Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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: The study was performed according to OECD and US EPA guidelines and according to GLP principles.
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:
other: OECD 422
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or the presence of sperm following a vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually into plastic maternity cages with nesting material, ground corncob bedding.
Duration of treatment / exposure:
males: 14 daily doses prior to mating; throughout mating period for a total of 31 doses
females: 14 daily doses prior to pairing, dosed through lactation day 3 (females that delivered) or post-mating day 25 (females that failed to deliver) for a total of 39-51 doses
Frequency of treatment:
daily
Duration of test:
until lactation day 4
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200, 500, 1000 mg/kg bw/day
Basis:
actual ingested
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of previous studies

Examinations

Maternal 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
- Sacrifice on lactation day #4
- Organs examined: according to guidelines
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No (dams delivered)
- Number of late resorptions: No (dams delivered)
Fetal examinations:
- External examinations: Yes, Fo animals delivered and the dams and her litter were euthanized on LD/PND 4. All pups were examined.
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.
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 if 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.
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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

See Table of Results attached.

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.