N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: [U.S. EPA OPPTS 890.1450 pubertal study

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: Attainment of puberty

Test material

Test material information

Constituent 1

Reference substance name:

N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

EC Number:

212-344-0

EC Name:

N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

Cas Number:

793-24-8

Molecular formula:

C18H24N2

IUPAC Name:

N1-(4-methylpentan-2-yl)-N4-phenylbenzene-1,4-diamine

Details on test material:

IUCLID4 Test substance: other TS: Purity = 91.0 %

Specific details on test material used for the study:

6-PPD
Lot no. NB40208100
Exp. date: 08-Feb-2016
CAS no. 793-24-8
WIL ID no. 14027E
State: Dark brown pellets
Date of receipt: 12-Sep-2014

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Juvenile female Sprague Dawley [Crl:CD(SD)] rats born from time-mated female rats were used as the test system on this study. This species and strain of animal has been recognized by the United States EPA as the preferred strain for the endocrine disruption screening battery. WIL Research has historical control data on the Crl:CD(SD) rat. The number of animals selected for this study (15 females/group) was based on the United States EPA Endocrine Disruptor Screening Program Test Guidelines (OPPTS 890.1450), which recommends assignment of 15 female pups per treatment group. Due to the strict guideline selection criteria for pups assigned to study, this was the minimum number of time-mated females and litters required to obtain at least 15 non-littermate female pups per treatment group for this study (and sufficient male pups for the concurrent pubertal assay in male rats WIL-158506).
Time-mated female Crl:CD(SD) rats (35 females) were received from Charles River Laboratories, Inc., Raleigh, NC, on 21-Nov-2014. The females were used to obtain juvenile females for this study, as well as juvenile males for a concurrent male pubertal assay (WIL-158506). The diet provided to the time-mated females prior to shipment was TestDiet® 5L79. At arrival, the time-mated female rats were on gestation day 11 (the day evidence of mating was confirmed was designated gestation day 0). Each female was examined by a qualified biologist on the day of receipt and animals were weighed and clinical observations were recorded. Each time-mated female was uniquely identified by a Monel® metal ear tag displaying the animal number. The rats were observed twice daily for mortality and changes in general appearance and behavior.
Time-mated females were individually housed in solid-bottom cages with nesting material (Aspen Bed™ 1, Cincinnati Lab Supply, Cincinnati, OH). Aspen bedding has been shown to be a suitable bedding for rat pups; it does not affect pup survival or significantly induce liver enzymes, unlike soft-pine bedding such as heat-treated pine wood bedding. Hardwood bedding, such as Aspen bedding, does not need to be heat-treated because it does not release oils and resins like soft-pine bedding. Therefore, the use of Aspen bedding (non-heat-treated) in the current study is not expected to impact the outcome of the study. The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research. The dams were housed in these cages until euthanasia on lactation day 21; weaned juveniles were housed 2 to 3 females per cage throughout the remainder of the study. The cage bedding was changed at least 3 times each week. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). This study was conducted simultaneously in the same animal room as WIL-158506, a pubertal assay study in males with the same test substance. The 2 studies were maintained in separate cage banks. The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
The basal diet used in this study, Harlan Laboratories 2016CM Teklad Global (16% Protein Rodent Diet), was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week.
Municipal water supplying the facility was sampled for contaminants according to WIL Research’s SOPs. The above mentioned diet was further analyzed for genistein equivalent (aglycone) content with results of an average of 5.00 ppm total isoflavones (genistein + daidzein + glycitein). The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 68.9°F to 71.6°F (20.5°C to 22.0°C) and mean daily relative humidity ranged from 33.7% to 55.4% during the study. Fluorescent lighting provided illumination for a 14:10-hour light:dark photoperiod (on at 0500 hours and off at 1900 hours local time). The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance, 6-PPD (1,3-dimethylbutyl-N’-phenyl-p-phenylenediamine), in the vehicle (corn oil) was administered orally by gavage to 2 groups of 15 juvenile/peripubertal female Crl:CD(SD) rats, obtained from non-treated time-mated
females, once daily during PND 22 to 42 or 43. Dosage levels were 250 and 500 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 15 juvenile/peripubertal females received the vehicle on a comparable
regimen.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Resuspension homogeneity and stability of the test substance formulations at target concentrations of 1 and 125 mg 6-PPD/mL following up to 10 days of room temperature
or refrigerated storage were established as part of a concurrent study (WIL-158513). Samples for homogeneity and/or concentration determinations were collected from the
top, middle, and bottom strata of the first test substance formulations and from the middle stratum of the first control group formulation. Samples for concentration analysis were
also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was
subjected to the appropriate analyses. All remaining samples were stored refrigerated (approximately 4°C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection (WIL-158513).

Duration of treatment / exposure:

21 days

Frequency of treatment:

Daily

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance, 6-PPD (1,3-dimethylbutyl-N’-phenyl-p-phenylenediamine), in the vehicle (corn oil) was administered orally by gavage to 2 groups of 15 juvenile/peripubertal female Crl:CD(SD) rats, obtained from non-treated time-mated females, once daily during PND 22 to 42 or 43. Dosage levels were 250 and 500 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 15 juvenile/peripubertal females received the vehicle on a comparable regimen.
All females selected for study were observed twice daily for mortality and moribundity from weaning through study termination. Clinical observations and body weights were recorded daily. All females were observed daily for vaginal opening. Once vaginal opening was observed, daily vaginal lavages were performed for each female to determine the stage of the estrous cycle. A complete necropsy was conducted on all rats found dead or that survived to the scheduled euthanasia on PND 42 or 43; selected organs were weighed and preserved. Hormone (T4 and TSH) and clinical pathology evaluations (serum chemistry) were conducted on all surviving animals on PND 42 or 43. In addition, histopathological evaluation of the thyroid, kidney, ovary, and uterus was performed at the scheduled necropsy on PND 42 or 43.

Examinations

Examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All females were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded on the day of randomization and daily (prior to test substance administration during the treatment period) through the day of euthanasia. Animals were also observed for signs of toxicity approximately 2 hours following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHTS
Individual body weights were recorded on the day of randomization, daily prior to test substance administration, and on the day of euthanasia. Group mean body weights were calculated for each of these days. In addition, mean body weight changes were calculated for each corresponding interval and also for PND 22-42. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

VAGINAL OPENING
Each female was observed for vaginal perforation beginning on PND 22. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded. The age and body weight on the day at which the vaginal lumen was first observed to open was recorded for each animal. However, if any animal within any group showed incomplete opening (persistent pin holes and/or threads) for greater than 3 days, a separate second analysis was conducted using the age at which incomplete opening was first observed. Examination of the females was continued daily until vaginal opening was present or until euthanasia.

ESTROUS CYCLICITY
Beginning on the day vaginal opening was observed, vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until necropsy. The age of first vaginal estrus after vaginal opening was recorded. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to diestrus [D] from estrus [E] or proestrus [P]). Estrous cycle length was determined by counting the number of days from the first D in a cycle to the first D in a subsequent cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data. The following definitions were used for determination of regular cycling, irregular cycling, non-cycling, and insufficient data in the female pubertal assay. Note that for a “complete cycle”, an animal must exhibit at minimum an E from the previous cycle, followed by one cycle (D to E), and a D from the start of the next cycle:

BLOOD COLLECTION FOR HORMONE AND SERUM CHEMISTRY ANALYSES
Immediately upon euthanasia by decapitation, trunk blood samples (minimum of 2 mL per animal) were collected for hormone analysis (thyroxine [T4] and thyroid stimulating hormone [TSH]) and serum chemistry. Serum was separated and divided approximately equally into 3 tubes. One tube of serum samples was used for serum chemistry evaluation. The remaining 2 tubes of serum samples for each animal were used for T4 and TSH analyses; the samples were stored frozen (≤-70°C) for subsequent analysis. An electrochemiluminescent immunoassay on the Cobas e411 (Roche Diagnostics, Indianapolis, IN) was used to analyze serum samples for the presence of T4. To determine the concentration of TSH in serum samples, the [125I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary) was used; the assay system relies on magnetic separation for quantitative measurement of TSH using a GAMMA-C12 gamma counter (Diagnostic Products Corporation, Los Angeles, CA). For both assays, multiple quality control samples were dispersed within the assay.

The following serum chemistry parameters were evaluated:

Albumin Total protein
Globulin [by calculation] Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total Bili) Urea nitrogen
Creatinine Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT) Glucose
Total cholesterol (Cholesterol) Calcium
Chloride Phosphorus
Potassium Sodium
Triglycerides (Triglyceride) Hemolysisa
Lipemia Icterus

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATIONS
Necropsies were performed on all juvenile females found dead or that survived to the scheduled necropsy on PND 42 or 43. The necropsy included examination of the external surface, all orifices, the external surface of the brain and the abdominal, thoracic, and pelvic cavities, including viscera. Tissues and any tissues with gross lesions were saved in 10% neutral-buffered formalin for possible future histopathologic examination. The carcasses were then discarded.

ORGAN WEIGHTS
Wet and blotted uterine weights were measured for all animals surviving to the scheduled necropsy. The uteri were harvested in the same sequence in which animals were dosed and euthanized. Each uterus was carefully dissected and trimmed, being careful to retain the luminal fluid. Each “wet” uterus was weighed intact (with the luminal fluid) to the nearest 0.1 mg, then opened longitudinally and blotted with filter paper to remove the luminal fluid. The blotted uterus was then weighed to the nearest 0.1 mg. Any loss in luminal content was recorded

In addition to the uterus, the following organs were weighed from all females at the scheduled necropsy:

Adrenal glands Kidneys
Liver Ovaries
Pituitary gland Thyroid

Tissues were weighed immediately to prevent drying out prior to weighing. All tissues were recorded to the nearest 0.1 mg, with the exception of the thyroid which was recorded to 0.01 mg and the liver and kidney which were recorded to 0.01 g. To minimize systematic bias in the weighing procedures, organ harvesting and weighing procedures were divided as equally as possible among the prosecting and weighing technicians, such that all animals from a group were not processed by a single individual.

HISTOLOGY AND MICROSCOPIC EXAMINATION
Following approximately 24 hours of fixation in 10% neutral-buffered formalin, the ovaries and uterus were rinsed and stored in 70% ethanol until histological processing. The thyroid glands, attached to the trachea, and kidneys were fixed for at least 24 hours in 10% neutral-buffered formalin. The kidneys were then placed in 70% ethanol until histological processing. The thyroid was then dissected from the trachea, blotted, weighed to the nearest 0.01 mg, and placed in 70% ethanol until histopathological processing. After fixation, protocol-specified tissues were shipped to WIL Research Hillsborough, for processing. The tissues were trimmed according to the SOPs of WIL Research Hillsborough and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research’s SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin, except as noted below. Microscopic examination was performed on the following tissues from all animals at the scheduled necropsy.

Thyroid Ovary
Kidney Uterus

The thyroid sections (minimum of 2 sections) were subjectively evaluated for follicular epithelial height and colloid area and any abnormalities/lesions were noted. Five random sections from an ovary were evaluated for follicular development and any abnormalities/lesions (such as ovarian atrophy). Uterine histology documented cases of hyper- or hypotrophy as characterized by changes in the uterine horn diameter and myometrial stroma or endometrial gland development. The stage of the estrous cycle of the female at the time of necropsy was taken into account in the final histological assessment, as ovarian and uterine cellular changes are dependent upon endocrine status. Stained histologic sections were examined by light microscopy and observations were entered in WTDMS™ by the pathologist. Histologic sections were of adequate size and quality for detailed evaluation. Histologic lesions were classified using standard published terminology to the extent possible. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Positive control:

no

Results and discussion

Details on results:

No test substance-related mortality or moribundity were noted at any dosage level. Two females in the 500 mg/kg/day group were found dead during the course of the study; however, mortality (1 female found dead) was also noted in the control group. In addition, 1 female in the 500 mg/kg/day group was found dead as the result of an intubation error. Test substance-related clinical findings included salivation prior to dosing and clear material around the mouth at approximately 2 hours following dose administration in the 250 and 500 mg/kg/day groups. In addition, yellow material around the urogenital area was noted sporadically for females in the 500 mg/kg/day group at the daily examinations and approximately 2 hours following dose administration. Incidences of dose expulsion were also sporadically noted for females in the 250 and 500 mg/kg/day groups at the time of dosing.
Test substance-related lower initial mean body weight gains were noted for females in the 250 and 500 mg/kg/day groups following the first 2 (250 mg/kg/day) or 3 (500 mg/kg/day) days of dose administration. As a result, mean body weights in these groups were up to 8.37% and 14.83% lower during the treatment period, respectively, than the control group. Mean body weight gains in these groups were generally similar to the control group throughout the remainder of the treatment period and when the entire treatment period was evaluated. Earlier ages at vaginal opening were noted for females in the 500 mg/kg/day group (33.2 days), compared to the concurrent control group (35.2 days), when complete and incomplete vaginal opening were evaluated. These results were considered test substance-related and adverse. In addition, lower mean body weights at the time of attainment were noted for females in the 250 (11.48%) and 500 (18.8%) mg/kg/day groups. A higher age at first estrus was observed in the 500 mg/kg/day group (39.2 days), compared to the control group (36.3 days). Due to the low number of females that were cycling in the 250 and 500 mg/kg/day groups, estrous cycle lengths could not be evaluated. The lower number of females that were cycling in these groups was partially due to the numbers of females with insufficient data in the 250 and 500 mg/kg/day group (3 and 4 females, respectively). Test substance administration was associated with higher total bilirubin and GGT in the 500 mg/kg/day group females and lower AST and triglycerides and higher serum cholesterol in the 250 and 500 mg/kg/day group females. In addition, there was test substance-related lower serum T4 and higher TSH in the 250 and 500 mg/kg/day group females. These effects were considered non-adverse. There was test substance-related pale liver in 2 of 15 females in the 500 mg/kg/day group. The pale livers correlated to a microscopic finding of hepatocellular vacuolation. Test substance administration was associated with lower uterus (blotted and unblotted) weights and higher kidney, liver, and thyroid gland weights in the 250 and 500 mg/kg/day group females and lower ovary weights in the 500 mg/kg/day group females.
Test substance-related microscopic findings included higher follicular cell height and lower colloid area in the thyroid glands of 250 and 500 mg/kg/day group females, vacuolation of the liver in the 500 mg/kg/day group females, an absence of corpora lutea with increased tertiary follicles (i.e., non-cycling) in the ovaries, and immature uterus of 500 mg/kg/day group females.
Liver vacuolation was considered a toxic effect, but this change was considered non-adverse. The non-cycling status of the ovary and the immature status of the uterus in the 500 mg/kg/day group females were considered adverse. The change in the ovaries and the uterus also correlated to changes in reproductive endpoints including age at attainment of vaginal opening, body weight at attainment of vaginal opening, and age at first estrus. There was test substance-related earlier age at attainment of vaginal opening in the 500 mg/kg/day group females when compared to controls; lower group mean body weight at attainment of vaginal opening in the 250 mg/kg/day group females and 500 mg/kg/day group females when compared to controls; and test substance related delayed age at first estrus in the 500 mg/kg/day females when compared to controls. Regardless of the direct or indirect pathogenesis, failure to ovulate is considered an adverse effect. The microscopic changes in the thyroid gland, higher liver weights, higher thyroid gland weights, lower levels of T4 and AST, higher levels of TSH, and higher cholesterol levels were non-adverse changes likely secondary to hepatomegaly. The cause of hepatomegaly was undetermined (because liver was not examined microscopically), but it likely was due to non-adverse metabolic enzyme induction.

Applicant's summary and conclusion

Conclusions:

Based on adverse effects noted on body weights, vaginal opening, estrous cyclicity, and the liver, thyroid gland, ovary, and uterus, there was evidence of possible test
substance-related endocrine-mediated effects on pubertal development and thyroid function in the juvenile/peripubertal female rat following oral administration of 6-PPD at
a dosage level of 500 mg/kg/day. In addition, based on the presence of lower uterine weights and lower body weight at the age of attainment of vaginal opening, there was
also evidence of possible test substance-related endocrine-mediated effects on pubertal evelopment in the juvenile/peripubertal female rat following oral administration of
6-PPD at a dosage level of 250 mg/kg/day. The cause for the non-cycling condition of the ovary and the immature appearance of the uterus in the 500 mg/kg/day group, and the
relationship between these histologic findings and the earlier onset of vaginal opening in the 500 mg/kg/day group and the lower uterine weights in the 250 and 500 mg/kg/day
groups are unclear. The precise nature of the endocrine disruption cannot be determined without a complete weight of evidence analysis taking into consideration results of other
endocrine disruption assays.

Executive summary:

SUMMARY

 

OBJECTIVE

 

The objective of the study was to assess the potential effects of the test substance on the endocrine system, by identifying effects on pubertal development and thyroid function in the juvenile/peripubertal female rat.

 

STUDY DESIGN

 

The test substance, 6-PPD (1,3-dimethylbutyl-N’-phenyl-p-phenylenediamine), in the vehicle (corn oil) was administered orally by gavage to 2 groups of 15 juvenile/peripubertal female Crl:CD(SD) rats, obtained from non-treated time-mated females, once daily during PND 22 to 42 or 43. Dosage levels were 250 and 500 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 15 juvenile/peripubertal females received the vehicle on a comparable regimen. All females selected for study were observed twice daily for mortality and moribundity from weaning through study termination. Clinical observations and body weights were recorded daily. All females were observed daily for vaginal opening. Once vaginal opening was observed, daily vaginal lavages were performed for each female to determine the stage of the estrous cycle. A complete necropsy was conducted on all rats found dead or that survived to the scheduled euthanasia on PND 42 or 43; selected organs were weighed and preserved. Hormone (T4 and TSH) and clinical pathology evaluations (serum chemistry) were conducted on all surviving animals on PND 42 or 43. In addition, histopathological evaluation of the thyroid, kidney, ovary, and uterus was performed at the scheduled necropsy on PND 42 or 43.

 

RESULTS

 

No test substance-related mortality or moribundity were noted at any dosage level. Two females in the 500 mg/kg/day group were found dead during the course of the study; however, mortality (1 female found dead) was also noted in the control group. In addition, 1 female in the 500 mg/kg/day group was found dead as the result of an intubation error. Test substance-related clinical findings included salivation prior to dosing and clear material around the mouth at approximately 2 hours following dose administration in the 250 and 500 mg/kg/day groups. In addition, yellow material around the urogenital area was noted sporadically for females in the 500 mg/kg/day group at the daily examinations and approximately 2 hours following dose administration. Incidences of dose expulsion were also sporadically noted for females in the 250 and 500 mg/kg/day groups at the time of dosing. Test substance-related lower initial mean body weight gains were noted for females in the 250 and 500 mg/kg/day groups following the first 2 (250 mg/kg/day) or 3 (500 mg/kg/day) days of dose administration. As a result, mean body weights in these groups were up to 8.37% and 14.83% lower during the treatment period, respectively, than the control group. Mean body weight gains in these groups were generally similar to the control group throughout the remainder of the treatment period and when the entire treatment period was evaluated.

Earlier ages at vaginal opening were noted for females in the 500 mg/kg/day group (33.2 days), compared to the concurrent control group (35.2 days), when complete and incomplete vaginal opening were evaluated. These results were considered test substance-related and adverse. In addition, lower mean body weights at the time of attainment were noted for females in the 250 (11.48%) and 500 (18.8%) mg/kg/day groups.

A higher age at first estrus was observed in the 500 mg/kg/day group (39.2 days), compared to the control group (36.3 days). Due to the low number of females that were cycling in the 250 and 500 mg/kg/day groups, estrous cycle lengths could not be evaluated. The lower number of females that were cycling in these groups was partially due to the numbers of females with insufficient data in the 250 and 500 mg/kg/day group (3 and 4 females, respectively). Test substance administration was associated with higher total bilirubin and GGT in the 500 mg/kg/day group females and lower AST and triglycerides and higher serum cholesterol in the 250 and 500 mg/kg/day group females. In addition, there was test substance-related lower serum T4 and higher TSH in the 250 and 500 mg/kg/day group females. These effects were considered non-adverse. There was test substance-related pale liver in 2 of 15 females in the 500 mg/kg/day group. The pale livers correlated to a microscopic finding of hepatocellular vacuolation. Test substance administration was associated with lower uterus (blotted and unblotted) weights and higher kidney, liver, and thyroid gland weights in the 250 and 500 mg/kg/day group females and lower ovary weights in the 500 mg/kg/day group females.

Test substance-related microscopic findings included higher follicular cell height and lower colloid area in the thyroid glands of 250 and 500 mg/kg/day group females, vacuolation of the liver in the 500 mg/kg/day group females, an absence of corpora lutea with increased tertiary follicles (i.e., non-cycling) in the ovaries, and immature uterus of 500 mg/kg/day group females. Liver vacuolation was considered a toxic effect, but this change was considered non-adverse. The non-cycling status of the ovary and the immature status of the uterus in the 500 mg/kg/day group females were considered adverse. The change in the ovaries and the uterus also correlated to changes in reproductive endpoints including age at attainment of vaginal opening, body weight at attainment of vaginal opening, and age at first estrus. There was test substance-related earlier age at attainment of vaginal opening in the 500 mg/kg/day group females when compared to controls; lower group mean body weight at attainment of vaginal opening in the 250 mg/kg/day group females and 500 mg/kg/day group females when compared to controls; and test substance related delayed age at first estrus in the 500 mg/kg/day females when compared to controls. Regardless of the direct or indirect pathogenesis, failure to ovulate is considered an adverse effect. The microscopic changes in the thyroid gland, higher liver weights, higher thyroid gland weights, lower levels of T4 and AST, higher levels of TSH, and higher cholesterol levels were non-adverse changes likely secondary to hepatomegaly. The cause of hepatomegaly was undetermined (because liver was not examined microscopically), but it likely was due to non-adverse metabolic enzyme induction.

 

CONCLUSIONS

 

Based on adverse effects noted on body weights, vaginal opening, estrous cyclicity, and the liver, thyroid gland, ovary, and uterus, there was evidence of possible test substance-related endocrine-mediated effects on pubertal development and thyroid function in the juvenile/peripubertal female rat following oral administration of 6-PPD at a dosage level of 500 mg/kg/day. In addition, based on the presence of lower uterine weights and lower body weight at the age of attainment of vaginal opening, there was also evidence of possible test substance-related endocrine-mediated effects on pubertal development in the juvenile/peripubertal female rat following oral administration of 6-PPD at a dosage level of 250 mg/kg/day. The cause for the non-cycling condition of the ovary and the immature appearance of the uterus in the 500 mg/kg/day group, and the relationship between these histologic findings and the earlier onset of vaginal opening in the 500 mg/kg/day group and the lower uterine weights in the 250 and 500 mg/kg/day groups are unclear. The precise nature of the endocrine disruption cannot be determined without a complete weight of evidence analysis taking into consideration results of other endocrine disruption assays.