Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-Sep-2015. Study initiation Date 28-Apr-2016. Experimental completion date
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Documentation regarding the purity and stability of the test item is on file with the Sponsor and WIL Research. A Certificate of Analysis for the test item was provided by the Sponsor and is presented in the attached Appendix B. The purity of the test item was 100%. The test item was stored at room temperature, protected from light, and was considered stable under these conditions. A reserve sample of the test item was collected and stored in the WIL Research Archives.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat.
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 29-Sep-2015. The animals were approximately 58 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was
implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 21 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL HOUSING
Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material . The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
Assignment of individual animals to social groups is presented in the attached Appendix D. During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding
material. Following the breeding period, the males were individually housed in solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in plastic maternity cages with bedding material. The dams and their litters were housed in these cages until euthanasia on PND 13 (pups) or lactation day 14 (dams). Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25. The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed
in groups of 2-3 in clean solid-bottom cages until euthanasia. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse
osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. All F0 animals (including animals not selected for clinical pathology evaluation) were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

ENVIRONMENTAL CONDITIONS
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in the attached Appendix E. Actual mean daily temperature ranged from 69.9°F to 70.7°F (21.1°C to 21.5°C) and mean daily relative humidity ranged from 35.5% to 61.0% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
During the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health, meeting acceptable body weight requirements, and females that exhibited a normal 4-5 day estrous cycle were selected for use in the computerized randomization procedure
based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony. The experimental design for the consisted of 4 test item-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose group were selected to be evaluated following a 15-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (study day 0), the males and females were approximately 11 weeks old. Male body weights
ranged from 332 g to 468 g and female body weights ranged from 220 g to 281 g on study day 0. The animals were approximately 13 weeks old when paired on study day 13; female body weights ranged from 224 g to 301 g on gestation day 0.

Route of administration:
oral: gavage
Details on route of administration:
The test item, in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily
Vehicle:
arachis oil
Details on oral exposure:
The vehicle used in preparation of the test item formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 2EA0347 and 2EH0290, exp. dates: 31-Jan-2016 and 31-Jul-2016, respectively).

The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots wereprepared for daily dispensation to the control group and stored at room temperature, protected from light. On each dosing day, aliquots were stirred in a water bath at 37°C ± 5°C for a minimum of 30 minutes prior to dosing and continuously throughout dosing.

Dosing formulations were prepared at the test item concentrations indicated in the following
table:
Group Number Treatment Dosage Level (mg/kg/day) Test Item Concentrationa (mg/mL)
1 Vehicle control 0 0
2 Test substance 100 20
3 Test substance 300 60
4 Test substance 500 100
5 Test substance 1000 200
a = The dosing formulations were not adjusted for purity.

Prior to formulation, the test item was melted in an incubator set to approximately 50°C. The test item formulations were prepared approximately weekly as single formulations for each dosage level, heated in a water bath at 50°C, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. On each dosing day, aliquots were stirred in a water bath at 37°C ± 5°C for a minimum of 30 minutes prior to dosing and continuously throughout dosing.
The first test item dosing formulations were visually inspected by the Study Director’s designee and were found to be visibly homogeneous and acceptable for administration.
Analytical verification of doses or concentrations:
yes
Remarks:
gas chromatography method with flame ionization detection
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test item formulations ranging in concentration from 20.0 to 220 mg/mL were previously established following 5 and 11 days of room temperature storage (Farris, 2015, WIL-168245).
Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first and last (in which all dosage levels were prepared) test item dosing formulations and from the middle stratum of the first and last control group dosing formulations. One set of samples from each collection was subjected to the appropriate analyses.
All remaining samples were stored at room temperature, protected from light, as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection

Analyses of Dosing Formulations Report: please see attached Appendix C
The analyzed dosing formulations were within WIL Research SOP range for suspensions (85% to 115%) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).


Results of the analyses of dosing formulations are summarized below.

Results of Homogeneity and Concentration Analyses
Group 2 Group 3 Group 4 Group 5
(20 mg/mL) (60 mg/mL) (100 mg/mL) (200 mg/mL)

Homogeneity Assessment of the 19-Oct-2015 Formulations

Mean Concentration (mg/mL) 17.8 52.7 85.2 172

RSD (%) 2.7 1.8 1.1 1.5

Mean % of Target 89.2 87.8 85.2 85.9

Homogeneity Assessment of the 07-Dec-2015 Formulations

Mean Concentration (mg/mL) 18.9 51.9 86.8 174

RSD (%) 3.9 2.5 3.1 1.8

Mean % of Target 94.7 86.5 86.8 87.0
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 100, 300, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen.
Control animals:
yes, concurrent vehicle
Details on study design:
SAMPLING AND ANALYSES
Homogeneity and stability of the test item formulations ranging in concentration from 20.0 to 220 mg/mL were previously established following 5 and 11 days of room temperature storage (Farris, 2015, WIL-168245).
Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first and last (in which all dosage levels were prepared) test item dosing formulations and from the middle stratum of the first and last control group dosing formulations. One set of samples from each collection was subjected to the appropriate analyses.
All remaining samples were stored at room temperature, protected from light, as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection

TEST SYSTEM, ANIMAL RECEIPT, AND ACCLIMATION PERIOD
Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat. The number of animals selected for this study (15 [Groups 1 and 5] or 10 rats/sex/group [Groups 2-4]) was the based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 28-Jul-2015, which recommends evaluation of at least 8 pregnant females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 females/group at termination. In addition, 5 animals/sex in the control and high-dose groups was necessary to assess the recovery, persistence, or progression of any toxic effects following the cessation of dosing.

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS, AND TREATMENT REGIMEN
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were
dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-63 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 49 doses for females, these animals remained on study for a 15-day recovery period. The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

The following table presents the study group assignment:
Group Number Treatment Dosage Level (mg/kg/day) Dose Volume (mL/kg) Number of Males Number of Females
1 Vehicle control 0 5 15a 15a
2 Test substance 100 5 10 10
3 Test substance 300 5 10 10
4 Test substance 500 5 10 10
5 Test substance 1000 5 15a 15a
a = 5 animals/sex in Groups 1 and 5 were necropsied following a 15-day recovery period. Recovery animals were not evaluated for reproductive performance.

Dosage levels were selected based on the results of a previous 14-day study in rats (Herberth, 2015, WIL-168243). In that study, the test item was administered to male and female rats for 14 consecutive days at dosage levels of 100, 300, and 1000 mg/kg/day. There were no significant clinical observations noted at any dosage level. Slightly lower mean body weight gains were noted in the 1000 mg/kg/day group males during the treatment period. At the scheduled necropsy, there were no significant macroscopic findings noted at any dosage level.

Based on these results, dosage levels of 100, 300, 500, and 1000 mg/kg/day were selected for the current study.
The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.


Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behavior, moribundity, mortality, and signs of overt toxicity. Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties. Due to social housing, some observations could not be attributed to a single animal; however, no social housing observations were noted.

BODY WEIGHTS
Individual male body weights were recorded weekly throughout the study until euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase). Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males and recovery phase females). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, 4, 7, 10, and 13. Mean body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation and lactation intervals (days 0-20 and 1-13, respectively). Weekly body weights for females with no evidence of mating are presented in the individual report tables until necropsy. When body weights could not be determined for an animal during a given interval (as females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOOD CONSUMPTION
Food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period).
Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period for animals selected for pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 10, and 13; food consumption was reported as g/animal/day and g/kg/day during gestation and lactation. Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia. Weekly food consumption for females with no evidence of mating is presented in the individual report tables until necropsy. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

ESTROUS CYCLES
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each breeding phase female for 14 days prior to test item administration and continuing until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating. Pretest estrous cycle data is presented in the attached Appendix F. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation.

BREEDING PROCEDURES
The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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

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


Male Fertility Index (%) = Total No of males siring a litter divided by total No of males used for mating x 100

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


Female Fertility Index (%) = No of females with confirmed pregnancy divided by total number of females used for mating x 100


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

PARTURITION
All females selected for pairing were allowed to deliver naturally and rear their young to PND 13. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

FOB ASSESSMENTS
FOB assessments were recorded for 5 animals/sex/group following 25 or 26 days of dose administration (study week 3; males selected for pairing) and on lactation day 13 (females; following separation from pups). The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991; and O’Donoghue, 1989). FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below (refer to attached Appendix G for a detailed description of the scoring criteria used for each observation and to the attached Appendix H for positive control/validation data):

HOME CAGE OBSERVATIONS
Posture
Convulsions/Tremors
Feces consistency
Biting
Palpebral (eyelid) closure

HANDLING OBSERVATIONS
Ease of removal from cage
Lacrimation/Chromodacryorrhea
Piloerection
Palpebral closure
Eye prominence
Red/Crusty deposits
Ease of handling animal in hand
Salivation
Fur appearance
Respiratory rate/character
Mucous membranes/Eye/Skin color
Muscle tone

OPEN FIELD OBSERVATIONS
Mobility
Rearing
Convulsions/Tremors
Grooming
Bizarre/Stereotypic behavior
Time to first step (seconds)
Gait
Arousal
Urination/Defecation
Gait score
Backing
Note: Open field observations were evaluated over a 2-minute observation period.

SENSORY OBSERVATIONS
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation

NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength
Hindlimb foot splay
Grip strength-hind and forelimb
Rotarod performance
Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al. (1979). The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

PHYSIOLOGICAL OBSERVATIONS
Catalepsy
Body temperature
Body weight

MOTOR ACTIVITY
Motor activity was assessed for 5 animals/sex/group following 25 or 26 days of dose administration (study week 3, males selected for pairing) or on lactation day 13 (females; following separation from pups). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as
a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams). Refer to attached Appendix H for motor activity validation and positive control data.




Sacrifice and pathology:
CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsies (study week 4 for males and lactation day 14 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; study day 42 for males and study day 63 for females). All F0 animals (including those males and females not scheduled for clinical pathology assessments) were fasted overnight prior to blood collection (see Deviations from the Protocol). Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry). Clinical pathology methods, procedures, and references are presented in the attached Appendix I.
The interpretation of the clinical pathology data was the responsibility of WIL Research (attached Appendix J). The following parameters were evaluated:

HEMATOLOGY AND COAGULATION
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLATELET)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
Reticulocyte count Percent (RETIC)
Absolute (RETIC ABSOLUTE)
Mean platelet volume (MPV)
Differential leukocyte count -Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Red cell distribution width (RDW)
Hemoglobin distribution width (HDW)
Platelet estimate a
Red cell morphology (RBC Morphology) a
( ) = Designates tabular abbreviation
a = Presented on individual tables if a manual differential was performed, and the manual data were accepted and reported instead of the automated differential data

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATION
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia. Females that delivered were euthanized on lactation day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating); uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). For females that delivered or had macroscopic evidence of implantation, the numbers of implantation sites or former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea
were also recorded for females with macroscopic evidence of implantation that did not deliver. Females not selected for pairing were euthanized following the 15-day recovery period. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):

Adrenal glands (2)
Levator ani plus bulbocavernosus (LABC) muscles
Aorta
Bone with marrow (sternebrae)
Ovaries and oviducts (2)
Brain
Pancreas
Coagulating glands (2)
Peripheral nerve (sciatic)
Cowper’s gland (2)
Pituitary gland
Eyes with optic nerve (2)a
Prostate gland
Gastrointestinal tract
Salivary gland [mandibular (2)]
Esophagus
Seminal vesicles (2)
Stomach
Skeletal muscle (rectus femoris)
Duodenum
Skin with mammary gland b
Jejunum
Spinal cord (cervical)
Ileum
Spleen
Cecum
Testes with epididymidesc (2) and vas deferens
Colon
Rectum
Thymus gland
Glans penis
Thyroids [with parathyroids, if present (2)]
Heart
Kidneys (2)
Trachea
Lymph node
Urinary bladder
Axillary (2)
Uterusd with cervix and vagina
Mandibular (2)
All gross lesions (all groups)
Mesenteric
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
a = Placed in Davidson’s solution.
b = For females; a corresponding section of skin was taken from the same anatomic area for males.
c = Placed in modified Davidson’s solution. Care was taken to ensure separation between left and right organs.
d = Uterus not retained if placed in 10% ammonium sulfide solution.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands Liver
Brain Ovaries with oviducts
Cowper’s gland (2)
Pituitary gland
Epididymidesa
Prostate gland
Glans penis
Spleen
Heart
Testesa
Kidneys
Thymus gland
Levator ani plus bulbocavernosus (LABC) muscles
Thyroids with parathyroids b
a = These paired organs were weighed separately.
b = Fixed in 10% neutral-buffered formalin prior to weighing.
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides. Microscopic examination was performed on all tissues listed in Section ANATOMIC PATHOLOGY. from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on all gross lesions from all animals in all groups at the primary necropsy. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as
appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc. The pathology report is presented in the attached Appendix J.


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

LITTER REDUCTION
To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. All selections were performed by computerized randomization. Blood samples for possible future thyroid hormone analysis were collected from 1 culled pup/sex/litter (pooled by litter) on PND 4 (see Section 5.12.); pups were euthanized by decapitation following blood collection and discarded. Remaining culled pups (not used forblood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

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

BODY WEIGHTS
Pups were individually weighed on PND 1, 4, 7, 10, and 13. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were designated as “NA” on the individual report tables.

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

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

CALCULATION OF LITTER PARAMETERS
Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0 divided by No. of Litters with Viable Pups PND 0

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

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) divided by No. of Litters Per Group x 100

Where N = PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10, 10-13, and 4 (post-selection)-13

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

THYROID HORMONE ANALYSIS
Blood samples for thyroid hormone analysis were collected as follow. Blood (approximately 1.0 mL) was collected via the retro-orbital sinus following isoflurane anesthesia from all F0 males and females on the day of scheduled euthanasia (study day 28 [primary] or 42 [recovery] for males and study day 63 [recovery] and lactation day 14 for females) (see Deviations from the Protocol). On PND 4, blood (as much as possible; pooled by litter) was collected via cardiac puncture under isoflurane inhalation from 1 culled pup/sex/litter (see Deviations from theProtocol). On PND 13, blood (approximately 1.0 mL) was collected via cardiac puncture under isoflurane inhalation from 1 pup/sex/litter. Blood was collected into tubes without anticoagulant and allowed to clot at room temperature. Serum was isolated in a refrigerated centrifuge and stored frozen (approximately -70ºC). The following thyroid hormone parameters were evaluated for F0 males and F1 PND 13 males and females: Thyroxine (T4)
The samples from the F0 females and F1 culled pups (PND 4) were not analyzed.
Statistics:
STATISTICAL METHODS
All analyses will be two-tailed for significance levels of 5% and 1%. All means will be presented with standard deviations. All statistical tests will be performed by a computer with appropriate programming as referenced below in section "any other information on materials & methods". Following completion of the mating period, data from nongravid females will be excluded from calculation of means and from comparative statistics. The litter, rather than the pup, will be considered as the experimental unit.

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit.

Clinical signs:
no effects observed
Description (incidence and severity):
F0 GENERATION
CLINICAL OBSERVATIONS AND SURVIVAL
See attached Summary Data: Table S1, Table S2, Table S3, Table S4, Table S5, Table S6 Individual Data: Table A1, Table A2, Table A3, Table A4, Table A5, Table A6, Table A7, Table A8
All F0 males and females survived to the scheduled necropsies. Increased incidences of clinical findings consisting of red material around the nose for F0 males and females in all test item-treated groups and red and/or clear material around the mouth for F0 males and females in the 300, 500, and 1000 mg/kg/day groups were noted approximately 1 hour following dose administration generally throughout the treatment period. Although these findings were considered test item-related, due to the absence of any other evidence of toxicity in the F0 animals, these findings were not considered adverse. Other clinical findings noted in the test item-treated groups, including hair loss on various body surfaces, were noted infrequently and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
MALES
Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.
See attached Summary Data: Table S7, Table S8, Table S9, Table S10; Figure 1, Figure 2 Individual Data: Table A9, Table A10, Table A11, Table A12

FEMALES
Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group F0 females were unaffected by test item administration during the pre-mating and recovery periods. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p<0.05) higher mean body weight gain was noted for the 1000 mg/kg/day group females compared to the control group when the overall pre-mating period (study days 0-13) was evaluated. In the absence of a corresponding effect on mean body weights, the difference in mean body weight gain in the 1000 mg/kg/day group was not attributed to the test item.
See attached Summary Data: Table S11, Table S12, Table S13, Table S14; Figure 3, Figure 4 Individual Data: Table A13, Table A14, Table A15, Table A16

GESTATION
Mean F0 maternal body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. Differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.
See attached Summary Data: Table S15, Table S16; Figure 5 individual Data: Table A17, Table A18

LACTATION
Mean F0 maternal body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.
See attached Summary Data: Table S17, Table S18; Figure 6 Individual Data: Table A19, Table A20

OFFSPRING BODY WEIGHTS
See attached Summary Data: Table S76, Table S77; Figure 15, Figure 16 Individual Data: Table A85, Table A86 Historical Control Data: Appendix N

Mean F1 male and female pup body weights and body weight changes during PND 1-13 in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected byparental administration of the test item. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) higher mean body weight gains were noted for the 300 and 1000 mg/kg/day group male pups compared to the control group during PND 4 to 7. In the absence of corresponding effects on absolute mean pup weights in this group, these transient differences were not attributed to the test item.
Food efficiency:
no effects observed
Description (incidence and severity):
FOOD CONSUMPTION

MALES
Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group F0 males was similar to that in the control group throughout the study. No statistically significant differences were observed.
See attached Summary Data: Table S19, Table S20 Individual Data: Table A21, Table A22

FEMALES
Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group F0 females was unaffected by test item administration during the pre-mating and recovery periods. None of the differences from the control group were statistically significant.
See attached Summary Data: Table S21, Table S22 Individual Data: Table A23, Table A24

GESTATION
Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.
see attached Summary Data: Table S23, Table S24 Individual Data: Table A25, Table A26

LACTATION
Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.
See attached Summary Data: Table S25, Table S26 Individual Data: Table A27, Table A28
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
HEMATOLOGY AND COAGULATION
See attached Summary Data: Table S45, Table S46, Table S47, Table S48 Individual Data: Table A47, Table A48, Table A49, Table A50 Pathology Report: Appendix J
There were no test item-related alterations in hematology and coagulation parameters. However, 1 statistically significant difference was observed when the control and test item-treated groups were compared. Higher mean hemoglobin distribution width (HDW) was noted in the 1000 mg/kg/day group F0 females at the primary necropsy. Measured erythrocyte parameters were similar to the control group, HDW values were similar to the control group at the recovery necropsy, and the difference was biologically irrelevant. Therefore, this finding was attributed to biological variability.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
SERUM CHEMISTRY
See attached Summary Data: Table S49, Table S50, Table S51, Table S52, Table S53, Table S54, Table S55, Table S56 Individual Data: Table A51, Table A52, Table A53, Table A54, Table A55, Table A56, Table A57, Table A58 Pathology Report: Appendix J

There were no test item-related alterations in serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower mean serum total protein was noted in the 1000 mg/kg/day group males at the recovery necropsy; other measured and calculated serum protein values were similar to the control group, values at the primary necropsy were similar to the control group, and the difference was attributed to biological variability. Lower mean serum glucose was noted in the 1000 mg/kg/day group F0 females at the recovery necropsy; values at the primary necropsy were similar to the control group, the magnitude difference was minimal, and the difference was attributed to biological variability.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See attached Summary Data: Table S27, Table S28 Individual Data: Table A29, Table A30 Scoring Criteria: Appendix G Historical Control Data: Appendix L

Home cage parameters were unaffected by test item administration. A significantly (p<0.05) increased incidence of the absence of fecal pellets was noted for males in the 100 mg/kg/day group compared to the control group. However, this did not occur in a dose-related manner and there were no corresponding effects on weekly food consumption noted at any dosage level. Therefore, this finding was not attributed to test item administration. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

See attached Summary Data: Table S29, Table S30 Individual Data: Table A31, Table A32 Scoring Criteria: Appendix G Historical Control Data: Appendix L
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

SENSORY OBSERVATIONS
See attached Summary Data: Table S33, Table S34 Individual Data: Table A35, Table A36 Scoring Criteria: Appendix G Historical Control Data: Appendix L
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

See attached NEUROMUSCULAR OBSERVATIONS Summary Data: Table S35, Table S36 Individual Data: Table A37, Table A38 Scoring Criteria: Appendix G Historical Control Data: Appendix L
Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

PHYSIOLOGICAL OBSERVATIONS
See attached Summary Data: Table S37, Table S38 Individual Data: Table A39, Table A40 Scoring Criteria: Appendix G Historical Control Data: Appendix L
Physiological parameters were unaffected by test item administration. A significantly (p<0.05) longer mean catalepsy time was noted in the 300 mg/kg/day group females compared to the control group on lactation day 13. In the absence of a dose-related response, the difference in catalepsy time was not attributed to the test item. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

MOTOR ACTIVITY
See attached Summary Data: Table S39, Table S40; Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14 Individual Data: Table A41, Table A42 Statistical Analysis Summary: Appendix K Historical Control Data: Appendix M
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all dosage levels when evaluated during study week 3 (males) or on lactation day 13 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, generally within the WIL historical control data ranges, and did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance -treated groups when the F0 animals were evaluated during study week 3 (males) or on lactation day 13 (females).

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
ORGAN WEIGHTS
See attached Summary Data: Table S65, Table S66, Table S67, Table S68 Individual Data: Table A67, Table A68, Table A69, Table A70, Table A71, Table A72, Table A73, Table A74, Table A75, Table A76, Table A77, Table A78 Pathology Report: Appendix J Historical Control Data: Appendix N

There were no test item-related effects on F0 male and female mean final body weights and organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test item-treated groups werecompared. Lower mean heart weight relative to final body weight was noted in the 100 mg/kg/day group F0 males at the primary necropsy. There was no dose-response relationship and the difference was of minimal magnitude; therefore, the finding was attributed to biological variability. Higher mean absolute brain weight was noted in the 1000 mg/kg/day group F0 females compared to the control group at the recovery necropsy. Weights were similar to the control group when corrected for body weight and mean brain weights at the primary necropsy were similar to the control group; therefore, the finding was attributed to biological variability.

ORGAN WEIGHTS (PND 13)
See attached Summary Data: Table S80 Individual Data: Table A89
There were no test item-related changes in mean thyroid/parathyroid weights for F1 pups at 100, 300, 500, and 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
THYROID HORMONE ANALYSIS (F0 MALES)
See attached Summary Data: Table S57, Table S58 Individual Data: Table A59, Table A60 Pathology Report: Appendix J
There were no test item-related effects on mean serum thyroxine (T4) values in F0 males at any dosage level at the primary and recovery necropsies.

MACROSCOPIC EXAMINATIONS
See attached Summary Data: Table S59, Table S60, Table S61, Table S62, Table S63, Table S64 Individual Data: Table A61, Table A62, Table A63, Table A64, Table A65, Table A66 Pathology Report: Appendix J Historical Control Data: Appendix N

No test item-related internal findings were observed at any dosage level in F0 females that failed to deliver, or males and females at the scheduled necropsy. Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, 500, and 1000 mg/kg/day group F0 females were similar to the control group values.

MICROSCOPIC EXAMINATIONS
See attached Summary Data: Table S69, Table S70 Individual Data: Table A61, Table A63, Table A64 Pathology Report: Appendix J
There were no test item-related microscopic findings noted in F0 males and females at any dosage level at the primary necropsy. The histologic changes noted were considered to be incidental findings or related to some aspect of experimental manipulation other than administation of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

NECROPSIES OF PUPS FOUND DEAD
See attached Summary Data: Table S81 Individual Data: Table A90 The numbers of F1 pups (litters) found dead during PND 0-13 numbered 2(2), 5(4), 2(2), 3(2), and 1(1) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively. No internal findings that could be attributed toparental test item administration were noted at the necropsies of the pups that were found dead. Aside from the presence or absence of milk in the stomach,
the following internal findings were noted. Pup no. 6751-01 in the 100 mg/kg/day group was noted with a developmental variation that consisted of an accessory lobule in the liver and pup no. 6721-01 in the control group had a malformation which consisted of an interventricular septal defect (entire septum absent).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
OPEN FIELD OBSERVATIONS
See attached Summary Data: Table S31, Table S32 Individual Data: Table A33, Table A34
Scoring Criteria: Appendix G Historical Control Data: Appendix L

Open field parameters were unaffected by test item administration. A significantly (p<0.01) lower mean rearing count was noted for females in the 500 mg/kg/day group compared to the control group on lactation day 13. This difference did not occur in a dose-related manner and there were no other effects on CNS activity noted at any dosage level. Therefore, the lower mean rearing count in the 500 mg/kg/day group females was not considered test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 3 (males) or on lactation day 13 (females).

REPRODUCTIVE PERFORMANCE
See attached Summary Data: Table S41, Table S42 Individual Data: Table A43, Table A44 Historical Control Data: Appendix N F0 male and female reproductive parameters are presented in the following table.


Text Table 2. Results of Reproductive Performance
Dosage Level (mg/kg/day) WIL HC*

Parameter (Mean) 0 100 300 500 1000 Mean (Range)
Male Mating Index 90.0 100.0 100.0 100.0 100.0 96.8 (84.0-100.0)
Female Mating Index 90.0 100.0 100.0 100.0 100.0 98.2 (86.7-100.0)
Male Fertility Index 90.0 100.0 100.0 90.0 90.0 91.5 (60.0-100.0)
Female Fertility Index 90.0 100.0 100.0 90.0 90.0 93.4 (60.0-100.0)
Male Copulation Index 100.0 100.0 100.0 90.0 90.0 94.8 (70.0-100.0)
Female Conception Index 100.0 100.0 100.0 90.0 90.0 95.2 (65.2-100.0)
Estrous Cycle Length (days) 4.1 4.1 4.2 4.3 4.8 4.3 (3.2-7.6)
Pre-Coital Interval (days) 2.6 3.4 2.3 2.3 2.8 2.9 (1.8-4.8)
* = WIL historical control data
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One, 0, 0, 1, and 1 males in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively, did not sire a litter. One, 0, 0, 1, and 1 females in these same respective groups were determined to be nongravid.
The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
See attached Summary Data: Table S43, Table S44 Individual Data: Table A45, Table A46 Historical Control Data: Appendix N
Mean gestation lengths in the 100, 300, 500, and 1000 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
Details on results:
F1 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
See attached Summary Data: Table S71, Table S72 Individual Data: Table A79, Table A80, Table A81 Historical Control Data: Appendix N
The mean number of F1 pups born, live litter size, and the percentage of males at birth in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival in these groups was unaffected by test item administration.

GENERAL PHYSICAL CONDITION
See attached Summary Data: Table S73 Individual Data: Table A82
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups (litters) that were found dead numbered 2(2), 5(4), 2(2), 3(2), and 1(1) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively. One pup each in the 300 and 500 mg/kg/day groups was missing and presumed to have been cannibalized.

ANOGENITAL DISTANCE
See attached Summary Data: Table S74, Table S75 Individual Data: Table A83, Table A84 Historical Control Data: Appendix N
Mean anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

AREOLAE/NIPPLE ANLAGEN
See attached Summary Data: Table S78 Individual Data: Table A87
The mean number of areolae/nipple anlagen in the F1 male pups was not affected by parental administration of the test item at any dosage level. Differences from the control group were not statistically significant.

THYROID HORMONE ANALYSIS (PND 13)
See attached Summary Data: Table S79 Individual Data: Table A88
There were no test item-related changes in mean serum T4 levels in F1 males and females at any dosage level on PND 13.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
dermal irritation
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
neuropathology
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: Under the conditions of this screening study, based on the lack of any adverse effects, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no
Conclusions:
Under the conditions of this screening study, based on the lack of any adverse effects, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive and systemic toxicity and F1 neonatal toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.
Executive summary:

OBJECTIVE

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

STUDY DESIGN

The test item in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 100, 300, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 11 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 49-63 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-cohabitation day 25 or post-mating day 25, respectively) for a total of 40-52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing recovery period. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 25-26 days of dose administration and for 5 females/group on lactation day 13. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (1/sex/litter; see Deviations from the Protocol). All F1 male pups were evaluated for areolae/nipple anlagen on PND 12. Remaining F1pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-dose groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0males and females at the primary and recovery necropsies; only male samples were analyzed. F0males were euthanized following completion of the mating period or 15-day recovery period and F0females were euthanized on lactation day 14 for females that delivered, post-cohabitation or post-mating day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0animals in the control and high-dose groups at the primary necropsy.

Results

All F0 males and females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test item-related clinical findings noted for F0 males and females were noted approximately 1 hour following dose administration generally throughout the dosing period and consisted of red material around the nose in all test item-treated groups and red and/or clear material around the mouth in the 300, 500, and 1000 mg/kg/day groups. In the absence of other signs of systemic toxicity (body weight and food consumption decrements) at any dosage level, these clinical findings were not considered adverse. No test item-related clinical findings were noted for F0males and females at the weekly examinations at any dosage level.

No test item-related effects were noted on parental body weights, body weight gains, or food consumption at any dosage level throughout the study. There were no test item-related effects noted during the FOB or motor activity evaluations for F0animals at any dosage level.

Mean F0 male and female mating, fertility, copulation, and conception indices in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration. There were no test item-related effects noted on mean estrous lengths or the mean numbers of implantation sites and unaccounted-for sites in F0 females at any dosage level.

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (hematology, coagulation, and serum chemistry) were noted for F0 males and females in the 100, 300, 500, and 1000 mg/kg/day groups during the dosing and recovery evaluations. Mean serum T4levels in F0 males were unaffected by test item administration.

Mean numbers of F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, pup body weights and body weight gains, anogenital distance, and areolae/nipple anlagen counts (males only) in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental test item administration. Necropsy findings for F1 pups that died were not suggestive of any association with maternal administration of the test item. There were no test item-related changes in mean serum T4 levels or mean thyroid/parathyroid weights in F1 males and females on PND 13.

Conclusion

Under the conditions of this screening study, based on the lack of any adverse effects, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0reproductive and systemic toxicity and F1 neonatal toxicity of test substance when administered orally by gavage to Crl:CD(SD) rats. 

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: 14-day range-finder study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 July 2015 to 17 November 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study report from a GLP 14-day range-finding study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
protocol deviations having no adverse effect on the study (animal age and heating of dose formulations during stirring)
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Sexually mature male and virgin female Sprague Dawley (Crl:CD(SD) rats were used as the test system on this study.
- Rats (24 males and 24 females) were received in good health from Charles River Laboratories Inc, Raleigh, NC, USA on 07 August 2015.
- The animals were approximately 49 days old upon receipt.
- Each animal was examined by a qualified biologist on the day of receipt.
- The day following receipt, all animals were weighed and clinical observations were recorded.
- Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsocapular region during the acclimation period.
- Animals were housed for an acclimation period of 10 days prior to the first day of treatment.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behaviour.

ANIMAL HOUSING
- Following receipt, all animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH, USA). Analyses of the bedding material were provided by the manufacturer and no contaminants were present at concentrations sufficient to interfere with the outcome of the study.
- Devices provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals' oral health were sanitised weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in the study (PMI Nutrition International, LLC Certified Rodent LabDiet 5002) was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitised once per week.
- Municipal water supplying the facility was sampled for contaminants in accordance with the WIL Research SOP.
- No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of the study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system and the basal diet were provided ad libitum throughout the acclimation period and during the study.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5 °F (22 ± 3 °C) and 50 ± 20 % respectively.
- Room temperature and relative humidity were monitored continuously and were scheduled for automatic collection on an hourly basis.
- Fluorescent lighting provided illumination for a 12-hour light (06:00 hours to 18:00 hours) and 12-hour dark photoperiod. The light status (on or off) was recorded every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot number 2EA0347; Expiry date 31 January 2016
Details on oral exposure:
PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations.
- Aliquots were prepared for daily dispensation to the control group and and stored at room temperature protected from light.
- On each dosing day, aliquots were stirred in a water bath at 37 °C ± 5 °C for a minimum of 30 minutes prior to dosing and continuously throughoutdosing.
- Dosing formulations of 0 mg/kg bw/day (0 mg/mL test item), 100 mg/kgbe/day (20 mg/mL test item), 300 mg/kg bw/day (60 mg/mL test item) and 1000 mg/kg bw/day (200 mg/mL) were not adjusted for purity.
- Prior to formulation, the test item was melted in an incubator set to approximately 50 °C.
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, heated in a water bath at 50 °C, divided into aliquots for daily dispensation, and stored at room temperature protected from light.
- Test item formulations were stirred continuously throughout the preparation and dose administration procedures.
- On each dosing day, aliquots were stirred in a water bath at 37 °C ± 5 °C for a minimum of 30 minutes prior to dosing and continuously throughout dosing.
- The first test item dosing formulations were visually inspected by the study director and were found to be visibly homogeneous and acceptable for administration.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test item formulations were administered orally by gavage via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula once daily for 14 consecutive days (study days 0-13).
- The dosage volume for all groups was 5 mL/kg.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg bw/day dose.
- All animals were dosed at approximately the same time each day.
- Dosage levels were selected by the sponsor and were intended to cover a wide dose range to assess tolerability up to the limit dose of 1000 mg/kg bw/day.
- Dose levels selected were not expected to produce mortality and were intended to provide dose-response information to be used in subsequent studies.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analyses to demonstrate homogeneity, concentration and stability of the test item formulations were not conducted as part of this non-GLP study.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities.
- At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerised randomisation procedure based on body weight stratification in a block design. At that time the individual body weights and corresponding animal identification numbers were entered into WTDMS and a printout containing the animal numbers, corresponding body weights and individual group assignments was generated. Animals were then arranged into groups according to the printout.
- Animals not assigned to the study were euthanised by carbon dioxide inhalation and discarded.
- The experimental design consisted of three test item-treated groups and one control group composed of 5 rats/sex/group.
- At initiation of dose administration (study day 0) the males and females were approximately 8 weeks old and weighed 276 to 314 g (males) and 192 to 217 g (females).
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Individual clinical observations were recorded daily (prior to the dose administration during the treatment period).
- Each male and female was also observed for signs of toxicity approximately one hour following dose administration.
- The absence or presence of findings was recorded for all animals.
- Some observations could not be attributed to a single animal due to social housing and, in these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHT
- Individual male and female body weights were recorded on study days 0, 4, 7, 11 and 14.
- Mean body weight changes were also calculated for the overall treatment period (study days 0-14).

FOOD CONSUMPTION
- Food consumption was recorded on study days 0, 4, 7, 11 and 14.
- Food consumption was measured on a per cage basis for the corresponding body weight intervals.
- Food consumption was normalised to the number of animals/cage and was reported as g/animal/day.
Sacrifice and pathology:
SCHEDULED EUTHANASIA
- Gross necropsy was conducted on all animals at the scheduled euthanasia on study day 14.
- All animals were euthanised by carbon dioxide inhalation.
- Necropsy included examination of the external surface of all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities, including viscera.
- Tissues were preserved in 10 % neutral buffered formalin only as deemed necessary by gross findings.
- All carcasses were discarded.
Other examinations:
ORGAN WEIGHTS
- The following organs were weighed from all animals at the scheduled necropsy: adrenal glands, brain, epididymides (paired organs weighed separately), heart, kidneys, liver, ovaries with oviducts, spleen, testes (paired organs weighed separately), thymus gland and thyroids with parathyroids (fixed in 10 % neutral buffered formalin prior to weighing).
- Unless otherwise noted, paired organs were weighed together.
- Absolute weights and organ to final body weight and brain weight ratios were reported.
Statistics:
STATISTICAL ANALYSIS
- Each mean was presented with the standard deviation (SD), standard error (SE) and the number of animals (N) used to calculate the mean.
- Statistical analyses were not conducted if the number of animals was two or less.
- All statistical tests were performed using WTDMS unless otherwise noted.
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test item-related group to the control group by sex.
- Body weights, body weight changes and absolute and relative organ weight values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett’s test (Dunnett, 1964) was used to compare the test item-related groups to the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not dose related (see below)
Mortality:
mortality observed, treatment-related
Description (incidence):
not dose related (see below)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
differences from control animals were slight and not statistically significant (see below)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
within historical control data ranges (see below)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings observed in the test item-treated groups occurred infrequently (see below)
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL OBSERVATIONS AND SURVIVAL
- All animals in the control, 100, 300 and 1000 mg/kg bw/day groups survived to the scheduled necropsy on study day 14.
- Clinical findings noted at the daily examinations or approximately one hour after dose administration occurred infrequently (including red material around the nose), at similar frequencies in the control group and/or in a manner that was not dose related.

BODY WEIGHTS
- Slightly lower mean body weights were noted in the 1000 mg/kg bw/day group males compared to the control group during study days 0 to 4 followed by a mean body weight gain similar to the control group during study days 4 to 7.
- A significantly (p < 0.05) lower mean body weight gain was noted for the 1000 mg/kg bw/day group males during study days 7 to 11. Mean body weight gains in this group were similar to the control group for the remainder of the treatment period (study days 11-14).
- As a result of the decrements in body weight gain, a slightly lower (not statistically significant) mean body weight gain was noted for the 1000 mg/kg bw/day group males when the overall treatment period (study days 0 to 14) was evaluated.
- In addition, mean body weights for the 1000 mg/kg bw/day group were slightly lower (3.8 %; not statistically significant) than the control group on study day 14.
- Mean body weights and body weight gains in the 100 and 300 mg/kg bw/day group males and 100, 300 and 1000 mg/kg bw/day group females were similar to that in the control group. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
- Mean food consumption, evaluated as g/animal/day, in the 100, 300 and 1000 mg/kg bw/day group males and females was similar to that in the control group.

ANATOMIC PATHOLOGY
- No remarkable internal findings were observed for males and females at any dosage level at the scheduled necropsy on study day 14.
- Macroscopic findings observed in the test item-treated groups occurred infrequently.

ORGAN WEIGHTS
- No remarkable effects on organ weights were noted at any dosage level.
- Significantly (p < 0.05 or p < 0.01) lower mean absolute and relative (to final body weight and brain weight) thymus weights were noted for males in the 100, 300 and 1000 mg/kg bw/day groups. However, these values were within the WIL Research historical control data ranges. No other statistically significant differences in organ weights were noted for males and females in the 100, 300 and 1000 mg/kg bw/day groups compared to the control groups.
Critical effects observed:
not specified
Conclusions:
No effects on survival or clinical condition were noted for males and females at any dosage level. At 1000 mg/kg bw/day, slightly lower mean body weights and body weight gains were noted together with lower mean absolute and relative (to final body and brain weights) thymus weights were noted for males. No evidence of toxicity was observed for males at 100 and 300 mg/kg bw/day or females at any dosage level. Based on these results, dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in male and female Crl:CD(SD) rats.
Executive summary:

GUIDELINE

The study was designed to determine dosage levels of the test item to be evaluated in a potential combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD 422) in rats.

METHODS

The test item was administered orally in arachis (peanut) oil by gavage to three groups of Crl:CD(SD) rats. Each group consisted of 5 males and 5 females and dosing took place daily for 14 consecutive days. Dosage levels were 100, 300 and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 5 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 8 weeks of age at the beginning of test item administration.

All animals were observed twice daily for mortality and morbundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. Complete necropsies were conducted on all animals on study day 14 and selected organs were weighed.

RESULTS

All animals survived to the scheduled necropsy on study day 14. No remarkable clinical observations were noted for males and females at the daily examination or approximately one hour following dose administration at any dosage level.

A slightly lower mean body weight gain was noted for males in the 1000 mg/kg bw/day group compared to the control group when the overall treatment period (study days 0 to 14) was evaluated. In addition, mean body weights in the 1000 mg/kg bw/day group males were slightly lower (3.8 %) compared to the control group on study day 14. Mean body weights and body weight gains in the 100 and 300 mg/kg bw/day males and 100, 300 and 1000 mg/kg bw/day group females were similart to the control group. Mean food consumption for males and females of all dosage levels was similar to the control group.

No remarkable macroscopic findings were noted for males and females at any dosage level at necropsy. Lower mean absolute and relative (to final body weight and brain weight) thymus weights were noted for males in the 100, 300 and 1000 mg/kg bw/day groups. No other differences in organ weights were noted for males and females when the test item-treated groups were compared to the control group.

CONCLUSION

No effects on survival or clinical condition were noted for males and females at any dosage level. At 1000 mg/kg bw/day, slightly lower mean body weights and body weight gains were noted together with lower mean absolute and relative (to final body and brain weights) thymus weights were noted for males. No evidence of toxicity was observed for males at 100 and 300 mg/kg bw/day or females at any dosage level. Based on these results, dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in male and female Crl:CD(SD) rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification