Registration Dossier

Administrative data

Endpoint:
screening for reproductive / 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:
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

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)
Sex:
male/female

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.
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.
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.
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
Details on study schedule:
Time of Necropsy
- F0 males: After final investigations completed (after at least five weeks of treatment).
- F0 females failing to produce a viable litter: Day 25 after mating.
- F0 females: Day 14 of lactation (following terminal blood sampling)
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

Parental animals: Observations and 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
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and evaluated microscopically to determine the stage of the estrous cycle of each female for 14 days prior to randomization, continuing until evidence of copulation was observed. The average cycle length was calculated and reported for complete estrous cycles beginning with the first day of dose administration. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
Sperm parameters (parental animals):
The weight of the testis were examined. The testes with epididymides were collected, treated, fixed and stained for microcopic examination.
Litter observations:
- 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.
- 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
- The anogenital distance of all F1 pups was measured on PND 1.
- To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4.
Postmortem examinations (parental animals):
Necropsy
All F0 adults were euthanized by carbon dioxide inhalation and were subject to a detailed necropsy. Males were euthanized following completion of the mating period. Females that delivered were euthanized on Lactation Day 14; one vehicle control female that failed to deliver was euthanized on Postcohabitation Day 25.

Macroscopic Examination
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia for males and on Lactation Day 13 for females. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulphide 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.

Organ Weights
The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands
Brain
Epididymides (paired organs were weighed separately)
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicles (with coagulating gland and fluid)
Spleen
Testes (paired organs were weighed separately)
Thymus gland
Thyroids with parathyroids

Histology and Microscopic Examinations
After fixation, specified tissues were trimmed, processed into paraffin blocks, sectioned, mounted on glass microscope slides, and stained with hematoxylin and eosin. PAS staining was used for the testes and epididymides. Microscopic examination was performed on all tissues from 5 animals/sex in the vehicle control and 1000 mg/kg/day groups at the scheduled necropsies. Gross lesions were examined from all animals in all groups.

Postmortem examinations (offspring):
Each litter was examined daily for survival, and all deaths were recorded and a daily record of litter size was maintained. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Tissues were preserved in 10% neutral-buffered formalin for gross findings.
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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:
no effects observed
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
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of estrous cycles in these groups were also comparable to the vehicle control group value.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dosage level. One male in the vehicle control group did not sire a litter and one female in this group was determined to be nongravid.

Effect levels (P0)

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:
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
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

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.