Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
.
Deviations:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
1,4-diisopropylbenzene
Lot no. 6F50825000
CAS no. 100-18-5
[WIL ID no. 15025F]
Clear, colorless liquid
date of receipt: 27-Aug-2015
purity: 99.8%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT, AND ACCLIMATION PERIOD
Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat. The number of animals selected for this study (10 rats/sex/group) was based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 28-Jul-2015, which recommends evaluation of at least 8 pregnant females per group. Give the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination. Crl:CD(SD) rats (50 males and 50 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 20-Aug-2015. The animals were approximately 60 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 12 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL HOUSING
Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. 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. Assignment of individual animals to social groups is presented in Appendix C. During cohabitation, rats were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in plastic maternity cages with bedding material. The dams and their litters were housed in these cages until euthanasia on PND 13 (pups) or lactation day 14 (dams). Females that failed to deliver were housed in plastic maternity cages until post-mating day 25. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). 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.

DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, 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. Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. 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, with the following exception. All F0 animals (including animals not selected for clinical pathology evaluations) were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

ENVIRONMENTAL CONDITIONS
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. These data are summarized in Appendix D. Actual mean daily temperature ranged from 69.9°F to 72.0°F (21.1°C to 22.2°C) and mean daily relative humidity ranged from 45.1% to 60.5% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were
transferred to the WIL Research colony. The experimental design for this study consisted of 3 test item-treated groups and 1 control group composed of 10 rats/sex/group. At the initiation of dose administration (study day 0), the males and females were approximately 10 weeks old. Male body weights ranged from 326 g to 396 g and female body weights ranged from 223 g to 278 g on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 230 g to 323 g on gestation day 0.

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS, AND TREATMENT REGIMEN
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The males were dosed during study days 0-27 or 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28-29 doses. The females were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-59 doses. The female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The dose volumes for Group 1, Group 2, Group 3, and Group 4 were 1.18, 0.12, 0.35, and 1.18 mL/kg, respectively. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
group treatment dosage level dosage volume number of animals per group
number (mg/kg/day) mL/kg) males females
1 Vehicle Control 0 1.18 10 10
2 1,4-DiPB 100 0.12 10 10
3 1,4-DiPB 300 0.35 10 10
4 1,4-DiPB 1000 1.18 10 10

Dosage levels were determined from the results of previous studies and were provided by the Sponsor after consultation with the Study Director. Additionally, the dosage levels selects for the current study test up to the limit dose of 1000 mg/kg/day. The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 13. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
Analytical verification of doses or concentrations:
no
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behavior, moribundity, mortality, and signs of overt toxicity. Individual clinical observations were recorded weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour 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. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties. Due to social housing, some observations could not be attributed to a single animal; however, no social housing observations were noted.

BODY WEIGHTS
Individual male body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until scheduled euthanasia. Individual female body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 10, and 13. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation and lactation intervals (days 0-20 and 1-13, respectively). When body weights could not be determined for an animal during a given interval (due to females entering gestation, 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

FOOD CONSUMPTION
Food consumption was recorded on the corresponding weekly body weight days until pairing. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 10, and 13; food consumption was reported as g/animal/day and g/kg/day during gestation and lactation. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOB ASSESSMENTS
FOB assessments were recorded for 5 animals/sex/group following 23 days of dose administration (study week 3; males) and on lactation day 13 (females; following separation from pups). The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991; and O’Donoghue, 1989). FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below (refer to Appendix E for a detailed description of the scoring criteria used for each observation and to Appendix F for positive control/validation data):

MOTOR ACTIVITY
Motor activity was assessed for 5 animals/sex/group following 23 days of dose administration (study week 3; males) and on lactation day 13 (females; following separation from pups). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams). Refer to Appendix F for motor activity validation and positive control data.

CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 or 29 for males and lactation day 14 for females). The same animals selected for FOB and motor activity assessments were selected for clinical pathology assessments. All F0 animals (including those not selected for clinical pathology assessments) were fasted overnight prior to blood collection with water available ad libitum. Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing
K2EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
Postmortem examinations (parental animals):
MACROSCOPIC EXAMINATION
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia (see Section 5.11.). Females that delivered were euthanized on lactation day 14 and blood samples were collected for possible thyroid hormone analysis immediately prior to euthanasia (see Section 5.11.). Females that failed to deliver were euthanized on post-mating day 25; uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Females with total litter loss were euthanized within 24 hours of litter loss. For females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed. Number of corpora lutea were also recorded for females necropsied during gestation through lactation day 4. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):

Adrenal glands (2)
Aorta
Levator ani plus bulbocavernosus (LABC)
muscles
Bone with marrow (sternebrae) Ovaries and oviducts (2)
Brain Pancreas
Coagulating glands (2) Peripheral nerve (sciatic)
Cowper’s gland (2) Pituitary gland
Eyes with optic nerve (2)a Prostate gland
Gastrointestinal tract Salivary gland [mandibular (2)]
Esophagus Seminal vesicles (2)
Stomach Skeletal muscle (rectus femoris)
Duodenum Skin with mammary glandb
Jejunum Spinal cord (cervical)
Ileum
Cecum
Spleen
Testes with epididymidesc (1) and vas
Colon
Rectum
deferens
Thymus gland
Glans penis Thyroids [with parathyroids, if present (2)]
Heart Trachea
Kidneys (2) Urinary bladder
Lymph node Uterusd with cervix and vagina
Axillary (2) All gross lesions
Mandibular (2)
Mesenteric
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by
inflation with fixative)

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands Liver
Brain Ovaries with oviducts
Cowper’s gland (2) Prostate gland
Epididymidesa Pituitary gland
Glans penis Spleen
Heart Testesa
Kidneys Thymus gland
Levator ani plus bulbocavernosus
(LABC) muscles
Thyroids with parathyroids

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides. Microscopic examination was performed on all tissues listed in Section 5.9.1. from all animals in the control and 1000 mg/kg/day groups at the scheduled necropsies. Gross lesions were examined from all groups. At the scheduled necropsy, the liver (males and females) and the adrenal cortex (males only) were examined from the 100 and 300 mg/kg/day group animals. 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.

Postmortem examinations (offspring):
LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead between PND 0-4 were necropsied using a fresh dissection technique, which included examination of the heart, brain by mid coronal slice, and major vessels (Stuckhardt and Poppe, 1984). A detailed gross necropsy was performed on any pup found dead after PND 4. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

LITTER REDUCTION
To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. All selections were performed by computerized randomization. Blood samples for possible future thyroid hormone analysis were collected from 1 culled pup/sex/litter (pooled by litter) on PND 4 (see Section 5.11.); pups were euthanized by decapitation following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanized by intraperitoneal injection of sodium pentobarbital, and discarded on PND 4.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Any abnormalities in nesting and nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and absolute distance relative to the cube root of body weight were reported for each pup. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

BODY WEIGHTS
Pups were individually weighed on PND 1, 4, 7, 10, and 13. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup 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 a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

SEX DETERMINATION
Pups were individually sexed on PND 0, 4, and 13.

ASSESSMENT OF AREOLAS/NIPPLE ANLAGEN
On PND 12, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae in accordance with methods described by Gray et al. (1999). A finding of “yes” and the number of nipples were recorded if nipples were present, and a finding of “no” was recorded if nipples were absent.

SCHEDULED EUTHANASIA
On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia (see Section 5.11.) from 1 pup/sex/litter; the thyroids (with parathyroids, if present) were weighed (following fixation) and placed in 10% neutral-buffered formalin for possible histopathological evaluation. Remaining pups (not used for blood collection) were discarded without examination.

THYROID HORMONE ANALYSIS
Blood samples for thyroid hormone analysis were collected as follows. Blood (approximately 1.0 mL) was collected via the retro-orbital sinus following isoflurane anesthesia from all F0 males and females on the day of scheduled euthanasia (study day 28 or 29 for males and lactation day 14 for females). On PND 4, blood (as much as possible; pooled by litter) was collected via cardiac puncture under isoflurane inhalation from 1 culled pup/sex/litter. On PND 13, blood (approximately 1.0 mL) was collected via cardiac puncture under isoflurane inhalation from 1 pup/sex/litter. Blood was collected into tubes containing no anticoagulant and allowed to clot at room temperature. Serum was isolated in a refrigerated centrifuge and stored frozen
(approximately -70°C). Thyroxine (T4) concentrations were evaluated for F0 males and F1 PND 13 males and females.

Statistics:
STATISTICAL ANALYSES
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit.

ANALYSES CONDUCTED BY WIL RESEARCH
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, pre-coital intervals, gestation length, numbers of former implantation sites, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and
relative organ weights, clinical pathology values, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), numbers of nipples/areolae, and FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL OBSERVATIONS AND SURVIVAL
Test item-related clinical observations of red and/or clear material around the mouth were noted for males and females in all test item-treated groups approximately 1 hour following dose administration. These observations were noted throughout the treatment period and occurred in a dose-related manner. However, the material observations did not persist to the weekly examinations and were considered nonadverse in the absence of other signs of systemic toxicity (body weight and food consumption decrements. There were no test item-related clinical observations noted at the weekly examinations. Observations noted in the test item-treated groups, including hair loss on various body surfaces and yellow material in the urogenital area, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

BODY WEIGHTS
MALES
Mean body weight gain in the 1000 mg/kg/day group males was similar to the control group during the pre-mating treatment period (study days 0-13) and during study days 13-21. A test item-related, significantly (p<0.01) lower mean body weight gain was noted in this group during study days 21-27 compared to the control group. As a result, a lower mean body weight gain was noted in the 1000 mg/kg/day group when the entire treatment period (study days 0-27) was evaluated; the difference from the control group was significant (p<0.05). The effect on mean body weight gain during the last week of treatment was not of sufficient magnitude to affect absolute mean body weights for this group, and therefore, was not considered adverse. Mean body weights and body weight gains in the 100 and 300 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.

BODY WEIGHTS
FEMALES (pre-mating)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

FOOD CONSUMPTION
MALES
Mean food consumption, evaluated as g/animal/day, in the 1000 mg/kg/day group males was significantly (p<0.01) lower (2 g/animal/day) than the control group during the first week of treatment (study days 0-7). This decrement was noted in the absence of an effect on mean body weight gain during this same interval. Mean food consumption in the 1000 mg/kg/day group was similar to the control group during study days 7-13, and therefore the effect on mean food consumption noted during the first week of test item administration was considered transient and not test item-related. Mean food consumption in the 100 and 300 mg/kg/day group males was similar to that in the control group throughout the study. None of the differences from the control group were statistically significant.

FOOD CONSUMPTION
FEMALES (pre-mating)
Mean food consumption, evaluated as g/animal/day, in the 100, 300, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

FEMALES (gestation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

FEMALES (lactation)
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. Differences from the control group were not statistically significant, occurred in a manner that was not
dose-related, and/or were transient.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

OPEN FIELD OBSERVATIONS
Open field parameters were unaffected by test item administration. A significantly (p<0.05) higher mean rearing count was noted in the 1000 mg/kg/day group males during study week 3 compared to the control group. However, in the absence of effects on any other effects on CNS activity, the higher rearing count was not considered to be test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. A significantly (p<0.05) lower mean hindlimb grip strength value was noted in the 300 mg/kg/day group males during study week 3 compared to the control group value. However, no dose-response was evident, and therefore this difference was not considered test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

MOTOR ACTIVITY
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 3 (males) and on lactation day 13 (females). Significantly (p≤0.008) lower mean total counts were noted in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval. However, the differences were the result of a greater habituation response in these groups relative to the control group during the first 20 minutes of testing. When rats are placed in a novel environment, exploratory behavior (resulting in a high level of activity counts) followed by habituation (resulting in a reduced level of activity counts) is an expected response. In addition, there were no statistically significant differences in the cumulative total counts at these dosage levels. Therefore, the lower activity counts in the 300 and 1000 mg/kg/day group males during the 11-20 minute subinterval were attributed to biologic variability and were not considered test item-related. All other values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL Research historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL Research historical control data ranges, and/or did not occur in a dose-related manner. Other than the greater habituation response described previously for the
300 and 1000 mg/kg/day males during the 11-20 minute subinterval, no remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 3 or lactation day 13.

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male in the 1000 mg/kg/day group did not sire a litter, as the female that this male was paired with was nongravid. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
The mean gestation length in the 1000 mg/kg/day group (22.3 days) was significantly (p<0.05) longer than the concurrent control group (21.7 days). The 1000 mg/kg/day group gestation length value was within the WIL Research historical control range (21.3 to 22.3 days) and was primarily attributed to a single female (no. 2852) that delivered on gestation day 24 (and subsequently had a total litter loss on lactation day 0). Therefore, the
longer mean gestation length at 1000 mg/kg/day was not considered test item-related. Mean gestation lengths in the 100 and 300 mg/kg/day groups (21.7 and 22.0 days, respectively) were similar to those in the concurrent control group. No signs of dystocia were noted in any group.

CLINICAL PATHOLOGY
SERUM CHEMISTRY
Test item-related higher cholesterol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and bile acids (1000 mg/kg/day group females), lower glucose (1000 mg/kg/day group males and females), higher phosphorus (300 and 1000 mg/kg/day group males, 1000 mg/kg/day group females) and higher urea nitrogen (1000 mg/kg/day group females) values were noted at study week 4 (males) or lactation day 14 (females). Minimal to mild elevations of cholesterol, ALP, ALT, and bile acids in 1000 mg/kg/day group females were coincident with test item-related hepatocellular hypertrophy; differences from the control group were significant (p<0.05 or p<0.01) for cholesterol, ALT, and bile acids. Minimally lower glucose was not associated with lower food consumption. Minimally to mildly higher phosphorus (significant at p<0.01 or p<0.05, males and p<0.05, females) and urea nitrogen (significant at p<0.01, females) were suggestive of dehydration but urinalysis was not performed in the present study. Glucose, phosphorous and urea changes were considered nonadverse given the magnitude of change. There were no other test item-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups
were compared. Lower urea nitrogen in the 100 mg/kg/day group males was not considered test item-related given the lack of a dose related response and a high control group mean and individual values.





MACROSCOPIC EXAMINATIONS
Test item-related dark red discoloration of the liver was noted in 100, 300, and 1000 mg/kg/day group males and correlated with angiectasis in single males in the 300 (no. 2827) and 1000 (no. 2797) mg/kg/day groups. Dark red discoloration in remaining animals in the 100, 300, and 1000 mg/kg/day groups correlated with sinusoidal congestion or was not associated with a relevant gross finding in 1 male (no. 2813) in the 1000 mg/kg/day group. The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values.

ORGAN WEIGHTS
Test item-related higher liver weights were noted in the 1000 mg/kg/day group males and females. Higher liver weights (absolute, relative to body and brain weight) were associated with test item-related hepatocellular hypertrophy and the weight relative to body weight was significant (p<0.01) in males. Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a non-test item-related higher body weight. The thyroid/parathryoid weight relative to brain weight was higher in 1000 mg/kg/day group females.
There were no other test item-related effects on organ weights. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Higher mean Cowper’s gland weights were noted in the 100 (absolute, relative to brain and body weight), 300 (absolute and relative to body weight), and 1000 (relative to body weight) mg/kg/day group males but were not considered test item-related given the lack of a dose-related response and lack of correlating microscopic changes.

MICROSCOPIC EXAMINATIONS
Test item-related microscopic findings were noted in the adrenal cortex of the 1000 mg/kg/day group males and liver of the 1000 mg/kg/day group males and females. Liver: Minimal to mild hepatocellular hypertrophy, characterized by increased amounts of cytoplasm, was noted in the centrilobular region of the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females. Angiectasis, characterized by focally extensive dilation of sinuosoids, was observed in a single male each in the 300 and 1000 mg/kg/day groups (male nos. 2827 and 2797, respectively) and correlated with dark red discoloration in the liver. Angiectasis was differentiated from routine terminal congestion that was noted in the control and
all test item-treated groups and was characterized by blood in the sinusoids. Adrenal cortex: There was an increased incidence and severity of cytoplasmic vacuolation of the adrenal cortex in the 1000 mg/kg/day group males. Vacuolation was characterized by variably sized, well demarcated vacuoles in the zona fasciculata and was not considered adverse. Vacuolation was also noted in females but vacuoles were uniform and there was no test item-related difference. There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Female no. 2852, which had a total litter loss, had mild myeloid hyperplasia of the bone marrow and a moderate increase in splenic extramedullary hematopoeisis which were both related to moderate uterine subacute inflammation with endometrial hyperplasia and mild mixed cell infiltrate in the vagina. Female no. 2846, which failed to conceive, was in early diestrus at the time of necropsy (study day 39).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10, 10-13, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 13 were unaffected by test item administration at all dosage levels. Differences form the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner. Slightly lower postnatal survival was noted during PND 0 relative to birth and when birth to PND 4 was evaluated in the 1000 mg/kg/day group; however, these differences were not statistically significant compared to the control group and were attributed to 1 female (no. 2852) in this group that had a total litter loss on PND 0.

GENERAL PHYSICAL CONDITION
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups (litters) that were found dead numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. One, 2, and 2 pups in the control, 300, and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

ANOGENITAL DISTANCE
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes during PND 1-13 in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental administration of the test item. No statistically significant differences from the control group were noted.

AREOLAE/NIPPLE ANLAGEN
Areolae/nipple anlagen in the F1 male pups was not affected by parental administration of the test item. The test item group values for males were not statistically significantly different from the control group.

THYROID HORMONE ANALYSIS (PND 13)
There were no test item-related changes in T4 levels in F1 males or females on PND 13.

ORGAN WEIGHTS (PND 13)
There were no test item-related changes in thyroid/parathyroid weights for F1 pups at 100, 300, and 1000 mg/kg/day.

NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead during PND 0-13 numbered 5(5), 9(5), 3(2), and 14(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. No internal findings that could be attributed to parental test item administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, the following internal findings were noted. Pup no. 2882-10 in the 1000 mg/kg/day group was noted with a malformation consisting of a right-sided aortic arch (aortic arch and descending aorta coursed to the right of the vertebral column; the right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]; and the left carotid and subclavian arteries arose from a common vessel from the aortic arch). Dark red subcutis contents were noted for pup no. 2871-01 in the 1000 mg/kg/day group. In the 100 mg/kg/day group, pup no. 2873-15 was noted with clear fluid in the abdominal cavity and renal papilla(e) not developed.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of 1,4-diisopropylbenzene when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test item-related clinical observations or effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones, or macroscopic/microscopic alterations in F0 males and females at any dosage levels. Therefore, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for postnatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.
Executive summary:

The objectives of the study were to evaluate the potential toxic effects of the test item, 1,4-diisopropylbenzene, when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

STUDY DESIGN: The test item, 1,4-diisopropylbenzene, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dose volumes of 0.12, 0.35, and 1.18 mL/kg. A concurrent control group of 10 rats/sex received the control item (deionized water) at a dose volume of 1.18 mL/kg on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28-29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 49-59 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following 23 days of dose administration and for 5 females/group on lactation day 13. All F0 females were allowed to deliver and rear their pups until lactation day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (1/sex/litter) but were not analyzed. All F1 male pups were evaluated for areolae/nipple anlagen on PND 12. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0 animals/sex/group just prior to scheduled necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females prior to euthanasia; only male samples were analyzed. F0 males were euthanized following completion of the mating period and F0 females were euthanized on lactation day 14 for females that delivered or post-mating day 25 for females that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and 1000 mg/kg/day groups. In addition, the adrenal cortex (males) and liver (males and females) were identified as potential target tissues and were examined from all animals.

RESULTS: All F0 males and females survived to the scheduled necropsies. Red and/or clear material around the mouth was noted in a dose-related manner for males and females in all test item-treated groups approximately 1 hour following dose administration throughout the treatment period. The aforementioned material observations did not persist to the weekly examinations and were

considered nonadverse in the absence of other signs of systemic toxicity (body weight and food consumption decrements). No test item-related clinical observations were noted for F0 males and females at the weekly examinations at any dosage level. Mean body weight gain in the 1000 mg/kg/day group males was similar to the control group during the pre-mating treatment period (study days 0-13) and during study days 13-21. A test item-related, lower mean body weight gain was noted in this group during study days 21-27 and when the entire treatment period (study days 0-27) was evaluated. However, the effect on mean body weight gain during the last week of treatment was not of sufficient magnitude to affect absolute mean body weights, and therefore, was not considered adverse. Mean body weights, body weight gains, and food consumption for F0 males at 100 and 300 mg/kg/day and females at

100, 300, and 1000 mg/kg/day were unaffected by test item administration throughout the study. No test item-related effects were noted during the FOB or motor activity evaluations at any dosage level. There was 1,4 -diisopropylbenzene-related minimal to mild nonadverse hepatocellular hypertrophy in the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females that was associated with higher liver weights in males and females at 1000 mg/kg/day and nonadverse elevations in alkaline phosphatase, alanine aminotransferase, bile acids, and cholesterol in females at 1000 mg/kg/day. Nonadverse angiectasis of the liver was noted in the 300 and 1000 mg/kg/day group males and correlated with gross observations of dark red discoloration. Increased incidence and severity of nonadverse adrenal cortical vacuolation was noted in the 1000 mg/kg/day group males. Other nonadverse findings included lower glucose values (1000 mg/kg/day group males and females), higher urea nitrogen values (1000 mg/kg/day group females), and higher phosphorus (300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females). There were no test

item-related changes in hematology and coagulation parameters for F0 males and females. T4 levels in F0 males were unaffected by test item administration. F0 male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. There were no test item-related effects on the mean number of implantation sites and unaccounted-for sites at any dosage level.

There were no test item-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 clinical observations, postnatal survival and growth, anogenital distance, or areolae/nipple anlagen. There were no test item-related macroscopic findings for F1 pups that were found dead at any dosage level. There were no test item-related changes in T4 levels or

thyroid/parathyroid weights in F1 males or females on PND 13.