Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date (date protocol signed by Study Director) 01-Apr-2016 Experimental termination/completion date (signed pathology report) 04-Jan-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
This study was conducted according to Charles River SOPs and the study protocol as approved by the Sponsor.
The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, 28-Jul-2015.

Test material

Reference
Name:
Unnamed
Test material form:
other: yellow semi-solid

Test animals

Species:
rat
Strain:
other: RccHan(WIST) rats
Details on species / strain selection:
Sexually mature male and virgin female Wistar Han (RccHan:WIST) rats were used as the test system on this study. The animal model, the Wistar Han (RccHan:WIST) rat, is recognized as appropriate for reproductive toxicity studies, has been used in previous studies with this test substance, and has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
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 Charles River.
Assignment of individual animals to social groups is presented in Appendix D. 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
solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 13. Females that failed to deliver were housed in solid-bottom cages until post-mating day 25.

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 Charles River. 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 Charles River SOPs. The results of the diet and water analyses are maintained at Charles River. 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.

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 73°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 E. Actual mean daily temperature ranged from 70.4°F to 74.9°F (21.3°C to 23.8°C) and mean daily relative humidity ranged from 42.1% to 67.9% 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Test Substance 1 = neat test sample
information is presented below.

Text Table 1
Test neat test sample Information
Exp. date: 24-Nov-2017 [ID no. 1503AC]
Physical Description Light brown gel
Date of Receipt 30-Nov-2015
Documentation regarding the purity and stability of test substance 1 is on file with the Sponsor and Charles River. A Certificate of Analysis for test substance 1 was provided by the Sponsor and is presented in the attached Appendix B. The purity of test substance 1 was 100%. Test substance 1 was stored at room temperature (18°C to 24°C), and was considered stable under this condition. A reserve sample of test substance 1 was collected and stored in the Charles River Archives.

Test Substance 2 = dilution sample contains 15% test material in mineral oil
Test substance 2 was 15% test item in Yubase 4 oil. Test substance 2 information is presented below.

Text Table 2
Test Substance 2 Information
Date of Receipt 04-Apr-2016
Exp. date: 31-Mar-2018 [ID no. 160107]
Physical Description Pale yellow, clear liquid

Documentation regarding the purity and stability of test substance 2 is on file with the Sponsor and Charles River. A Certificate of Analysis for test substance 2 was provided by the Sponsor and is presented in the attached Appendix B. The purity of test substance 2 was 100%. Test substance 2 was stored at room temperature (18°C to 24°C), and was considered stable under this condition. A reserve sample of test substance 2 was collected and stored in the Charles River Archives.

Test Substance 3 =
Test substance 3 was 1.5% test substance in Yubase 4 oil. Test substance 3 information is presented below.

Text Table 3 = dilution sample 1.5% test material in mineral oil) = dilution sample 1.5% test material in mineral oil
Test Substance 3 Information
Date of Receipt 04-Apr-2016
Exp. date: 31-Mar-2018 [ID no. 160108]
Physical Description Pale yellow, clear liquid

Documentation regarding the purity and stability of test substance 3 is on file with the Sponsor and Charles River. A Certificate of Analysis for test substance 3 was provided by the Sponsor and is presented in the attached Appendix B. The purity of test substance 3 was 100%. Test substance 3 was stored at room temperature (18°C to 24°C), and was considered stable under this condition. A reserve sample of test substance 3 was collected and stored in the Charles River Archives.

Vehicle
The vehicle used in preparation of the test substance formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 1EH0533 and 2EL0356, exp.
dates: 30-Jun-2016 and 31-Dec-2016, respectively).

Preparation
The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.
Dosing formulations were prepared at the test substance concentrations indicated in the following table:

Text Table 4
Dose Formulation Concentration

Group Number Treatment Dosage Level (mg/kg/day) Test Substance Concentration (a) (mg/mL)
1 Vehicle Control 0 0
2 neat test sample 35 8.75
3 neat test sample 100 25
4 neat test sample 350 87.5
5 dilution sample contains 15% test material in mineral oil 160 40
6 dilution sample 1.5% test material in mineral oil) = dilution sample 1.5% test material in mineral oil 160 40
(a) The dosing formulations were not adjusted for purity.

The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.






Details on mating procedure:
Estrous Cycles
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 10 days prior to test substance administration and continuing until evidence of copulation was observed. Pretest estrous cycle data is presented in the attached Appendix F. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. 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 mean individual estrous cycle length calculation.

Breeding Procedures
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.

Mating, fertility, and copulation/conception indices were calculated as follows:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) divided by Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = No. of Males Siring a Litter divided by Total No. of Males Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter divided by No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy divided by Total No. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy divided by No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100


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:
yes
Details on analytical verification of doses or concentrations:
Sampling and Analyses
Homogeneity and stability of the test substance formulations at concentrations of 2.00 and 200 mg neat test sample/mL, 40 mg dilution sample contains 15% test material in mineral oil/mL, and 40 mg dilution sample 1.5% test material in mineral oil/mL were previously
established following a minimum of 12 hours and 5 and 10 days of room temperature storage.1 Therefore, homogeneity and stability analyses were not conducted on the current study.
The previous study1 summarized that the 40 mg dilution sample 1.5% test material in mineral oil/mL formulations did not meet the protocol-specified acceptance criteria for the 5- and 10-day room temperature stability assessments. The 5- and 10-day room temperature resuspension homogeneity assessments at this same concentration did meet the protocol-specified acceptance criteria in the previous study.
The same 40 mg dilution sample 1.5% test material in mineral oil/mL aliquot, stored at room temperature for 10 days, was used to assess both stability and resuspension homogeneity in the previous study. Although the stability assessment, based on the results of the samples collected from the middle stratum, was 86.9% of the time-zero value, the samples collected from the top and bottom strata of this same aliquot, and used for assessment of resuspension homogeneity, resulted in a mean concentration that was 103% of the time-zero value. If all samples collected from this stability/resuspension homogeneity aliquot are averaged, the mean concentration of dilution sample 1.5% test material in mineral oil in the vehicle results in a post-storage concentration that is 91.7% of the time-zero value which meets the acceptance criteria for stability assessments (>90% of the pre-storage value). Therefore, the dilution sample 1.5% test material in mineral oil formulations used on the current study were considered stable for a period of 10 days following room temperature storage which covers the conditions of use on the current study.
Samples for concentration analysis were collected from the middle stratum of the first, approximate middle, and 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 at room temperature as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol detection.1 Details about the methodology and results of these analyses are presented in the attached Appendix C, and the results are summarized in Section “RESULTS Analyses of Dosing Formulations”.
Duration of treatment / exposure:
Organization of Test Groups, Dosage Levels, and Treatment Regimen
The vehicle and test substance 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 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 12) for a total of 48-61 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39-49 doses. The dose volume for all groups was 4 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose (see the attached Appendix A - Study Protocol and Deviations). All animals were dosed at approximately the same time each day.
The following table presents the study group assignment:
Study Group Assignments


Group Number Treatment Dosage Level Dose Volume Number of Animals
Number (mg/kg/day) (mL/kg) Males / Females
1 Vehicle Control 0 4 10 10
2 neat test sample 35 4 10 10
3 neat test sample 100 4 10 10
4 neat test sample 350 4 10 10
5 dilution sample contains 160 4 10 10
15% test material in mineral oil
6 dilution sample contains 160 4 10 10
1.5% test material in mineral oil
Frequency of treatment:
Dosage levels were selected based on the results of a previous 28-day toxicity study in rats.2 In the previous study, male and female rats were dosed with neat test sample for 28 consecutive days at dosage levels of 0, 35, 350, and 600 mg/kg/day. Mortality was observed in 3 females in the
600 mg/kg/day group. Treatment-related findings noted at 350 mg/kg/day included decreased food consumption, increased liver and kidney weights, and sporadic changes in serum chemistry parameters. As a result, a high dosage level of 350 mg/kg/day neat test sample was chosen for the current study. The dosage level of 160 mg/kg/day for the dilution sample contains 15% test material in mineral oil and dilution sample 1.5% test material in mineral oil groups was chosen to assess the toxicity of the lubricant additives that would be put on the market.
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 study schedule:
Assignment of Animals to Treatment Groups
During 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, meeting acceptable body weight requirements, and exhibiting normal 4-5 day estrous cycles (females) 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 Charles River rat colony or euthanized by carbon dioxide inhalation and discarded.
The experimental design consisted of 5 test substance-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 11 weeks old. Male body weights ranged from 244 g to 324 g and female body weights ranged from 178 g to 217 g on study day 0 (see the attached Appendix A - Study Protocol and Deviations). The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 196 g to 243 g on gestation day 0.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle Control
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Remarks:
Neat test sample
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Neat test sample
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
Neat test sample
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Remarks:
Dilution sample contains 15% test material in mineral oil
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Remarks:
Dilution sample 1.5% test material in mineral oil
No. of animals per sex per dose:
10 males & 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
STUDY DESIGN

Acclimation period

10 F0 animals/sex/group (1-6) dosed once daily for at least 14 days prior to mating

Clinical observations, body weights, and food consumption recorded at appropriate intervals

Vaginal lavages performed daily to determine stage of the estrous cycle, beginning 10 days prior to randomization and continuing until evidence of mating observed

F0 animals paired to produce F1 litters

Necropsies performed on females that were found dead or euthanized in extremis


F0 animals dosed daily throughout the mating period and post-mating interval through the day prior to euthanasia

F0 females allowed to deliver and rear their pups to PND 13

F1 pup clinical observations and body weights recorded at appropriate intervals; anogenital distance measured on PND 1

Necropsy of F1 pups that were found dead


Standardization of F1 litters on PND 4; blood samples for possible thyroid hormone evaluation collected from
2 culled pups/litter

Evaluation of developmental parameters in F1 males (areolae/nipple anlagen) on PND 13


F1 pups euthanized on PND 13; 1 pup/sex/litter examined for gross
abnormalities; blood samples for thyroid hormone evaluation collected and thyroid (with parathyroids) collected from
1 pup/sex/litter and placed in 10% neutral-buffered formalin

F0 adults necropsied following completion of the mating period (males) or on lactation day 13 or post-mating day 25 (females);
blood samples for thyroid hormone evaluation collected; selected organs weighed; histopathological evaluation of selected tissues from the control and high-dose groups (Groups 4, 5, and 6)

Positive control:
No

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 moribundity and mortality. Individual clinical observations were recorded daily and individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period) (see the attached Appendix A - Study Protocol and Deviations). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration (see the attached Appendix A - Study Protocol and Deviations). 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. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

Body Weights
Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia (see the attached Appendix A - Study Protocol and Deviations). Individual female body weights were recorded weekly 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 dosing period (males and females) and for the entire dosing period (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 0 (when
possible), 1, 4, 7, 10, and 13. Mean body weights and corresponding mean body weight changes are presented for each of these intervals and for the entire 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 an unscheduled death, as females enter 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 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 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 an unscheduled death, 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 designated as “NA” on the individual report tables.

Oestrous cyclicity (parental animals):
Estrous Cycles
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 10 days prior to test substance administration and continuing until evidence of copulation was observed. Pretest estrous cycle data is presented in the attached Appendix F. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. 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 mean individual estrous cycle length calculation.
Litter observations:
F1 Litter Parameters

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 from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.4 If a skeletal anomaly was suspected, the pup was eviscerated with 100% ethyl alcohol and stained with Alizaran Red S;5 the pups were then examined for skeletal malformations and developmental variations. 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 2 culled pups/sex/litter (pooled by litter) on PND 4 (see Section 5.9.); pups were euthanized by decapitation following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

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 the 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 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae. A finding of “yes” and the number of nipples were recorded if nipples were present, and finding of “no” was recorded if nipples were absent.

Calculation of Litter Parameters

Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0 divided by No. of Litters with Viable Pups PND 0


Postnatal Survival Between Birth and
PND 0 or PND 4
(% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) divided by No. of Litters Per Group x 100


Postnatal Survival for All
Other Intervals (% Per Litter) =Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) divided by No. of Litters Per Group x 100
Where N = PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10,10-13, and 4 (post-selection)-13

Scheduled Euthanasia
On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected from 1 pup/sex/litter immediately prior to euthanasia for thyroid hormone analysis (see Section 5.9.), a gross necropsy was performed, and the thyroids (with parathyroids, if present) were collected and placed in 10% neutral-buffered formalin for possible histopathological examination (see the attached Appendix A - Study Protocol and Deviations). 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 jugular vein from all F0 males and females on the day of scheduled euthanasia (study day 28 for males and lactation day 13 for females). On PND 4, blood (as much as possible; pooled by litter) was collected via cardiac puncture under isoflurane inhalation from 2 culled pups/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 without anticoagulant and allowed to clot at room temperature. Serum was isolated in a refrigerated centrifuge and stored frozen (-65ºC to -85 ºC).
The following thyroid hormone parameters were evaluated for F0 males and F1 PND 13 males and females:
Thyroxine (Total T4)

The samples from the F0 females were not analyzed. Samples from the F1 culled pups (PND 4) were analyzed (see the attached Appendix A - Study Protocol and Deviations).
Clinical pathology methods, procedures, and references are presented in the attached Appendix H. The interpretation of the hormone data was the responsibility of Charles River (the attached Appendix G).
Postmortem examinations (parental animals):
Anatomic Pathology

Macroscopic Examination
A complete necropsy was conducted on all F0 parental animals found dead, euthanized
in extremis, or at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period and females that delivered were euthanized on lactation day 13; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia (see Section 5.9.). 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.3 For females that delivered or had macroscopic evidence of implantation, the numbers of implantation sites or 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. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females with evidence of mating that failed to deliver. 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; see the attached Appendix A - Study Protocol and Deviations):

Text Table 6 Tissue Collection

Brain Pituitary gland
Coagulating glands (2) Prostate gland
Kidneys (2) Seminal vesicles (2)
Liver (sections of 2 lobes) Testes with epididymides a (1) and vas deferens
Mammary glands Thyroids [with parathyroids, if present (2)]
Stomach Uterus b with cervix and vagina
Ovaries and oviducts (2) All gross lesions c
a Testes and epididymides were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
b Uterus not retained if placed in 10% ammonium sulfide solution.
c Representative sections from corresponding organs from a sufficient number of control group animals were retained for comparison, if possible.

Organ Weights
The following organs were weighed from all F0 animals at the scheduled necropsies:

Text Table 7
Organs Weighed at Necropsy

Adrenal glands Liver
Brain Ovaries (with oviducts)
Bulbourethral (Cowper’s) gland a Pituitary gland
Epididymides b Seminal vesicles (with coagulating gland and fluid) a
Glans penis a Spleen
Heart Testes b
Kidneys Thymus gland
Levator ani plus bulbocavernosus (LABC) Thyroids with parathyroids c
muscle complex a
a These organs were weighed and placed in 10% neutral-buffered formalin for possible future histopathologic examination.
b These paired organs were weighed separately. Care was taken to ensure separation between the left and right organs.
c Tissues were weighed after fixation in 10% neutral-buffered formalin.
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data. The organs for which weights could not be determined were designated as “NA” on the individual report tables (see the attached Appendix A - Study Protocol and Deviations).

Histology and Microscopic Examinations
After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River 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 "Macroscopic Examination". from all animals in the control, 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day

dilution sample 1.5% test material in mineral oil groups at the scheduled necropsies. In addition, the nonglandular stomach was identified as a potential target tissue for neat test sample in males and was examined from all males administered neat test sample. 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. The pathology report is presented in the attached Appendix G.
Postmortem examinations (offspring):
Histology and Microscopic Examinations
After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River 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 "Macroscopic Examination". from all animals in the control, 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day

dilution sample 1.5% test material in mineral oil groups at the scheduled necropsies. In addition, the nonglandular stomach was identified as a potential target tissue for neat test sample in males and was examined from all males administered neat test sample. 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. The pathology report is presented in the attached Appendix G
Statistics:
Please see "Statistical Analyses" in any other information section

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Observations and Survival
Summary Data see attached : Table S1, Table S2, Table S3, Table S4, Table S5, Table S6
Individual Data see attached: Table A1, Table A2, Table A3, Table A4, Table A5, Table A6, Table A7
In the 350 mg/kg/day neat test sample group, female no. 9409 was euthanized in extremis on study day 8 following clinical observations that included rales and gasping. In addition, female
no. 9408 in this same group was found dead on gestation day 19. The cause of death was undetermined in both animals. The mortality and moribundity observed in the 350 mg/kg/day group F0 females was not attributed to neat test sample administration due to the absence of any other evidence of toxicity in this group. All other F0 males and females in the control, 35, 100, and 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day dilution sample 1.5% test material in mineral oil groups survived to the scheduled necropsies.
Increased incidences of rales and clear and/or red material around the mouth were noted for
F0 males in the 350 mg/kg/day neat test sample group and F0 females in the 100 and 350 mg/kg/day neat test sample groups approximately 1 hour following dose administration generally throughout the treatment period. Rales were also noted for F0 males and females in these groups to a much lesser degree occasionally at the daily examinations and/or detailed physical examinations.
Although these findings were considered test substance-related, they were generally noted in a subset of animals, the overall incidence versus the total number of doses administered was quite low, and in the absence of any other evidence of toxicity in the F0 animals, these observations were not considered adverse. Other clinical observations noted in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups at the daily examinations, weekly detailed physical examinations, and 1 hour post-dosing observations, including red material around the nose and yellow material around the urogenital area, were noted infrequently and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Clinical Observations and Survival
Summary Data see attached : Table S1, Table S2, Table S3, Table S4, Table S5, Table S6
Individual Data see attached: Table A1, Table A2, Table A3, Table A4, Table A5, Table A6, Table A7
In the 350 mg/kg/day neat test sample group, female no. 9409 was euthanized in extremis on study day 8 following clinical observations that included rales and gasping. In addition, female
no. 9408 in this same group was found dead on gestation day 19. The cause of death was undetermined in both animals. The mortality and moribundity observed in the 350 mg/kg/day group F0 females was not attributed to neat test sample administration due to the absence of any other evidence of toxicity in this group. All other F0 males and females in the control, 35, 100, and 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day dilution sample 1.5% test material in mineral oil groups survived to the scheduled necropsies.
Increased incidences of rales and clear and/or red material around the mouth were noted for
F0 males in the 350 mg/kg/day neat test sample group and F0 females in the 100 and 350 mg/kg/day neat test sample groups approximately 1 hour following dose administration generally throughout the treatment period. Rales were also noted for F0 males and females in these groups to a much lesser degree occasionally at the daily examinations and/or detailed physical examinations.
Although these findings were considered test substance-related, they were generally noted in a subset of animals, the overall incidence versus the total number of doses administered was quite low, and in the absence of any other evidence of toxicity in the F0 animals, these observations were not considered adverse. Other clinical observations noted in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups at the daily examinations, weekly detailed physical examinations, and 1 hour post-dosing observations, including red material around the nose and yellow material around the urogenital area, were noted infrequently and/or in a manner that was not dose-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Summary Data see attached: Table S7, Table S8; Figure 1 Individual Data: Table A8, Table A9
No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects on mean body weights and body weight gains were noted for F0 males throughout the study.

Mean body weight gain in the 350 mg/kg/day neat test sample group was significantly (p<0.01) lower than the control group during study days 7-13, resulting in significantly (p<0.05 or p<0.01) lower mean body weight gains in this group when the pre-mating period (study days 0-13) and entire dosing period (study days 0-27) were evaluated. In addition, a significantly (p<0.01) lower mean body weight gain was noted for F0 males in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group compared to the control group during study days 21-27 which resulted in a significantly (p<0.05) lower mean body weight gain for this group when the entire dosing period was evaluated. These differences were not of sufficient magnitude to affect absolute mean body weights in the
350 mg/kg/day neat test sample group and mean body weights in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group were unaffected throughout the treatment period; therefore, they were not considered test substance-related.

Females

Weekly
Summary Data see attached: Table S9, Table S10; Figure 2 Individual Data: Table A10, Table A11
No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on mean body weights and body weight gains for F0 females during the pre-mating period. None of the differences from the control group were statistically significant.

Gestation
Summary Data see attached: Table S11, Table S12; Figure 3 Individual Data: Table A12, Table A13
No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on mean body weights or body weight gains for F0 females in the test substance-treated groups during gestation. None of the differences from the control group were statistically significant.

Lactation
Summary Data see attached: Table S13, Table S14; Figure 4 Individual Data: Table A14, Table A15
No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on mean body weights or body weight gains for F0 females in the test substance-treated groups during lactation.
Significantly (p<0.05 or p<0.01) lower mean body weight gains were noted for F0 females in the 100 mg/kg/day neat test sample and 160 mg/kg/day dilution sample 1.5% test material in mineral oil groups during lactation days 7-10; however, these differences were transient, there was not a dose-response, and/or mean body weights were unaffected. No other 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):
Males
Summary Data see attached: Table S15 Individual Data: Table A16
Mean food consumption, evaluated as g/animal/day, in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated F0 males was unaffected by test substance administration throughout the study. The only significant (p<0.05) difference from the control group was lower mean food consumption in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group during study days 7-13. This difference was of minimal magnitude and there were no corresponding effects on mean body weight gain and therefore it was not attributed to dilution sample 1.5% test material in mineral oil administration.

Females

Weekly
Summary Data see attached: Table S16 Individual Data: Table A17
Mean food consumption, evaluated as g/animal/day, in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated F0 females was unaffected by test substance administration during the pre-mating period. Significant (p<0.05 or p<0.01) differences from the control group were
sporadic, had no corresponding effect on mean body gains, and therefore were not attributed to test substance administration.

Gestation
Summary Data see attached: Table S17 Individual Data: Table A18
Mean maternal food consumption, evaluated as g/animal/day, in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated F0 females was unaffected by test substance administration during gestation. The only significant (p<0.05) difference from the control group was higher mean food consumption in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group during gestation days 7-11. This difference was transient, there was no corresponding effect on mean body weight gain, and therefore it was not attributed to dilution sample 1.5% test material in mineral oil administration.

Lactation
Summary Data see attached: Table S18 Individual Data: Table A19
Mean maternal food consumption, evaluated as g/animal/day, in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated F0 females was unaffected by test substance administration during lactation. The only significant (p<0.05) differences from the control group were lower mean food consumption in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group during lactation days 1-4 and 7-10. However, these differences were transient, did not affect the overall lactation period (days 1-13), and there were no corresponding effects on mean body weight gains. Therefore, the lower mean food consumption noted in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group was not attributed to dilution sample contains 15% test material in mineral oil administration.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic Examinations
Pathology Report: see the attached Appendix G
Test substance-related microscopic findings were noted in the nonglandular stomach of the 100 and 350 mg/kg/day neat test sample group F0 males. Squamous epithelial hyperplasia was characterized by focally-extensive thickened, proliferative stratified squamous epithelial mucosa with rete peg formation and papillary projections in some animals. Affected epithelium was covered by increased layers of keratin (hyperkeratosis). The subjacent submucosa often contained increased neutrophils and mononuclear cells. Test substance-related mixed cell inflammation and/or squamous epithelial hyperplasia and hyperkeratosis of the nonglandular stomach noted in the 100 and 350 mg/kg/day neat test sample group F0 males was considered to be a response to neat test sample-related gastric irritation, and was considered to be nonadverse.12 Text Table 11 summarizes test substance-related microscopic changes.

Text Table 11
Incidence of Selected Histopathologic Findings, Scheduled Necropsy


Dosage (mg/kg/day): Males Females
Dosage (mg/kg/day): 0 35a 100a 350a 160b 160c 0 35a 100a 350a 160b 160c
Stomach, nonglandular d 10 10 10 10 10 10 NA 1 8 8 10
Hyperkeratosis 0 0 3 6 0 0 0 - 0 0 0 0
Minimal - - 3 2 - - - - - - - -
Mild - - 0 2 - - - - - - - -
Moderate - - 0 2 - - - - - - - -
Hyperplasia, squamous epithelium 1 0 3 6 0 0 0 - 0 0 0 0
Minimal 1 - 3 2 - - - - - - - -
Mild 0 - 0 3 - - - - - - - -
Moderate 0 - 0 1 - - - - - - - -
Inflammation, mixed cell 0 0 0 4 0 0 0 - 0 0 0 0
Minimal - - - 3 - - - - - - - -
Mild - - - 1 - - - - - - - -

a neat test sample
b dilution sample contains 15% test material in mineral oil
c dilution sample 1.5% test material in mineral oil
d Number of tissues examined from each group. NA = Not applicable.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation

other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ Weights
Pathology Report:see the attached Appendix G
There were no test substance-related alterations in final body weights or in organ weights. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Higher mean bulbourethral gland and levator ani plus bulbocavernosus (LABC) muscle complex weights (absolute) were noted in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group F0 males. In addition, higher mean LABC muscle complex weights (absolute and relative to final body and brain weights) were noted in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group F0 males. Most individual bulbourethral gland and LABC muscle complex weights were within the concurrent control group range of weights and the magnitude difference was minimal.
Higher mean ovaries/oviducts weights (absolute and relative to final body and brain weights) were noted in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group F0 females. There were no microscopic correlates and mean weights were within the Charles River Ashland historical control database range. Higher mean kidney weights relative to body weight were noted in the 350 mg/kg/day neat test sample group F0 males. Absolute weights were similar to the control group and kidney weights were within the Charles River Ashland historical control database range. Higher mean liver weights relative to final body weight were noted in the 350 mg/kg/day neat test sample group F0 males. Most individual weights were within the concurrent control group range, all individual weights were within the Charles River Ashland historical control database range, and there were no microscopic correlates. Lower mean spleen weights were noted in the 350 mg/kg/day

neat test sample group (relative to brain weight) and the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group (absolute and relative to brain weight) F0 males. Most individual weights were within the range of concurrent control weights and all were within the Charles River Ashland historical control database range. Higher mean thymus weights (absolute and relative to final body and brain weights) were noted in the 160 mg/kg/day dilution sample 1.5% test material in mineral oil group females. Mean thymus gland weights in the control and test substance-treated group females were lower than the historical control range. Therefore, the statistically significant differences in organ weights were not considered to be test substance-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic Examinations
Summary Data see the attached: Table S23 Individual Data: Table A24 Pathology Report: & the attached Appendix G
There were 2 unscheduled deaths in the 350 mg/kg/day neat test sample group females. Female no. 9409 was euthanized in extremis on study day 8 and female no. 9408 was found dead on gestation day 19. Female no. 9409 was euthanized prior to mating, and had no test
substance-related macroscopic or microscopic findings. Female no. 9408 had a gravid uterus with 3 dead fetuses in the left horn and 8 dead fetuses in the right horn. Increased mucification was noted in the vagina. The cause of death was undetermined in both animals. The relationship of early deaths to administration of neat test sample was considered uncertain, given that both deaths occurred in the high dose group.

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substances at the scheduled necropsies.
The mean numbers of unaccounted-for sites and implantation sites in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were similar to the control group values, with the following exception. A significantly (p<0.05) higher number of unaccounted-for sites was noted in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group compared to the control group. However, in the absence of any test substance-related effects on reproductive performance (i.e., number of pups born), this difference was not attributed to dilution sample contains 15% test material in mineral oil administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic Examinations
Pathology Report: see the attached Appendix G
Test substance-related microscopic findings were noted in the nonglandular stomach of the 100 and 350 mg/kg/day neat test sample group F0 males. Squamous epithelial hyperplasia was characterized by focally-extensive thickened, proliferative stratified squamous epithelial mucosa with rete peg formation and papillary projections in some animals. Affected epithelium was covered by increased layers of keratin (hyperkeratosis). The subjacent submucosa often contained increased neutrophils and mononuclear cells. Test substance-related mixed cell inflammation and/or squamous epithelial hyperplasia and hyperkeratosis of the nonglandular stomach noted in the 100 and 350 mg/kg/day neat test sample group F0 males was considered to be a response to neat test sample-related gastric irritation, and was considered to be nonadverse.12 Text Table 11 summarizes test substance-related microscopic changes.

Text Table 11
Incidence of Selected Histopathologic Findings, Scheduled Necropsy


Dosage (mg/kg/day): Males Females
Dosage (mg/kg/day): 0 35a 100a 350a 160b 160c 0 35a 100a 350a 160b 160c
Stomach, nonglandular d 10 10 10 10 10 10 NA 1 8 8 10
Hyperkeratosis 0 0 3 6 0 0 0 - 0 0 0 0
Minimal - - 3 2 - - - - - - - -
Mild - - 0 2 - - - - - - - -
Moderate - - 0 2 - - - - - - - -
Hyperplasia, squamous epithelium 1 0 3 6 0 0 0 - 0 0 0 0
Minimal 1 - 3 2 - - - - - - - -
Mild 0 - 0 3 - - - - - - - -
Moderate 0 - 0 1 - - - - - - - -
Inflammation, mixed cell 0 0 0 4 0 0 0 - 0 0 0 0
Minimal - - - 3 - - - - - - - -
Mild - - - 1 - - - - - - - -

a neat test sample
b dilution sample contains 15% test material in mineral oil
c dilution sample 1.5% test material in mineral oil
d Number of tissues examined from each group. NA = Not applicable.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation

other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Reproductive Performance
Summary Data see attached: Table S19, Table S20 Individual Data: Table A20, Table A21

F0 male and female reproductive parameters are presented in the following table.

Text Table 10
Results of Reproductive Performance

Dosage Level (mg/kg/day)
neat test sample dilution sample contains 15% test material in mineral oil dilution sample 1.5% test material in mineral oil
Parameter 0 35 100 350 160 160
Male Mating Index (%) 100.0 100.0 100.0 100.0 100.0 100.0
Female Mating Index (%) 100.0 100.0 100.0 100.0 100.0 100.0
Male Fertility Index (%) 100.0 90.0 80.0 100.0 90.0 100.0
Female Fertility Index (%) 100.0 90.0 80.0 100.0 90.0 100.0
Male Copulation Index (%) 100.0 90.0 80.0 100.0 90.0 100.0
Female Conception Index (%) 100.0 90.0 80.0 100.0 90.0 100.0
Estrous Cycle Length (days) 4.1 4.0 4.8 4.3 4.1 4.1
Pre-Coital Interval (days) 2.6 2.3 3.6 2.9 2.3 4.4
No neat test sample-. dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects on reproductive performance were noted in the test substance-treated groups. No statistically significant differences were noted between the control and test substance-treated groups. One and 2 males in the 35 and
100 mg/kg/day neat test sample groups, respectively, and 1 male in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group did not sire a litter. One, 2, and 1 females in these same respective groups were determined to be nongravid.
The mean numbers of days between pairing and coitus in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-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.

Gestation Length and Parturition
Summary Data see attached: Table S21, Table S22 Individual Data: Table A22, Table A23
Mean gestation lengths in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Thyroid Hormone Analysis (F0 Males)
Pathology Report: SEE the attached Appendix G
There were no test substance-related alterations in total thyroxine (T4) values noted for F0 males in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
Gestation Length and Parturition
Summary Data see attached: Table S21, Table S22 Individual Data: Table A22, Table A23
Mean gestation lengths in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
No neat test sample-. dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects on reproductive performance were noted in the test substance-treated groups. No statistically significant differences were noted between the control and test substance-treated groups.

Details on results (P0)

In the 350 mg/kg/day neat test sample group, 1 F0 female was euthanized in extremis on study day 8 following clinical observations of rales and gasping. In addition, 1 F0 female in this group was found dead on gestation day 19; no remarkable clinical or macroscopic findings were noted for this female. The cause of death/moribundity in the 350 mg/kg/day group F0 females could not be determined at necropsy. The mortality and moribundity observed in the 350 mg/kg/day group

F0 females was not attributed to neat test sample administration due to the absence of any other evidence of toxicity in this group. All other F0 males and females in the neat test sample -, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups survived to the scheduled necropsies.
neat test sample -related clinical observations of rales and increased incidences of red and clear material around the mouth were noted for F0 males in the 350 mg/kg/day group and F0 females in the 100 and 350 mg/kg/day groups at approximately 1 hour following dose administration generally throughout the treatment period; occasionally these findings persisted to the daily examinations on the following day. These findings were not considered adverse. No other neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related clinical observations were noted.
No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on F0 male and female body weights, body weight gains, or food consumption throughout the study. F0 estrous cycle length, reproductive performance (mating, fertility, copulation, and conception indices), gestation length, and the process of parturition were unaffected by neat test sample, dilution sample contains 15% test material in mineral oil, and dilution sample 1.5% test material in mineral oil administration. No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on F1 pup growth, viability, clinical condition, anogenital distance, or areolae/nipple anlagen (males only).
There were no neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related macroscopic findings or alterations in organ weights. No test substance-related effects on T4 values were noted in F0 males and F1 male pups.
Test substance-related mixed cell inflammation and/or squamous epithelial hyperplasia and hyperkeratosis of the nonglandular stomach noted in the 100 and 350 mg/kg/day neat test sample group F0 males was considered to be a response to neat test sample-related gastric irritation, and was considered to be nonadverse. No other test substance-related microscopic findings were noted for F0 males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups.
For F1 pups, no neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related organ weight alterations or macroscopic findings were noted.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the lack of adverse neat test sample-related effects at any dosage level,
Remarks on result:
other: Under the conditions of this screening study, based on lack of adverse neat test sample-related effects @ any dosage level,350 mg/kg/day (the highest dosage level of neat test sample tested) was considered to be the NOAEL
Key result
Dose descriptor:
NOAEL
Effect level:
160 other: 160 mg/kg/day dilution sample contains 15% test material in mineral oil
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were noted following administration of 160 mg/kg/day dilution sample contains 15% test material in mineral oil
Remarks on result:
other: See remarks
Remarks:
for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity when administered orally by gavage to RccHan(WIST) rats. In addition, no adverse effects were noted following administration of 160 mg/kg/day dilution sample contains 15% test material in mineral oil or 160 mg/kg/day dilution sample 1.5% test material in mineral oil; therefore, these dosage levels were considered to be the NOAEL for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity.
Key result
Dose descriptor:
NOAEL
Effect level:
160 other: 160 mg/kg/day dilution sample containing 1.5% test materia
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were noted following administration of 160 mg/kg/day dilution sample containing 1.5% test material in mineral oil
Remarks on result:
other: please see remarks section
Remarks:
no adverse effects were noted following administration of 160 mg/kg/day dilution sample containing 1.5% test material in mineral oil therefore, this dosage level was considered to be the NOAEL for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
General Physical Condition
Summary Data see attached: Table S26 Individual Data: see attached Table A28
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 substances. Pups (litters) that were found dead numbered 0(0), 0(0), 1(1), 1(1), 1(1), and 0(0) in the control, 35,
100, and 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day dilution sample 1.5% test material in mineral oil groups, respectively. Four, 1, and 9 pups in the control, 100 mg/kg/day neat test sample, and
160 mg/kg/day dilution sample 1.5% test material in mineral oil groups, respectively, were missing and presumed to have been cannibalized.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
PND 0 Litter Data and Postnatal Survival
Summary Data see attached: Table S24, Table S25
Individual Data: see attached Table A25, Table A26, Table A27
The mean number of pups born, live litter size, and the percentage of males at birth in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were similar to the control group values. Postnatal survival in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups was unaffected by test substance administration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring Body Weights
Summary Data see attached: Table S29, Table S30; Figure 5, Figure 6 Individual Data: Table A31, Table A32
Mean male and female pup body weights and body weight changes during PND 1-13 in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were unaffected by parental administration of the test substance. Differences from the control group were slight and not statistically significant, with the following exceptions. Statistically lower mean body weight gains were noted for F1 males and females in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group during
PND 7-10 and for F1 males in the 100 mg/kg/day neat test sample group during PND 10-13. Due to the lack of corresponding effects on mean absolute body weights, these decrements were not considered adverse.
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:
no effects observed
Description (incidence and severity):
Scheduled Pup Necropsies (PND 13)
Summary Data see attached: Table S33, Table S34 Individual Data: Table A35, Table A36
No internal findings were noted at the necropsy of pups euthanized on PND 13. Although not protocol-specified, the thyroid/parathyroids from PND 13 pups were weighed (see the attached Appendix A - Study Protocol and Deviations). There were no test substance-related alterations in thyroid/parathyroid weights noted for F1 males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups on PND 13.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Although not protocol-specified, the thyroid/parathyroids from PND 13 pups were weighed (see the attached Appendix A - Study Protocol and Deviations). There were no test substance-related alterations in thyroid/parathyroid weights noted for F1 males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups on PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Scheduled Pup Necropsies (PND 13)
Summary Data see attached: Table S33, Table S34 Individual Data: Table A35, Table A36
No internal findings were noted at the necropsy of pups euthanized on PND 13. Although not protocol-specified, the thyroid/parathyroids from PND 13 pups were weighed (see the attached Appendix A - Study Protocol and Deviations). There were no test substance-related alterations in thyroid/parathyroid weights noted for F1 males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups on PND 13.
Histopathological findings:
no effects observed
Description (incidence and severity):
No internal findings were noted at the necropsy of pups euthanized on PND 13
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Anogenital Distance
Summary Data see attached: Table S27, Table S28 Individual Data: Table A29, Table A30
Mean anogenital distances (absolute and relative to the cube root of pup body weight) in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Areolae/Nipple Anlagen
Summary Data see attached: Table S31 Individual Data: Table A33
No retained nipples were noted in F1 male pups at any dosage level. The test substance-treated group values were not statistically different from the control group values.

Thyroid Hormone Analysis (PND 4 and 13)
Summary Data see attached: Table S31A Individual Data: Table A33A Pathology Report: the attached Appendix G
There were no test substance-related alterations in total T4 values noted for F1 males in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups on PND 13. Although not required by the protocol, serum samples from PND 4 culled pups were analyzed (see the attached Appendix A - Study Protocol and Deviations). There were no test substance-related alterations in total T4 values noted for F1 males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-treated groups on PND 4.

Necropsies of Pups Found Dead
Summary Data see attached: Table S32 Individual Data: Table A34
The numbers of pups (litters) found dead during PND 0-13 numbered 0(0), 0(0), 1(1), 1(1), 1(1),
and 0(0) in the control, 35, 100, and 350 mg/kg/day neat test sample, 160 mg/kg/day dilution sample contains 15% test material in mineral oil, and 160 mg/kg/day dilution sample 1.5% test material in mineral oil groups, respectively. Aside from the absence or presence of milk in the stomach, the following internal findings were noted. Pup no. 9391-01 in the 160 mg/kg/day dilution sample contains 15% test material in mineral oil group had malformations that consisted of a bent tail (distal portion with no apparent skeletal origin), an absent kidney and ureter, and a vertebral anomaly with associated rib anomaly (fused, misshapen, and malproportioned centra and arches and fused and misshapen sternebrae and ribs). In addition, pup no. 9412-07 in the 350 mg/kg/day neat test sample group had a developmental variation that consisted of a distended ureter.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
other: Parental animals
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the lack of adverse neat test sample-related effects at any dosage level,
Remarks on result:
other: Under the conditions of this screening study, based on lack of adverse neat test sample-related effects @ any dosage level,350 mg/kg/day (the highest dosage level of neat test sample tested) was considered to be the NOAEL
Key result
Dose descriptor:
other: F0 systemic toxicity, F0 reproductive toxicity
Generation:
other: F0
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see Remarks

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
350 mg/kg bw/day (actual dose received)
Treatment related:
no
Relation to other toxic effects:
not specified
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Under the conditions of this screening study, based on the lack of adverse neat test sample-related effects at any dosage level, a dosage level of 350 mg/kg/day (the highest dosage level of neat test sample tested) was considered to be the no-observed-adverse-effect level (NOAEL) for

F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity when administered orallyby gavage to RccHan(WIST) rats. In addition, no adverse effects were noted following administration of 160 mg/kg/day dilution sample contains 15% test material in mineral oil or 160 mg/kg/day dilution sample 1.5% test material in mineral oil; therefore, thesedosage levels were considered to be the NOAEL for F0systemic toxicity, F0rproduc tivetoxicity, and F1neonatal toxicity.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, based on the lack of adverse neat test sample-related effects at any dosage level, a dosage level of 350 mg/kg/day (the highest dosage level of neat test sample tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity when administered orally by gavage to RccHan(WIST) rats. In addition, no adverse effects were noted following administration of 160 mg/kg/day dilution sample contains 15% test material in mineral oil or 160 mg/kg/day dilution sample 1.5% test material in mineral oil; therefore, these dosage levels were considered to be the NOAEL for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity.
Executive summary:

 Objective

The objective of the study was to provide preliminary information on the potential adverse effects of the test substances on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0generation and the development of offspring from conception through day 13 of postnatal life.

 

StudyDesign

The test substances, neat test sample, dilution sample contains 15% test material in mineral oil, and dilution sample 1.5% test material in mineral oil, in the vehicle (arachis [peanut] oil) were administered orally by gavage once daily to 5 groups of RccHan(WIST) rats, each group consisting of 10 males and 10 females. Dosage levels were 35, 100, and 350 mg/kg/day for the neat test sample -treated groups (Groups 2-4) and 160 mg/kg/day for the dilution sample contains 15% test material in mineral oil- and

dilution sample 1.5% test material in mineral oil-treated groups (Groups 5 and 6, respectively). A concurrent control group of

10 rats/sex received the vehicle on a comparable regimen. The dose volume was 4 mL/kg for all groups. Males and females were approximately 11 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating and were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 12 for a total of

48-61 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39-49 doses.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. All F0females were allowed to deliver and rear their pups until lactation day 13. F1clinical 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 (2/litter). All F1male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanized on PND 13; from 1 pup/sex/litter, blood samples for thyroid hormone analysis were collected, pups were examined for gross abnormalities, and the thyroid (with parathyroids) was collected. Blood samples for thyroid hormone analysis were collected from F0males and females; only male samples were analyzed.F0males were euthanized following completion of the mating period and F0females wereeuthanized on lactation day 13 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 high-dose groups (Groups 4, 5, and 6).

 

 Results

In the 350 mg/kg/day neat test sample group, 1 F0female was euthanizedin extremison study day 8 following clinical observations of rales and gasping. In addition, 1 F0 female in this group was found dead on gestation day 19; no remarkable clinical or macroscopic findings were noted for this female. The cause of death/moribundity in the 350 mg/kg/day group F0females could not be determined at necropsy. The mortality and moribundity observed in the 350 mg/kg/day group


F0 females was not attributed to neat test sample administration due to the absence of any other evidence of toxicity in this group. All other F0males and females in the neat test sample -, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups survived to the scheduled necropsies.

neat test sample -related clinical observations of rales and increased incidences of red and clearmaterial around the mouth were noted for F0males in the 350 mg/kg/day group and F0females inthe 100 and 350 mg/kg/day groups at approximately 1 hour following dose administration generally throughout the treatment period; occasionally these findings persisted to the daily examinations on the following day. These findings were not considered adverse. No other neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related clinical observations were noted.

No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on F0male and female body weights, body weight gains, or food consumption throughout the study. F0estrous cycle length, reproductive performance (mating, fertility, copulation, and conception indices), gestation length, and the process of parturition were unaffected by neat test sample, dilution sample contains 15% test material in mineral oil, and dilution sample 1.5% test material in mineral oil administration. No neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related effects were noted on F1 pup growth, viability, clinical condition, anogenital distance, or areolae/nipple anlagen (males only).

There were no neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related macroscopic findings or alterations in organ weights. No test substance-related effects on T4values were noted inF0males and F1male pups.

Test substance-related mixed cell inflammation and/or squamous epithelial hyperplasia and hyperkeratosis of the nonglandular stomach noted in the 100 and 350 mg/kg/day neat test sample group F0males was considered to be a response to neat test sample-related gastric irritation, and was considered to be nonadverse. No other test substance-related microscopic findings were noted for F0males and females in the neat test sample-, dilution sample contains 15% test material in mineral oil-, and dilution sample 1.5% test material in mineral oil-treated groups.

For F1pups, no neat test sample-, dilution sample contains 15% test material in mineral oil-, or dilution sample 1.5% test material in mineral oil-related organ weight alterations or macroscopic findings were noted.

 

 Conclusion

Under the conditions of this screening study, based on the lack of adverse neat test sample-related effects at any dosage level, a dosage level of 350 mg/kg/day (the highest dosage level of neat test sample tested) was considered to be the no-observed-adverse-effect level (NOAEL) for

F0 systemic toxicity, F0reproductive toxicity, and F1 neonatal toxicity when administered orallyby gavage to RccHan(WIST) rats. In addition, no adverse effects were noted following administration of 160 mg/kg/day dilution sample contains 15% test material in mineral oil or 160 mg/kg/day dilution sample 1.5% test material in mineral oil; therefore, thesedosage levels were considered to be the NOAEL for F0 systemic toxicity, F0 reproductivetoxicity, and F1 neonatal toxicity.