1H-Isoindol-1-one, 2,3-dihydro-3,3-bis(4-hydroxyphenyl)-2-phenyl-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

In addition to the main study, systemic exposure to the substance was evaluated in adult female rats, prior to and during gestation (the exposure phase).

GLP compliance:

yes

Remarks:

WIL Research, 1407 George Road Ashland, OH 44805-8946

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (F0) approximately 10 weeks old
- Weight at study initiation: For the main study phase, male body weights ranged from 343 g to 381 g and female body weights ranged from 204 g to 258 g on study day 0. For the exposure phase, female body weights ranged from 220 g to 271 g on study day 0.
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The main study phase females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH)
- Diet: Basal diet (PMI Nutrition International, LLC Certified Rodent, LabDiet® Meal 5002), ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 13 days prior to the first day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ranged from 21.3 to 22.8
- Humidity (%): ranged from 29.4% to 51.8%
- Air changes (per hr): minimum of 10 fresh air changes
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

VEHICLE:
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg dosage volume
- Lot/batch no.: corn oil, NF lot nos. 2CI0307 and 2CK0155

Details on mating procedure:

- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group.
- Length of cohabitation: until positive evidence of mating or completion of the mating period
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. The day when evidence of mating was identified was termed gestation day 0
- After successful mating each pregnant female of the main study phase was caged: plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH)
- If evidence of copulation was not detected after 14 days of pairing, any main study phase females that had not shown evidence of mating were also placed in plastic maternity cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Validated high performance liquid chromatography method with ultraviolet absorbance detection (LM No. 202.OCO1.01; Freer, 2006, WIL-202043) was used for analytical verification.
The results met the acceptance criteria for homogeneity, i.e., the relative standard deviation for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration), and concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to 115% of the target concentration. No test substance was detected in the analyzed vehicle administered to the control group.

Duration of treatment / exposure:

Main study phase
Males were dosed for 14 days prior to pairing through 1 day prior to scheduled euthanasia for a total of 28-29 doses.
Females were dosed for 14 days prior to pairing through lactation day 3 for a total of 39-52 doses.
Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses.
Exposure phase
Females were dosed for 14 days prior to pairing through gestation day 15 for a total of 30-36 doses.
Males were not dosed.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Main Study Phase: 12
Exposure Phase *: 8
*Males in the exposure phase were not administered the vehicle or test substance and were used for breeding purposes only

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were determined from results of a previous 28-day oral (gavage) toxicity study (Eapen, 2005, WIL-202042) and were provided by the Sponsor Representative. In that study, male and female Sprague Dawley rats received the test substance in corn oil at 100, 300, and 1000 mg/kg/day for 28 consecutive days. All animals survived to scheduled necropsy and there were no test substance related clinical observations.
In that study, 1000 mg/kg/day was identified as the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of the test substance to rats for 28 consecutive days. Based on these data, 1000 mg/kg/day was selected as the high dose for the current study. Dosage levels of 100 and 300 mg/kg/day were selected to further characterize potential dose-response relationships.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
In both study phases, all rats were observed twice daily for moribundity and mortality

DETAILED CLINICAL OBSERVATIONS:
In both study phases, individual detailed physical examinations were recorded weekly. Each test substance-treated male and female was also observed for signs of toxicity 1-2 hours following dose administration. 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. In addition, any changes or abnormalities (if any) in nesting or nursing behavior were recorded.

BODY WEIGHT:
Individual male body weights were recorded weekly throughout the study except for males assigned to the exposure assessment phase, for which body weights were only recorded once prior to randomization.
Individual female body weights were recorded weekly until copulation. Once evidence of mating was observed, main study phase 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. For exposure phase females, body weights were recorded on gestation days 0, 4, 7, 11, and 14.

FOOD CONSUMPTION (non feeding study)
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the breeding period and was not recorded for toxicokinetic phase males. Once evidence of mating was observed, main study phase female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. For exposure phase females, food consumption was recorded on gestation days 0, 4, 7, 11, and 14. Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.

OTHER:
BREEDING PROCEDURES
Each mating pair was examined daily for evidence of mating.

TOXICOKINETICS (EXPOSURE PHASE)
Blood samples for toxicokinetics were collected from females on study day 0 and gestation day 15 prior to dose administration and at approximately 1, 2, 4, 8, and 24 hours after dose administration.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nesting and nursing behavior were recorded. The pups were individually weighed on PND 1 and 4 and were individually sexed on PND 0 and 4.

GROSS EXAMINATION OF DEAD PUPS:
Each litter was examined daily for survival and all deaths were recorded. 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.

Postmortem examinations (parental animals):

SACRIFICE
- All surviving male animals:
Males were euthanized following completion of the mating period.

- All surviving maternal animals:
Females that delivered were euthanized on lactation day 4. One female that failed to deliver was euthanized on post-mating day 25.

GROSS NECROPSY
A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination. The numbers of former implantation sites and corpora lutea were recorded. Uteri with no macroscopic evidence of implantation were opened and early implantation loss was investigated. Necropsy included examination of the external surface, all orifices, the external surface of the brain and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies: Brain, Epididymides *, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Testes *.
* = These paired organs were weighed separately. Care was taken to ensure separation between the left and right organs.

Microscopic examination was performed on the tissues listed below from all animals in the control and 1000 mg/kg/day groups at the scheduled necropsies and from any animals found dead. In addition, gross lesions from animals in all groups were examined.
Cervix
Coagulating gland
Epididymides *
Mammary glands (females only)
Ovaries
Pituitary gland
Prostate gland
Seminal vesicles
Testes *
Uterus
Vagina
Vas deferens
All gross (internal) lesions
*= Sections of 2-4 microns of the testis (transverse) and epididymis (longitudinal) were stained in PAS and hematoxylin in addition to routine hematoxylin and eosin staining.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, surviving F1 rats were euthanized and the carcasses were discarded without examination.

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.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test 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 (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test (Steel and Torrie, 1980).

Reproductive indices:

Mating, fertility, and copulation/conception indices were calculated for main study phase 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 (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 / 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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test substance-related mortality or moribundity was noted in this study. A single F0 female in the 300 mg/kg/day group was found dead on gestation day 21. A cool and pale body, labored or decreased respiration, and/or red material around the ventral abdominal and urogenital areas were noted for this female on the day prior to and day of death. In addition, this female consumed little feed and exhibited a marked body weight loss during gestation days 17-20. This female had 13 dead fetuses in utero and 1 dead fetus in the vagina. Histologic findings for this animal included chronic active inflammation and bacterial colonies in uterus, suggesting that dystocia associated with a bacterial pathogen was the cause of death. However, no mortality or signs of dystocia were noted in the 1000 mg/kg/day group. Therefore, this death was not attributed to test substance administration.
Test substance-related clinical findings of soft stool were noted sporadically for F0 males in the 300 and 1000 mg/kg/day groups and F0 females in the 100, 300, and 1000 mg/kg/day groups at 1-2 hours following dose administration. These findings were not considered adverse.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Test substance-related statistically significantly (during gestation days 11-14) lower mean body weight gains, with corresponding effects on food consumption, were noted for F0 females in the 1000 mg/kg/day group during gestation days 11-17. As a result, mean body weights in this group were lower (up to 8.7%) than the control group throughout the remainder of gestation (days 17 and 20) and lactation (days 1 and 4). No test substance related effects on mean body weights, body weight gains, or food consumption were noted for F0 females in the 100 and 300 mg/kg/day groups or males at any dosage level.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. One male in the 1000 mg/kg/day group did not sire a litter. The one female in the 1000 mg/kg/day group with whom he was paired had evidence of mating but did not deliver.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The differences were not statistically significant.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test substance-related alterations in organ weights were noted at any dosage level.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One female in the 300 mg/kg/day group was found dead on gestation day 21. This female had 13 dead fetuses in utero and 1 dead fetus in the vagina with no apparent malformations. Internally, this female had a small, mildly brown-pigmented spleen. However, no mortality or signs of dystocia occurred at the 1000 mg/kg/day dose level; therefore, the occurrence at 300 mg/kg/day was not considered test substance-related.
No test substance-related internal findings were observed in the 1000 mg/kg/day group females that failed to deliver or had total litter loss or at any dosage level in males and females at the scheduled necropsy. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.
The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values.

HISTOPATHOLOGY (PARENTAL ANIMALS)
One female in the 300 mg/kg/day group was found dead on gestation day 21. Histologic findings included chronic active inflammation and bacterial colonies in the uterus, suggesting that dystocia associated with a bacterial pathogen was the cause of death of this animal. However, similar results were not noted in the 1000 mg/kg/day group. Therefore, this death was not attributed to test substance administration. All other females survived to the scheduled necropsies.
Substance-related microscopic findings were not observed in the reproductive organs of any male or female rats assigned to this study. All findings were consistent with spontaneous, background lesions of no relationship with the test substance.

OTHER FINDINGS (PARENTAL ANIMALS)
Toxicokinetics
All female rats dosed orally with the test substance were systemically exposed. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. On gestation day 15, systemic exposure to the test substance increased less than dose-proportionally over the 100 to 1000 mg/kg/day range. Exposure the test substance, in terms of AUClast, was higher on gestation day 15 than on study day 0 at 1000 mg/kg/day, but was similar on both evaluation days at 100 and 300 mg/kg/day. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively. Peak plasma the test substance concentrations were reached at 8, 24, and 8 hours post-dosing on study day 0 and at 8, 4, and 8 hours post-dosing on gestation day 15 at 100, 300, and 1000 mg/kg/day, respectively. the test substance concentrations were generally at or near Cmax or fluctuated from 1 through 24 hours post-dosing, except for animals dosed at 100 mg/kg/day on study day 0, where concentrations increased through 8 hours, then decreased through 24 hours post-dosing. Concentration data for the terminal elimination phase was inadequate to determine half-life. Plasma the test substance concentrations in control group animals were below the limit of quantitation (BLQ) in all samples collected at all time points on study day 0 and gestation day 15.

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproductive performance at any dosage level

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects at any dosage level

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Lower mean body weight and food consumption during gestation

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
The number of pups born and live litter size were unaffected by test substance administration. Slightly lower (not statistically significant) mean postnatal survival was noted in the 1000 mg/kg/day group during PND 0-1, PND 1-4, and from birth to PND 4 when compared to the concurrent control group; the values during PND 0-1 and PND 1-4 were below the minimum mean values in the WIL Research historical control data. However, these decreases were attributed to a single total litter loss in this group on PND 2. Therefore, the slightly lower postnatal survival in the 1000 mg/kg/day group was not considered test substance-related.

CLINICAL SIGNS (OFFSPRING)
In the 1000 mg/kg/day group pups, test substance-related clinical findings of pale and cool body were noted. No other test substance-related offspring clinical findings were noted. Although pale body was also noted for pups in the 300 mg/kg/day group, this finding was noted at a low incidence in this group and also for a single pup in the control group. Pups that were found dead numbered 6, 4, 5, and 15 in the control, 100, 300, and 1000 mg/kg/day groups, respectively. The slightly higher number of pups found dead in the 1000 mg/kg/day group was attributed to a single total litter loss of
13 pups on PND 2, and was not considered test substance-related. One and 7 pups in the 300 and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

BODY WEIGHT (OFFSPRING)
Slightly lower mean birth weights were noted for F1 males and females in the 1000 mg/kg/day group. Slightly lower mean body weight gains for F1 pups at 1000 mg/kg/day during PND 1-4 resulted in lower mean F1 male and female body weights in this group on PND 4. Despite slightly lower mean body weight gains during PND 1-4 in the 100 and 300 mg/kg/day groups, mean offspring body weights in these groups remained similar to the control group on PND 4; therefore, the lower mean body weight gains during PND 1-4 in these groups were not considered adverse.

SEXUAL MATURATION (OFFSPRING)
No examined

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
The numbers of pups (litters) found dead during PND 0-4 numbered 6(3), 4(2), 5(3), and 15(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, internal findings were limited to renal papilla(e) not fully developed for a single pup in the 100 mg/kg/day group and renal papilla(e) not fully developed and/or distended ureter(s) for a single pup in the 1000 mg/kg/day group. However, these findings occurred infrequently and were not considered test substance-related.

HISTOPATHOLOGY (OFFSPRING)
Not examined

OTHER FINDINGS (OFFSPRING)
-

Reproductive effects observed:

not specified

Conclusions:

Due to the lack of effects on reproductive performance at any dosage level, 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 reproductive toxicity of the test substance when administered orally by gavage to rats. Due to the effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. No adverse effects were noted for F0 males at any dosage level; therefore, the NOAEL for F0 male systemic toxicity was 1000 mg/kg/day, the highest dosage level evaluated.
All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

Executive summary:

The substance has been studied in a GLP-compliant reproduction/developmental toxicity screening test performed in accordance with OECD guideline 421. Groups of 12 male and 12 female Sprague-Dawley rats were administered the substance as a suspension in corn oil at dose levels of 100, 300, and 1000 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle alone. Males received 14 daily doses prior to mating. Females received 14 daily doses prior to pairing and were dosed through lactation day 3. All F0 females were allowed to deliver and rear their pups until lactation day 4.

In addition, an exposure phase was conducted with 8 female rats/group administered the test substance at dosage levels of 0, 100, 300, and 1000 mg/kg/day for 14 days prior to pairing through gestation day 15. An additional eight males were assigned to each exposure phase group and were used for breeding purposes only. On study day 0 and gestation day 15, blood samples for determination of plasma drug concentration were collected from females prior to dose administration and at 1, 2, 4, 8, and 24 hours following dose administration. Pregnancy status was determined for each female on gestation day 16.

Under the conditions of this screening study, due to the lack of effects on reproductive performance at any dosage level, 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 reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Due to the effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. No adverse effects were noted for F0 males at any dosage level; therefore, the NOAEL for F0 male systemic toxicity was 1000 mg/kg/day, the highest dosage level evaluated.

All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

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:

The test substance was examined according to OECD guideline 421 (Reproduction / developmental Toxicity Screening Test) and GLP to provide information on potential adverse effects on reproduction.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance has been studied in a GLP-compliant reproduction/developmental toxicity screening test performed in accordance with OECD guideline 421. Groups of 12 male and 12 female Sprague-Dawley rats were administered the substance as a suspension in corn oil at dose levels of 100, 300, and 1000 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle alone. Males received 14 daily doses prior to mating. Females received 14 daily doses prior to pairing and were dosed through lactation day 3. All F0 females were allowed to deliver and rear their pups until lactation day 4.

In addition, an exposure phase was conducted with 8 female rats/group administered the test substance at dosage levels of 0, 100, 300, and 1000 mg/kg/day for 14 days prior to pairing through gestation day 15. An additional eight males were assigned to each exposure phase group and were used for breeding purposes only. On study day 0 and gestation day 15, blood samples for determination of plasma drug concentration were collected from females prior to dose administration and at 1, 2, 4, 8, and 24 hours following dose administration. Pregnancy status was determined for each female on gestation day 16.

Under the conditions of this screening study, due to the lack of effects on reproductive performance at any dosage level, 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 reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Due to the effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. No adverse effects were noted for F0 males at any dosage level; therefore, the NOAEL for F0 male systemic toxicity was 1000 mg/kg/day, the highest dosage level evaluated.

All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

Short description of key information:
Due to the lack of effects on reproductive performance at any dosage level, 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 reproductive toxicity of the test substance when administered orally by gavage to rats. Due to the effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. No adverse effects were noted for F0 males at any dosage level; therefore, the NOAEL for F0 male systemic toxicity was 1000 mg/kg/day, the highest dosage level evaluated.
All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

Justification for selection of Effect on fertility via oral route:
Only study available, GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Effects on developmental toxicity

Description of key information

Due to the lower mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. The NOAEL for neonatal toxicity was 300 mg/kg/day, based on adverse clinical findings and lower mean body weight in the 1000 mg/kg/day group. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

other: OECD 421 (Reproduction / Developmental Toxicity Screening Test

GLP compliance:

yes

Remarks:

WIL Research, 1407 George Road Ashland, OH 44805-8946

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (F0) approximately 10 weeks old
- Weight at study initiation: For the main study phase, male body weights ranged from 343 g to 381 g and female body weights ranged from 204 g to 258 g on study day 0. For the exposure phase, female body weights ranged from 220 g to 271 g on study day 0.
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The main study phase females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH)
- Diet: Basal diet (PMI Nutrition International, LLC Certified Rodent, LabDiet® Meal 5002), ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 13 days prior to the first day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ranged from 21.3 to 22.8
- Humidity (%): ranged from 29.4% to 51.8%
- Air changes (per hr): minimum of 10 fresh air changes
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

VEHICLE:
- Concentration in vehicle: 20, 60 and 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg dosage volume
- Lot/batch no.: corn oil, NF lot nos. 2CI0307 and 2CK0155

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Validated high performance liquid chromatography method with ultraviolet absorbance detection (LM No. 202.OCO1.01; Freer, 2006, WIL-202043) was used for analytical verification.
The results met the acceptance criteria for homogeneity, i.e., the relative standard deviation for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration), and concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to 115% of the target concentration. No test substance was detected in the analyzed vehicle administered to the control group.

Details on mating procedure:

- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group.
- Length of cohabitation: until positive evidence of mating or completion of the mating period
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. The day when evidence of mating was identified was termed gestation day 0
- After successful mating each pregnant female of the main study phase was caged: plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH)
- If evidence of copulation was not detected after 14 days of pairing, any main study phase females that had not shown evidence of mating were also placed in plastic maternity cages.

Duration of treatment / exposure:

Main study phase
Males were dosed for 14 days prior to pairing through 1 day prior to scheduled euthanasia for a total of 28-29 doses.
Females were dosed for 14 days prior to pairing through lactation day 3 for a total of 39-52 doses.
Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses.
Exposure phase
Females were dosed for 14 days prior to pairing through gestation day 15 for a total of 30-36 doses.
Males were not dosed.

Frequency of treatment:

Once daily

Duration of test:

Up to 52 doses

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Main Study Phase: 12
Exposure Phase *: 8
*Males in the exposure phase were not administered the vehicle or test substance and were used for breeding purposes only

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were determined from results of a previous 28-day oral (gavage) toxicity study (Eapen, 2005, WIL-202042) and were provided by the Sponsor Representative. In that study, male and female Sprague Dawley rats received the test substance in corn oil at 100, 300, and 1000 mg/kg/day for 28 consecutive days. All animals survived to scheduled necropsy and there were no test substance related clinical observations.
In that study, 1000 mg/kg/day was identified as the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of the test substance to rats for 28 consecutive days. Based on these data, 1000 mg/kg/day was selected as the high dose for the current study. Dosage levels of 100 and 300 mg/kg/day were selected to further characterize potential dose-response relationships.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
In both study phases, all rats were observed twice daily for moribundity and mortality

DETAILED CLINICAL OBSERVATIONS:
In both study phases, individual detailed physical examinations were recorded weekly. Each test substance-treated male and female was also observed for signs of toxicity 1-2 hours following dose administration. 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. In addition, any changes or abnormalities (if any) in nesting or nursing behavior were recorded.

BODY WEIGHT:
Individual male body weights were recorded weekly throughout the study except for males assigned to the exposure assessment phase, for which body weights were only recorded once prior to randomization.
Individual female body weights were recorded weekly until copulation. Once evidence of mating was observed, main study phase 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. For exposure phase females, body weights were recorded on gestation days 0, 4, 7, 11, and 14.

FOOD CONSUMPTION (non feeding study)
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the breeding period and was not recorded for toxicokinetic phase males. Once evidence of mating was observed, main study phase female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. For exposure phase females, food consumption was recorded on gestation days 0, 4, 7, 11, and 14. Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.

OTHER:
BREEDING PROCEDURES
Each mating pair was examined daily for evidence of mating.

TOXICOKINETICS (EXPOSURE PHASE)
Blood samples for toxicokinetics were collected from females on study day 0 and gestation day 15 prior to dose administration and at approximately 1, 2, 4, 8, and 24 hours after dose administration.

SACRIFICE
- All surviving male animals:
Males were euthanized following completion of the mating period.

- All surviving maternal animals:
Females that delivered were euthanized on lactation day 4. One female failed to deliver and was euthanized on post-mating day 25.

GROSS NECROPSY
A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination. The numbers of former implantation sites and corpora lutea were recorded. Uteri with no macroscopic evidence of implantation were opened and early implantation loss was investigated. Necropsy included examination of the external surface, all orifices, the external surface of the brain and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies: Brain, Epididymides *, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Testes *.
* = These paired organs were weighed separately. Care was taken to ensure separation between the left and right organs.

Microscopic examination was performed on the tissues listed below from all animals in the control and 1000 mg/kg/day groups at the scheduled necropsies and from any animals found dead. In addition, gross lesions from animals in all groups were examined.
Cervix
Coagulating gland
Epididymides *
Mammary glands (females only)
Ovaries
Pituitary gland
Prostate gland
Seminal vesicles
Testes *
Uterus
Vagina
Vas deferens
All gross (internal) lesions
*= Sections of 2-4 microns of the testis (transverse) and epididymis (longitudinal) were stained in PAS and hematoxylin in addition to routine hematoxylin and eosin staining.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nesting and nursing behavior were recorded. The pups were individually weighed on PND 1 and 4 and were individually sexed on PND 0 and 4.

GROSS EXAMINATION OF DEAD PUPS:
Each litter was examined daily for survival and all deaths were recorded. 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. On PND 4, surviving F1 rats were euthanized and the carcasses were discarded without examination.

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.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test 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 (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test (Steel and Torrie, 1980).

Indices:

Mating, fertility, and copulation/conception indices were calculated for main study phase 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


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 (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 / 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

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test substance-related statistically significantly (during gestation days 11-14) lower mean body weight gains, with corresponding effects on food consumption, were noted for F0 females in the 1000 mg/kg/day group during gestation days 11-17. No adverse effects were noted during gross pathological and histopathological examinations.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
The number of pups born and live litter size were unaffected by test substance administration. Slightly lower (not statistically significant) mean postnatal survival was noted in the 1000 mg/kg/day group during PND 0-1, PND 1-4, and from birth to PND 4 when compared to the concurrent control group; the values during PND 0-1 and PND 1-4 were below the minimum mean values in the WIL Research historical control data. However, these decreases were attributed to a single total litter loss in this group on PND 2. Therefore, the slightly lower postnatal survival in the 1000 mg/kg/day group was not considered test substance-related.

CLINICAL SIGNS (OFFSPRING)
In the 1000 mg/kg/day group pups, test substance-related clinical findings of pale and cool body were noted. No other test substance-related offspring clinical findings were noted. Although pale body was also noted for pups in the 300 mg/kg/day group, this finding was noted at a low incidence in this group and also for a single pup in the control group. Pups that were found dead numbered 6, 4, 5, and 15 in the control, 100, 300, and 1000 mg/kg/day groups, respectively. The slightly higher number of pups found dead in the 1000 mg/kg/day group was attributed to a single total litter loss of 13 pups on PND 2, and was not considered test substance-related. One and 7 pups in the 300 and 1000 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

BODY WEIGHT (OFFSPRING)
Slightly lower mean birth weights were noted for F1 males and females in the 1000 mg/kg/day group. Slightly lower mean body weight gains for F1 pups at 1000 mg/kg/day during PND 1-4 resulted in lower mean F1 male and female body weights in this group on PND 4. Despite slightly lower mean body weight gains during PND 1-4 in the 100 and 300 mg/kg/day groups, mean offspring body weights in these groups remained similar to the control group on PND 4; therefore, the lower mean body weight gains during PND 1-4 in these groups were not considered adverse.

GROSS PATHOLOGY (OFFSPRING)
The numbers of pups (litters) found dead during PND 0-4 numbered 6(3), 4(2), 5(3), and 15(5) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, internal findings were limited to renal papilla(e) not fully developed for a single pup in the 100 mg/kg/day group and renal papilla(e) not fully developed and/or distended ureter(s) for a single pup in the 1000 mg/kg/day group. However, these findings occurred infrequently and were not considered test substance-related.

Abnormalities:

not specified

Developmental effects observed:

not specified

OTHER FINDINGS (PARENTAL ANIMALS)

Toxicokinetics

All female rats dosed orally with the test substance were systemically exposed. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. On gestation day 15, systemic exposure to the test substance increased less than dose-proportionally over the 100 to 1000 mg/kg/day range. Exposure the test substance, in terms of AUClast, was higher on gestation day 15 than on study day 0 at 1000 mg/kg/day, but was similar on both evaluation days at 100 and 300 mg/kg/day. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively. Peak plasma the test substance concentrations were reached at 8, 24, and 8 hours post-dosing on study day 0 and at 8, 4, and 8 hours post-dosing on gestation day 15 at 100, 300, and 1000 mg/kg/day, respectively. the test substance concentrations were generally at or near Cmax or fluctuated from 1 through 24 hours post-dosing, except for animals dosed at 100 mg/kg/day on study day 0, where concentrations increased through 8 hours, then decreased through 24 hours post-dosing. Concentration data for the terminal elimination phase was inadequate to determine half-life. Plasma the test substance concentrations in control group animals were below the limit of quantitation (BLQ) in all samples collected at all time points on study day 0 and gestation day 15.

Conclusions:

Due to the lower mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. The NOAEL for developmental toxicity was 300 mg/kg/day, based on adverse clinical findings and lower mean body weight in the 1000 mg/kg/day group.

Executive summary:

The substance has been studied in a GLP-compliant reproduction/developmental toxicity screening test performed in accordance with OECD guideline 421. Groups of 12 male and 12 female Sprague-Dawley rats were administered the substance as a suspension in corn oil at dose levels of 100, 300, and 1000 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle alone. Males received 14 daily doses prior to mating. Females received 14 daily doses prior to pairing and were dosed through lactation day 3. All F0 females were allowed to deliver and rear their pups until lactation day 4.

In addition, an exposure phase was conducted with 8 female rats/group administered the test substance at dosage levels of 0, 100, 300, and 1000 mg/kg/day for 14 days prior to pairing through gestation day 15. An additional eight males were assigned to each exposure phase group and were used for breeding purposes only. On study day 0 and gestation day 15, blood samples for determination of plasma drug concentration were collected from females prior to dose administration and at 1, 2, 4, 8, and 24 hours following dose administration. Pregnancy status was determined for each female on gestation day 16.

Under the conditions of this screening study, due to effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. The NOAEL for developmental toxicity was 300 mg/kg/day, based on adverse clinical findings and effects on body weight in the 1000 mg/kg/day group.

All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The test substance was examined according to OECD guideline 421 (Reproduction / developmental Toxicity Screening Test) and GLP to provide information on potential adverse effects on reproduction.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance has been studied in a GLP-compliant reproduction/developmental toxicity screening test performed in accordance with OECD guideline 421. Groups of 12 male and 12 female Sprague-Dawley rats were administered the substance as a suspension in corn oil at dose levels of 100, 300, and 1000 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle alone. Males received 14 daily doses prior to mating. Females received 14 daily doses prior to pairing and were dosed through lactation day 3. All F0 females were allowed to deliver and rear their pups until lactation day 4.

In addition, an exposure phase was conducted with 8 female rats/group administered the test substance at dosage levels of 0, 100, 300, and 1000 mg/kg/day for 14 days prior to pairing through gestation day 15. An additional eight males were assigned to each exposure phase group and were used for breeding purposes only. On study day 0 and gestation day 15, blood samples for determination of plasma drug concentration were collected from females prior to dose administration and at 1, 2, 4, 8, and 24 hours following dose administration. Pregnancy status was determined for each female on gestation day 16.

Under the conditions of this screening study, due to effects on mean body weight and food consumption for F0 females during gestation at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 300 mg/kg/day. The NOAEL for developmental toxicity was 300 mg/kg/day, based on adverse clinical findings and effects on body weight in the 1000 mg/kg/day group. The reviewer concluded that these effects are most likely to be caused by the females not providing enough care to their pups, and thus are a result of (secondary) maternal toxicity.

All female rats dosed orally with the test substance were systemically exposed to the test substance. Exposure to the test substance, in terms of AUClast and Cmax, was similar at all dosage levels on study day 0, but increased as dosage increased from 100 to 1000 mg/kg/day on gestation day 15. Accumulation ratios were 1.2, 1.6, and 3.0 at 100, 300, and 1000 mg/kg/day, respectively.

Justification for selection of Effect on developmental toxicity: via oral route:
Only study available, GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Justification for classification or non-classification

The test substance does not meet classification criteria under EU Directive 67/548/EEC for reproductive toxicity.

The test substance does not meet classification criteria under Regulation (EC) No 1272/2008 for reproductive toxicity.