Registration Dossier

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Administrative data

Endpoint:
developmental toxicity
Remarks:
Combined repeated dose with reproduction/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

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

Test material

Constituent 1
Test material form:
liquid
Details on test material:
- Purity: 100%

Test animals

Species:
rat
Strain:
other: Sprague Dawley [Crl:CD(SD)]
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, U.S.A. (males) and St. Constant, Quebec, Canada (females).
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 10.5 weeks
- Weight at study initiation: 322-373 g for males; 218-276 g for females
- Fasting period: All males were fasted prior to clinical pathology blood collection when food, but not water, was withheld.
- Housing: Following receipt and until pairing, all F0 animals were housed 2 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®). During cohabitation, rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in clean, solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in solid-bottom cages with bedding material until the scheduled necropsies.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 50% ± 20%
- Air changes (per hr): 10
- Photoperiod: 12 hour light / 12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Preparation: The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures. The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first dosing formulations were visually inspected and were found to be visibly homogeneous and acceptable for administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of the first 10, 50, and 100 mg/mL dosing formulations; samples for concentration analysis were also collected from the middle stratum of the first control group formulation. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from the first 10 and 100 mg/mL dosing formulations following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the approximate middle and last dosing formulations (including the control group) prepared during the in-life phase of the study. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature, protected from light, as back-up. All analyses were conducted by high performance liquid chromatography method with ultraviolet absorbance detection. The analyzed dosing formulations were within the test facility SOP range for suspensions (85% to 115%) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. Each female was cohabited with 1 male in a solid-bottom cage. 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 solid-bottom cages with bedding material. 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.

During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-49 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined from results of previous studies and were provided by the Sponsor. In a previous study, male and female rats (5/sex/group) were dosed at 0, 100, 500, and 1000 mg/kg/day from study day 0 to study day 13. There were no adverse effects attributed to test item administration; therefore, the same dosage levels were selected for the definitive study.
- Animal assignment: Computerized randomization

Examinations

Maternal examinations:
Clinical observations and mortality: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Mortality and all signs of overt toxicity were recorded on the day observed. Individual detailed physical examinations were recorded daily (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 2 hours following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties. Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

Body weights: Individual male body weights were recorded weekly throughout the study, beginning 1 week prior to the initiation of test item administration, and prior to the scheduled euthanasia. Individual female body weights were recorded weekly, beginning 1 week prior to the initiation of test item administration, until evidence of copulation was observed or until euthanasia (for female without evidence of mating). Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire treatment period (males only). Once evidence of mating was observed, female body weights were recorded on presumed gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4.

Macroscopic Examination: A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. The female with evidence of mating that failed to deliver was euthanized on post-mating day 25. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

Histology and Microscopic Examination: Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups at the scheduled necropsies, as well as the F0 male that was found dead. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate.
Fetal examinations:
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. The carcass of each pup was then discarded.
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical observation on PND 1 and 4. Any abnormalities in nesting and nursing behavior were recorded.
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, as females enter gestation/lactation, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA”.
Pups were individually sexed on PND 0 and 4.
Statistics:
Refer to any other information on materials and methods for statistical analysis information.
Indices:
Mean live litter size, postnatal survival between birth and PND 0 or PND 4 (% per litter), postnatal survival for all other intervals (% per litter)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related clinical findings were noted approximately 2 hours following dose administration for males and females in the 500 and/or 1000 mg/kg/day groups. Red material around the mouth was noted for 9 of 10 males and 8 of 10 females in the 1000 mg/kg/day group compared to 0 animals in the control group; this finding was noted during study days 10-26 (males) and gestation days 1-15 (females). In addition, clear material around the mouth was noted for 6 of 10 males and 7 of 10 females in the 1000 mg/kg/day group, and to a lesser extent (4 males) in the 500 mg/kg/day group; this finding was noted during study days 14-25 (males) and gestation days 2-12 (females). These material findings did not persist to the next detailed physical examination, and therefore were not considered adverse. There were no test item-related findings noted at the weekly detailed physical examinations for F0 males and females at any dosage level and at 2 hours following dose administration for females in the 500 mg/kg/day group and males and females in the 100 mg/kg/day group. Other clinical observations noted in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival was unaffected by test item administration for F0 males and females at all dosage levels. One F0 male in the 100 mg/kg/day group was found dead on study day 28. The death was considered to be accidental (gavage error), and not test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower (55.6%, not statistically significant) mean body weight gain was noted in the 1000 mg/kg/day group during gestation days 0-4 compared to the control group. Mean body weight gains in the 1000 mg/kg/day group were similar to the control group during the remainder of gestation, as well as when the entire gestation treatment period (gestation days 0-20) was evaluated compared to the control group. However, mean body weights in this group, although not statistically significant, were 5.5% to 7.2% lower than the control group during gestation days 4-17; the difference on gestation day 14 was significant (p<0.05). The lower mean body weights noted in the 1000 mg/kg/day group were most likely a continuation of the lower mean body weights noted in this group during the premating period, but were also affected by the lower mean body weight gain following the initiation of the gestation treatment period. Mean body weights and body weight gains for females in the 100 and 500 mg/kg/day groups were unaffected by test item administration during gestation.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in hematology and coagulation parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. A significantly (p <0.01) higher mean absolute monocyte count was noted in the 1000 mg/kg/day group males. However, the higher mean value was due to a single outlier and all other individual animal values were within the range of the test facility historical control data. A significantly (p <0.05) higher absolute basophil count was also noted in the 1000 mg/kg/day group males. All individual animal values were within the test facility historical control data range, and therefore the difference was not attributed to the test item.
Statistically significant findings that involved percentage leukocyte differential counts were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights. However, a significantly (p<0.05) higher liver weight relative to final body weight value was noted in the 1000 mg/kg/day group males when compared with the control group. The margin of change was small, the absolute organ weight value was not statistically significantly different from the control group, and there were no corresponding microscopic findings. Therefore, the change was not considered to be associated with administration of the test item.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Description (incidence and severity):
The mean numbers of implantation sites (13.5 sites) in the 1000 mg/kg/day group were lower than the control group values (16.1 sites). This difference was significant (p<0.05).
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Pups (litters) that were found dead numbered 3(3), 9(6), 2(2), and 4(3) in the control, 100, 500, and 1000 mg/kg/day groups, respectively. Two (2), 0(0), 2(2), 0(0) pups (litters) in these same respective groups were missing and presumed to have been cannibalized. At necropsy, aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects observed at highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (Male/Female): 1000 mg/kg/day (Reproductive and neonatal toxicity)
NOAEL (Female): 500 mg/kg/day (systemic toxicity) (based on the effects on body weight)
Executive summary:

The objective of this study was evaluating the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The protocol was designed in general accordance with the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

The test item, in the vehicle (corn oil) was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females. Dosage levels were 100, 500, and 1000 mg/kg/day administered at a dosage volume of 10 mL/kg. A concurrent control group of 10 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10.5 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-49 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. All F0 animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females 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. Found dead pups were necropsied, while surviving pups were euthanized and discarded without examination on PND 4. Clinical pathology evaluations (haematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group (the same animals used for FOB/motor activity assessments) at necropsy. F0 males were euthanized following completion of the mating period. F0 females were euthanized on lactation day 4 (for females that delivered) or post-mating day 25 (for the female that failed to deliver). 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-dose groups and from any F0 animal that died spontaneously.

Survival was unaffected by test item administration for F0 males and females at all dosage levels. One F0 male in the 100 mg/kg/day group was found dead on study day 28. The cause of death for this male was not determined microscopically; however, based on macroscopic findings of lungs that were not fully collapsed and contained dark red areas, which correlated microscopically with acute hemorrhage and was considered to represent an agonal change, the death was considered to be accidental (gavage error), and not test item-related. To further support this, there were no deaths in the higher dose groups. All other animals survived to the scheduled necropsies.

Clear material around the mouth was noted for the majority of F0 males and females in the 1000 mg/kg/day group, and to a lesser extent in the 500 mg/kg/day group males, approximately 2 hours following dose administration. Red material around the mouth was also observed for the majority of F0 males and females in the 1000 mg/kg/day group at approximately 2 hours following dose administration. The aforementioned material findings for F0 males and females in the 500 and/or 1000 mg/kg/day groups were noted sporadically throughout the respective treatment periods and considered non-adverse because they did not persist to the next detailed physical examination. No test item-related clinical findings were noted for F0 males and females at any dosage level at the detailed physical examinations or for F0 males and females in the 100 mg/kg/day group and F0 females in the 500 mg/kg/day group approximately 2 hours following dose administration.

A test item-related lower (76.5%) mean body weight gain and corresponding reduction (27.3%) in mean food consumption were noted for the 1000 mg/kg/day group F0 females during the first week of test item administration (study days 0-7), which resulted in a lower (39.3%) mean body weight gain when the entire pre-mating period (study days 0-13) was evaluated. The effect on mean body weight gain was of sufficient magnitude to result in lower (5.9% and 4.5%) mean body weights for the 1000 mg/kg/day group F0 females on study days 7 and 13, respectively; therefore, the differences were considered to be adverse. Mean body weight gain for F0 females in the 1000 mg/kg/day group was lower (55.6%) than the control group following the initiation of the gestation treatment period (gestation days 0-4) compared to the control group, but similar to the control group throughout the remainder of gestation (gestation days 4-20) and during lactation days 1-4. However, mean body weights in the 1000 mg/kg/day group remained generally lower (up to 7.2%) than the control group during the gestation and lactation treatment periods, in part due to the lower mean body weight gain during gestation days 0-4, as well as the aforementioned effects noted during the pre-mating period. Mean body weights, body weight gains, and food consumption for F0 males at all dosage levels and for F0 females in the 100 and 500 mg/kg/day groups were unaffected by test item administration.

No test item-related effects were noted during the FOB assessments or motor activity evaluations at any dosage level for F0 males and females.

F0 male and female mating and fertility, male copulation, and female conception indices, the mean number of days between pairing and coitus, gestation lengths, and the process of parturition were unaffected by test item administration at all dosage levels.

There were no test item-related clinical pathology (haematology, coagulation, and serum chemistry) alterations or histologic changes in F0 males and females. In addition, no test item-related macroscopic or microscopic findings or effects on organ weights for F0 males and females in any group or effects on the mean numbers of implantation sites, unaccounted-for sites, and corpora lutea at any dosage level.

Mean F1 offspring body weights and body weight gains were unaffected by parental test item administration at all dosage levels. In addition, the mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival were unaffected by parental test item administration. None of the clinical findings for F1 pups or macroscopic findings for the F1 pups found dead were attributed to the test item.

Under the conditions of this screening study, no test item-related effects were noted on F0 reproductive endpoints, including male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity was considered to be 1000 mg/kg/day (the highest dosage level evaluated) when the test item was administered orally by gavage to Crl:CD(SD) rats. The NOAEL for neonatal toxicity was considered to be 1000 mg/kg/day based on the lack of effects on the F1 pups at all dosage levels. A test item-related lower mean body weight gain with corresponding reduced mean food consumption were observed for the 1000 mg/kg/day group F0 females during the first week of treatment, which resulted in lower mean body weights in this group during the pre-mating period, as well as during gestation and lactation. These effects were considered adverse. There were no adverse clinical findings noted for F0 males or females at any dosage level and no effects on mean body weights, body weight gains, and food consumption for F0 males at all dosage levels or F0 females at 100 and 500 mg/kg/day. In addition, there were no test item-related effects on clinical pathology, organ weights, and macroscopic and histopathological findings at any dosage level. Based on the effects on body weight and food consumption for F0 females at 1000 mg/kg/day, the NOAEL for F0 female systemic toxicity was considered to be 500 mg/kg/day. In the absence of toxicity noted for F0 males at all dosage levels, the NOAEL for F0 male systemic toxicity was considered to be 1000 mg/kg/day.