Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-21 to 2013-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): OS48604M
- Physical state: Light brown, clear, viscous liquid
- Lot/batch No.: 1X4 LT
- Stability under test conditions: Considered stable under these conditions
- Storage condition of test material: room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) rats were received in good health from Charles River Laboratories, Inc. on 22 May 2012; 75 females received from the Raleigh, NC facility, 38 males were received from the Portage, MI facility, and 37 males were received from the Kingston, NY facility.

The animals were approximately 63 days old upon receipt.
The animals were housed for an acclimation period of 10 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes of all males were palpated at least once.

Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The breeding phase rats (12/sex/group) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams and their litters were housed in these cages until euthanasia on PND 4 (pups) or lactation day 5 (dams). Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.

Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. All males and lactation day 5 females in the treatment phase were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Actual mean daily temperature ranged from 70.1°F to 71.4°F (21.2°C to 21.9°C) and mean daily relative humidity ranged from 45.5% to 54.4% during the study.

Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): stability and solubility
- Concentration in vehicle: 0, 20, 40, 100, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 2AF0861 and 2AJ0197, exp. dates: 1 June 2013 and 14 September 2013
Details on mating procedure:
The 12 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 housed in the home cage of the male. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and 8-day room temperature stability were established at concentrations of 50 and 200 mg/mL in a previous study (Toot, Draft, WIL-168199). Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 20 mg/mL dosing formulation. In addition, samples for stability and resuspension homogeneity determinations were collected from the top and bottom strata of this same dosing suspension following room temperature storage for 8 days. Samples for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the control group). One set of samples from the first and last dosing formulations was subjected to the appropriate analyses. The remaining set of samples was stored frozen (approximately -20°C) until discarded following acceptance of the analytical results. Following the completion of the dosing period, additional formulations were prepared at 20 and 200 mg/mL to further assess stability of the test item in corn oil. Samples were collected from the middle stratum of these formulations on the day of preparation (time zero) and following 13 days of frozen storage (approximately -20°C).

Validated gas chromatography method with flame ionization detection.
A GC method using FID for the determination of OS48604M concentration in formulations containing corn oil and test item ranging in concentration from
10.0 to 400 mg/mL was validated in a previous study (Toot, Draft, WIL-168199). In the present study. A formulation prepared at a target test item concentration of 20 mg/mL was analyzed to assess test item homogeneity/concentration acceptability and, following 8 days of room temperature storage, resuspension homogeneity and stability.
Additionally, formulations prepared at target test item concentrations ranging from 20 to 200 mg/mL, following 13 days of frozen (approximately -20°C) storage, were analyzed to assess test item stability. Finally, formulations used for dose administration were analyzed to verify test item concentration acceptability.

Method
Instrument: Varian CP-3800 GC equipped with an flame ionization detector, autosampler, and Dionex Chromeleion® software version 6.8 or Varian MS Workstation software
Column: Zebron ZB-5HT Inferno, 30 m × 0.25 mm, 0.25-μm film-thickness
Injector Temperature: 200°C
Injection Volume: 1.0 μL, split
Split Ratio: 10:1
Carrier Gas: Helium
Flow Rate: 1.5 mL/minute
Temperature Program: Initial temperature 50°C, hold for 3.0 minutes
Ramp at 25°C/minute to 300°C, hold for 2.0 minutes
Detector: Flame ionization detector
Detector Temperature: 310°C
Retention Time: Approximately 6.1 minutes for OS48604M
Run Time: 15.0 minutes
Retention time of the test item was approximately 6.1 minutes

Calibration
A calibration stock solution was prepared at a concentration of 1.00 mg OS48604M/mL as follows. Approximately 10 mg of OS48604M (WIL ID no. 120017; no correction for purity) was accurately weighed in a tared glass weigh funnel and transferred to a 10-mL volumetric flask with rinses of acetone. The preparation was mixed as necessary to achieve complete dissolution of the test item. Additional acetone was added to obtain the desired concentration.
Calibration standards: 100, 125, 150, 175, and 200 μg/mL

QC
QC samples were prepared to simulate the processing of formulation samples at concentrations of 10.0, 200, and 400 mg/mL (nominal QC concentrations) by combining aliquots of the QC stock solution, vehicle (corn oil), and acetone in polypropylene tubes. The processed samples were mixed with vortex action. Portions of the samples were further diluted as necessary with acetone in amber autosampler vials.

Formulation sampling
Quadruplicate formulation samples were collected using a syringe and dosing cannula and placed in polypropylene tubes. Two samples from each quadruplicate set were processed for analysis. The remaining 2 samples (back-up samples) were stored frozen (approximately -20°C). Formulation samples were processed by adding acetone and mixing with vortex action. Samples were further diluted with acetone in amber autosampler vials.

Specificity
Assay specificity/selectivity was confirmed when GC/FID analysis of processed vehicle samples revealed that there were no significant peaks (with S/N >10) at or near the retention time for the test item (approximately 6.1 minutes)

Homogeneity
The analyzed formulation met the acceptance criteria for homogeneity, i.e., the RSD for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration), concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to
115% of the target concentration, and resuspension homogeneity, i.e., the RSD for the mean concentration was ≤10%.

Test item stability
The mean post-storage concentration was 94.5% of the pre-storage value.
The 13-day frozen (approximately -20°C) mean post-storage concentrations ranged from 92.2% to95.0% of the pre-storage values.

Test item concentrations

Mean Concentration, mg/mL (% of Target)
Date of Preparation Group 1 Group 2 Group 3 Group 4 Group 5
(0 mg/mL) (20 mg/mL) (40 mg/mL) (100 mg/mL) (200 mg/mL)
31 May 2012 ND 21.4 (107) 43.8 (109) 112 (112) 214 (107)
12 July 2012 ND 20.8 (104) 42.6 (107) --- 203 (101)
19 July 2012 --- --- --- 98.2 (98.2) ---
No test item was detected in the analyzed vehicle administered to the control group
Duration of treatment / exposure:
30 days
Frequency of treatment:
Once/day
Details on study schedule:
The males selected for mating were dosed during study days 0-30 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a
total of 31 doses.
The females selected for mating were dosed during study days 0 through 2 days prior to euthanasia (14 days prior to pairing through lactation day 3) for a
total of 39-51 doses; however, these females should also have been dosed on lactation day 4 but were not administered the test item.
Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or postcohabitation day 25) for a total of 39-52 doses.
Males not selected for pairing remained on study for a 14-day non-dosing post-treatment period following 28 doses.
Females not selected were dosed through study day 39 (the day prior to the first necropsy of paired females on lactation day 5), for a total of 40 doses.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200, 500 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Vehicle and high dose: 17/sex/group
Treatment groups: 12/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the results of previous studies.
All animals were dosed at approximately the same time each day.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- for moribundity and mortality, and for signs of toxicity 2 hours following dose administration. Females expected to deliver were observed for dystocia or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for males and weekly for females until evidence of copulation. Female body weights were recorded on GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.
FOOD CONSUMPTION
- Food consumption for each animal determined as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymides weight, seminal vesicle weight; microscopic examinations of testes, epididymides, seminal vesicles, coagulating glands and prostate gland.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- all pups

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals on lactation day 4 (females that delivered) or on post-mating day 25 (females that failed to deliver)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including external surface, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Moreover the number and location of corpora lutea and implantation sites were examined , and uteri were investigated for early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to guidelines were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age and discarded.
Statistics:
Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates' correction factor. A parametric one-way ANOVA to determine intergroup differences was applied to mean parental BW, BW changes and food consumption, offspring BW and BW changes, gestation length, numbers of implantation sites, number of pups born, live litter size on PND 0, corpora lutea, organ weights (absolute and relative) and pre-coital intervals. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Kruskal-Wallis nonparametric ANOVA was used to determine intergroup-differences for mean litter proportions of males at birth and postnatal survival. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn's test was used to compare the test substance-treated groups to the control group. Histopathological findings of treated groups were compared to the control group by the Fisher's Exact test.
Reproductive indices:
Mating, fertility, and copulation/conception indices were calculated as follows:

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

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

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

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

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100
Offspring viability indices:
Litter parameters were defined as follows:

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

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

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

Where N= PND 0-1 and 1-4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details of results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details of results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details of results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details of results

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
See details of results
Reproductive performance:
no effects observed
Description (incidence and severity):
See details of results

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One male each in the 500 and 1000 mg/kg/day groups was found dead. The male in the 1000 mg/kg/day group was found dead on study day 8 prior to dose administration and had an unkempt appearance, red material on the urogenital area, forelimbs, nose, mouth, and ventral abdomen, yellow material on the urogenital area, and decreased defecation within 3 days of death. This male lost 114 g of body weight and consumed only 5 g/day of feed during study days 0-7. Pale, enlarged thyroid glands were noted for this male at necropsy. In the 500 mg/kg/day group, one male was found dead on study day 31 prior to dose administration. The only clinical finding for this male consisted of clear material around the mouth observed 1 hour following dose administration 9 days prior to death. Dark red areas in the lungs were noted for this male at necropsy; there was no evidence of esophageal perforation. Test item-related microscopic findings for these males (hepatocellular vacuolation [both males] and hepatocellular cytomegaly and thyroid follicular cell hyperplasia [1000 mg/kg/day male]) were similar to those observed in the 1000 mg/kg/day group males at the scheduled necropsy. Therefore, the deaths in the 500 and 1000 mg/kg/day group males were attributed to the test item.
One female in the 500 mg/kg/day group and one female in the 1000 mg/kg/day group were euthanized in extremis on gestation day 22 and lactation day 0, respectively, due to dystocia; coagulative necrosis of the liver was noted microscopically and contributed to the condition of these females. Pale, cool body was noted during parturition for the female in the 500 mg/kg/day group on the day of euthanasia. Dystocia, also noted in other females and was attributed to the test item. In addition, microscopic findings for these females included test item-related coagulative necrosis of the liver; this finding was considered the cause of moribundity. Two females in the control group were found dead on lactation days 5 and 0, respectively; a cause of death could not be determined for either female. No clinical findings were noted prior to death for these females. At necropsy, dark red areas in the lungs were noted for one control group female and lungs that were not fully collapsed, dark red contents in the jejunum, and dark red areas in the thymus gland were noted for the other control group female. There was no evidence of esophageal perforation for either of these females.
All other animals survived to the scheduled necropsies.

Test item-related clinical findings were noted in males and females in all test item-treated groups at the time of dosing and/or 1 hour following dose administration; the onset (earlier at higher dosage levels), number of affected animals, and frequency of findings generally occurred in a dose-related manner. The most frequently observed findings (red/clear material around the mouth/nose and yellow material around the urogenital area) were generally noted throughout the treatment period, at least sporadically, at ≥200 mg/kg/day once observed. Salivation noted at ≥200 mg/kg/day or evidence thereof (clear material around the mouth/nose) at ≥100 mg/kg/day were attributed to potentially irritating properties of the test item and were not considered adverse. Red material around the mouth at ≥ 200 mg/kg/day and red material around the nose, and yellow material on the urogenital area at 1000 mg/kg/day were not considered adverse. Soft stool at 1000 mg/kg/day was considered adverse. Although males only received 31 doses (treatment period males) or 28 doses (1000 mg/kg/day post-treatment period males), more males were affected and/or had generally earlier onset of findings compared to the females, which received 39-51 doses.
Following the 14-day post-treatment period, clinical observations noted in the 1000 mg/kg/day group males and females (hair loss or scabbing on the neck or forelimbs and red material around the nose) were noted in single animals and similarly in the control group.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
Mean body weight losses and/or lower mean body weight gain and food consumption were observed in the 500 and 1000 mg/kg/day group males generally throughout the treatment period. As a result, mean body weights in these males were 9.2% and 13.4% lower, respectively, than the control group on study day 30. Mean body weights and body weight gain in the 1000 mg/kg/day during the post-treatment period remained lower than the control; however, mean body weight gain was slightly improved. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100 and 200 mg/kg/day group males. No test item-related effects on food consumption were noted in females at any dosage level during the pre-mating period.
Lower mean food consumption was noted in the 1000 mg/kg/day group females generally throughout gestation, and lower mean body weight gains were noted only late in gestation (days 14-20), resulting in a mean body weight that was 5.8% lower than the control group on gestation day 20. The effects on mean body weight gain late in gestation in the 1000 mg/kg/day group females were attributed to the test item-related lower mean number of pups and an increase in the mean number of unaccounted-for sites, rather than maternal toxicity. No test item-related effects on mean body weights and body weight gain were noted in these females during lactation or in the 1000 mg/kg/day group post-treatment phase females. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100, 200, and 500 mg/kg/day group females throughout the treatment period.

CLINICAL CHEMISTRY, HAEMATOLOGY, URINALYSIS
Test item-related clinical pathology findings in male rats included the following. Longer mean prothrombin and activated partial thromboplastin times and higher mean urine volumes and lower urine osmolality were noted at >100 mg/kg/day. Minimally to slightly higher mean albumin, total protein, and alkaline phosphatase levels were noted at 500 and 1000 mg/kg/day. Slightly lower mean erythrocyte and eosinophil counts, triglyceride, chloride, phosphorus, and potassium levels and slightly higher mean reticulocyte counts and red blood cell distribution width, mean globulin, total bilirubin, creatinine, and cholesterol, were noted at 1000 mg/kg/day. Test item-related findings were considered to be adverse for prothrombin at >200 mg/kg/day and for activated partial thromboplastin time at 1000 mg/kg/day. These findings resolved in the 1000 mg/kg/day group males during the recovery period with the exception of slightly lower mean erythrocyte and eosinophil counts and alanine aminotransferase level. Nonadverse, test item-related findings in female rats consisted of minimally to slightly higher mean albumin levels at >100 mg/kg/day, higher mean cholesterol levels and lower mean alanine transferase levels at >200 mg/kg/day, lower erythrocyte counts, and mean hemoglobin and hematocrit levels and urine osmolality and higher mean total bilirubin at >500 mg/kg/day, and slightly higher mean red blood cell distribution width, and alkaline phosphatase levels, and slightly lower mean triglyceride levels at 1000 mg/kg/day.
Following the post-treatment period, percent and absolute mean reticulocyte counts were slightly lower than the control group in the 1000 mg/kg/day females. The mean hemoglobin distribution width in the 1000 mg/kg/day females was slightly lower than controls on study day 54 and was considered to be secondary to the lower mean reticulocyte counts.

ORGAN WEIGHTS
Test item-related effects on organ weights consisted of lower mean thymus in the 200, 500, and 1000 mg/kg/day group males, lower mean spleen and heart weights in the 500 and 1000 mg/kg/day group males, lower brain weight in the 1000 mg/kg/day group males, higher mean liver weights in the 500 and 1000 mg/kg/day group males, and higher thyroid/parathyroid weights in 500 and 1000 mg/kg/day group males and females at the treatment period necropsy. Mean liver and thyroid gland weights remained higher in the 1000 mg/kg/day group females at the post-treatment necropsy.

GROSS PATHOLOGY
No test item-related macroscopic findings were noted at the necropsy of F0 animals that were found dead, euthanized in extremis, or at the scheduled necropsies.

HISTOPATHOLOGY
Test item-related microscopic findings were noted in the liver and thyroid of the 200, 500, and 1000 mg/kg/day groups. These findings corresponded to higher organ weights and consisted of midzonal hepatocellular vacuolation, hepatocellular cytomegaly, and thyroid follicular cell hyperplasia. Liver lesions were more severe in males than in females for all test item-treated groups. The severity of hepatocellular vacuolation and thyroid follicular cell hyperplasia in the 1000 mg/kg/day group males and females was less at the post-treatment period necropsy.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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. Males that did not sire a litter numbered 1, 0, 0, 1, and 0 in the control, 100, 200, 500, and 1000 mg/kg/day groups, respectively. One female each in the control and 1000 mg/kg/day groups had evidence of mating but did not deliver; both of these females 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. None of the differences were statistically significant.
Mean gestation lengths in the 100, 200, 500, and 1000 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. One female in the 500 mg/kg/day group and 2 females in the 1000 mg/kg/day were noted with dystocia. Because the severity of dystocia-related findings required euthanasia of 1 female each in the 500 and 1000 mg/kg/day groups, this finding was attributed to the test item. No evidence of dystocia was observed in the 100 and 200 mg/kg/day groups.
No test item-related effects were noted on male and female mating and fertility, male copulation, or female conception indices, or gestation lengths at any dosage level.
A statistically significantly higher mean number of unaccounted-for sites was noted in the 1000 mg/kg/day group females compared to the control group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Some organ weight differences were due to an effect of final body weight and were not test item-related including left and right epididymides and left and right testes for the 500 and 1000 mg/kg/day group males

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive performance
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive performance observed
Dose descriptor:
LOAEL
Remarks:
Reproductive performance
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of pups born and live litter size on PND 0 in the 1000 mg/kg/day group (11.1 and 10.8 per dam, respectively) were lower than the concurrent control group values (13.5 and 13.5 per dam, respectively). The differences were not statistically significant; however, these values were also lower than the minimum mean values in the WIL reproductive historical control data (11.7 and 11.6 per dam, respectively). The lower mean number of pups born in the 1000 mg/kg/day group was due to one litter that had only 4 pups born (5 implantation sites and 8 corpora lutea). Because the mean number of unaccounted-for sites in the 1000 mg/kg/day group was higher than the control group with no remarkable differences in the mean numbers of corpora lutea and implantation sites , the lower mean number of pups born and live litter size on PND 0 in the 1000 mg/kg/day group were attributed to the test item. The mean percentage of males in this group was similar to the concurrent control group. Mean postnatal survival in the 1000 mg/kg/day group during PND 0-1, 1-4, and from
birth to PND 4 (77.4%, 87.0%, and 69.0% per litter, respectively) was lower than the concurrent control group (100.0%, 95.5%, and 86.0% per litter, respectively). The values were also below or equal to the minimum mean values in the WIL reproductive historical control data (94.6%, 87.0%, and 83.8% per litter, respectively). The lower postnatal survival in the 1000 mg/kg/day group was considered to be test item-related.
The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 200, and 500 mg/kg/day groups were similar to the control group values. Postnatal survival in these groups were unaffected by test item administration.

CLINICAL SIGNS (OFFSPRING)
The general physical condition of all F1 pups in this study were unaffected by test item administration. Pups (litters) that were found dead numbered 2(2), 4(3), 4(4), 5(2), and 30(8) in the control, 100, 200, 500, and 1000 mg/kg/day groups, respectively. Five (3), 0(0), 1(1), 6(3), and 7(3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Fifteen (1) and 3(1) pups (litters) in the control and 1000 mg/kg/day groups were euthanized due to death of the dam.

BODY WEIGHT (OFFSPRING)
Mean male and female pup body weights and body weight changes in the 100, 200, 500, and 1000 mg/kg/day groups were unaffected by test item administration during PND 1-4.
No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING)
Two (2), 4(3), 4(4), 4(2), and 30(8) pups (litters) in the control, 100, 200, 500, and 1000 mg/kg/day groups, respectively, were found dead during PND 0-4. Aside from the absence (indicating stillborn pups) or presence of milk in the stomach, the following findings were noted in a single pup in the 1000 mg/kg/day. One pup in this group had mandibular micrognathia (the mandibles were smaller than normal and fused), situs inversus (lateral transposition of the trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidney, adrenal glands, and intestine), and distended ureters.
There were no findings that could be attributed to the test item observed in pups that were euthanized due to the death of the dam or found dead due to mechanical injury. Milk was not present in the stomach of all pups that were euthanized due to the death of the dam, and the pup in the 500 mg/kg/day (found dead due to mechanical injury) had a fractured parietal bone.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Neonatal toxicity
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

See attached Document for Tables of Results

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no effects on F0 reproductive performance were noted at any dosage level. However, dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity of OS48604M when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for neonatal toxicity was considered to be 500 mg/kg/day based on lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups.
An expert review of the data from the OECD 422 screening study with repeat oral dosing for 28-days indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms. The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOEL for systemic toxicity.
Executive summary:

Test Guidance

OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity

Screening Test in Rats, with Recovery.

Method and material

The test item, OS48604M, in the vehicle (corn) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and 2 mid-dosage groups (Groups 2, 3, and 4, respectively) each consisted of 12 males and 12 females, and the high-dosage

group (Group 5) consisted of 17 males and females. Dosage levels were 100, 200, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group (Group 1) of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 31 doses. Twelve females/group selected for pairing received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-51 doses; however, these females should also have been dosed on lactation day 4 but were not administered the test item. The females 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 the males and 40 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F0 females 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, and urinalysis) were performed on 6 F0 animals/sex/group at the treatment period necropsy and on all animals at the post-treatment period necropsy. The F0 males were euthanized following completion of the mating period, and the F0 females were euthanized on lactation day 5. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were euthanized following the 14-day non-dosing 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 high-dosage groups at the treatment period necropsy. In addition, the liver and thyroid glands from all animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.

Results

One male each in the 500 and 1000 mg/kg/day groups were found dead on study days 31 and 8, respectively. The male in the 1000 mg/kg/day group lost 114 g of body weight and had an unkempt appearance, red material on the urogenital area, forelimbs, ventral abdomen, nose and mouth, and decreased defecation prior to death. Findings for the 500 mg/kg/day group male were limited to red material around the mouth 1 week prior to death. These deaths were attributed to the test item. One female each in the 500 and 1000 mg/kg/day groups were euthanized in extremis due to test item-related dystocia on gestation day 22 and lactation day 0, respectively. Microscopic findings for these females included test item-related coagulative necrosis of the liver; this finding was considered the cause of moribundity. All other animals survived to the scheduled necropsies. Test item-related clinical findings noted in the 100, 200, 500, and 1000 mg/kg/day group males and females consisted of salivation prior to dose administration or clear material around the mouth and/or nose. These findings were attributed to irritating properties of the test item and were not considered adverse. Other test item-related clinical findings noted in the 200, 500, and 1000 mg/kg/day groups consisted of red material around the mouth. In the 1000 mg/kg/day group males and females, yellow material on the urogenital area, red material around the nose, and soft stool were also noted. These findings were considered adverse. Mean body weight losses and/or lower mean body weight gain and food consumption (generally statistically significant) were observed in the 500 and 1000 mg/kg/day group males generally throughout the treatment period. As a result, mean body weights in these males were statistically significantly lower (9.2% and 13.4%, respectively) than the control group on study day 30. Mean body weights and body weight gain in the 1000 mg/kg/day during the post-treatment period remained lower than the control; however, mean body weight gain was improving slightly. Lower mean food consumption throughout gestation and lower mean body weight gains late in gestation (days 14-20) were noted in the 1000 mg/kg/day group females, resulting in a mean body weight that was 5.8% lower than the control group on gestation day 20; some of the differences were statistically significant. The effects on mean body weight gain during gestation in the 1000 mg/kg/day group females was attributed to the test item-related lower mean number of pups and an increase in the mean number of unaccounted-for sites, not to maternal toxicity. No test item-related effects on mean body weights or body weight gains were observed in the 1000 mg/kg/day group females during the post-treatment period. Test item-related clinical pathology findings (occasionally statistically significant) in male rats included the following. Longer mean prothrombin and activated partial thromboplastin times and higher mean urine volumes and lower urine osmolality were noted at > 100 mg/kg/day. Minimally to slightly higher mean albumin, total protein, and alkaline phosphatase levels were noted at 500 and 1000 mg/kg/day. Slightly lower mean erythrocyte and eosinophil counts, alanine aminotransferase, triglyceride, chloride, phosphorus, and potassium levels and slightly higher mean reticulocyte counts and red blood cell distribution width, mean globulin, total bilirubin, creatinine, and cholesterol, were noted at 1000 mg/kg/day. Test item-related findings were considered to be adverse for prothrombin > 200 mg/kg/day and for activated partial thromboplastin time at 1000 mg/kg/day. These findings resolved in the 1000 mg/kg/day group males during the recovery period with the exception of slightly lower mean erythrocyte and eosinophil counts. Nonadverse, test item-related findings (occasionally statistically significant) in female rats consisted of minimally to slightly mean higher albumin levels at > 100 mg/kg/day, higher mean cholesterol levels and lower mean alanine transferase levels at > 200 mg/kg/day, lower erythrocyte counts, and mean hemoglobin and hematocrit levels and urine osmolality and higher mean total bilirubin levels at > 500 mg/kg/day, and slightly higher mean red blood cell distribution width, and alkaline phosphatase levels, and slightly lower mean triglyceride levels at 1000 mg/kg/day. Following the post-treatment period, mean percent and absolute reticulocyte counts were slightly lower than the control group in the 1000 mg/kg/day group females. The mean hemoglobin distribution width in the 1000 mg/kg/day group females was slightly lower than controls on study day 54 and was considered to be secondary to the lower mean reticulocyte counts. No test item-related effects were noted on male and female mating and fertility, male copulation, or female conception indices, or gestation lengths at any dosage level. One and 2 females in the 500 and 1000 mg/kg/day groups, respectively, were noted with dystocia, resulting in the euthanasia of the 500 mg/kg/day group female and 1 of the 1000 mg/kg/day group females. Dystocia was attributed to the test item. Findings for these females that were euthanized included active vaginal hemorrhage, weakness, pallor, body cool/cold to the touch, hypoactivity, and torpid/tense abdomen. The female in the 500 mg/kg/day did not deliver any pups; however, dead fetuses and an early resorption in utero. The female in the 1000 mg/kg/day group that was euthanized delivered 3 pups, but left them unattended; this female also had dead fetuses and an early resorption in utero. The other female in the 1000 mg/kg/day group noted with dystocia was pale and cool to the touch on lactation day 2 and was euthanized with total litter loss on that day (after delivering 13 pups on lactation day 0). A statistically significantly higher mean number of unaccounted-for sites was noted in the 1000 mg/kg/day group females compared to the control group. A lower mean number of F1 pups born and live litter size on PND 0 were noted in the 1000 mg/kg/day group; the differences from the control group were not statistically significant. The lower mean number of pups born corresponded in part with the higher mean number of unaccounted-for sites observed in the F0 females in this group. Statistically significantly lower mean postnatal survival noted in the 1000 mg/kg/day from birth to PND 4 group was attributed to the test item. No test item-related macroscopic findings were noted at the necropsy of F0 animals that were found dead, euthanized in extremis, or at the scheduled necropsies.

Test item-related effects on organ weights consisted of lower mean thymus weights in the 200, 500, and 1000 mg/kg/day group males, lower mean spleen, and heart weights in the 500 and 1000 mg/kg/day group, lower mean brain weight in the 1000 mg/kg/day group males, higher mean liver weights in the 500 and 1000 mg/kg/day group males, and higher thyroid/parathyroid weights in 200, 500, and 1000 mg/kg/day group males and females. Some of the differences from the control group were statistically significant. Mean liver and thyroid gland weights remained higher (generally statistically significant) in the 1000 mg/kg/day group females at the post-treatment necropsy. Corresponding test item-related microscopic findings consisted of midzonal hepatocellular vacuolation, hepatocellular cytomegaly in the 200, 500, and/or 1000 mg/kg/day group males and females and thyroid follicular cell hyperplasia in the 200, 500, and 1000 mg/kg/day group males and females. The differences from the control group were generally statistically significant. The severity of hepatocellular vacuolation and thyroid follicular cell hyperplasia in the 1000 mg/kg/day group males and females was less at the post-treatment period necropsy; however, the differences from the control group generally remained statistically significant. There was no correlation between these microscopic findings and the clinical pathology results.

Conclusions

Under the conditions of this screening study, no effects on F0 reproductive performance were noted at any dosage level. However, dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity of OS48604M when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for neonatal toxicity was considered to be 500 mg/kg/day based on lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups.

An expert review of the data from the OECD 422 screening study with repeat oral dosing for 28-days indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms. The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOEL for systemic toxicity.