was euthanized due to a total litter loss on Lactation Day 1 and was noted with clinical observations of decreased defecation, soft feces, hunched posture, and red, yellow, and/or brown material on various body surfaces at the daily examinations and/or approximately 3 hours postdose from Gestation Day 22 through the day of euthanasia. At necropsy, this female was noted with enlarged kidneys, which correlated with microscopic findings compatible with Caroli Syndrome (an incidental disease process associated with enlarged polycystic kidneys). The cause of dystocia and total litter loss was nonetheless considered to be attributed to test substance administration.

Lastly, in the 50 mg/kg/day group, Female No. 4575 was euthanized due to dystocia and a total litter loss on Lactation Day 0. This female was noted with clinical observations of hunched posture and red material around the mouth and urogenital area on the day of euthanasia. At necropsy, this female had 2 conceptuses retained in utero and was noted with a small spleen and a microscopic finding of vacuolation of the liver. These effects on parturition across all test substance groups were considered test substance-related and adverse.
With the exception of the aforementioned females, all other males and females in the control, 50, 100, and 200 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
In the 200 mg/kg/day group males, statistically significantly lower mean body weight gains were generally noted throughout the dosing period and when the premating period (Study Days 0–13) and entire dosing period (Study Days 0–27) were evaluated compared to the control group. As a result, mean absolute body weights were 5.0 % to 8.7 % lower than the control group during Study Days 13–27; the difference was statistically significant on Study Day 27. The effects on body weight and body weight gain in the 200 mg/kg/day group males were considered test substance related and adverse based on the sustained effects on mean body weight gain throughout the dosing period (Study Days 0–27).
In the 100 mg/kg/day group, lower mean body weight gains were generally noted during the dosing period; differences were statistically significant during Study Days 13–21 and when the entire dosing period (Study Days 0–27) was evaluated compared to the control group. As a result, mean absolute body weights were 5.1 % to 6.2 % lower (not statistically significant) than the control group during Study Days 21–27. The effects on mean body weight and body weight gain in the 100 mg/kg/day group males were considered test substance-related but likely nonadverse based on the lower magnitude of change versus the control group.
Mean body weights and body weight gains in the 50 mg/kg/day group males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant.

Females

Weekly
Mean body weights and body weight gains in the 50, 100, and 200 mg/kg/day group females were unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.

Gestation
In the 200 mg/kg/day group, statistically significantly lower mean body weight gains were noted during Gestation Days 0–4 and 17–20, and when the entire gestation period (Gestation Days 0–20) was evaluated compared to the control group. As a result, a statistically significantly lower (7.3 %) mean absolute body weight was noted in this group compared to the control group on Gestation Day 20. The lower mean absolute body weights on Gestation Day 20 at 200 mg/kg/day were considered secondary to the maternal toxicity and likely contributed to the lower mean F1 pup birth weights noted in this group.
Mean body weights and body weight gains in the 50 and 100 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation
Mean body weights and body weight gains in the 50, 100, and 200 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food Consumption
Males
In the 200 mg/kg/day group males, statistically significantly lower mean food consumption, evaluated as g/animal/day, was noted during Study Days 0–7 compared to the control group and corresponded to the lower mean body weight gains noted in this group and was considered adverse. Mean food consumption in the 200 mg/kg/day group males was comparable to the control group during Study Days 7–13.
Mean food consumption in the 50 and 100 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.

Females
Weekly
Mean food consumption, evaluated as g/animal/day, in the 50, 100, and 200 mg/kg/day group females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.

Gestation
Mean maternal food consumption, evaluated as g/animal/day, in the 50, 100, and 200 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant, with the following exceptions. In the 50 mg/kg/day group, statistically significantly higher mean food consumption compared to the control group was noted during Gestation Days 4–11 and when the entire gestation period (Gestation Days 0–20) was evaluated. However, these differences were not noted in a dose related manner, and therefore were not considered test substance-related.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, in the 50, 100, and 200 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Statistically significantly lower (18.3 % and 20.3 %) mean T4 concentrations were noted in the 100 and 200 mg/kg/day group, respectively, compared to the concurrent control group; however, the values (44,950 and 43,827 pg/mL, respectively) were within the range of values in the Charles River Ashland historical control data (39,010 to 97,270 pg/mL; version 2019.02). There were also no corresponding effects on thyroid gland weights or histopathological findings, and therefore the changes were not considered test substance-related. Mean T4 concentration in the 50 mg/kg/day group males was comparable to the concurrent control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. All females were determined to be gravid.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. All females were determined to be gravid.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance administration. One (1), 23(7), 15(3), and 74(12) pups (litters) in the control, 50, 100, and 200 mg/kg/day groups, respectively, were found dead or euthanized due to death of dam. Clinical observations of gasping, a pale and/or cool body, and/or pale extremities were noted for 3 (2) pups (litters) in the 200 mg/kg/day group on PND 0 or 1, the day prior to death. An additional F1 pup (No. 4562-02) was noted with a pale and cool body on PND 0 and was euthanized at the scheduled euthanasia. These clinical observations were not attributed to F0 maternal administration of the test substance because they were noted infrequently and/or in a manner that was not dose-related. One (1), 3(3), 0(0), and 6(4) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of pups born at 50, 100, and 200 mg/kg/day, live litter size at 50 and 100 mg/kg/day, and the percentage of males at birth at 50, 100, and 200 mg/kg/day were similar to the control group values. Postnatal survival in the 50 and 100 mg/kg/day groups were unaffected by test substance administration. In the 200 mg/kg/day group, the mean number of pups born was similar to that in the control group. However, lower (statistically significant) mean postnatal survival was noted in this group beginning on PND 0 (relative to the number born) to PND 4 (pre-selection) compared to the concurrent control group and was considered test substance-related and adverse. Postnatal survival in the 200 mg/kg/day group was comparable to the control group thereafter (PND 4–13). The increased total litter losses in the 200 mg/kg/day were also considered test substance-related and adverse and likely contributed to the lower mean pup survival rates.
In addition, 1 and 4 females in the 50 and 200 mg/kg/day groups, respectively, were noted with total litter losses (100 % pup death) between Lactation Days 0 and 2. The dam was euthanized following the death of the last pup in the litter.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On PND 1, mean male and female pup body weights in the 200 mg/kg/day group were 10.1 % and 12.2 % lower, respectively, than the concurrent control group; the difference was statistically significant for the females. Mean body weight gains for the F1 males and females in this group were comparable to the control group during PND 1–7. Statistically significantly lower mean body weight gains were noted for F1 males and females in the 200 mg/kg/day group during PND 7–13 compared to the control group, resulting in mean absolute body weights that were 8.5 % to 10.1 % lower on PND 10 and 13, respectively, for the F1 males and 6.6 % to 8.3 % lower on PND 10 and 13, respectively, for the F1 females compared to the control group; the difference was statistically significant for the F1 males on PND 13. The lower mean male and female pup body weights in the 200 mg/kg/day group were considered test substance-related and adverse.
Mean male and female pup body weights and body weight changes during PND 1–13 in the 50 and 100 mg/kg/day groups were unaffected by parental administration of the test substance. Statistically significantly lower mean body weight gains were noted in the F1 males and females in the 100 mg/kg/day group during PND 7–10 compared to the control group; however, this difference was transient in nature and had no effect on mean absolute body weight, and therefore was not attributed to parental test substance administration. No other statistically significant differences from the control group were noted.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid hormone values (T4) in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 50, 100, and 200 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areolae/nipple anlagens in the F1 male pups were unaffected by parental administration of the test substance when evaluated on PND 13. The substance article treated group values were not statistically different from the control group values; there were no retained areolae/nipple anlagens.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled Deaths
One (1), 23 (7), 15 (3), and 74 (12) pups (litters) in the control, 50, 100, and 200 mg/kg/day groups, respectively, were found dead or euthanized in extremis. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized due to death of the dam. Aside from the presence or absence of milk in the stomach, internal findings were limited to a developmental variation of renal papillae not fully developed (Woo and Hoar Grade 1) for 2 (1) pups (litters) each in the 100 and 200 mg/kg/day groups. No other internal findings were noted.
Scheduled Necropsy
No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 13 Internal findings limited to a fractured left fibula for Pup No. 4574-13 in the 100 mg/kg/day group. No other internal findings were noted.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Results of Homogeneity Analyses

 

Group 2

(10 mg/mL)

Group 4

(40 mg/mL)

Homogeneity Assessment of the 14 Jun 2019 Formulations

Mean Concentration

(mg/mL)

10.3

40.9

RSD (%)

0.76

1.2

Mean Concentration % of Target

103

102

 
Results of Concentration Analyses

 

Mean Concentration, mg/mL (% of Target)

Date of

Preparation

Group 2

(10 mg/mL)

Group 3

(20 mg/mL)

Group 4

(40 mg/mL)

14 Jun 2019

10.4 (104)

20.3 (101)

41.0 (103)

01 Jul 2019

9.39 (93.9)

19.7 (98.4)

39.5 (98.9)

15 Jul 2019

10.4 (104)

20.4 (102)

41.1 (103)

05 Aug 2019

10.5 (105)

21.3 (106)

41.6 (104)

 

Summary of Unscheduled Euthanasia (F0Females)

Female No.

Type of Euthanasia/Day

Clinical Observations

Relevant Macroscopic and Microscopica Findings

Cause of Euthanasia

50 mg/kg/day

4575

TLL/LD 0

Hunched posture (LD 0);
red material around the mouth and urogenital area (LD 0)

2 conceptuses retained in utero
Spleen – small
Liver – vacuolationa

Dystocia and a total litter loss

100 mg/kg/day

4544

EIE/LD 0

Unkempt appearance (LD 0);
pale body (LD 0)b;
cool body (LD 0)b;
pale extremities (LD 0)b;
cool extremities (LD 0)b

Brain – pale
Kidney – pale
Liver – pale
Pituitary gland – pale
Liver – centrilobular necrosisa
Kidney – vacuolation and acute degeneration of tubulesa

Prolonged labor (dystocia)

200 mg/kg/day

4553

EIE/GD 23

Unkempt appearance (GD 21–23);
partial closure of the eyes (GD 21 and 23);
red material around the nose, forelimb(s), and/or mouth
(GD 21–23);
brown material around the anogenital area (GD 23 and 23)

Kidney – pale
Kidney – vacuolationa
Liver – centrilobular hypertrophy, vacuolationa

Prolonged labor (dystocia)


 

Female No.

Type of Euthanasia/Day

Clinical Observations

Relevant Macroscopic and MicroscopicaFindings

Cause of Euthanasia

200 mg/kg/day (continued)

4528

TLL/LD 2

Unkempt appearance
(LD 0–2);
hunched posture (LD 1–2)
red material around the nose, forelimb(s), urogenital area, and/or mouth (GD 21 through LD 2);
brown material around anogenital area (GD 22 through LD 2);
yellow material on the dorsal trunk (GD 21)

Adrenal glands – enlarged
Thymus gland – small
Kidneys – vacuolationa
Liver – centrilobular hypertrophya

Dystocia and a total litter loss

4558

TLL/LD 0

Unkempt appearance (LD 0);
red material around the left eye, nose, urogenital area, forelimb(s), ventral trunk, and/or mouth (GD 22 through LD 0)

Adrenal glands – enlarged
Thymus gland – small
Adrenal glands – vacuolation and hypertrophy of the adrenal cortexa
Kidney - vacuolationa

Dystocia and a total litter loss

4571

TLL/LD 2

Red material around the nose (GD 21 and LD 0)

Kidney – vacuolationa
Liver – centrilobular hypertrophya

Total litter loss

4547

TLL/LD 1

Hunched posture (LD 1)
red material around the eyes, nose, forelimb(s), urogenital area, and/or ventral trunk (GD 21 through LD 1);
decreased defecation (GD 22);
soft feces (GD 22);
yellow material around the forelimb(s) and/or lateral trunk (GD 22 and LD 1);
brown material around anogenital and/or urogenital area (GD 22 and LD 1)

Kidneys – enlarged
Kidneys – findings consistent with Caroli Syndromea

Total litter loss

 

TLL = Total litter loss; EIE = Euthanized in extremis; GD = Gestation Day; LD = Lactation Day.

a    Microscopic finding.

b   Clinical observation noted by the veterinary staff.

 

Results of Reproductive Performance

Parameter

Dosage Level (mg/kg/day)

CRL HCa

Mean (Range)

0

50

100

200

Male Mating Index (%)

100.0

100.0

100.0

100.0

98.0 (83.3–100.0)

Female Mating Index (%)

100.0

100.0

100.0

100.0

98.0 (83.3–100.0)

Male Fertility Index (%)

100.0

100.0

100.0

100.0

93.9 (80.0–100.0)

Female Fertility Index (%)

100.0

100.0

100.0

100.0

93.9 (80.0–100.0)

Male Copulation Index (%)

100.0

100.0

100.0

100.0

95.7 (80.0–100.0)

Female Conception Index (%)

100.0

100.0

100.0

100.0

95.7 (80.0–100.0)

Estrous Cycle Length (days)

4.2

4.0

3.9

4.0

4.2 (3.9–5.2)

Pre-Coital Interval (days)

2.7

3.4

3.8

2.7

2.7 (1.4–4.5)

 

a Charles River Ashland historical control data (version 2019.04)

 

Adverse Maternal Findings (F0Females)

Female No.

Type
of Death/Day

Remarkable Clinical Observations

Cause of Death/Moribundity

50 mg/kg/day

 

4575

TLL/LD 0

LD 0: Hunched posture and red material around the mouth and urogenital area; 2 retained conceptuses

Total Litter Loss; Dystocia

100 mg/kg/day

 

4544

EE/LD 0

LD 0: unkempt appearance, pale/cool body, and pale/cool extremities

Dystocia

200 mg/kg/day

 

4528

TLL/LD 2

GD 21 - LD 2: unkempt appearance, hunched posture, and red, yellow, and/or brown material on various surfaces

Dystocia; Total Litter Loss

4553

EE/GD 23

GD 21–23: unkempt appearance, partial closure of eye(s), and red and brown material on various body surfaces

Dystocia

4547

TLL/LD 1

GD 22 - LD 1: hunched posture and clear, red, yellow, and brown material on various body surfaces

Total Litter Loss

4558

TLL/LD 0

GD 22 - LD 0: unkempt appearance, red and brown material on various body surfaces

Dystocia; Total Litter Loss

4571

TLL/LD 2

GD 21 and LD 0: red material on nose

Total Litter Loss

 

TLL= Total litter loss, N/A = not applicable.

EE= Euthanized in extremis; GD= Gestation Day; LD= Lactation Day.

 

 

Summary of Organ Weight Data – Scheduled Euthanasia1

 

Males

Females

Group

2

3

4

2

3

4

Dose (mg/kg/day)

50

100

200

50

100

200

No. Animals per Group

10

10

15

9

9

10

Liver (No. Weighed)a

(10)

(10)

(15)

(9)

(9)

(10)

    Absolute value

17.93

18.30

20.15**

19.52

20.91**

22.52**

    % of body weightb

3.800*

3.995**

4.486**

5.406

5.744**

6.193**

    % of brain weight

837.040

856.935

929.420**

956.988

1060.289**

1152.296**

Thymus (No. Weighed)

(10)

(10)

(15)

(9)

(9)

(10)

    Absolute value

0.4307

0.3776

0.3084**

0.2531

0.1765

0.2350

    % of body weightb

0.092

0.083

0.069**

0.070

0.048

0.065

    % of brain weight

20.111

17.689

14.223**

12.348

8.906

12.021

Adrenal Gland (No. Weighed)

(10)

(10)

(15)

(9)

(9)

(10)

    Absolute value

0.0671

0.0748

0.0708

1.023

0.1110*

0.1144*

    % of body weightb

0.014

0.016*

0.016

0.028

0.030*

0.032**

    % of brain weight

3.142

3.497

3.265

4.998

5.623**

5.846**

1    Table does not include data from females with total litter loss.

a  All values expressed as percent difference of control group means.  

b= % of body weight for F0females in lactation may have significant biological variability.

    Based upon statistical analysis of group means, values highlighted in bold are considered 7-PPD-related changes. *=p<0.05; **=p<0.01

 

Test Substance-related Microscopic Findings – F0Females with Total Litter Loss

Group/Sex

Animal Number

LD/Fate

Microscopic Features

7-PPD-related?

2F

4575

LD 0

Scheduled Euthanasia

Liver – Vacuolation

Yes

4F

4528

LD 2

Scheduled Euthanasia

Kidney – Vacuolation

 

Liver - Centrilobular Hypertrophy

Yes

4F

4558

LD 0

Scheduled Euthanasia

Adrenal cortex – Vacuolation and Hypertrophy

 

Kidney - Vacuolation

Yes

4F

4571

LD 2

Scheduled Euthanasia

Kidney - Vacuolation

 

Liver - Centrilobular Hypertrophy

Yes

4F

4547

LD 1

Scheduled Euthanasia

Liver - Cystic biliary ducts; Vacuolation

 

Kidney - Multiple cysts; Vacuolation

Yes

 

 

Summary of Microscopic Findings – Scheduled Euthanasiaa

 

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

50

100

200

0

50

100

200

No. Animals Examined

10

10

10

15

10

9

9

10

Liver (No. Examined)

10

10

10

15

10

9

9

10

Vacuolation

(1)a

(3)

(8)

(9)

(0)

(4)

(4)

(2)

     Minimal

0

3

7

7

0

3

3

2

     Mild

1b

0

1

2

0

1

1

0

1    Table does not include microscopic findings from females with total litter loss

a  Numbers in parentheses represent the number of animals with the finding.

b    Vacuolation in this control male was centrilobular and considered to be of a different nature and pathogenesis than the vacuolation in treated animals.

 

F1Postnatal Survival

Day

Dosage Level (mg/kg/day)

CRL HCa

Mean (Range)

0

50

100

200

PND 0 (relative to number born)

99.3

81.8

98.9

78.5++

98.3 (88.1–100.0)

PND 0–1

99.2

99.3

100.0

85.7

98.9 (89.1–100.0)

PND 1–4

100.0

98.2

99.2

83.3

98.3 (87.5–100.0)

PND 4–7

100.0

100.0

100.0

100.0

99.5 (96.7–100.0)

Birth to PND 4

98.5

79.7

98.1

58.7++

95.7 (84.5–100.0)

PND 4–13

100.0

100.0

100.0

100.0

99.6 (95.8–100.0)

 

a Charles River Ashland historical control data (version 2019.04).

++ = Statistically significantly different from the control group at 0.01.

 

F0Total Litter Losses

Female No.

Dosage Level (mg/kg/day)

Day of Litter Loss

4575

50

Lactation Day 0

4528

200

Lactation Day 2

4547

200

Lactation Day 1

4558

200

Lactation Day 0

4571

200

Lactation Day 2

 

Applicant's summary and conclusion

Conclusions:
Adverse effects on body weights and organ weights changes (higher liver weights and lower thymus weights) were noted for F0 males at 200 mg/kg/day, and therefore a dosage level of 100 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male systemic toxicity of 7-PPD when administered orally by gavage to Crl:CD(SD) rats. There was no evidence of F0 male reproductive toxicity at any dosage level tested; therefore, the NOAEL for F0 male reproductive toxicity was considered to be 200 mg/kg/day. Based on higher liver and adrenal gland weights and histopathologic findings in F0 females at 200 mg/kg/day, the NOAEL for female systemic toxicity was considered to be 100 mg/kg/day. Based on total litter losses noted at 200 mg/kg/day, longer mean gestation lengths at 100 and 200 mg/kg/day, and evidence of dystocia leading to the euthanasia of 1 female each in the 50, 100, and 200 mg/kg/day groups, the NOAEL for F0 female reproductive toxicity was considered to be < 50 mg/kg/day. Lower postnatal survival and lower mean F1 pup birth weights, body weights, and body weight gains were observed in the 200 mg/kg/day group, and therefore the NOAEL for F1 neonatal toxicity was considered to be 100 mg/kg/day.
Executive summary:

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (a total of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (a total of 49–60 doses).

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.

One female each in the 100 and 200 mg/kg/day group were euthanized due to prolonged labor and possible dystocia on Lactation Day 0 and Gestation Day 23, respectively. At necropsy, these females were noted with macroscopic findings of a pale brain, kidney, liver, and/or pituitary gland and microscopic findings of centrilobular hypertrophy and/or necrosis of the liver and vacuolation and/or acute degeneration of the tubules in the kidney. The dystocia and consequent moribund condition of these females was considered test substance-related. In addition, 1 and 4 females in the 50 and 200 mg/kg/day groups, respectively, were noted with total litter losses between Lactation Days 0 and 2; the 50 mg/kg/day female had 11 dead pups on Postnatal Day 0, but also 2 retained conceptuses, which was also indicative of dystocia in the dam. These total litter losses were also considered test substance-related and adverse and correlated with lower mean F1postnatal survival in the 200 mg/kg/day group. All F0males and remaining F0females survived to the scheduled necropsy. Test substance-related increased incidences of clear material around the mouth and/or ventral neck were noted approximately 3 hours postdose for males in the 200 mg/kg/day group and for females in the 100 and 200 mg/kg/day groups. These clinical observations generally did not persist to the daily examinations and were not considered adverse.

For F0males, lower mean body weight gains were noted generally throughout the study in the 100 and 200 mg/kg/day group with corresponding lower mean food consumption noted in the 200 mg/kg/day group during Study Days 0–7 compared to the control group. A lower mean body weight gain was noted in the 200 mg/kg/day group when the premating period (Study Days 0–13) was evaluated, and lower mean body weight gains were noted in the 100 and 200 mg/kg/day groups when the entire dosing period (Study Days 0–27) was evaluated compared to the control group. As a result, mean absolute body weights were 5.1% to 6.2% lower in the 100 mg/kg/day group during Study Days 21–27 and were 5.0 % to 8.7 % lower in the 200 mg/kg/day group during Study Days 13–27 compared to the control group. The alterations in body weights for males in the 100 and 200 mg/kg/day groups was considered test substance‑related and adverse at 200 mg/kg/day; the changes at 100 mg/kg/day were considered nonadverse based on the lower magnitude of change. Mean absolute body weights, body weight gains, and food consumption in the 50 mg/kg/day group males were similar to those in the control group. For F0females, mean absolute body weights, body weight gains, and food consumption in all test substance-treated groups were unaffected by test substance administration during the premating period (Study Days 0–13). During gestation, lower mean body weight gains were noted in the 200 mg/kg/day group during Gestation Days 0–4 and 17–20 in the absence of effects on food consumption, resulting in a mean absolute body weight that was 7.3 % lower than the control group on Gestation Day 20. The effects on gestation body weights and body weight gains at 200 mg/kg/day were considered test substance‑related. Mean body weights, body weight gains, and food consumption during gestation in the 50 and 100 mg/kg/day groups, and during lactation in all test substance‑treated groups were unaffected by test substance administration.

There were no test substance-related effects on reproductive performance (mating, fertility, copulation, and/or conception indices), precoital interval, and estrous cycle length for F0males and females at any dosage level.

Test substance-related longer mean gestation lengths were noted for F0females in the 100 and 200 mg/kg/day groups compared to the control group and was considered adverse. The mean gestation length in the 50 mg/kg/day group was unaffected by test substance administration, however, 1 female was noted with dystocia, as evidenced by 2 retained conceptuses on Lactation Day 0, which was also considered test substance related and adverse based on similar findings of dystocia at 100 and 200 mg/kg/day.

There were no test substance-related effects on T4concentration in F0males at any dosage level. At necropsy, there were no macroscopic or microscopic observations in the thyroid gland or any alteration in thyroid gland weights in F0 males or females.

Administration of the test substance was associated with higher liver weights in 200 mg/kg/day group F0males and 100 and 200 mg/kg/day group F0females, higher adrenal gland weights in 100 and 200 mg/kg/day group F0females, lower thymus weights in 200 mg/kg/day group Fmales, and non-adverse microscopic findings of vacuolation in the liver (50, 100 and 200 mg/kg/day group F0males and females), and adrenal cortical hypertrophy (200 mg/kg/day F0 females).

In the 200 mg/kg/day group, a lower mean litter proportion of postnatal survival was noted on PND 0 (relative to the number born) and when the birth to PND 4 (pre-selection) interval was evaluated compared to the control group. The effect on postnatal survival in the 200 mg/kg/day group were considered test substance-related and adverse. The mean live litter size and postnatal survival in the 50 and 100 mg/kg/day groups, and the mean number of pups born and percentage of males in all test substance-treated groups were unaffected by F0parental test substance administration.

There were no clinical observations in the F1pups that could be attributed to F0parental test substance administration at any dosage level.

In the 200 mg/kg/day group, mean F1male and female body weights on PND 1 were 10.1 % and 12.2 % lower, respectively than the control group. Mean body weight gains for these pups were similar to the control group during PND 1–7. Although mean pup body weights were comparable to controls on PND 7, lower mean body weight gains were noted for the F1 males and females during PND 7–13, resulting in mean absolute body weights for F1males and females that were 6.6 % to 10.1 % lower than the control group on PND 10 and 13. The effects on F1pup body weights at 200 mg/kg/day were considered test substance-related and adverse. Mean F1pup birth weights, absolute body weights, and body weight gains in the 50 and 100 mg/kg/day groups were unaffected by F0parental test substance administration.

Mean anogenital distance (absolute and relative to cube root of body weight) for F1males and females and areolae/nipple anlagen retention for F1males were unaffected by F0parental test substance administration at all dosage levels.

There were no test substance-related effects on T4concentration for F1male and female pups at any dosage level on PND 13. At necropsy, there were no macroscopic observations and no effects on thyroid gland weights in the F1pups that could be attributed to F0parental test substance administration.

Adverse effects on body weights and organ weights changes (higher liver weights and lower thymus weights) were noted for F0males at 200 mg/kg/day, and therefore a dosage level of 100 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0male systemic toxicity of 7-PPD when administered orally by gavage to Crl:CD(SD) rats. There was no evidence of F0male reproductive toxicity at any dosage level tested; therefore, the NOAEL for F0male reproductive toxicity was considered to be 200 mg/kg/day. Based on higher liver and adrenal gland weights and histopathologic findings in F0females at 200 mg/kg/day, the NOAEL for female systemic toxicity was considered to be 100 mg/kg/day. Based on total litter losses noted at 200 mg/kg/day, longer mean gestation lengths at 100 and 200 mg/kg/day, and evidence of dystocia leading to the euthanasia of 1 female each in the 50, 100, and 200 mg/kg/day groups, the NOAEL for F0female reproductive toxicity was considered to be < 50 mg/kg/day. Lower postnatal survival and lower mean F1pup birth weights, body weights, and body weight gains were observed in the 200 mg/kg/day group, and therefore the NOAEL for F1neonatal toxicity was considered to be 100 mg/kg/day.