Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-02-26 to 2018-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Quinazolin-4(1H)-one
EC Number:
207-735-8
EC Name:
Quinazolin-4(1H)-one
Cas Number:
491-36-1
Molecular formula:
C8H6N2O
IUPAC Name:
4-Hydroxyquinazoline
Test material form:
solid
Specific details on test material used for the study:
Batch No.: 20170707
Purity: 99.13%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley® rat is the system of choice because this strain of rat has been widely accepted and used for developmental and reproductive toxicity studies throughout industry. The current state of scientific knowledge does not provide acceptable alternatives to the use of live animals to accomplish the objectives of this study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
-Animal Requirements
Number of Animals: 96 (48 virgin male and 48 virgin female)
Number of Groups: 4 (1 control + 3 dose levels)
Number of Animals per Group: 24 (12 male, 12 female)
Species/Strain: Crl Sprague-Dawley CD® IGS rats
Body Weight, Age and Sex: Male and female rats were ordered from the supplier on the basis of age and birth date. Male and female rats had different birth dates in order to avoid brother-sister mating. Male and female rats were 8-9 weeks of age at the time of arrival at Product Safety Labs. Body weights were recorded the day after receipt and did not exceed ± 20% of the mean weight for each sex.
Supplier: Charles River Laboratories, Inc. Rats were shipped in filtered cartons by truck.

-Environment:
Room Temperature and Relative Humidity Range: 19-23°C and 3562%, respectively.
Acclimation: The animals were conditioned to the housing facilities for six days prior to testing.
Feed: 2016 Certified Envigo Teklad Global Rodent Diet® was stored in a dedicated temperature and humidity monitored feed storage site and was available ad libitum during acclimation and throughout the study.
Water: filtered tap water was available ad libitum from individual bottles attached to the cages or from an automatic watering access system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
The test substance was mixed weight to volume (w/v) in polyethylene glycol 300 (PEG 300). Fresh formulations containing 2, 6, and 20 mg/mL of the test substance were prepared daily. The formulations were stirred at ambient temperature until a visually homogenous mixture was achieved. Preparation of the test substance was documented in the raw data.
Individual doses were calculated based on the most recent body weight for all rats (i.e., mg/kg/day) over the course of the study. All doses were administered volumetrically at 5 mL/kg. Prepared formulations were discarded following daily administration.
Details on mating procedure:
After two weeks of dosing, animals were randomly mated by placing one female in the breeding cage of a male from the same dose group. A record of mating pairs was maintained.
The cohabitation period consisted of a maximum of 14 days. Vaginal smears were performed daily on females during mating and the presence or absence of sperm or vaginal plug was recorded. Females considered to have successfully mated were removed from the cage of the male, assigned a GD 0, and housed individually. Female rats that had not mated with the first seven days of cohabitation were randomly paired with an alternate male rat that had successfully mated. These female rats remained in cohabitation with the alternate male for a maximum of seven additional days. If successful mating had not occurred after 14 days of cohabitation, females were assigned a GD 0 and were evaluated as if in gestation thereafter. After cohabitation, any female with no positive evidence of mating was individually housed in their home cage, containing nesting material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose preparations were sampled at the beginning (as part of the homogeneity assessment), near the middle, and again at near the end of the study for verification of dose concentration. Samples were collected from preparations at each concentration of test substance and from the vehicle control.
Duration of treatment / exposure:
32 days
Frequency of treatment:
daily (7 days/week)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
12
Control animals:
yes
yes, concurrent vehicle
Details on study design:
Males and females were dosed while housed separately for a 14-day pre-mating period, followed by dosing through a 14-day cohabitation period. Upon determination of pregnancy or following the prescribed mating period, females were removed to a separate cage and continue to be dosed through the gestational period of pregnancy and Day 13 of lactation (the day prior to terminal sacrifice).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for viability. Cage-side observations of all animals were performed daily during the acclimation, pre-mating, mating, post-mating, gestation, and lactation periods. All findings were recorded.
On Day 1, prior to the first treatment with the test substance, and approximately weekly during the pre-mating, mating, post-mating periods (if applicable) for males and females, and approximately weekly for the females during gestational and lactation periods, a detailed observation was conducted while handling the animal, generally on days that the animals are weighed and food consumption measurements are taken. Potential signs noted included, but were not limited to: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Toward the end of the gestation period, females were examined twice daily for signs of parturition. The mated females were allowed to give birth (F1) and the gestation length was calculated.
Female rats were evaluated for adverse clinical signs during parturition. Maternal behavior (e.g. nursing, handling of pups), was observed on LD 0, LD 4, and LD 13, with any abnormal behavior recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All male rats were weighed weekly during the pre-mating, mating, and post-mating periods, as well as prior to terminal sacrifice.
All female rats were weighed approximately weekly (during pre-mating and mating and gestation periods), within 24 hours of parturition (LD 0 or LD 1) and approximately weekly thereafter, as well as at terminal sacrifice (LD 14). Females showing no evidence of mating at the end of the cohabitation period were assigned GD 0 and body weights were measured approximately weekly.

FOOD CONSUMPTION AND FOOD EFFICIENCY: Yes
Individual food consumption was measured and recorded to coincide with body weight measurements during pre-mating, gestation and lactation. Food consumption was not measured
during cohabitation. Food efficiency was calculated and reported for all applicable intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

Mating:
After two weeks of dosing, animals were randomly mated by placing one female in the breeding cage of a male from the same dose group. A record of mating pairs was maintained.
The cohabitation period consisted of a maximum of 14 days. Vaginal smears were performed daily on females during mating and the presence or absence of sperm or vaginal plug was recorded.
Oestrous cyclicity (parental animals):
Estrus cycles were monitored daily during the pre-mating (beginning on Day 2) and mating periods until evidence of mating, by vaginal smears to evaluate for regular cyclicity. A vaginal smear was also collected at termination to determine the stage of estrus at sacrifice.
Litter observations:
The litter was the experimental until for evaluation where appropriate. Mean and standard deviations were calculated independently for all test groups. Similarly, mortality and sex was analyzed as number per litter. Inferential comparisons, for quantitative data, were performed on litter size, viability, litter weight, ano-genital distance, and number of nipples/areolae. Clinical Observations:
The day on which all pups were delivered was designated as LD 0. The litters were examined as soon as possible after delivery, and parameters evaluated included, but were not limited to: the number of stillborn pups, live births, runts (pups that are significantly smaller than the corresponding control groups), and any gross abnormalities. From LD 0 through LD 4, pups were counted and general appearance was recorded twice daily, including but not limited to viability and presence of milk bands.
Individual observations were conducted during the post-natal period on LD 0, LD 4, and LD 13.
These observations included, but were not limited to: litter size and weight, as well as the determination of sex.
The ano-genital distance (AGD) of each pup was measured on LD 4 and was normalized to a measure of pup body weight. The number of nipples/areolae in male pups was counted on LD 13.

Body Weight:
Litters were weighed within 24 hours of parturition (LD 0 or LD 1), on LD 4 and LD 13 (the day prior to terminal sacrifice).
Postmortem examinations (parental animals):
SACRIFICE
At terminal sacrifice, all survivors were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. All male rats were sacrificed following a minimum treatment of four weeks. Female rats (with or without surviving pups) were sacrificed on LD 14.

GROSS NECROPSY
A gross necropsy of all males and females included an initial examination of the external surfaces and orifices, as well as the cranial, thoracic, and abdominal cavities. All abnormalities, including gross lesions and palpable masses were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histological examination was performed on the ovaries, epididymides and testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of animals from both the control and high dose groups (Groups 1 and 4, respectively), including the unscheduled death male were processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E).
Postmortem examinations (offspring):
SACRIFICE
All surviving pups were sacrificed on LD 14 by CO2 followed by exsanguination.

GROSS NECROPSY
All pups were examined externally for gross abnormalities. The thorax, abdomen, and pelvis of each pup were examined internally.
Statistics:
Product Safety Labs performed statistical analysis of quantitative data collected during the in-life phase of the study as well as organ weight data. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05. Male and female rats were evaluated separately.
Statistical analysis was conducted using the following software applications: Provantis® version 9, Tables and Statistics, Instem LSS, Staffordshire UK.
Reproductive indices:
-In-Life Data: For all in-life endpoints that were identified as multiple measurements of continuous data over time (e.g., body weight parameters, food consumption, and food efficiency), treatment and control groups were compared using a two-way analysis of variance (ANOVA), testing the effects of both time and treatment, with methods accounting for repeated measures in one independent variable (time; Motulsky, 2014). Significant interactions observed between treatment and time as well as main effects were further analyzed by a post hoc multiple comparisons test (e.g., Dunnett’s test; Dunnett, 1964 and 1980) of the individual treated groups to control.
-Organ Weight Data: When warranted by sufficient group sizes, all endpoints with single measurements of continuous data within groups (e.g., organ weight and relative organ weight) were evaluated for homogeneity of variances (Bartlett, 1937) and normality. Where homogeneous variances and normal distribution was observed, treated and control groups were compared using a one-way ANOVA. When the one-way ANOVA was significant, a comparison of the treated groups to control was performed with a multiple comparisons test (e.g., Dunnett’s test; Dunnett, 1964 and 1980). Where variance was considered significantly different, groups were compared using a non-parametric method (e.g., Kruskal-Wallis non-parametric analysis of variance; Kruskal-Wallis, 1952). When nonparametric analysis of variance was significant, a comparison of treated groups to control will be performed (e.g., Dunn’s test; Dunn, 1964).
Offspring viability indices:
The litter was the experimental until for evaluation where appropriate. Mean and standard deviations were calculated independently for all test groups. Similarly, mortality and sex was analyzed as number per litter. Inferential comparisons, for quantitative data, were performed on litter size, viability, litter weight, ano-genital distance, and number of nipples/areolae.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs in males or females attributable to the administration of test substance. Clinical signs of soiled coat (yellow) in the ano-genital area, interpreted to be secondary to test substance administration, were observed for individual Group 4 animals during the mating, gestation and/or lactation periods.
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:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Male and female fertility, and fecundity indices, were decreased in Group 4 animals administered test substance, as compared to concurrent control, due to a lower number of pregnant females. The Male mating index was below PSL historical control data at all dose levels, and further reduced in the mid dose group. However, without a trending dose response relationship, and no correlating reproductive functionality and pathology observations, changes in mating and fertility endpoints are not considered to be an adverse effect.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations in the filial generation.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test substance-related differences in the average pup body weight per litter.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One control Group 1 pup had a short tail; one Group 2 pup had an umbilical hernia; 3 pups from Group 2 (1 litter) and 1 pup from Group 4 were found dead (too autolyzed to evaluate); No milk band present was observed at necropsy for all groups with 1 (1 litter), 7 (3 litters), 1 (1 litter), and 18 (3 litters), pups in Groups 1-4, respectively, this finding was not accompanied by a significant changes in litter viability or body weight and is considered to have no toxicological relevance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for this substance was 100 mg/kg/day for male and female Sprague-Dawley rats in this repeated dose oral toxicity study with reproduction/developmental testing.
Executive summary:

The test is performed with OECD Guideline 421. The objective of this study was to evaluate the potential effects on male and female reproduction and/or development, resulting from repeated exposure to the test item.

A repeated oral dose gavage toxicity study with a reproduction/developmental toxicity test was conducted in Crl: CD®(SD) IGS rats to provide information on male and female toxicity and reproductive performance, including gonadal function, mating behavior, conception, development of conceptus, and parturition and lactation following administration of test item.

There were no mortalities and no clinical signs in males or females considered to be attributed to the administration of the test substance. 

The test substance administration produced no significant changes in any of the gestational or birth parameters evaluated during the course of the present study including gestational length, gestation index, number of implantation sites, corpora lutea, average resorption (early and late) numbers and pre-implantation and post-implantation loss. Earlier stage of gestation indicators such as total litter size, live birth index, and number of stillborn pups were comparable between treatment Groups and control animals.

There were no toxicologically relevant clinical or necropsy findings observed in the filial generation. There were no test substance-related differences in the average pup body weight per litter. Pups from all litters increased in weight during the lactation phase. Litter weight gain was comparable to control values.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for this substance was 100 mg/kg/day for male and female Sprague-Dawley rats in this repeated dose oral toxicity study with reproduction/developmental testing.

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