Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2017 to 10 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS number: Not yet known
EC Number: Not yet known
Specific details on test material used for the study:
Physical Description: Dark brown, clear liquid
Purity: 91%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots in corn oil for daily dispensation, and stored refrigerated (2°C to 8°C), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material formulations prepared at concentrations of 20, 60, and 200 mg/mL were analyzed by a validated high performance liquid chromatography method using ultraviolet absorbance detection at a wavelength of 260 nm. The analytical results met the applicable protocol-specified acceptance criteria. No test substance was detected in the analyzed vehicle administered to the control group.
Details on mating procedure:
Males and females were paired on a 1:1 basis within each treatment group following 14 days of treatment and were cohabitated in a solid-bottom cage containing bedding material until positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed Gestation Day 0 and the animals were separated.
Duration of treatment / exposure:
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 13 for a total of 49–53 doses.
Frequency of treatment:
Once daily
Duration of test:
A minimum of 28 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Charles River, 2017 (range-finding)).

Examinations

Maternal examinations:
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 Males
Weeks 1-3: Twice daily
Last week of dosing (Study Day 26): 5 males/group only

F0 Females
Weeks 1-4:Twice daily
Last week of dosing (Lactation Day 13): 5 females/group only
Lactation Day 14

Detailed observations were recorded at the following times in relation to dose administration: Approximately 1.5 hours after dosing

Parturition
All females were allowed to deliver naturally and rear their young to PND 13. 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.
Ovaries and uterine content:
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulphide solution for detection of early implantation loss. For females that delivered or had macroscopic evidence of implantation, the numbers of implantation sites for former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through Lactation Day 4.
Fetal examinations:
Each litter was examined daily for survival, and all deaths were recorded. Intact offspring that were found dead were necropsied via a dissection technique, which included examination of the heart and major vessels. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia from 1 pup/sex/litter; gross necropsies were conducted and the thyroids (with parathyroids, if present) were placed in 10% neutral-buffered formalin for possible histopathological examination.

Litter Reduction
To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. Blood samples for possible future thyroid hormone analysis were collected from at least 2 culled pups/litter (pooled by litter) on PND 4; pups were euthanized by an intraperitoneal injection of sodium pentobarbital following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

Clinical Observations
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Any abnormalities in nesting and nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 113; the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup.

Body Weights
Pups were individually weighed on PND 1, 4, 7, 10, and 13.

Sex Determination
Pups were individually sexed on PND 0, 4, and 13.
- Assessment of Areolas/Nipple Anlagen: On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae in accordance with previous established methods. The number of nipples was recorded if nipples were present, and a zero was recorded if nipples were absent.
Statistics:
Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the vehicle control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor. Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, estrous cycle length, precoital intervals, gestation length, numbers of former implantation sites, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), number of nipples/areolae, and FOB data values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the vehicle control group. FOB parameters that yield scalar or descriptive data in the test substance-treated groups were compared to the vehicle control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the vehicle control group.
Historical control data:
Historical Control Data (HCD) was presented in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations noted in the test substance-treated groups, including red and yellow material on various body surfaces and hair loss on the forelimbs, were noted infrequently, similarly in the vehicle control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant differencse from the vehicle control group were an increased mean potassium level and decreased mean sodium level in the 100 mg/kg/day group F0 males and a lower mean bile acid level in the 300 mg/kg/day group F0 males. There was no dose-response relationship, no similar occurrence in the opposite sex, and/or the changes were of minimal magnitude. These differences from the vehicle control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Differences from the vehicle control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One (1), 3(3), 5(4), and 4(4) pups (litters) in the vehicle control, 100, 300, and 1000 mg/kg/day groups, respectively, were found dead. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean male and female F1 pup body weights and body weight changes during PND 1–13 in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental administration of the test substance. Differences from the vehicle control group were slight and not statistically significant with the exception of significantly (p< 0.05 or p< 0.01) higher mean body weight gains in the 300 and 1000 mg/kg/day group F1 males and female during PND 4-7, which resulted in a significantly (p < 0.05) higher mean body weight for F1 males in the 1000 mg/kg/day group on PND 7. These differences were transient, and therefore not considered test substance-related.
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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
- Anogenital Distance: Differences from the vehicle control group were slight and not statistically significant.
- Areolae/Nipple Anlagen: Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Internal findings included a cleft palate (skeletally, palatine plates were not joined along the entire length), fused sternebrae (nos. 2 through 4 and nos. 5 and 6), and an accessory skull bone in one pup in the 1000 mg/kg/day group. No other internal findings were noted.
Visceral malformations:
no effects observed
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
No adverse effects were noted in F0 adults or F1 pups at any dosage level, therefore, 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive and systemic toxicity and F1 neonatal toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.
Executive summary:

The potential toxic effects of the test substance at dosage levels of 100, 300 and 1000 mg/kg bw/day were investigated in an OECD 422 study, performed under GLP conditions. The test substance or vehicle control (corn oil) was administered to rats via gavage once daily to 4 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females.

All F0 males and females in the vehicle control, 100, 300, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test substance-related clinical observations were noted for the 100, 300, and 1000 mg/kg/day group F0 males and females during the weekly detailed physical examinations, at the daily examinations, or approximately 1.5 hours following dose administration.

No test substance-related effects were noted on F0 male and female body weights, body weight gains, or food consumption at any dosage level throughout the study. Mean F0 male and female reproductive performance (mating, fertility, and copulation/conception indices) and the process of parturition in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration. The mean number of days between pairing and coitus, gestation length, and estrous cycle length were unaffected by test substance administration. There were no test substance-related effects noted on the mean numbers of former implantation sites and unaccounted-for sites in F0 females at any dosage level. There were no test substance-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

There were no test substance-related effects on F0 organ weights, hematology and serum chemistry parameters, and serum T4 levels (males only) in the 100, 300, and 1000 mg/kg/day groups. No test substance-related gross necropsy observations or microscopic findings were noted for F0 males and females at any dosage level.

Mean numbers of F1 pups born, live litter size, and percentage of males at birth were unaffected by parental test substance administration. There were no test substance-related effects on postnatal survival and no test substance-related clinical observations were noted for F1 pups at any dosage level. F1 pup body weights and body weight gains in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental test substance administration.

Under the conditions of this screening study, no adverse effects were noted in F0 adults or F1 pups at any dosage level. Therefore, 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive and systemic toxicity and F1 neonatal toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.