Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0 animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item
administration.
- Weight at study initiation: 142.4 to 190.6 g; female group mean range 137.4 to 165.1 g.
- Fasting period before study: Not applicable.
- Housing: During the acclimation period (F0 animals only) and throughout the study, all F0 and F1 parental animals were housed
individually, except during the mating period, in solid-bottom polycarbonate cages with stainless steel wire lids. The rats were paired
for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period),
the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. Beginning on pre-natal
Day 21, the selected F1 pups were individually housed until the start of the mating period.
- Diet: Purina Certified Pelleted Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO) was available ad libitum in the wire cage
lids. Available information on the diet did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Water (tap water) was available ad libitum via plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes.
Available information on the water did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Acclimation period: Seven days

ENVIRONMENTAL CONDITIONS
- Temperature: (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hr dark / hrs light):12 hours continuous light and 12 hours darkness
IN-LIFE DATES: First day of treatment: 13 October 2009 - Final necropsy day was 07 July 2010.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared as solutions at concentrations of 0 (vehicle), 25, 100, and 375 mg/mL were formulated 10 times (approximately every 4 weeks), dispensed into 30-mL amber glass bottles with Teflon-lined lids for use as daily dose aliquots, and stored at room temperature protected from light. Concentrations of 25 and 375 mg/mL in the same vehicle have been shown to be homogeneous and stable for 42 days when stored refrigerated or at room temperature protected from light (Analytical Chemistry report. Report date: 10 March 2010). The test item formulations (but not the vehicle formulation) were stirred continuously throughout dose administration.

Prior to use on study, samples (~25 mL each) for homogeneity determination were collected from the top, middle, and bottom strata of each test item formulation from 3 of the 10 formulation dates (06 October 2009, 26 January 2010, and 15 June 2010). Samples (~25 mL each) for concentration analysis were also collected from the middle stratum of each dosing formulation (including the vehicle formulation) from these same 3 formulation dates. All analyses were conducted using a validated gas chromatography method with flame ionization detection (AM-0212131-002.0). Details about the methodology and results of these analyses are presented in Appendix 1, and summarized as follows:

Based on these results, the analyzed dosing formulations were found to contain the amount of test item prescribed in the protocol (±10% of nominal) and were homogeneous. Therefore, the protocol-specified dosages of test item were administered to the animals.

-DIET PREPARATION
Not applicable
- Rate of preparation of diet (frequency): Not applicable
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: No data
- VEHICLE: Corn oil
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: 0, 25, 100, and 375 mg/mL
- Amount of vehicle (if gavage): 2 ml/kg
- Lot/batch no. (if required): Not applicable
Details on mating procedure:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group).
- Length of cohabitation: Following positive evidence of mating (or at the completion of the 2-week mating period).
- Proof of pregnancy: Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after
pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were
evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs.
Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- After a set number of days of unsuccessful pairing replacement of first male by another male with proven fertility.: Not applicable
- Further matings after two unsuccessful attempts: Not applicable
- After mating each pregnant female was returned to individual caging: Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method has been satisfactorily validated and details about the methodology and results of these analyses are presented in detail.
The results are summarized as follows.
Three sets of dose formulation samples were analyzed for concentration verification and homogeneity assessment for the test item in corn oil formulations had previously been evaluated for homogeneity , in long-term storage, and animal room dosing stabilities in the concentration range from 25 to 375 mg/mL and the detailed analytical procedures were reported in an Analytical Chemistry Report, Report Date 10 March 2010. The formulation analyses reported here were performed following an validated analytical method to verify the test item concentration and homogeneity in corn oil formulations prepared on October 6, 2009 and January 26 and June 15, 2010. All dose formulations analyzed were found to be within ±10% of the nominal concentration and were homogeneous.

ANALYTICAL METHOD
The validated analytical method used to analyze study samples is outlined as follows:
For each dose formulation sample, triplicate 1 mL aliquots were in 10-mL volumetric flasks and diluted to volume with n-hexane. Processed formulation samples were then analysed on a gas chromatograph with a flame ionization detector along with a series of vehicle standards used to generate a calibration curve. Vehicle standards were prepared by mixing blank formulations samples. Test item concentrations were calculated using a least square s linear regression equation that fit the relationship between the nominal concentration s of vehicle standard and the detector response (ie the sum of integrated areas of selected peaks within the chromatogram). The formulation sample concentrations were determined in mg/g and converted to mg/mL measuring the formulation densities.
Duration of treatment / exposure:
Duration of treatment / exposure
All male and female for the F0 generation and F1 generation were administered the test item or vehicle control, daily for at least 70 consecutive days prior to mating. The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
A complete gross necropsy was conducted after death for any parental (F0 and F1) or offspring (F1 and F2) animals found in moribund condition or dead on study. Organs were not weighed for moribund animals. Histopathological examination of study specified organs were performed for all high dose and control parental (F0 and F1). Additionally, reproductive organs of the low and mid dose animals suspected of reduced fertility were subjected to histopathological evaluation. For any organ(s) demonstrating possible treatment-related changes in the high dose group versus the organ(s) of interest were evaluated histopathologically from successively lower concentration dose groups until a clear no observed adverse effect level (NOAEL) was established. However, at the request of the Sponsor, no further evaluation of the lungs from lower dose groups was conducted.
Frequency of treatment:
The test and vehicle control item formulations were administered to the F0 and F1 males and females and once daily for a minimum of 70 consecutive days prior to mating and for males continued throughout mating to the day prior to termination. The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through to the day prior to termination. The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero andthrough nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22).
Details on study schedule:
Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0 and F1 generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. A concurrent control group of 25 and 28/sex for the F0 and F1 generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia. The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21). The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22). The first day of dosing was designated as Day 1 of the study. Animals were paired on a 1 male: 1 female basis within each dose group. The rats were paired for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids.
Remarks:
Doses / Concentrations:
50 mg/kg/day (25 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day (100 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day (mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
F0 generation
0 mg/kg/day (Control) - 25 per sex
50 mg/kg/day - 25 per sex
200 mg/kg/day - 25 per sex
750 mg/kg/day - 25 per sex

F1 generation
0 mg/kg/day (Control) - 28 per sex
50 mg/kg/day - 28 per sex
200 mg/kg/day - 28 per sex
750 mg/kg/day - 28 per sex
Details on study design:
- Dose selection rationale: Dosage levels were selected based on repeat-dose studies on similar substances and a 90-day repeat-dose study on 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' and 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'. In addition to repeat-dose studies on conventional petroleum-based diesel, a number of repeat-dose studies on individual components of the test substance were available. These studies do not give any indication of unexpected effects and are in line with the observations in 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' studies. The selected route of administration was oral (gavage) by the Sponsor because this is a possible route of human exposure. The number of animals selected for this study was the minimum required to yield statistically and scientifically meaningful data and was consistent with regulatory agency expectations.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- See attached Summary Tables 6, 14, 20, 25, 41, 49, 55, 60 and Individual Data: Appendix 2, Tables 4, 10, 17, 35, 41, 45 and 48
- Mortality
Observations were made twice daily at least 6 hours apart.
- Clinical examinations
- Time schedule: Clinical observations were conducted and recorded at least once daily, approximately 1-2 hours post dosing, throughout the course
of the study.

BODY WEIGHT: - See attached Summary Tables 2, 3, 10, 11, 16, 17, 21, 22, 37, 38, 45, 46, 51, 52, 56 and 57 and Appendix 2, Tables 2, 8, 15, 31,
32, 33, 39, 43 and 46.
- Time schedule for examinations:
Individual body weights of the male F0 and F1 rats were determined and recorded on the first day of dosing and weekly through the
pre breed exposure and mating periods.
The body weights of male F0 and F1 rats were recorded in the same manner after the mating period until scheduled euthanasia.
The body weights of female F0 and F1 rats were recorded in the same manner until evidence of mating was observed.
During gestation, sperm-positive (presumed pregnant) females were weighed on gestational days (GD) 0, 7, 14, and 20.
Dams producing litters were weighed on lactational days 0, 4, 7, 14, and 21.

FOOD CONSUMPTION AND EFFICIENCY:
- See attached Summary Tables 4, 5, 12, 13, 18, 19, 23, 24, 39, 40, 47, 48, 53, 54, 58 and 59 and Individual Data: Appendix 2,
Tables 3, 9, 16, 34, 40, 44and 47.
Individual F0 and F1 male and female feed consumption was measured weekly until mating.
Feed consumption was not recorded during the 2-week mating period.
Following mating, male feed consumption was measured on a weekly basis until the scheduled necropsy.
During pregnancy of F0 and F1 females, feed consumption was recorded on gestation days 0, 7, 14, and 20.
During lactation of F1 and F2 litters, maternal feed consumption was measured on 0, 4, 7, 14, and 21.
- Food efficiency:
Percent food efficiency (body weight gained as a percentage of food consumed) was also calculated

WATER CONSUMPTION:
Not applicable

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Not applicable
- Time schedule for collection of blood:
Not applicable
- Anaesthetic used for blood collection:
Not applicable.
- Animals fasted:
Not applicable.
- How many animals:
Not applicable.

URINALYSIS:
Not applicable

NEUROBEHAVIOURAL EXAMINATION:
Not applicable.
- Time schedule for examinations:
Not applicable.
Behavioural assessment)
Not applicable.
(Functional Performance Tests)
Not applicable.
(Sensory Reactivity)
Not applicable.
- Dose groups that were examined:
Not applicable.

OTHER:
- MATING
Animals were paired on a 1 male: 1 female basis
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- see attached Summary Tables 15, 32 and 50 and Individual Data: Appendix 2, Tables 11, 28, 42 and 59

PREGNANCY AND PARTURITION
During the period of expected parturition, the females were observed at least twice daily for initiation and completion of parturition. On the day parturition was completed (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 completed.
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1
males as appropriate.
Litter observations:
Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1 and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
Postmortem examinations (parental animals):
Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from the F0 and F1 parental animals were examined microscopically.
Postmortem examinations (offspring):
Non selected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21.
Statistics:
STATISTICAL METHODS
See Attachment “"Report Text" pages 34 to 35.
Reproductive indices:
For the Mating, fertility, and gestational indices see attached ""Report Text" page 25.
Offspring viability indices:
See attached Report Text Pages 1 to 74, Results Tables and Appendices.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29.
There were no deaths attributed to test substance administration noted through the F0 prebreed exposure period (Days 0 through 70). Seven unscheduled deaths (3 found dead and 4 euthanized for humane reasons) occurred on study through Day 70. Male No. 153 (Group 4) and Female No. 152 (Group 4) were found dead on Day 1 and Day 68, respectively. The cause of death for these animals was not conclusively determined from the gross necropsy. Red staining around the nose and left eye (Male No. 153) or nose and mouth (Female No. 152), mottled (Male No. 153, correlated with alveolus hemorrhage) or discolored/dark (Female No. 152) lungs, and reddish, dark fluid in the intestines (Female No. 152 only) indicate that aspiration of the test item is the most likely cause of death of these animals. Female No. 154 (Group 4) was found dead on Day 27, approximately 1.2 hours postdose, due to an apparent dosing error, as evidenced by red fluid around the nose and mouth, lungs that failed to collapse and were discolored (dark), and fluid expelled from the trachea with slight pressure applied to lungs.

Male No. 7 (Group 1), Male No. 185 (Group 4), and Female No. 124 (Group 3) were euthanized on Days 56, 66, and 70, respectively. Gross observations at necropsy (mottled/discolored lungs, red/dark fluid in stomach and gastrointestinal tract, red staining around mouth, and/or red fluid in mouth) indicate most likely aspiration of the test item, leading to the clinical signs that prompted humane euthanasia of these animals; microscopic lung findings for Male No. 7 and 185 further support this conclusion. In addition, Male No. 143 (Group 3) was euthanized for humane reasons on Day 16 due to bilateral lesions on the neck that worsened despite veterinary intervention. All other animals survived to Day 70. There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were
noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Two males (Nos. 115 and 127, Group 3) exhibited neck lesions that
required veterinary intervention; the neck lesions healed by Day 70.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33 Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29.
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There was an increased incidence of rooting in animals given 750 mg/kg/day and of chromodacryorrhea (eye) in animals given the 200 and 750 mg/kg/day compared with the control group. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Male No. 127 (Group 3; terminated as scheduled on Day 106) exhibited a neck lesion that required veterinary intervention on Days 97 to 105; this same animal had previous neck lesions requiring veterinary intervention that had healed by Day 72.

For the F0 females, there were no deaths attributed to the test substance administration during the mating, gestation, and lactation periods (Days 70 to 128); there were 3 unscheduled F0 female deaths during this time. Female Nos. 38 (Group 1) and 180 (Group 4) were severely injured by their mating partner and were therefore euthanized on Day 72 for humane reasons (both were sperm negative on Day 72 prior to euthanasia). There were no macroscopic necropsy findings for these 2 females other than the wound inflicted by the mating partner. Female No. 166 (Group 4) was euthanized for humane reasons on Day 95 (GD 22) due to dystocia with concomitant hypothermia. Macroscopic necropsy findings for Female No. 166 included pale, mottled, mildly enlarged adrenal glands and mottled lungs (all lobes), and there were 18 visible implant sites that corresponded to 18 pups (3 live and 15 dead) in the uterus.

From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F0 females, there were occasional observations of struggling during dosing in animals given 200 and 750 mg/kg/day, and there were very occasional observations of rooting post dosing and salivating prior to dosing in up to 2 females given 750 mg/kg/day during the gestation period (GD 0 through 23). Likewise, there were occasional observations of animals struggling during dosing in the groups given 200 and 750 mg/kg/day during the lactation period. One dam (No. 118) given 200 mg/kg/day had an eye injury and swollen nose from LD 17 to 21 that were not test item-related. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26 Individual Data: Appendix 2, Tables 6, 11, 18, 19.
No test item-related effects on F0 reproductive performance were observed at any dosage level. There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 69.6%, 80.0%, 95.8%, and 90.9% and female mating indices were 70.8%, 80.0%, 95.8%, and 90.9% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data for male and female mating indices ranged from 87.5% to 100% and 88% to 100%, respectively. Following a thorough investigation, which included a detailed review of all the environmental data during the study and discussions with the animal vendor, the reason for the low mating indices in the control and low-dose groups remained unknown; however, 2 males (Nos. 3 and 41) were infertile based on andrology parameters, thus partially accounting for the lower mating indices in the control group. Male fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% and female fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% in the same respective groups. All fertility indices were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for F0 females.

All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (3.8 to 4.0 days) or number of cycles (4.4 to 4.6) in test item-treated groups compared with control values (3.9 days and 4.4, respectively). The mean number of days between pairing and coitus (i.e., the precoital interval) in the test itemtreated groups was similar to the control group values of 2.4 days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 26 Individual Data: Appendix 2, Table 19.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. The mean gestation lengths in the groups given 50, 200, and 750 mg/kg/day were 22.4, 22.2, and 22.4 days, respectively, compared to the mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 94.4%, 95.5%, and 89.5% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range of 87% to 100%. There were corresponding increases and decreases in the stillbirth index and live birth index, respectively, for groups given 200 and 750 mg/kg/day. These slight changes in the live born gestation index, stillbirth index, and live birth index in the group given 750 mg/kg/day were due to Female No. 166 being euthanized on GD 22 due to dystocia and Female No. 200 having an all-stillborn litter. Likewise, the slight changes in these same indices for the group given 200 mg/kg/day were due to Female No. 128 having a presumed all-stillborn litter, and the slight decrease in live born gestation index for the group given 50 mg/kg/day was due to Female No. 86 not delivering any pups, though having 5 visible implant sites at necropsy. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent post implantation loss, pups delivered (live, dead, and total), and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Slight, nondose-related increases in feed consumption (g/day and g/kg/day) were noted for males and females at several intervals (occasionally statistically significant) for all test item-treated groups when compared with the control group; however, these changes in feed consumption did not result in or correlate with statistically significant changes in body weights, body weight changes, or food efficiency compared with the controls. There were no adverse test item-related effects on F0 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. A statistically significant increase in feed consumption (g/day and g/kg/day) for one interval (Days 84 to 91) was noted for the F0 males given 750 mg/kg/day; however, this change in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or food efficiency compared with the controls. In general, there were no notable test item-related effects on body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F0 females that were never found sperm positive or that never
delivered a live litter. Occasional statistically significant changes were observed in these parameters, but were not considered biologically significant as they were sporadic and not dose related.

GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation, there were no adverse test item-related changes in F0 female body weights and feed consumption (g/day and g/kg/day). Compared with the concurrent control group, there was a statistically significant decrease (~17%) in percent food efficiency from GD 7 to 14 for the females given 750 mg/kg/day, which correlated with a decrease (~16%) in body weight change that was not statistically significant for this same group during the same interval. The following interval (GD 14 to 20) also showed a slight, though not statistically significant, decrease in percent food efficiency and body weight change (~10% and ~8%, respectively) for the females given 750 mg/kg/day compared with the concurrent control group. Thus, percent food efficiency and body weight change were slightly reduced (~8% and ~7%, respectively) between GD 0 and 20 but did not achieve statistical significance for the females given 750 mg/kg/day compared with the control group; thus, these changes were considered incidental to treatment and not adverse.

LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no notable test item-related or statistically significant changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.

ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6.
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant. As per the protocol, assessments using the frozen right testis, as well as sperm morphology evaluations, were conducted initially on the control and high-dose groups only. As there were no test itemrelated effects in the group given 750 mg/kg/day, evaluation of the lower dose groups was not conducted for these endpoints.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males given 200 and 750 mg/kg/day. At the F0 male necropsy, 7 males given 750 mg/kg/day were noted as having pale lungs (cranial lobe, left or bilateral), and this gross finding correlated with an increase in both absolute and relative lung weights in this dose group. Two males given 200 mg/kg/day also had pale lungs (cranial lobe, left); however, there was no significant increase in lung weights in this dose group. The thymus of 2 males (Nos. 187 and 199) given 750 mg/kg/day had multiple, pinpoint, red foci. There was an increased incidence of mottled kidneys (bilateral) in the males given 200 and 750 mg/kg/day (10 of 24 and 9 of 23 animals, respectively) compared with the control and low-dose groups (5 of 24 and 5 of 25 animals, respectively). The testes of 2 control males (Nos. 3 and 41) were found to be reduced in size (bilateral) and flaccid (bilateral), and the epididymides of Male No. 3 were also reduced in size. Neither of these males (Nos. 3 and 41) sired a litter and both had few motile or progressively motile sperm (thus the reason for the larger standard error in these parameters for the control group compared with the test item-treated groups). All other macroscopic gross findings were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. There were no test item-related macroscopic findings for the F0 females necropsied on LD (≡PND) 21. All macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was an increase in both absolute and relative lung weights (38% and 33%, respectively) in F0 males given 750 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (14.0% and 12.8%, respectively) in F0 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic (males) and microscopic (males and females) lung findings in rats given 750 mg/kg/day. Although there was a statistically significant increase (approximately 10%) in the absolute lung weight for F0 males given 50 mg/kg/day, there was no significant increase in relative lung weight and no macroscopic lung findings in this same group, as well as no significant changes in absolute and relative lung weights for F0 females given 50 mg/kg/day and F0 males and females given 250 mg/kg/day; therefore, the change in absolute lung weight for F0 males given 50 mg/kg/day was considered spurious.

MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3.
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 males and females given 750 mg/kg/day compared with the controls. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test itemexposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning.

There did not appear to be any significant difference between male and female lungs with regard to the appearance of interstitial/alveolus inflammation. Another similar pulmonary finding (alveolar macrophage aggregation, alveolus) was noted in control male and female lungs and
was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle. An equivocal, non-adverse histopathological lesion was identified in the kidneys. Renal tubule mineralization was observed in the kidneys of 7 of 23 males given 750 mg/kg/day. This lesion was characterized by the presence of multifocal mineralized renal tubules (proximal convoluted tubules) in the outer cortex. Similar appearing tubules were not seen in any of the 24 male F0 controls. The renal tubules were characterized as basophilic tubules with mineralization of both the nucleus and cytoplasm affected. Some tubules were strongly mineralized and others less.

There was no apparent cause for the mineralization and no histopathological reaction to the mineralized tubules was noted. In the study pathologist’s experience, these tubules appeared similar to artifactual changes occasionally observed as a result of tissue fixation and/or processing. Since mineralized tubules may be an artifactual change and were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal. Other changes in the kidneys such as chronic progressive nephropathy and renal tubule regeneration were not clearly increased in either incidence or in severity and were therefore not deemed test item-related. The microscopic examination of reproductive tissues for F0 animals suspected of reduced fertility in the low- and mid-dose groups did not have any relationship to test item administration. A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Lack of adverse treatment-related effects observed at the highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No developmental effects in offspring
Remarks on result:
other: Generation: F1 and F2 (migrated information)
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
LITTER DATA F1
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 26, Individual Data: Appendix 2, Tables 19, 20.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21(for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant. The slight reduction (which was not statistically significant) in the 4-day survival index per litter in the group given 750 mg/kg/day was because the entire litter for Female No. 198 was found dead (3 pups) or euthanized (12 pups) prior to PND 2 due to poor clinical condition (including no milk band, cold to touch, reduced activity, and loss of skin elasticity) due to the lack of care for the pups by the dam.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 28, Individual Data: Appendix 2, Tables 20, 23.
The numbers of F1 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F1 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 4, 6, 12, and 19 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 198) accounted for 15 of the 19 pups in the group given 750 mg/kg/day. In addition, 3, 3, 2 and 0 pups in the same respective groups were either missing (and presumed dead) or cannibalized.

ANOGENITAL DISTANCE
Summary Data: Table 27, Individual Data: Appendix 2, Table 21.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F1 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 27, Individual Data: Appendix 2, Table 22.
Mean male and female pup body weights and body weight changes in the groups given 50, 200, and 750 mg/kg/day were unaffected by test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 28, 29, Individual Data: Appendix 2, Tables 23, 24.
The numbers of pups (litters) stillborn, found dead, or euthanized from PND 0 through the selection of the F1 generation on PND 21 numbered 4(4), 8(5), 12(6), and 19(5) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of pups that were stillborn, found dead, or euthanized. The combined incidence of bilateral and unilateral hydronephrosis in stillborn pups was not different from the control group, nor was the incidence of hydronephrosis seen in pups necropsied as scheduled on PND 21 different in test item-treated groups compared with the control group; therefore, the increased pup and litter incidence of bilateral hydronephrosis in stillborn pups in both the 200 and 750 mg/kg/day dose groups compared with the control group was not considered test item-related. There were several F1 pups found dead and euthanized moribund in the 200 mg/kg/day and/or 750 mg/kg/day groups, with observations of no milk in the stomach, indicating the pups were not nursing. There were no F1 pups found dead in the control group and no F1 pups euthanized moribund in the control and 200 mg/kg/day dose groups, so a direct comparison of the incidenceof this macroscopic finding across groups in unscheduled death pups is not possible. The one low-dose pup euthanized moribund also had no milk in the stomach and loss of skin elasticity noted at necropsy. Of the 12 pups euthanized moribund in the high-dose group (all from Dam No. 198), 3 had distended ureter (either unilateral or bilateral); however, the incidence of this finding in the culled pups from all dose groups was similar, and thus this finding was not considered to be test item-related.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 2, Table 26.
At the PND 21 necropsy of F1 weanlings, there were no findings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.

ORGAN WEIGHTS
Summary Data: Table 30, Individual Data: Appendix 2, Table 25.
There were no statistically significant changes in F1 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 9% to 10% and 4% to 5%, respectively) were observed in F1 pups in the group given 750 mg/kg/day compared with the control group. The spleen weights in F1pups from the high-dose group in this study fall within the historical control data ranges for this parameter and were therefore considered an equivocal, non-adverse finding.

MICROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 3.
There were no microscopic findings in the F1 PND 21 weanlings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.

F1 DEVELOPMENTAL LANDMARKS
BALANOPREPUTIAL SEPARATION
Summary Data: Table 36, Individual Data: Appendix 2, Table 31.
The test item-related increase in attainment of balanopreputial separation in males given 750 mg/kg/day was not considered adverse, as the day of attainment falls within the historical control ranges for this parameter. The mean ages of attainment of balanopreputial separation were 43.3, 43.2, and 43.2 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 41.6 in the control group, and were all statistically significantly longer than the control group, though in a nondose-responsive manner and within the historical control range for the mean age of attainment of balanopreputial separation of 40.8 to 43.6 days. Mean body weights at the age of attainment were 254.0, 249.8, and 245.4 g in the same respective groups, compared with 238.9 g in the control group, and were not statistically significant. When adjusted with body weight as a covariate, the adjusted mean ages of attainment of balanopreputial separation were 43.0, 43.1, and 43.3 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 42.0 in the control group, and although dose-responsive, only the group given 750 mg/kg/day was statistically significantly longer than the control group. This test item-related increase in adjusted age of attainment of balanopreputial separation was not considered adverse, as 43.3 days falls within the historical control ranges for this parameter of 40.7 to 43.8 days. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group with the longest delay in attainment of balanopreputial separation were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse.

VAGINAL PATENCY
Summary Data: Table 36, Individual Data: Appendix 2, Table 32.
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test item administration. The mean ages of attainment of vaginal patency were 31.3, 30.6, and 30.0 days in the groups given 50, 200, and 750 mg/kg/day, respectively compared with 29.9 days in the control group. Mean body weights at the age of attainment were 117.5, 111.0, and 105.5 g in the same respective groups compared with 108.6 g in the control group. The mean age for the group given 50 mg/kg/day was statistically significant, though was not considered test item-related as there was no dose-response and the adjusted mean age was not statistically significantly different from the control group.

GENERATION F1
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 35, 41, 43, 49, 68, Individual Data: Appendix 2, Tables 30, 35, 38, 41, 60.
There were no deaths directly attributed to the test substance administration noted through the F1 weaning and prebreed exposure period (Days -20 through 70). Seven unscheduled deaths (6 found dead and 1 euthanized for humane reasons) occurred through Day 70. Male No. 1147 (Group 2, 50 mg/kg/day) was found dead on Day -2 (PND 36) after being noted as lethargic the previous day. Male No. 1305 (Group 4, 750 mg/kg/day) was also found dead on Day -2 (PND 38). The cause of death for these 2 males could not be determined upon necropsy; there were no obvious signs of gavage error and some autolysis was present. Group 4 Male Nos. 1329 and 1351 were found dead on Days 2 (PND 41) and 3 (PND 40), respectively. Both males had hydronephrosis of the right kidney and dark red lungs. The lungs from Male No. 1351 failed to collapse and had pale areas around the edges of all lobes. Slight pressure on the lungs released bubbles from the trachea, suggesting the cause of death of Male No. 1351 was a gavage error. Based on the appearance of the lungs at necropsy, Male No. 1329 may have died due to aspiration of the dose formulation. Group 4 Female Nos. 1346 and 1354 were found dead on Days 18 (PND 56) and 30 (PND 67), respectively. Female No. 1346 had struggled during dosing the day prior to being found dead. At necropsy, the only finding was red substance in the nose and mouth. The cause of death of Female No. 1346 could not be determined upon necropsy. Female No. 1354 was noted as lethargic at the postdose observation time and died a short time later. At necropsy, slight hydronephrosis of the right kidney was seen, and the lungs were found to be red and failed to collapse fully. Slight pressure on the lungs released fluid and bubbles from the trachea, suggesting the cause of death of Female No. 1354 was gavage error. Female No. 1348 (Group 4, 750 mg/kg/day) was euthanized for humane reasons on Day 54 (PND 91) on welfare grounds. At necropsy, reddish fluid from nose and mouth, reddish fluid in the intestines, very little food present in the stomach, and dark red lungs that did not fully collapse were noted, suggesting the apparent cause of death was gavage error. All other animals survived to Day 70. There was a slightly increased incidence of rooting in male and female F1 animals given 750 mg/kg/day and of chromodacryorrhea (eye) in females given 200 mg/kg/day and 750 mg/kg/day compared with the control group. These observations are not adverse. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were limited to unscheduled death animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND
LACTATION PERIODS
Summary Data: Tables 41, 43, 49, 55, 60, 68, Individual Data: Appendix 2, Tables 35, 38, 41, 45, 48, 60.
For the F1 males, there was one death during the mating/postmating period (Days 70 through 107). Male No. 1325 (Group 4, 750 mg/kg/day) was noted as lethargic and gasping for breath prior to dying on Day 78. At necropsy, the lungs were mottled and failed to fully collapse; when slight pressure was applied to the lungs, bubbles were released from the trachea. The apparent cause of death of this animal was either a direct dosing error or inadvertent aspiration of the test item leading to a chronic aspiration pneumonia. All clinical findings in the F1 male, test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For the F1 females, there were no deaths directly attributed to the test substance administration during the mating, gestation, and lactation periods. Female No. 1352 (Group 4, 750 mg/kg/day) was found dead on Day 96 (LD 1) with the macroscopic necropsy finding of dark red substance present in the uterus. On the previous day (LD 0), clinical signs for Animal No. 1352 included piloerection, dystocia, labored breathing, lethargy, and excess red substance present; as the animal was not hypothermic and was actively delivering pups at the time of evaluation by the veterinarian, the veterinarian recommendation was for continued monitoring. From Day 71 through Day 113, there were no test item-related clinical observations in the F1 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F1 females, there were no test item-related clinical observations during the gestation period (GD 0 through 23) or lactation period (LD 0 through 21). One dam (No. 1316) given 750 mg/kg/day a mass/sore on the chest for the entire lactation period and from days 9 (mass) or 18 (sore) through 21 of gestation. The area was not seen at terminal necropsy and thus was not saved for evaluation. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 50, 61
Individual Data: Appendix 2, Tables 42, 49, 50
No test item-related effects on F1 reproductive performance were observed at any dosage level. There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 92.9%, 92.6%, 85.7%, and 88.0% and female mating indices were 92.9%, 92.9%, 85.7%, and 88.0% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data male and female F1 mating indices ranged from 70.0% to 100% and 50% to 100%, respectively. Male fertility indices were 96.2%, 92.0%, 95.8% and 81.8% and female fertility indices were 96.2%, 92.3%, 95.8% and 81.8% in the same respective groups. Although the fertility index for males and females was lower in the group given 750 mg/kg/day, all fertility indicies were within the historical control data ranges of 76.2% to 100% for F1 males and 70.8% to 100% for F1 females. All test item-treated F1 females evaluated during the final 3 weeks of the 10-week pre breed period were noted to be cycling compared with 89.3% of the control females noted to be cycling.
There was no statistically significant difference in the number and percent of test item-treated females with abnormal or irregular cycles compared with the control group. There were no differences in mean cycle length (4.1 to 4.3 days) and number of cycles (4.0 to 4.4) in test item treated groups compared with control values (4.0 days and 4.6, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group values of 2.8 days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 61, Individual Data: Appendix 2, Table 50.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Gestation indices were 100%, 100%, and 88.9% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 96.0%. The historical control data range for F1 gestational index is 94.1% to 100%. The slight, though not statistically significant, decrease in the liveborn gestation index in the group given 750 mg/kg/day was due to Female Nos. 1324 and 1344 having implantation sites but not delivering a litter. There was also one control female (No. 1024) that had implantation sites but did not deliver a litter. Therefore, these observations are not considered test item related. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 37, 38, 39, 40, 45, 46, 47, 48, Individual Data: Appendix 2, Tables 33, 34, 39, 40. There were no adverse, test item-related effects on F1 body weights or feed consumption during the prebreed exposure period (Days 0 to 70). The increased (7% to 11%) body weights for F1 females given 50 mg/kg/day, though statistically significant at every interval from Day 0 to Day 70, was not considered test item-related since there was no dose relationship and only occasional corresponding significant increases in body weight change, food consumption (g/day only), and food efficiency. Although other occasional statistically significant changes in body weight, body weight change, food consumption, and food efficiency were noted when compared with the control group, including the slightly decreased (4.4% and 5.4%) percent food efficiency for the overall interval from Days 0 to 70 for F1 males given 50 and 750 mg/kg/day, respectively, these changes were not considered the result of test item administration since these changes were sporadic, slight, not dose related, and generally had no effect on the other body weight and/or food consumption parameters. There were no adverse test item-related effects on F1 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. Statistically significant decreases in body weight change and percent food efficiency for one interval (Days 91 to 98) were noted for the F1 males given 750 mg/kg/day; however, these changes did not result in or correlate with statistically significant effects on body weight or feed consumption (g/day and g/kg/day) compared with the controls for this same interval and were thus considered nonadverse. The statistically significant increases in body weight change and/or percent food efficiency during the postmating holding period, for F1 males given the lower dose levels, were sporadic and not dose related, and therefore not considered test item related. There were no statistically significant changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F1 females that were never found sperm positive or that never delivered a live litter.

GESTATION
Summary Data: Tables 51, 52, 53, 54, Individual Data: Appendix 2, Tables 43, 44.
There were no test item-related changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group during gestation. Although body weights for females given 50 mg/kg/day were statistically significantly increased at each interval, there were no corresponding statistically significant increased in body weight change, feed consumption, or percent food efficiency. The change in body weights was not considered test item-related as there was no dose response.

LACTATION
Summary Data: Tables 56, 57, 58, 59, Individual Data: Appendix 2, Tables 46, 47.
During lactation (LD 0 to 21), there were no notable test item-related changes in F1 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group. The one statistically significant change (an increase in body weight for females given 50 mg/kg/day on LD 21) was not considered test item-related as the change was sporadic, showed no dose response, and did not correlate with any changes in any other body weight or food parameters.

ANDROLOGY
Summary Data: Table 42, Individual Data: Appendix 2, Table 37
There was an equivocal, non-adverse, statistically significant increase in the percent of abnormal sperm in the F1 males given 750 mg/kg/day compared with the control group; however, the historical control data range of 1.29% to 6.1% encompasses the 2.29% seen in the high dose in this study. There was an increase in the mean number of sperm with blunt heads (indicating the absence of the acrosome), sperm with small heads, and sperm with heads only or tails only. One particular male in the high dose group (Animal No. 1339) had a very high percentage of sperm with heads only; this breakage was possibly test item-related and Female No. 1336, mated with this male, failed to become pregnant. No test item-related effects were observed on the F1 spermatogenesis endpoints of mean testicular and epididymal sperm numbers and sperm production rate, motility, and progressive motility in males at any dosage concentration. Differences from the control group were slight and were not statistically significant for these parameters.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 42, 43, 61, 67, 68, Individual Data: Appendix 2, Tables 36, 38, 50, 58, 59, 60.
The only test item-related macroscopic findings in F1 parental animals were observed in the lungs of males and females given 750 mg/kg/day. At the F1 male scheduled necropsy, a total of 11 males given 750 mg/kg/day were noted as having lung findings, including small dark red spots and mottled (alone or with other findings such as firm, tan, and/or small dark spots), and these gross findings correlated with an increase in both absolute and relative lung weights in this dose group. There was an increased incidence of mottled kidneys (bilateral) in the males given 750 mg/kg/day (10 of 24) compared with the control group (4 of 28). All other macroscopic gross findings in F1 males were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. At the scheduled necropsies on LD (≡PND) 21, a total of 4 F1 females given 750 mg/kg/day were noted as having lung findings including mottled, firm, tan, for 2 animals and left cranial lobe, tan, meaty for 2 animals; these gross findings correlated with an increase in absolute lung weights in this dose group. There were 2 F1 females given 750 mg/kg/day that had a fluid-filled cyst in the uterus at necropsy; the relationship to test item administration of this finding is unknown. All other macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 42, 67, Individual Data: Appendix 2, Tables 36, 58.
There was an increase in both absolute and relative lung weights (39.5% and 37.7%, respectively) in F1 males given 750 mg/kg/day. Absolute and relative lung weights were also increased [12.9% (statistically significant) and 6.9% (not statistically significant), respectively] in F1 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic and microscopic lung findings in rats given 750 mg/kg/day.

MICROSCOPIC EXAMINATION
Summary Data: Tables 44, 67, 69, Individual Data: Appendix 3.
Like the F0 males and females, chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F1 males and females given 750 mg/kg/day compared with the controls. All 24 F1 males and 24 F1 females given 750 mg/kg/day that survived until scheduled euthanasia had this test item-related finding. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. Also like the F0 animals, alveolar macrophage aggregation, alveolus was noted in F1 control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle. Test item-related slight increases in incidence of renal tubule degeneration/necrosis and renal tubule hyaline droplets were seen in the F1 males given 750 mg/kg/day. Degeneration/necrosis was characterized by the presence of multifocal, cortical proximal convoluted tubules which were lined by epithelial cells that were slightly vacuolated with nuclear pyknosis and containing occasional sloughed necrotic cells. This change was very subtle and recorded as minimal. In a few instances, lightly staining
eosinophilic droplets were increased in proximal convoluted tubules of the same animals. This morphologic appearance suggested hydrocarbon-induced α2μ-globulin male rat nephropathy. Hyaline droplets were not observed in the treated females. Kidneys from selected males given the vehicle or 750 mg/kg/day from both the F0 and F1 generations (10 males/group/generation) were stained with Mallory Heidenhain stain which is used to characterize α2μ-globulin in renal tubules. As expected due to the age of these rats, renal tubules from all of these selected animals contained variable numbers of tubule droplets which stained positive and had the characteristic distribution and features of α2μ-globulin (male rats of this age contain α2μ-globulin). The droplets were subjectively scored on a scale of 1 to 5 +. No apparent increase in positive tubule droplets was present in the F0 males given 750 mg/kg/day compared with the F0 control males. However, the mean average score of the positive tubule droplets was increased in the F1 males given 750 mg/kg/day compared with the F1 control males, confirming that these kidney findings were the result of α2μ-globulinergic nephropathy, which is known to occur in male rats following exposure to hydrocarbons.Other changes in the kidneys, such as chronic progressive nephropathy and renal tubule regeneration, were not clearly increased in either incidence or in severity and were therefore not deemed test item-related. For F1 males and females in the low- and mid-dose groups suspected of reduced fertility, there were no test item-related microscopic findings in the reproductive organs/tissues examined. Two males given 50 mg/kg/day and 3 males given 200 mg/kg/day had inflammation of the prostate gland; however, 7 males given the vehicle control and 9 males given the high dose of 750 mg/kg/day had a similar finding; thus, this finding was not considered test item-related. One male given 200 mg/kg/day showed seminiferous tubule atrophy in the testis. One female given 50 mg/kg/day had dilatation of the uterus and one other had an ovarian cyst while one female given 200 mg/kg/day had ovarian atrophy. These microscopic findings were not observed in F1 males and females given the high dose of 750 mg/kg/day and may have contributed to the reduced fertility for these particular animals. A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat. Ovarian follicle counts were not statistically significantly different in the F1 females given 750 mg/kg/day compared with the control group; therefore, counts were not conducted on the lower dose groups.

F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 61, Individual Data: Appendix 2, Tables 50, 51.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21 (for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 63, Individual Data: Appendix 2, Tables 51, 54.
The numbers of F2 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F2 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 17, 17, 25, and 17 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 1352 found dead on LD 1) accounted for 10 of the 17 pups in the group given 750 mg/kg/day. In addition, 3, 3, 3 and 0 pups in the same respective groups were missing (and presumed dead).

ANOGENITAL DISTANCE
Summary Data: Table 62, Individual Data: Appendix 2, Table 52.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F2 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 62, Individual Data: Appendix 2, Table 53.
Mean male and female pup body weights and body weight changes in the 50, 200, and 750 mg/kg/day groups were unaffected by parental test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 63, 64, Individual Data: Appendix 2, Tables 54, 55. The number of F2 pups (litters) stillborn, found dead, or euthanized from PND 0 through PND 21 were 17(11), 16(13), 25(13), and 17(4) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of F2 pups that were stillborn, found dead, or euthanized. Findings in the stillborn, found dead, or euthanized pups from test item-treated dams were similar to the findings in stillborn, found dead, or euthanized pups from vehicle-treated dams, culled PND 4 pups, and/or scheduled death PND 21 F2 pups.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 2, Table 57. At the PND 21 necropsy of F2 weanlings, there were no test item-related findings. Hydronephrosis of the right kidney was noted at a similar incidence in males in the control and test item-treated groups.

ORGAN WEIGHTS
Summary Data: Table 65, Individual Data: Appendix 2, Table 56.
There were no statistically significant changes in F2 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 7% to 8% and 4% to 7%, respectively) were observed in the F2 male and female pups in the group given 750 mg/kg/day compared with the control group. Given the small magnitude of the change, the lack of statistical significance, and since the weights fall within the historical control data ranges, these changes were considered equivocal and not adverse.

MICROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 3.
Dilatation of the pelvis of the kidney was noted for all male pups with a macroscopic finding of hydronephrosis; thus, this finding was noted at a similar incidence in the control and test item treated groups and was therefore not considered test item-related.
Key result
Dose descriptor:
NOAEL
Generation:
other: F1 and F2
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility/Developmental Toxicity
Reproductive effects observed:
not specified

OBJECTIVE AND METHODS

The objective of this study was to characterize the effects of the test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrus cycle, mating behavior, conception, gestation, parturition, lactation, and weaning of the F1generation, as well as the neonatal survival, growth, and development of the F1and F2offspring. In addition, the systemic toxicity of the test substance was partially characterized in this study by evaluation of several nonreproductive tissues. Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0and F1generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 50, 200, and 750 mg/kg/day for the F0and F1generations. A concurrent control group of 25 and 28/sex for the F0and F1generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. F0animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration. The test item was administered to offspring selected to become the F1parental generation following weaning, beginning on postnatal day (PND) 22. The F0and F1males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0and F1females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia. Animals were observed at least twice daily for mortality. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for determination of estrous cycles beginning 21 days prior to pairing. All F0and F1females were allowed to deliver and rear their pups until weaning on lactation day (LD≡PND) 21. Clinical observations, body weights, and sexes for F1and F2pups were recorded at appropriate intervals. For both generations (F1and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0animals were selected on PND 21 to constitute the F1generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1rats. Nonselected F1pups were necropsied on PND 21, and F2pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1and F2pups that were necropsied on PND 21. Each F0and F1parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0and F1males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1females in the control and high-dose groups. Designated tissues from all F0and F1parental animals were examined microscopically.

Conclusions:
SUMMARY OF RESULTS
Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test item related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test article related increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes.

Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy.

There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the low and mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarbon induced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS
The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
Executive summary:

SUMMARY OF RESULTS AND CONCLUSION

Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test itemrelated effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test articlerelated increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes. Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy. There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the lowand mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0 animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item
administration.
- Weight at study initiation: 142.4 to 190.6 g; female group mean range 137.4 to 165.1 g.
- Fasting period before study: Not applicable.
- Housing: During the acclimation period (F0 animals only) and throughout the study, all F0 and F1 parental animals were housed
individually, except during the mating period, in solid-bottom polycarbonate cages with stainless steel wire lids. The rats were paired
for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period),
the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. Beginning on pre-natal
Day 21, the selected F1 pups were individually housed until the start of the mating period.
- Diet: Purina Certified Pelleted Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO) was available ad libitum in the wire cage
lids. Available information on the diet did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Water (tap water) was available ad libitum via plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes.
Available information on the water did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Acclimation period: Seven days

ENVIRONMENTAL CONDITIONS
- Temperature: (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hr dark / hrs light):12 hours continuous light and 12 hours darkness
IN-LIFE DATES: First day of treatment: 13 October 2009 - Final necropsy day was 07 July 2010.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared as solutions at concentrations of 0 (vehicle), 25, 100, and 375 mg/mL were formulated 10 times (approximately every 4 weeks), dispensed into 30-mL amber glass bottles with Teflon-lined lids for use as daily dose aliquots, and stored at room temperature protected from light. Concentrations of 25 and 375 mg/mL in the same vehicle have been shown to be homogeneous and stable for 42 days when stored refrigerated or at room temperature protected from light (Analytical Chemistry report. Report date: 10 March 2010). The test item formulations (but not the vehicle formulation) were stirred continuously throughout dose administration.

Prior to use on study, samples (~25 mL each) for homogeneity determination were collected from the top, middle, and bottom strata of each test item formulation from 3 of the 10 formulation dates (06 October 2009, 26 January 2010, and 15 June 2010). Samples (~25 mL each) for concentration analysis were also collected from the middle stratum of each dosing formulation (including the vehicle formulation) from these same 3 formulation dates. All analyses were conducted using a validated gas chromatography method with flame ionization detection (AM-0212131-002.0). Details about the methodology and results of these analyses are presented in Appendix 1, and summarized as follows:

Based on these results, the analyzed dosing formulations were found to contain the amount of test item prescribed in the protocol (±10% of nominal) and were homogeneous. Therefore, the protocol-specified dosages of test item were administered to the animals.

-DIET PREPARATION
Not applicable
- Rate of preparation of diet (frequency): Not applicable
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: No data
- VEHICLE: Corn oil
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: 0, 25, 100, and 375 mg/mL
- Amount of vehicle (if gavage): 2 ml/kg
- Lot/batch no. (if required): Not applicable
Details on mating procedure:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group).
- Length of cohabitation: Following positive evidence of mating (or at the completion of the 2-week mating period).
- Proof of pregnancy: Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after
pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were
evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs.
Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- After a set number of days of unsuccessful pairing replacement of first male by another male with proven fertility.: Not applicable
- Further matings after two unsuccessful attempts: Not applicable
- After mating each pregnant female was returned to individual caging: Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method has been satisfactorily validated and details about the methodology and results of these analyses are presented in detail.
The results are summarized as follows.
Three sets of dose formulation samples were analyzed for concentration verification and homogeneity assessment for the test item in corn oil formulations had previously been evaluated for homogeneity , in long-term storage, and animal room dosing stabilities in the concentration range from 25 to 375 mg/mL and the detailed analytical procedures were reported in an Analytical Chemistry Report, Report Date 10 March 2010. The formulation analyses reported here were performed following an validated analytical method to verify the test item concentration and homogeneity in corn oil formulations prepared on October 6, 2009 and January 26 and June 15, 2010. All dose formulations analyzed were found to be within ±10% of the nominal concentration and were homogeneous.

ANALYTICAL METHOD
The validated analytical method used to analyze study samples is outlined as follows:
For each dose formulation sample, triplicate 1 mL aliquots were in 10-mL volumetric flasks and diluted to volume with n-hexane. Processed formulation samples were then analysed on a gas chromatograph with a flame ionization detector along with a series of vehicle standards used to generate a calibration curve. Vehicle standards were prepared by mixing blank formulations samples. Test item concentrations were calculated using a least square s linear regression equation that fit the relationship between the nominal concentration s of vehicle standard and the detector response (ie the sum of integrated areas of selected peaks within the chromatogram). The formulation sample concentrations were determined in mg/g and converted to mg/mL measuring the formulation densities.
Duration of treatment / exposure:
Duration of treatment / exposure
All male and female for the F0 generation and F1 generation were administered the test item or vehicle control, daily for at least 70 consecutive days prior to mating. The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
A complete gross necropsy was conducted after death for any parental (F0 and F1) or offspring (F1 and F2) animals found in moribund condition or dead on study. Organs were not weighed for moribund animals. Histopathological examination of study specified organs were performed for all high dose and control parental (F0 and F1). Additionally, reproductive organs of the low and mid dose animals suspected of reduced fertility were subjected to histopathological evaluation. For any organ(s) demonstrating possible treatment-related changes in the high dose group versus the organ(s) of interest were evaluated histopathologically from successively lower concentration dose groups until a clear no observed adverse effect level (NOAEL) was established. However, at the request of the Sponsor, no further evaluation of the lungs from lower dose groups was conducted.
Frequency of treatment:
The test and vehicle control item formulations were administered to the F0 and F1 males and females and once daily for a minimum of 70 consecutive days prior to mating and for males continued throughout mating to the day prior to termination. The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through to the day prior to termination. The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero andthrough nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22).
Details on study schedule:
Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0 and F1 generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. A concurrent control group of 25 and 28/sex for the F0 and F1 generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia. The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21). The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22). The first day of dosing was designated as Day 1 of the study. Animals were paired on a 1 male: 1 female basis within each dose group. The rats were paired for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day (25 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day (100 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day (mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
F0 generation
0 mg/kg/day (Control) - 25 per sex
50 mg/kg/day - 25 per sex
200 mg/kg/day - 25 per sex
750 mg/kg/day - 25 per sex

F1 generation
0 mg/kg/day (Control) - 28 per sex
50 mg/kg/day - 28 per sex
200 mg/kg/day - 28 per sex
750 mg/kg/day - 28 per sex
Details on study design:
- Dose selection rationale: Dosage levels were selected based on repeat-dose studies on similar substances and a 90-day repeat-dose study on 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' and 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'. In addition to repeat-dose studies on conventional petroleum-based diesel, a number of repeat-dose studies on individual components of the test substance were available. These studies do not give any indication of unexpected effects and are in line with the observations in 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' studies. The selected route of administration was oral (gavage) by the Sponsor because this is a possible route of human exposure. The number of animals selected for this study was the minimum required to yield statistically and scientifically meaningful data and was consistent with regulatory agency expectations.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- See attached Summary Tables 6, 14, 20, 25, 41, 49, 55, 60 and Individual Data: Appendix 2, Tables 4, 10, 17, 35, 41, 45 and 48
- Mortality
Observations were made twice daily at least 6 hours apart.
- Clinical examinations
- Time schedule: Clinical observations were conducted and recorded at least once daily, approximately 1-2 hours post dosing, throughout the course
of the study.

BODY WEIGHT: - See attached Summary Tables 2, 3, 10, 11, 16, 17, 21, 22, 37, 38, 45, 46, 51, 52, 56 and 57 and Appendix 2, Tables 2, 8, 15, 31,
32, 33, 39, 43 and 46.
- Time schedule for examinations:
Individual body weights of the male F0 and F1 rats were determined and recorded on the first day of dosing and weekly through the
pre breed exposure and mating periods.
The body weights of male F0 and F1 rats were recorded in the same manner after the mating period until scheduled euthanasia.
The body weights of female F0 and F1 rats were recorded in the same manner until evidence of mating was observed.
During gestation, sperm-positive (presumed pregnant) females were weighed on gestational days (GD) 0, 7, 14, and 20.
Dams producing litters were weighed on lactational days 0, 4, 7, 14, and 21.

FOOD CONSUMPTION AND EFFICIENCY:
- See attached Summary Tables 4, 5, 12, 13, 18, 19, 23, 24, 39, 40, 47, 48, 53, 54, 58 and 59 and Individual Data: Appendix 2,
Tables 3, 9, 16, 34, 40, 44and 47.
Individual F0 and F1 male and female feed consumption was measured weekly until mating.
Feed consumption was not recorded during the 2-week mating period.
Following mating, male feed consumption was measured on a weekly basis until the scheduled necropsy.
During pregnancy of F0 and F1 females, feed consumption was recorded on gestation days 0, 7, 14, and 20.
During lactation of F1 and F2 litters, maternal feed consumption was measured on 0, 4, 7, 14, and 21.
- Food efficiency:
Percent food efficiency (body weight gained as a percentage of food consumed) was also calculated

WATER CONSUMPTION:
Not applicable

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Not applicable
- Time schedule for collection of blood:
Not applicable
- Anaesthetic used for blood collection:
Not applicable.
- Animals fasted:
Not applicable.
- How many animals:
Not applicable.

URINALYSIS:
Not applicable

NEUROBEHAVIOURAL EXAMINATION:
Not applicable.
- Time schedule for examinations:
Not applicable.
Behavioural assessment)
Not applicable.
(Functional Performance Tests)
Not applicable.
(Sensory Reactivity)
Not applicable.
- Dose groups that were examined:
Not applicable.

OTHER:
- MATING
Animals were paired on a 1 male: 1 female basis
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- see attached Summary Tables 15, 32 and 50 and Individual Data: Appendix 2, Tables 11, 28, 42 and 59

PREGNANCY AND PARTURITION
During the period of expected parturition, the females were observed at least twice daily for initiation and completion of parturition. On the day parturition was completed (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 completed.
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1
males as appropriate.
Litter observations:
Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1 and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
Postmortem examinations (parental animals):
Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from the F0 and F1 parental animals were examined microscopically.
Postmortem examinations (offspring):
Non selected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21.
Statistics:
STATISTICAL METHODS
See Attachment “"Report Text" pages 34 to 35.
Reproductive indices:
For the Mating, fertility, and gestational indices see attached ""Report Text" page 25.
Offspring viability indices:
See attached Report Text Pages 1 to 74, Results Tables and Appendices.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29.
There were no deaths attributed to test substance administration noted through the F0 prebreed exposure period (Days 0 through 70). Seven unscheduled deaths (3 found dead and 4 euthanized for humane reasons) occurred on study through Day 70. Male No. 153 (Group 4) and Female No. 152 (Group 4) were found dead on Day 1 and Day 68, respectively. The cause of death for these animals was not conclusively determined from the gross necropsy. Red staining around the nose and left eye (Male No. 153) or nose and mouth (Female No. 152), mottled (Male No. 153, correlated with alveolus hemorrhage) or discolored/dark (Female No. 152) lungs, and reddish, dark fluid in the intestines (Female No. 152 only) indicate that aspiration of the test item is the most likely cause of death of these animals. Female No. 154 (Group 4) was found dead on Day 27, approximately 1.2 hours postdose, due to an apparent dosing error, as evidenced by red fluid around the nose and mouth, lungs that failed to collapse and were discolored (dark), and fluid expelled from the trachea with slight pressure applied to lungs.

Male No. 7 (Group 1), Male No. 185 (Group 4), and Female No. 124 (Group 3) were euthanized on Days 56, 66, and 70, respectively. Gross observations at necropsy (mottled/discolored lungs, red/dark fluid in stomach and gastrointestinal tract, red staining around mouth, and/or red fluid in mouth) indicate most likely aspiration of the test item, leading to the clinical signs that prompted humane euthanasia of these animals; microscopic lung findings for Male No. 7 and 185 further support this conclusion. In addition, Male No. 143 (Group 3) was euthanized for humane reasons on Day 16 due to bilateral lesions on the neck that worsened despite veterinary intervention. All other animals survived to Day 70. There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were
noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Two males (Nos. 115 and 127, Group 3) exhibited neck lesions that
required veterinary intervention; the neck lesions healed by Day 70.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33 Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29.
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There was an increased incidence of rooting in animals given 750 mg/kg/day and of chromodacryorrhea (eye) in animals given the 200 and 750 mg/kg/day compared with the control group. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Male No. 127 (Group 3; terminated as scheduled on Day 106) exhibited a neck lesion that required veterinary intervention on Days 97 to 105; this same animal had previous neck lesions requiring veterinary intervention that had healed by Day 72.

For the F0 females, there were no deaths attributed to the test substance administration during the mating, gestation, and lactation periods (Days 70 to 128); there were 3 unscheduled F0 female deaths during this time. Female Nos. 38 (Group 1) and 180 (Group 4) were severely injured by their mating partner and were therefore euthanized on Day 72 for humane reasons (both were sperm negative on Day 72 prior to euthanasia). There were no macroscopic necropsy findings for these 2 females other than the wound inflicted by the mating partner. Female No. 166 (Group 4) was euthanized for humane reasons on Day 95 (GD 22) due to dystocia with concomitant hypothermia. Macroscopic necropsy findings for Female No. 166 included pale, mottled, mildly enlarged adrenal glands and mottled lungs (all lobes), and there were 18 visible implant sites that corresponded to 18 pups (3 live and 15 dead) in the uterus.

From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F0 females, there were occasional observations of struggling during dosing in animals given 200 and 750 mg/kg/day, and there were very occasional observations of rooting post dosing and salivating prior to dosing in up to 2 females given 750 mg/kg/day during the gestation period (GD 0 through 23). Likewise, there were occasional observations of animals struggling during dosing in the groups given 200 and 750 mg/kg/day during the lactation period. One dam (No. 118) given 200 mg/kg/day had an eye injury and swollen nose from LD 17 to 21 that were not test item-related. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26 Individual Data: Appendix 2, Tables 6, 11, 18, 19.
No test item-related effects on F0 reproductive performance were observed at any dosage level. There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 69.6%, 80.0%, 95.8%, and 90.9% and female mating indices were 70.8%, 80.0%, 95.8%, and 90.9% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data for male and female mating indices ranged from 87.5% to 100% and 88% to 100%, respectively. Following a thorough investigation, which included a detailed review of all the environmental data during the study and discussions with the animal vendor, the reason for the low mating indices in the control and low-dose groups remained unknown; however, 2 males (Nos. 3 and 41) were infertile based on andrology parameters, thus partially accounting for the lower mating indices in the control group. Male fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% and female fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% in the same respective groups. All fertility indices were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for F0 females.

All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (3.8 to 4.0 days) or number of cycles (4.4 to 4.6) in test item-treated groups compared with control values (3.9 days and 4.4, respectively). The mean number of days between pairing and coitus (i.e., the precoital interval) in the test itemtreated groups was similar to the control group values of 2.4 days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 26 Individual Data: Appendix 2, Table 19.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. The mean gestation lengths in the groups given 50, 200, and 750 mg/kg/day were 22.4, 22.2, and 22.4 days, respectively, compared to the mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 94.4%, 95.5%, and 89.5% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range of 87% to 100%. There were corresponding increases and decreases in the stillbirth index and live birth index, respectively, for groups given 200 and 750 mg/kg/day. These slight changes in the live born gestation index, stillbirth index, and live birth index in the group given 750 mg/kg/day were due to Female No. 166 being euthanized on GD 22 due to dystocia and Female No. 200 having an all-stillborn litter. Likewise, the slight changes in these same indices for the group given 200 mg/kg/day were due to Female No. 128 having a presumed all-stillborn litter, and the slight decrease in live born gestation index for the group given 50 mg/kg/day was due to Female No. 86 not delivering any pups, though having 5 visible implant sites at necropsy. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent post implantation loss, pups delivered (live, dead, and total), and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Slight, nondose-related increases in feed consumption (g/day and g/kg/day) were noted for males and females at several intervals (occasionally statistically significant) for all test item-treated groups when compared with the control group; however, these changes in feed consumption did not result in or correlate with statistically significant changes in body weights, body weight changes, or food efficiency compared with the controls. There were no adverse test item-related effects on F0 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. A statistically significant increase in feed consumption (g/day and g/kg/day) for one interval (Days 84 to 91) was noted for the F0 males given 750 mg/kg/day; however, this change in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or food efficiency compared with the controls. In general, there were no notable test item-related effects on body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F0 females that were never found sperm positive or that never
delivered a live litter. Occasional statistically significant changes were observed in these parameters, but were not considered biologically significant as they were sporadic and not dose related.

GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation, there were no adverse test item-related changes in F0 female body weights and feed consumption (g/day and g/kg/day). Compared with the concurrent control group, there was a statistically significant decrease (~17%) in percent food efficiency from GD 7 to 14 for the females given 750 mg/kg/day, which correlated with a decrease (~16%) in body weight change that was not statistically significant for this same group during the same interval. The following interval (GD 14 to 20) also showed a slight, though not statistically significant, decrease in percent food efficiency and body weight change (~10% and ~8%, respectively) for the females given 750 mg/kg/day compared with the concurrent control group. Thus, percent food efficiency and body weight change were slightly reduced (~8% and ~7%, respectively) between GD 0 and 20 but did not achieve statistical significance for the females given 750 mg/kg/day compared with the control group; thus, these changes were considered incidental to treatment and not adverse.

LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no notable test item-related or statistically significant changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.

ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6.
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant. As per the protocol, assessments using the frozen right testis, as well as sperm morphology evaluations, were conducted initially on the control and high-dose groups only. As there were no test itemrelated effects in the group given 750 mg/kg/day, evaluation of the lower dose groups was not conducted for these endpoints.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males given 200 and 750 mg/kg/day. At the F0 male necropsy, 7 males given 750 mg/kg/day were noted as having pale lungs (cranial lobe, left or bilateral), and this gross finding correlated with an increase in both absolute and relative lung weights in this dose group. Two males given 200 mg/kg/day also had pale lungs (cranial lobe, left); however, there was no significant increase in lung weights in this dose group. The thymus of 2 males (Nos. 187 and 199) given 750 mg/kg/day had multiple, pinpoint, red foci. There was an increased incidence of mottled kidneys (bilateral) in the males given 200 and 750 mg/kg/day (10 of 24 and 9 of 23 animals, respectively) compared with the control and low-dose groups (5 of 24 and 5 of 25 animals, respectively). The testes of 2 control males (Nos. 3 and 41) were found to be reduced in size (bilateral) and flaccid (bilateral), and the epididymides of Male No. 3 were also reduced in size. Neither of these males (Nos. 3 and 41) sired a litter and both had few motile or progressively motile sperm (thus the reason for the larger standard error in these parameters for the control group compared with the test item-treated groups). All other macroscopic gross findings were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. There were no test item-related macroscopic findings for the F0 females necropsied on LD (≡PND) 21. All macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was an increase in both absolute and relative lung weights (38% and 33%, respectively) in F0 males given 750 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (14.0% and 12.8%, respectively) in F0 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic (males) and microscopic (males and females) lung findings in rats given 750 mg/kg/day. Although there was a statistically significant increase (approximately 10%) in the absolute lung weight for F0 males given 50 mg/kg/day, there was no significant increase in relative lung weight and no macroscopic lung findings in this same group, as well as no significant changes in absolute and relative lung weights for F0 females given 50 mg/kg/day and F0 males and females given 250 mg/kg/day; therefore, the change in absolute lung weight for F0 males given 50 mg/kg/day was considered spurious.

MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3.
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 males and females given 750 mg/kg/day compared with the controls. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test itemexposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning.

There did not appear to be any significant difference between male and female lungs with regard to the appearance of interstitial/alveolus inflammation. Another similar pulmonary finding (alveolar macrophage aggregation, alveolus) was noted in control male and female lungs and
was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle. An equivocal, non-adverse histopathological lesion was identified in the kidneys. Renal tubule mineralization was observed in the kidneys of 7 of 23 males given 750 mg/kg/day. This lesion was characterized by the presence of multifocal mineralized renal tubules (proximal convoluted tubules) in the outer cortex. Similar appearing tubules were not seen in any of the 24 male F0 controls. The renal tubules were characterized as basophilic tubules with mineralization of both the nucleus and cytoplasm affected. Some tubules were strongly mineralized and others less.

There was no apparent cause for the mineralization and no histopathological reaction to the mineralized tubules was noted. In the study pathologist’s experience, these tubules appeared similar to artifactual changes occasionally observed as a result of tissue fixation and/or processing. Since mineralized tubules may be an artifactual change and were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal. Other changes in the kidneys such as chronic progressive nephropathy and renal tubule regeneration were not clearly increased in either incidence or in severity and were therefore not deemed test item-related. The microscopic examination of reproductive tissues for F0 animals suspected of reduced fertility in the low- and mid-dose groups did not have any relationship to test item administration. A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Lack of adverse treatment-related effects observed at the highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No developmental effects in offspring
Remarks on result:
other: Generation: F1 and F2 (migrated information)

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)

LITTER DATA F1
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 26, Individual Data: Appendix 2, Tables 19, 20.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21(for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant. The slight reduction (which was not statistically significant) in the 4-day survival index per litter in the group given 750 mg/kg/day was because the entire litter for Female No. 198 was found dead (3 pups) or euthanized (12 pups) prior to PND 2 due to poor clinical condition (including no milk band, cold to touch, reduced activity, and loss of skin elasticity) due to the lack of care for the pups by the dam.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 28, Individual Data: Appendix 2, Tables 20, 23.
The numbers of F1 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F1 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 4, 6, 12, and 19 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 198) accounted for 15 of the 19 pups in the group given 750 mg/kg/day. In addition, 3, 3, 2 and 0 pups in the same respective groups were either missing (and presumed dead) or cannibalized.

ANOGENITAL DISTANCE
Summary Data: Table 27, Individual Data: Appendix 2, Table 21.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F1 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 27, Individual Data: Appendix 2, Table 22.
Mean male and female pup body weights and body weight changes in the groups given 50, 200, and 750 mg/kg/day were unaffected by test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 28, 29, Individual Data: Appendix 2, Tables 23, 24.
The numbers of pups (litters) stillborn, found dead, or euthanized from PND 0 through the selection of the F1 generation on PND 21 numbered 4(4), 8(5), 12(6), and 19(5) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of pups that were stillborn, found dead, or euthanized. The combined incidence of bilateral and unilateral hydronephrosis in stillborn pups was not different from the control group, nor was the incidence of hydronephrosis seen in pups necropsied as scheduled on PND 21 different in test item-treated groups compared with the control group; therefore, the increased pup and litter incidence of bilateral hydronephrosis in stillborn pups in both the 200 and 750 mg/kg/day dose groups compared with the control group was not considered test item-related. There were several F1 pups found dead and euthanized moribund in the 200 mg/kg/day and/or 750 mg/kg/day groups, with observations of no milk in the stomach, indicating the pups were not nursing. There were no F1 pups found dead in the control group and no F1 pups euthanized moribund in the control and 200 mg/kg/day dose groups, so a direct comparison of the incidenceof this macroscopic finding across groups in unscheduled death pups is not possible. The one low-dose pup euthanized moribund also had no milk in the stomach and loss of skin elasticity noted at necropsy. Of the 12 pups euthanized moribund in the high-dose group (all from Dam No. 198), 3 had distended ureter (either unilateral or bilateral); however, the incidence of this finding in the culled pups from all dose groups was similar, and thus this finding was not considered to be test item-related.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 2, Table 26.
At the PND 21 necropsy of F1 weanlings, there were no findings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.

ORGAN WEIGHTS
Summary Data: Table 30, Individual Data: Appendix 2, Table 25.
There were no statistically significant changes in F1 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 9% to 10% and 4% to 5%, respectively) were observed in F1 pups in the group given 750 mg/kg/day compared with the control group. The spleen weights in F1pups from the high-dose group in this study fall within the historical control data ranges for this parameter and were therefore considered an equivocal, non-adverse finding.

MICROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 3.
There were no microscopic findings in the F1 PND 21 weanlings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.

F1 DEVELOPMENTAL LANDMARKS
BALANOPREPUTIAL SEPARATION
Summary Data: Table 36, Individual Data: Appendix 2, Table 31.
The test item-related increase in attainment of balanopreputial separation in males given 750 mg/kg/day was not considered adverse, as the day of attainment falls within the historical control ranges for this parameter. The mean ages of attainment of balanopreputial separation were 43.3, 43.2, and 43.2 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 41.6 in the control group, and were all statistically significantly longer than the control group, though in a nondose-responsive manner and within the historical control range for the mean age of attainment of balanopreputial separation of 40.8 to 43.6 days. Mean body weights at the age of attainment were 254.0, 249.8, and 245.4 g in the same respective groups, compared with 238.9 g in the control group, and were not statistically significant. When adjusted with body weight as a covariate, the adjusted mean ages of attainment of balanopreputial separation were 43.0, 43.1, and 43.3 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 42.0 in the control group, and although dose-responsive, only the group given 750 mg/kg/day was statistically significantly longer than the control group. This test item-related increase in adjusted age of attainment of balanopreputial separation was not considered adverse, as 43.3 days falls within the historical control ranges for this parameter of 40.7 to 43.8 days. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group with the longest delay in attainment of balanopreputial separation were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse.

VAGINAL PATENCY
Summary Data: Table 36, Individual Data: Appendix 2, Table 32.
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test item administration. The mean ages of attainment of vaginal patency were 31.3, 30.6, and 30.0 days in the groups given 50, 200, and 750 mg/kg/day, respectively compared with 29.9 days in the control group. Mean body weights at the age of attainment were 117.5, 111.0, and 105.5 g in the same respective groups compared with 108.6 g in the control group. The mean age for the group given 50 mg/kg/day was statistically significant, though was not considered test item-related as there was no dose-response and the adjusted mean age was not statistically significantly different from the control group.

GENERATION F1
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 35, 41, 43, 49, 68, Individual Data: Appendix 2, Tables 30, 35, 38, 41, 60.
There were no deaths directly attributed to the test substance administration noted through the F1 weaning and prebreed exposure period (Days -20 through 70). Seven unscheduled deaths (6 found dead and 1 euthanized for humane reasons) occurred through Day 70. Male No. 1147 (Group 2, 50 mg/kg/day) was found dead on Day -2 (PND 36) after being noted as lethargic the previous day. Male No. 1305 (Group 4, 750 mg/kg/day) was also found dead on Day -2 (PND 38). The cause of death for these 2 males could not be determined upon necropsy; there were no obvious signs of gavage error and some autolysis was present. Group 4 Male Nos. 1329 and 1351 were found dead on Days 2 (PND 41) and 3 (PND 40), respectively. Both males had hydronephrosis of the right kidney and dark red lungs. The lungs from Male No. 1351 failed to collapse and had pale areas around the edges of all lobes. Slight pressure on the lungs released bubbles from the trachea, suggesting the cause of death of Male No. 1351 was a gavage error. Based on the appearance of the lungs at necropsy, Male No. 1329 may have died due to aspiration of the dose formulation. Group 4 Female Nos. 1346 and 1354 were found dead on Days 18 (PND 56) and 30 (PND 67), respectively. Female No. 1346 had struggled during dosing the day prior to being found dead. At necropsy, the only finding was red substance in the nose and mouth. The cause of death of Female No. 1346 could not be determined upon necropsy. Female No. 1354 was noted as lethargic at the postdose observation time and died a short time later. At necropsy, slight hydronephrosis of the right kidney was seen, and the lungs were found to be red and failed to collapse fully. Slight pressure on the lungs released fluid and bubbles from the trachea, suggesting the cause of death of Female No. 1354 was gavage error. Female No. 1348 (Group 4, 750 mg/kg/day) was euthanized for humane reasons on Day 54 (PND 91) on welfare grounds. At necropsy, reddish fluid from nose and mouth, reddish fluid in the intestines, very little food present in the stomach, and dark red lungs that did not fully collapse were noted, suggesting the apparent cause of death was gavage error. All other animals survived to Day 70. There was a slightly increased incidence of rooting in male and female F1 animals given 750 mg/kg/day and of chromodacryorrhea (eye) in females given 200 mg/kg/day and 750 mg/kg/day compared with the control group. These observations are not adverse. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were limited to unscheduled death animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND
LACTATION PERIODS
Summary Data: Tables 41, 43, 49, 55, 60, 68, Individual Data: Appendix 2, Tables 35, 38, 41, 45, 48, 60.
For the F1 males, there was one death during the mating/postmating period (Days 70 through 107). Male No. 1325 (Group 4, 750 mg/kg/day) was noted as lethargic and gasping for breath prior to dying on Day 78. At necropsy, the lungs were mottled and failed to fully collapse; when slight pressure was applied to the lungs, bubbles were released from the trachea. The apparent cause of death of this animal was either a direct dosing error or inadvertent aspiration of the test item leading to a chronic aspiration pneumonia. All clinical findings in the F1 male, test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For the F1 females, there were no deaths directly attributed to the test substance administration during the mating, gestation, and lactation periods. Female No. 1352 (Group 4, 750 mg/kg/day) was found dead on Day 96 (LD 1) with the macroscopic necropsy finding of dark red substance present in the uterus. On the previous day (LD 0), clinical signs for Animal No. 1352 included piloerection, dystocia, labored breathing, lethargy, and excess red substance present; as the animal was not hypothermic and was actively delivering pups at the time of evaluation by the veterinarian, the veterinarian recommendation was for continued monitoring. From Day 71 through Day 113, there were no test item-related clinical observations in the F1 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F1 females, there were no test item-related clinical observations during the gestation period (GD 0 through 23) or lactation period (LD 0 through 21). One dam (No. 1316) given 750 mg/kg/day a mass/sore on the chest for the entire lactation period and from days 9 (mass) or 18 (sore) through 21 of gestation. The area was not seen at terminal necropsy and thus was not saved for evaluation. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 50, 61
Individual Data: Appendix 2, Tables 42, 49, 50
No test item-related effects on F1 reproductive performance were observed at any dosage level. There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 92.9%, 92.6%, 85.7%, and 88.0% and female mating indices were 92.9%, 92.9%, 85.7%, and 88.0% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data male and female F1 mating indices ranged from 70.0% to 100% and 50% to 100%, respectively. Male fertility indices were 96.2%, 92.0%, 95.8% and 81.8% and female fertility indices were 96.2%, 92.3%, 95.8% and 81.8% in the same respective groups. Although the fertility index for males and females was lower in the group given 750 mg/kg/day, all fertility indicies were within the historical control data ranges of 76.2% to 100% for F1 males and 70.8% to 100% for F1 females. All test item-treated F1 females evaluated during the final 3 weeks of the 10-week pre breed period were noted to be cycling compared with 89.3% of the control females noted to be cycling.
There was no statistically significant difference in the number and percent of test item-treated females with abnormal or irregular cycles compared with the control group. There were no differences in mean cycle length (4.1 to 4.3 days) and number of cycles (4.0 to 4.4) in test item treated groups compared with control values (4.0 days and 4.6, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group values of 2.8 days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 61, Individual Data: Appendix 2, Table 50.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Gestation indices were 100%, 100%, and 88.9% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 96.0%. The historical control data range for F1 gestational index is 94.1% to 100%. The slight, though not statistically significant, decrease in the liveborn gestation index in the group given 750 mg/kg/day was due to Female Nos. 1324 and 1344 having implantation sites but not delivering a litter. There was also one control female (No. 1024) that had implantation sites but did not deliver a litter. Therefore, these observations are not considered test item related. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 37, 38, 39, 40, 45, 46, 47, 48, Individual Data: Appendix 2, Tables 33, 34, 39, 40. There were no adverse, test item-related effects on F1 body weights or feed consumption during the prebreed exposure period (Days 0 to 70). The increased (7% to 11%) body weights for F1 females given 50 mg/kg/day, though statistically significant at every interval from Day 0 to Day 70, was not considered test item-related since there was no dose relationship and only occasional corresponding significant increases in body weight change, food consumption (g/day only), and food efficiency. Although other occasional statistically significant changes in body weight, body weight change, food consumption, and food efficiency were noted when compared with the control group, including the slightly decreased (4.4% and 5.4%) percent food efficiency for the overall interval from Days 0 to 70 for F1 males given 50 and 750 mg/kg/day, respectively, these changes were not considered the result of test item administration since these changes were sporadic, slight, not dose related, and generally had no effect on the other body weight and/or food consumption parameters. There were no adverse test item-related effects on F1 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. Statistically significant decreases in body weight change and percent food efficiency for one interval (Days 91 to 98) were noted for the F1 males given 750 mg/kg/day; however, these changes did not result in or correlate with statistically significant effects on body weight or feed consumption (g/day and g/kg/day) compared with the controls for this same interval and were thus considered nonadverse. The statistically significant increases in body weight change and/or percent food efficiency during the postmating holding period, for F1 males given the lower dose levels, were sporadic and not dose related, and therefore not considered test item related. There were no statistically significant changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F1 females that were never found sperm positive or that never delivered a live litter.

GESTATION
Summary Data: Tables 51, 52, 53, 54, Individual Data: Appendix 2, Tables 43, 44.
There were no test item-related changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group during gestation. Although body weights for females given 50 mg/kg/day were statistically significantly increased at each interval, there were no corresponding statistically significant increased in body weight change, feed consumption, or percent food efficiency. The change in body weights was not considered test item-related as there was no dose response.

LACTATION
Summary Data: Tables 56, 57, 58, 59, Individual Data: Appendix 2, Tables 46, 47.
During lactation (LD 0 to 21), there were no notable test item-related changes in F1 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group. The one statistically significant change (an increase in body weight for females given 50 mg/kg/day on LD 21) was not considered test item-related as the change was sporadic, showed no dose response, and did not correlate with any changes in any other body weight or food parameters.

ANDROLOGY
Summary Data: Table 42, Individual Data: Appendix 2, Table 37
There was an equivocal, non-adverse, statistically significant increase in the percent of abnormal sperm in the F1 males given 750 mg/kg/day compared with the control group; however, the historical control data range of 1.29% to 6.1% encompasses the 2.29% seen in the high dose in this study. There was an increase in the mean number of sperm with blunt heads (indicating the absence of the acrosome), sperm with small heads, and sperm with heads only or tails only. One particular male in the high dose group (Animal No. 1339) had a very high percentage of sperm with heads only; this breakage was possibly test item-related and Female No. 1336, mated with this male, failed to become pregnant. No test item-related effects were observed on the F1 spermatogenesis endpoints of mean testicular and epididymal sperm numbers and sperm production rate, motility, and progressive motility in males at any dosage concentration. Differences from the control group were slight and were not statistically significant for these parameters.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 42, 43, 61, 67, 68, Individual Data: Appendix 2, Tables 36, 38, 50, 58, 59, 60.
The only test item-related macroscopic findings in F1 parental animals were observed in the lungs of males and females given 750 mg/kg/day. At the F1 male scheduled necropsy, a total of 11 males given 750 mg/kg/day were noted as having lung findings, including small dark red spots and mottled (alone or with other findings such as firm, tan, and/or small dark spots), and these gross findings correlated with an increase in both absolute and relative lung weights in this dose group. There was an increased incidence of mottled kidneys (bilateral) in the males given 750 mg/kg/day (10 of 24) compared with the control group (4 of 28). All other macroscopic gross findings in F1 males were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. At the scheduled necropsies on LD (≡PND) 21, a total of 4 F1 females given 750 mg/kg/day were noted as having lung findings including mottled, firm, tan, for 2 animals and left cranial lobe, tan, meaty for 2 animals; these gross findings correlated with an increase in absolute lung weights in this dose group. There were 2 F1 females given 750 mg/kg/day that had a fluid-filled cyst in the uterus at necropsy; the relationship to test item administration of this finding is unknown. All other macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 42, 67, Individual Data: Appendix 2, Tables 36, 58.
There was an increase in both absolute and relative lung weights (39.5% and 37.7%, respectively) in F1 males given 750 mg/kg/day. Absolute and relative lung weights were also increased [12.9% (statistically significant) and 6.9% (not statistically significant), respectively] in F1 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic and microscopic lung findings in rats given 750 mg/kg/day.

MICROSCOPIC EXAMINATION
Summary Data: Tables 44, 67, 69, Individual Data: Appendix 3.
Like the F0 males and females, chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F1 males and females given 750 mg/kg/day compared with the controls. All 24 F1 males and 24 F1 females given 750 mg/kg/day that survived until scheduled euthanasia had this test item-related finding. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. Also like the F0 animals, alveolar macrophage aggregation, alveolus was noted in F1 control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle. Test item-related slight increases in incidence of renal tubule degeneration/necrosis and renal tubule hyaline droplets were seen in the F1 males given 750 mg/kg/day. Degeneration/necrosis was characterized by the presence of multifocal, cortical proximal convoluted tubules which were lined by epithelial cells that were slightly vacuolated with nuclear pyknosis and containing occasional sloughed necrotic cells. This change was very subtle and recorded as minimal. In a few instances, lightly staining
eosinophilic droplets were increased in proximal convoluted tubules of the same animals. This morphologic appearance suggested hydrocarbon-induced α2μ-globulin male rat nephropathy. Hyaline droplets were not observed in the treated females. Kidneys from selected males given the vehicle or 750 mg/kg/day from both the F0 and F1 generations (10 males/group/generation) were stained with Mallory Heidenhain stain which is used to characterize α2μ-globulin in renal tubules. As expected due to the age of these rats, renal tubules from all of these selected animals contained variable numbers of tubule droplets which stained positive and had the characteristic distribution and features of α2μ-globulin (male rats of this age contain α2μ-globulin). The droplets were subjectively scored on a scale of 1 to 5 +. No apparent increase in positive tubule droplets was present in the F0 males given 750 mg/kg/day compared with the F0 control males. However, the mean average score of the positive tubule droplets was increased in the F1 males given 750 mg/kg/day compared with the F1 control males, confirming that these kidney findings were the result of α2μ-globulinergic nephropathy, which is known to occur in male rats following exposure to hydrocarbons.Other changes in the kidneys, such as chronic progressive nephropathy and renal tubule regeneration, were not clearly increased in either incidence or in severity and were therefore not deemed test item-related. For F1 males and females in the low- and mid-dose groups suspected of reduced fertility, there were no test item-related microscopic findings in the reproductive organs/tissues examined. Two males given 50 mg/kg/day and 3 males given 200 mg/kg/day had inflammation of the prostate gland; however, 7 males given the vehicle control and 9 males given the high dose of 750 mg/kg/day had a similar finding; thus, this finding was not considered test item-related. One male given 200 mg/kg/day showed seminiferous tubule atrophy in the testis. One female given 50 mg/kg/day had dilatation of the uterus and one other had an ovarian cyst while one female given 200 mg/kg/day had ovarian atrophy. These microscopic findings were not observed in F1 males and females given the high dose of 750 mg/kg/day and may have contributed to the reduced fertility for these particular animals. A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat. Ovarian follicle counts were not statistically significantly different in the F1 females given 750 mg/kg/day compared with the control group; therefore, counts were not conducted on the lower dose groups.

F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 61, Individual Data: Appendix 2, Tables 50, 51.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21 (for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 63, Individual Data: Appendix 2, Tables 51, 54.
The numbers of F2 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F2 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 17, 17, 25, and 17 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 1352 found dead on LD 1) accounted for 10 of the 17 pups in the group given 750 mg/kg/day. In addition, 3, 3, 3 and 0 pups in the same respective groups were missing (and presumed dead).

ANOGENITAL DISTANCE
Summary Data: Table 62, Individual Data: Appendix 2, Table 52.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F2 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 62, Individual Data: Appendix 2, Table 53.
Mean male and female pup body weights and body weight changes in the 50, 200, and 750 mg/kg/day groups were unaffected by parental test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 63, 64, Individual Data: Appendix 2, Tables 54, 55. The number of F2 pups (litters) stillborn, found dead, or euthanized from PND 0 through PND 21 were 17(11), 16(13), 25(13), and 17(4) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of F2 pups that were stillborn, found dead, or euthanized. Findings in the stillborn, found dead, or euthanized pups from test item-treated dams were similar to the findings in stillborn, found dead, or euthanized pups from vehicle-treated dams, culled PND 4 pups, and/or scheduled death PND 21 F2 pups.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 2, Table 57. At the PND 21 necropsy of F2 weanlings, there were no test item-related findings. Hydronephrosis of the right kidney was noted at a similar incidence in males in the control and test item-treated groups.

ORGAN WEIGHTS
Summary Data: Table 65, Individual Data: Appendix 2, Table 56.
There were no statistically significant changes in F2 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 7% to 8% and 4% to 7%, respectively) were observed in the F2 male and female pups in the group given 750 mg/kg/day compared with the control group. Given the small magnitude of the change, the lack of statistical significance, and since the weights fall within the historical control data ranges, these changes were considered equivocal and not adverse.

MICROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 3.
Dilatation of the pelvis of the kidney was noted for all male pups with a macroscopic finding of hydronephrosis; thus, this finding was noted at a similar incidence in the control and test item treated groups and was therefore not considered test item-related.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
other: F1 and F2
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility/Developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

OBJECTIVE AND METHODS

The objective of this study was to characterize the effects of the test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrus cycle, mating behavior, conception, gestation, parturition, lactation, and weaning of the F1generation, as well as the neonatal survival, growth, and development of the F1and F2offspring. In addition, the systemic toxicity of the test substance was partially characterized in this study by evaluation of several nonreproductive tissues. Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0and F1generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 50, 200, and 750 mg/kg/day for the F0and F1generations. A concurrent control group of 25 and 28/sex for the F0and F1generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. F0animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration. The test item was administered to offspring selected to become the F1parental generation following weaning, beginning on postnatal day (PND) 22. The F0and F1males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0and F1females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia. Animals were observed at least twice daily for mortality. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for determination of estrous cycles beginning 21 days prior to pairing. All F0and F1females were allowed to deliver and rear their pups until weaning on lactation day (LD≡PND) 21. Clinical observations, body weights, and sexes for F1and F2pups were recorded at appropriate intervals. For both generations (F1and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0animals were selected on PND 21 to constitute the F1generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1rats. Nonselected F1pups were necropsied on PND 21, and F2pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1and F2pups that were necropsied on PND 21. Each F0and F1parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0and F1males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1females in the control and high-dose groups. Designated tissues from all F0and F1parental animals were examined microscopically.

Applicant's summary and conclusion

Conclusions:
SUMMARY OF RESULTS
Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test item related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test article related increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes.

Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy.

There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the low and mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarbon induced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS
The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
Executive summary:

SUMMARY OF RESULTS AND CONCLUSION

Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test itemrelated effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test articlerelated increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes. Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy. There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the lowand mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.