Tall Oil Fatty Acids, compounds with 2-(2-aminoethoxy) ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproductive toxicity potential of the test item was assessed in accordance with OECD Guideline 422.  Under the conditions of this screening study, no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no‑observed-adverse-effect level (NOAEL) for reproductive toxicity of test material when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 100, 300, 500, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2015 to 01 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 01-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 14 days prior to the first day of treatment.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed O'Cobs; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analysed by the manufacturer for contaminants. Analyses of the nesting 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 took place and, 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 housed in solid-bottom cages until the scheduled necropsy.
- Following positive evidence of mating, the females were individually housed in plastic maternity cages with nesting material.
- The dams and their litters were housed in these cages until euthanasia on lactation day 4.
- 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.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research’s 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 exception that all males and recovery phase females 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.
- Actual mean daily temperature ranged from 70.5°F to 74.2°F (23.4°C to 22.0°C) and mean daily relative humidity ranged from 38.0% to 52.0% 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.
- 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 and meeting acceptable body weight requirements was selected for use in the computerized 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. 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 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 10 weeks old.
- Male body weights ranged from 333 g to 401 g and female body weights ranged from 218 g to 249 g on study day 0.
- The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 226 g to 286 g on gestation day 0.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on 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 stored at room temperature, protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, 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.
- The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

Details on mating procedure:

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.

PARTURITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 4.
- During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
- On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test item formulations ranging in concentration from 1 to 220 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 6 and 12 days of room temperature.
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first test item dosing formulations and from the middle stratum of the first and last control group dosing formulation. .
- One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Duration of treatment / exposure:

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMES
- 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 the day prior to 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 3) for a total of 39-44 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 39-52 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 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/day dose. All animals were dosed at approximately the same time each day.

Frequency of treatment:

Once daily

Details on study schedule:

- An overview of study design is given in the attached scheme.
- Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Group 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 5

No. of animals per sex per dose:

- Group 1: 15 males and 15 females
- Group 2: 10 males and 10 females
- Group 3: 10 males and 10 females
- Group 4: 10 males and 10 females
- Group 5: 15 males and 15 females

Five animals/sex in Groups 1 and 5 were necropsied following a 15-day recovery period.
Recovery animals were not evaluated for reproductive performance.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
- The dosage levels were selected based on the results of a previous 14-day study in rats. In the previous study, the test substance 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 signs noted at any dosage level in the previous study. Slightly lower mean body weight gains were noted in the 1000 mg/kg/day males during the treatment period. There were no significant macroscopic findings noted at any dosage level at the scheduled necropsy.
- The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Rationale for animal assignment:
- An overview of study design is given in the attached scheme.
- 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 based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22 Mar 1996, 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, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination.
- In addition, 5 animals/sex in the control and high-dose groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behaviour, 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 and prior to the scheduled euthanasia.
- Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase or without evidence of mating).
- 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 and 4.
- When body weights could not be determined for an animal during a given interval (due to a weighing error, as females enter gestation, 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
- Individual 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 in grams/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 and 4; 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 necropsy.
- Food consumption was reported as g/animal/day and g/kg/day for the corresponding gestation and lactation body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.
- The time periods when food consumption values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4; males selected for pairing) and on lactation day 4 (females).
- 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 scored for home cage parameters, handling observations, open field observations, sensory observations and neuromuscular observations and physiological observations (see Appendix F, attached).

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (study week 4, males selected for pairing) or on lactation day 4 (females).
- 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).

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsy (study week 4 for males and lactation day 4 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 53 for females).
- All males and recovery phase females were fasted overnight prior to blood collection. 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 EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
- The parameters evaluated were 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 and Red cell morphology (RBC Morphology).

SERUM CHEMISTRY
- The parameters evaluated were Albumin, Total protein, Globulin [by calculation] Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Bile acids and Appearance (including degree of hemolysis, lipemia, and icterus).

Oestrous cyclicity (parental animals):

Not investigated

Sperm parameters (parental animals):

Not investigated

Litter observations:

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 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- 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.

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 and 4.
- Any abnormalities in nesting and nursing behavior were recorded.

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

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

Postmortem examinations (parental animals):

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 15-day recovery period.
- 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 macriscopic 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, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded.
- The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed.
- Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through lactation day 4.
- 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.
- The following tissues and organs were placed in 10 % neutral-buffered formalin except where noted: Adrenal glands (2), Aorta, Bone with marrow (sternbrae), Brain, Coagulating glands (2), Eyes with optic nerve (2) placed in Davidson’s solution, Gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), Heart, Kidneys (2), Liver (sections of 2 lobes), Lymph node (axillary (2), mandibular (2), mesenteric), Lungs (including bronchi, fixed by inflation with fixative), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Salivary gland [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland (for females; a corresponding section of skin was taken from the same anatomic area for males), Spinal cord (cervical), Spleen, Testes with epididymides (2) [placed in modified Davidson’s solution with care taken to ensure separation between left and right organs] and vas deferens, Thymus gland, Thyroids (2) [with parathyroids, if present], Trachea, Urinary bladder, Uterus with cervix and vagina [any uterus that was placed in 10 % ammonium sulphide solution for detection of implantation sites was discarded and not preserved in 10 % neutral-buffered formalin] and all gross lesions [all groups].

ORGAN WEIGHTS
- The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (paired organs weighed separately), Heart, Kidnesys, Liver, Ovaries with oviducts, Spleen, Testes (fixed in 10 % neutral-buffered formalin prior to weighing), Thymus gland and Thyroids with parathyroids (fixed in 10 % neutral-buffered formalin prior to weighing).

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol specified tissues were trimmed according to WIL Research’s SOPs and the protocol.
- Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research’s SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from 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.
- Some tissues were not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Postmortem examinations (offspring):

EUTHANASIA
- On PND 4, surviving F1 rats were euthanised via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

- 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. 
- Statisitcial analyses were not conducted if the number of animals was 2 or less.
- Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical analyses were not conducted on F0weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit. 

Reproductive indices:

- Calculation methods for mating, fertility, and copulation/conception indices are attached.

Offspring viability indices:

- Litter parameter definitions are attached.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings of red material around the nose and mouth and clear material around the mouth were noted for males and females at all dosage levels at approximately 1 hour following dose administration generally throughout the treatment period. These findings were generally noted in a dose-related manner and occurred sparingly in the 100 mg/kg/day group. In addition, incidences of rales were noted sporadically for males and females in the 1000 mg/kg/day group at approximately 1 hour following dose administration. These findings were considered test item-related, but due to the absence of any other evidence of toxicity in these groups, these findings were not considered adverse. Further, these findings did not persist to the weekly detailed physical exams performed prior to dose administration. Other clinical findings noted in the test item-treated groups, including hair loss on various body surfaces, were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 500 mg/kg/day group was found dead on lactation day 4 following blood collection. No mortality was noted in the 1000 mg/kg/day group, and due to the lack of any other evidence of toxicity for this female and the close proximity of this death to blood collection, this death was not attributed to test item administration. All other males and females survived to the scheduled necropsies.

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 males were unaffected by test item administration throughout the study. Differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.
- Females: Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating and recovery periods. Higher mean body weight gains were noted for females in the 1000 mg/kg/day group when the pre-mating period (study days 0-13) was evaluated (significant at p<0.05) and when the entire dosing period (study days 0-38) was evaluated (not statistically significant). As a result of the higher mean body weight gains during the pre-mating period, a significantly (p<0.05) higher mean body weight was noted for females in the 1000 mg/kg/day group on study day 35. In addition, body weights for these females were higher (not statistically significant) throughout the recovery period. However, in the absence of any evidence of toxicity, these changes were considered incidental. Other differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Males: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating and recovery periods. Mean food consumption for females in the 300, 500, and 1000 mg/kg/day groups was significantly (p<0.05 and p<0.01) higher than the control group during study days 7-13; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in hematology and coagulation parameters noted at the scheduled necropsies. Statistically significant differences in hematology parameters noted at recovery (higher platelet, lymphocyte, and basophil values in the 1000 mg/kg/day group males and higher hematocrit and absolute neutrophil values in the 1000 mg/kg/day group females) were not considered to be test item-related because values were similar to the control groups at the primary necropsy and were within the WIL historical control database range. Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in serum chemistry parameters. However, statistically significantly lower serum bile acid values in the 100, 300, 500, and 1000 mg/kg/day group females were observed at the primary necropsy when the control and test item-treated groups were compared. The differences were not considered to be test item-related because of a lack of dose response relationship and the change was in a direction of no known toxicologic importance. At the recovery necropsy, serum bile acid values in test item-treated males and females were similar to the control groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histologic changes noted at the primary necropsy. Histologic changes noted in control and test item-treated groups were considered to be incidental findings not related to administration of the test item. However, 1 microscopic findings deserves mention. Atypical renal tubular hyperplasia was noted in a single 1000 mg/kg/day group male at the primary necropsy. The finding was multifocal, unilateral, and restricted to areas of chronic progressive hyperplasia. There were no other proliferative renal changes noted microscopically in the 1000 mg/kg/day group animals. The finding was considered to be a spontaneous finding and not test item related. There were no test item-related alterations in the prevalence, severity, or histologic character of other spontaneous or incidental tissue alterations.

Reproductive performance:

no effects observed

GESTATION
- Mean 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.
- The higher mean body weight gains during the pre-mating period and slightly higher (not statistically significant) mean body weight gains for females in the 1000 mg/kg/day group generally throughout gestation resulted in higher mean body weights for females in the 1000 mg/kg/day group throughout gestation (statistically significant [p<0.05] on gestation days 4 and 11); however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, did not occur
in a dose-related manner, and/or were transient in nature.

LACTATION
- Mean 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.
- The higher mean body weight gains during the pre-mating period and higher mean body weights during gestation in the 1000 mg/kg/day group resulted in significantly (p<0.01) higher mean body weights on lactation days 1 and 4; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.

FOOD CONSUMPTION
- Males: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating and recovery periods. Mean food consumption for females in the 300, 500, and 1000 mg/kg/day groups was significantly (p<0.05 and p<0.01) higher than the control group during study days 7-13; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

GESTATION
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during gestation.
- Mean food consumption for females in the 1000 mg/kg/day group was significantly (p<0.05 and p<0.01) higher than the control group during gestation days 4-7, 7-11 (g/animal/day only), and 0-20 (g/animal/day only); this corresponded to the slightly higher mean body weight gains noted for these females during gestation. However, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

LACTATION
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. A significant (p<0.05) increased incidence of the absence of fecal pellets was noted for males in the 500 mg/kg/day group. However, this did not occur in a dose-related manner and there were no corresponding effects on weekly food consumption. Therefore, this result was considered incidental and is not evidence of an adverse effect. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

OPEN FIELD OBSERVATIONS
Open field 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 4 (males) or on lactation day 4 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

MOTOR ACTIVITY
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 4 (males) or on lactation day 4 (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, with the following exception. Significantly lower mean cumulative ambulatory motor activity counts were noted for females at all dosage levels; however, no dose response pattern was evident and a pattern of habituation was evident. Therefore these differences were attributed to high values in the control group and were considered incidental and are not evidence of an adverse effect. Further, a lower level of habituation was noted in the control group animals during the last 30 minutes of the testing session. Other differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose -related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 4 (males) or on lactation day 4 (females).

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male in each of the 100 and 1000 mg/kg/day groups did not sire a litter. One female in each of 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; differences were not statistically significant.

GESTATION LENGTH AND PARTURITION
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.

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:

other: No treatment-related effects reported for reproduction or the survival, growth and development of the offspring.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- The mean number of 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.
- 3 pups were found dead in the control group, 1 pup was found dead in the 300 mg/kg bw/day group, 2 pups were found dead in the 500 mg/kg bw/day and 3 pups were found dead in the 1000 mg/kg bw/day group. Four and 2 pups in the 300 and 1000 mg/kg/day groups, respectively, was missing and presumed to have been cannibalized.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean male and female pup body weights and body weight changes during PND 1-4 in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental administration of the test item.
- A significantly (p<0.05) higher male pup weight was noted in the 100 mg/kg/day group on PND 4; however, this did not occur in a dose-related manner.
- No other statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead during PND 0-4 numbered 3(3), 0(0), 1(1), 2(2), and 3(2) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively.
- Aside from the presence or absence of milk in the stomach, the only other internal finding was renal papillae not fully developed for 1 pup in the 500 mg/kg/day group. However, this did not occur in a dose-related manner.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects were observed on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The repeated dose toxicity of the test item via the oral route was assessed in accordance with OECD Guideline 422. 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 material when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

GUIDELINE

The study, conducted in accordance with OECD 422, was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

 

METHODS

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 1000 mg/kg/day group (Group 5) consisted of 15 rat/sex and the 100, 300, and 500 mg/kg/day groups (Groups 2, 3, and 4, respectively) consisted of 10 rats/sex. The dosage volume was 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test item administration. Ten males/group 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. Ten females/group received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-44 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 39-52 doses. The extra 5 males and 5 females in the control and 1000 mg/kg/day groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for female, these animals were assigned to a 15-day non‑dosing recovery period, respectively. 

 

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 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology serum chemistry) were performed on 5 F0 animals/sex/group at primary necropsy and from 5 F0 animals/sex in the control and 1000 mg/kg/day groups at the recovery necropsy. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, on 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 F0 animals in the control and 1000 mg/kg/day groups at the primary necropsy.

 

RESULTS

No test item-related mortality or moribundity was noted at any dosage level. Clinical findings of red material around the nose and mouth and clear material around the mouth were noted for males and females at all dosage levels at approximately 1 hour following dose administration generally throughout the treatment period. These findings were generally noted in a dose-related manner and occurred sparingly in the 100 mg/kg/day group. In addition, incidences of rales were noted sporadically for males and females in the 1000 mg/kg/day group at approximately 1 hour following dose administration. These findings were considered test item-related, but due to the absence of any other evidence of toxicity in these groups, these findings were not considered adverse.

 

No test item-related effects were noted at any dosage level on parental body weights, body weight gains, or food consumption throughout the study. Reproductive performance (mating, fertility, copulation, conception, gestation length, and the process of parturition) were unaffected by test item administration at all dosage levels. In addition, functional observational battery parameters and motor activity were unaffected by test item administration at all dosage levels.

 

No test item-related macroscopic or microscopic findings were noted at any dosage level. Clinical pathology parameters and organ weights were unaffected by test item administration.

 

Survival, growth, and clinical condition of F1 pups were unaffected by parental test item administration at all dosage levels. No test item-related macroscopic findings were observed for pups found dead at any dosage level.

 

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 F0 reproductive and systemic toxicity and F1 neonatal toxicity of OS343777 when administered orally by gavage to Crl:CD(SD) rats.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch grade 1, combined 28-day repeated dose oral (gavage) toxicity study with reproduction/developmental toxicity screeening test in rats, with recovery

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The developmental toxicity potential of the test item was assessed in accordance with OECD Guideline 422.  No test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level when the substance was administered to maternal animals at 100, 300, 500 or 1000 mg/kg bw/day.  As such, the NOAEL for neonatal toxicity was 1000 mg/kg bw/day. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2015 to 01 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 01-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 14 days prior to the first day of treatment.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed O'Cobs; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analysed by the manufacturer for contaminants. Analyses of the nesting 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 took place and, 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 housed in solid-bottom cages until the scheduled necropsy.
- Following positive evidence of mating, the females were individually housed in plastic maternity cages with nesting material.
- The dams and their litters were housed in these cages until euthanasia on lactation day 4.
- 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.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research’s 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 exception that all males and recovery phase females 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.
- Actual mean daily temperature ranged from 70.5°F to 74.2°F (23.4°C to 22.0°C) and mean daily relative humidity ranged from 38.0% to 52.0% 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.
- 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 and meeting acceptable body weight requirements was selected for use in the computerized 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. 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 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 10 weeks old.
- Male body weights ranged from 333 g to 401 g and female body weights ranged from 218 g to 249 g on study day 0.
- The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 226 g to 286 g on gestation day 0.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

arachis oil

Details on 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 stored at room temperature, protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, 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.
- The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test item formulations ranging in concentration from 1 to 220 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 6 and 12 days of room temperature.
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first test item dosing formulations and from the middle stratum of the first and last control group dosing formulation. .
- One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Details on mating procedure:

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.

PARTURITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 4.
- 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.

Duration of treatment / exposure:

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMES
- 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 the day prior to 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 3) for a total of 39-44 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 39-52 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 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/day dose. All animals were dosed at approximately the same time each day.

Frequency of treatment:

Once daily

Duration of test:

39 - 52 doses

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Group 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 5

No. of animals per sex per dose:

- Group 1: 15 males and 15 females
- Group 2: 10 males and 10 females
- Group 3: 10 males and 10 females
- Group 4: 10 males and 10 females
- Group 5: 15 males and 15 females

Five animals/sex in Groups 1 and 5 were necropsied following a 15-day recovery period.
Recovery animals were not evaluated for reproductive performance..

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
- The dosage levels were selected based on the results of a previous 14-day study in rats. In the previous study, the test substance 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 signs noted at any dosage level in the previous study. Slightly lower mean body weight gains were noted in the 1000 mg/kg/day males during the treatment period. There were no significant macroscopic findings noted at any dosage level at the scheduled necropsy.
- The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Rationale for animal assignment:
- An overview of study design is given in the attached scheme.
- 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 based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22 Mar 1996, 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, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination.
- In addition, 5 animals/sex in the control and high-dose groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration and on lactation day 4.
- 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 scored for home cage parameters, handling observations, open field observations, sensory observations and neuromuscular observations and physiological observations (see Appendix F, attached).

DETAILED CLINICAL OBSERVATIONS: Yes
- All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behaviour, moribundity, mortality and signs of overt toxicity.
- Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period).
- Each 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 WEIGHT: Yes
- Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase or without evidence of mating).
- 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 and 4.
- When body weights could not be determined for an animal during a given interval (due to a weighing error, as females enter gestation, 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: Yes
- Individual 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 in grams/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 and 4; 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 necropsy.
- Food consumption was reported as g/animal/day and g/kg/day for the corresponding gestation and lactation body weight change intervals. 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

POST-MORTEM EXAMINATIONS: Yes
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation.
- Females that delivered were euthanized on lactation day 4.
- 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 macriscopic 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, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded.
- The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed.
- Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through lactation day 4.
- 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.
- The following tissues and organs were placed in 10 % neutral-buffered formalin except where noted: Adrenal glands (2), Aorta, Bone with marrow (sternbrae), Brain, Coagulating glands (2), Eyes with optic nerve (2) placed in Davidson’s solution, Gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), Heart, Kidneys (2), Liver (sections of 2 lobes), Lymph node (axillary (2), mandibular (2), mesenteric), Lungs (including bronchi, fixed by inflation with fixative), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Salivary gland [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland (for females; a corresponding section of skin was taken from the same anatomic area for males), Spinal cord (cervical), Spleen, Thymus gland, Thyroids (2) [with parathyroids, if present], Trachea, Urinary bladder, Uterus with cervix and vagina [any uterus that was placed in 10 % ammonium sulphide solution for detection of implantation sites was discarded and not preserved in 10 % neutral-buffered formalin] and all gross lesions [all groups].

ORGAN WEIGHTS
- The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (paired organs weighed separately), Heart, Kidnesys, Liver, Ovaries with oviducts, Spleen, Thymus gland and Thyroids with parathyroids (fixed in 10 % neutral-buffered formalin prior to weighing).

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol specified tissues were trimmed according to WIL Research’s SOPs and the protocol.
- Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research’s SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from 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.
- Some tissues were not examined due to the tissue not being in the plane of section, not present at trimming, etc.

OTHER: Yes
MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration or on lactation day 4 (females).
- 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).

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsy (lactation day 4 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 53 for females).
- All recovery phase females were fasted overnight prior to blood collection. 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 EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
- The parameters evaluated were 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 and Red cell morphology (RBC Morphology).

SERUM CHEMISTRY
- The parameters evaluated were Albumin, Total protein, Globulin [by calculation] Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Bile acids and Appearance (including degree of hemolysis, lipemia, and icterus).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes

Fetal 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 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- 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.

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 and 4.
- Any abnormalities in nesting and nursing behavior were recorded.

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

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

EUTHANASIA
- On PND 4, surviving F1 rats were euthanised via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

- 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. 
- Statisitcial analyses were not conducted if the number of animals was 2 or less.
- Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical analyses were not conducted on F0weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit. 

Indices:

- Litter parameter definitions are attached.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings of red material around the nose and mouth and clear material around the mouth were noted for females at all dosage levels at approximately 1 hour following dose administration generally throughout the treatment period. These findings were generally noted in a dose-related manner and occurred sparingly in the 100 mg/kg/day group. In addition, incidences of rales were noted sporadically for females in the 1000 mg/kg/day group at approximately 1 hour following dose administration. These findings were considered test item-related, but due to the absence of any other evidence of toxicity in these groups, these findings were not considered adverse. Further, these findings did not persist to the weekly detailed physical exams performed prior to dose administration. Other clinical findings noted in the test item-treated groups, including hair loss on various body surfaces, were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 500 mg/kg/day group was found dead on lactation day 4 following blood collection. No mortality was noted in the 1000 mg/kg/day group, and due to the lack of any other evidence of toxicity for this female and the close proximity of this death to blood collection, this death was not attributed to test item administration. All other males and females survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Females: Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating and recovery periods. Higher mean body weight gains were noted for females in the 1000 mg/kg/day group when the pre-mating period (study days 0-13) was evaluated (significant at p<0.05) and when the entire dosing period (study days 0-38) was evaluated (not statistically significant). As a result of the higher mean body weight gains during the pre-mating period, a significantly (p<0.05) higher mean body weight was noted for females in the 1000 mg/kg/day group on study day 35. In addition, body weights for these females were higher (not statistically significant) throughout the recovery period. However, in the absence of any evidence of toxicity, these changes were considered incidental. Other differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Females: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating and recovery periods. Mean food consumption for females in the 300, 500, and 1000 mg/kg/day groups was significantly (p<0.05 and p<0.01) higher than the control group during study days 7-13; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in hematology and coagulation parameters noted at the scheduled necropsies. Statistically significant differences in hematology parameters noted at recovery (higher platelet, lymphocyte, and basophil values in the 1000 mg/kg/day group males and higher hematocrit and absolute neutrophil values in the 1000 mg/kg/day group females) were not considered to be test item-related because values were similar to the control groups at the primary necropsy and were within the WIL historical control database range. Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in serum chemistry parameters. However, statistically significantly lower serum bile acid values in the 100, 300, 500, and 1000 mg/kg/day group females were observed at the primary necropsy when the control and test item-treated groups were compared. The differences were not considered to be test item-related because of a lack of dose response relationship and the change was in a direction of no known toxicologic importance. At the recovery necropsy, serum bile acid values in test item-treated males and females were similar to the control groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in final body or organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Group mean final body weights were higher in the 1000 mg/kg/day group females at the primary necropsy. Higher thymus weights were noted in the 300 (absolute and relative to body and brain weights) and 1000 (absolute) mg/kg/day group females at the primary necropsy; there were no dose response effects or microscopic correlates, and the findings were attributed to biological variability. Thymus weights were similar to control groups at recovery.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One female in the 500 mg/kg/day group was found dead on lactation day 4 following blood collection. No significant macroscopic findings were noted for this female at necropsy and the death was not attributed to test item administration.

No test item-related internal findings were observed at any dosage level in females that failed to deliver or 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 groups were similar to the control group values.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histologic changes noted at the primary necropsy. Histologic changes noted in control and test item-treated groups were considered to be incidental findings not related to administration of the test item. There were no test item-related alterations in the prevalence, severity, or histologic character of other spontaneous or incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

BODY WEIGHTS
- LACTATION
- Mean 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.
- The higher mean body weight gains during the pre-mating period and higher mean body weights during gestation in the 1000 mg/kg/day group resulted in significantly (p<0.01) higher mean body weights on lactation days 1 and 4; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, did not occur in a dose-related manner, and/or were transient in nature.

FOOD CONSUMPTION
- Females: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating and recovery periods. Mean food consumption for females in the 300, 500, and 1000 mg/kg/day groups was significantly (p<0.05 and p<0.01) higher than the control group during study days 7-13; however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

- LACTATION
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. There were no other statistically significant differences for the test item-treated groups when compared to the control group during on lactation day 4 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on lactation day 4 (females).

OPEN FIELD OBSERVATIONS
Open field 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 on lactation day 4 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on lactation day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on lactation day 4 (females).

MOTOR ACTIVITY
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated on lactation day 4 (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, with the following exception. Significantly lower mean cumulative ambulatory motor activity counts were noted for females at all dosage levels; however, no dose response pattern was evident and a pattern of habituation was evident. Therefore these differences were attributed to high values in the control group and were considered incidental and are not evidence of an adverse effect. Further, a lower level of habituation was noted in the control group animals during the last 30 minutes of the testing session. Other differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose -related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated on lactation day 4 (females).

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): GESTATION LENGTH AND PARTURITION
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.

BODY WEIGHTS
GESTATION
- Mean 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.
- The higher mean body weight gains during the pre-mating period and slightly higher (not statistically significant) mean body weight gains for females in the 1000 mg/kg/day group generally throughout gestation resulted in higher mean body weights for females in the 1000 mg/kg/day group throughout gestation (statistically significant [p<0.05] on gestation days 4 and 11); however, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, did not occur
in a dose-related manner, and/or were transient in nature.

FOOD CONSUMPTION
GESTATION
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during gestation.
- Mean food consumption for females in the 1000 mg/kg/day group was significantly (p<0.05 and p<0.01) higher than the control group during gestation days 4-7, 7-11 (g/animal/day only), and 0-20 (g/animal/day only); this corresponded to the slightly higher mean body weight gains noted for these females during gestation. However, in the absence of any evidence of toxicity, these changes were considered incidental and are not evidence of an adverse effect. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One female in each of the 100 and 1000 mg/kg/day 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; differences were not statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No treatment-related effects on mating, fertility, gestation length or litter size in the 100, 300, 500 and 1000 mg/kg bw/day groups.

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Mean male and female pup body weights and body weight changes during PND 1-4 in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental administration of the test item.
- A significantly (p<0.05) higher male pup weight was noted in the 100 mg/kg/day group on PND 4; however, this did not occur in a dose-related manner.
- No other statistically significant differences from the control group were noted.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- The mean number of pups born and live litter size 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.
- 3 pups were found dead in the control group, 1 pup was found dead in the 300 mg/kg bw/day group, 2 pups were found dead in the 500 mg/kg bw/day and 3 pups were found dead in the 1000 mg/kg bw/day group. Four and 2 pups in the 300 and 1000 mg/kg/day groups, respectively, was missing and presumed to have been cannibalized.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- The percentage of males at birth in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values.

Visceral malformations:

no effects observed

Description (incidence and severity):

- Aside from the presence or absence of milk in the stomach, the only other internal finding was renal papillae not fully developed for 1 pup in the 500 mg/kg/day group. However, this did not occur in a dose-related manner.

Other effects:

no effects observed

Description (incidence and severity):

- 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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects were observed on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The repeated dose toxicity of the test item via the oral route was assessed in accordance with OECD Guideline 422. 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 material when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

GUIDELINE

The study, conducted in accordance with OECD 422, was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

 

METHODS

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 1000 mg/kg/day group (Group 5) consisted of 15 rat/sex and the 100, 300, and 500 mg/kg/day groups (Groups 2, 3, and 4, respectively) consisted of 10 rats/sex. The dosage volume was 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test item administration. Ten males/group 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. Ten females/group received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-44 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 39-52 doses. The extra 5 males and 5 females in the control and 1000 mg/kg/day groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for female, these animals were assigned to a 15-day non‑dosing recovery period, respectively. 

 

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 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology serum chemistry) were performed on 5 F0 animals/sex/group at primary necropsy and from 5 F0 animals/sex in the control and 1000 mg/kg/day groups at the recovery necropsy. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, on 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 F0 animals in the control and 1000 mg/kg/day groups at the primary necropsy.

 

RESULTS

No test item-related mortality or moribundity was noted at any dosage level. Clinical findings of red material around the nose and mouth and clear material around the mouth were noted for males and females at all dosage levels at approximately 1 hour following dose administration generally throughout the treatment period. These findings were generally noted in a dose-related manner and occurred sparingly in the 100 mg/kg/day group. In addition, incidences of rales were noted sporadically for males and females in the 1000 mg/kg/day group at approximately 1 hour following dose administration. These findings were considered test item-related, but due to the absence of any other evidence of toxicity in these groups, these findings were not considered adverse.

 

No test item-related effects were noted at any dosage level on parental body weights, body weight gains, or food consumption throughout the study. Reproductive performance (mating, fertility, copulation, conception, gestation length, and the process of parturition) were unaffected by test item administration at all dosage levels. In addition, functional observational battery parameters and motor activity were unaffected by test item administration at all dosage levels.

 

No test item-related macroscopic or microscopic findings were noted at any dosage level. Clinical pathology parameters and organ weights were unaffected by test item administration.

 

Survival, growth, and clinical condition of F1 pups were unaffected by parental test item administration at all dosage levels. No test item-related macroscopic findings were observed for pups found dead at any dosage level.

 

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 F0 reproductive and systemic toxicity and F1 neonatal toxicity of OS343777 when administered orally by gavage to Crl:CD(SD) rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch grade 1, combined 28-day repeated dose oral (gavage) toxicity study with reproduction/developmental toxicity screeening test in rats, with recovery

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The NOAEL for reproductive toxicity was 1000 mg/kg/day based the absence of test item-related effects F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs when the substance was administered to maternal animals at dose levels of 100, 300, 500 and 1000 mg/kg bw/day (OECD 422). As a result, the test item is not classified for reproductive or developmental toxicity under the terms of Regulation (EC) No 1272/2008.