Registration Dossier

Administrative data

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
Study period:
1997-02-16 to 1997-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: An OECD guideline study, submitted to authorities.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(22 Mar 1996)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Japan Inc.
- Age at study initiation: 8 weeks old
- Weight at study initiation: ranging from 305 to 327 g for males and 201 to 225 g for females
- Fasting period before study: no
- Housing: except a mating period and a period from day 17 of gestation to day 4 of nurture, the animals were reared individually in metal mesh cages
- Diet: a solid diet (radiation sterilization diet NMF: Oriental Yeast Co., Ltd.), ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5 to 25.5°C
- Humidity: 41 to 66 %
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12-hour light / 12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The administration volume was set to 0.5 mL/100 g to body weight. The administration liquid volume per individual was calculated based on a body weight measurement on each day in case of males. In case of females, the administration liquid volume per individual was calculated based on each body weight measurement in the pre-mating and mating periods, and then on day 0, day 7, day 14, and day 21 of gestation and on day 0 of nurture in the lactation period.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The administration period was 49 days by males starting 14 days before mating to 1 day before autopsy and 54 days by females starting 14 days before mating, mating period (14 days at maximum), gestation period, until day 3 of nurture (day before autopsy).
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosage levels were selected based on results of a range-finding study (2 weeks oral administartion of 100, 300 and 1000 mg/kg bw/d of the test substance to rats). A high dose in the main test was set to 500 mg/kg bw/d which was approximate to a geometric mean of 1000 mg/kg bw/d and 300 mg/kg bw/d and which was estimated to represent toxicity signs but no deaths or serious pains. Subsequently, by subtracting it by the common ratio of about 3, a medium dose was set to 150 mg/kg bw/d, and a low dose was set to 50 mg/kg bw/d.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times per days. Before administration, immediately after administration, and 2 hours after administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 times per days. Before administration, immediately after administration, and 2 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was measured on the day of the start of administration (before administration and on day 1 of administration), day 4, day 8, day 11, day 15, day 22, day 29, day 36, day 43, day 49 of administration, and the day of autopsy (day 50 after the start of administration).

FOOD CONSUMPTION
- Time schedule for examinations: Food consumption remaining quantity was measured on each of the same days as the days of the body weight measurements except the mating period and the day of autopsy.
- The quantity of food consumed per day was calculated from the difference between the remaining quantity and the feed quantity on the previous day.




Oestrous cyclicity (parental animals):
For observations of each estrous cycle, vaginal smear was collected every day from the following day of the start of administration to confirmation of copulation and was examined microscopically. During the administration period before mating, the vaginal smear images were classified into an early estrus period, an estrus period, a late estrus period, and an estrus recess period, and a count of estrus and mean duration of cycles (estrous cycle) were examined. In the mating period, presence or absence of sperms in vaginal smear was examined.
Sperm parameters (parental animals):
Not examined.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical observations, body weight, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 49 days after the beginning of the administration
- Maternal animals: All surviving animals 54 days after the beginning of the administration. The females were allowed to deliver and rear their pups until day 4 after parturition.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were prepared for microscopic examination and weighed:
brain, sciatic nerve, heart, lung including bronchus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, pituitary, thyroid including glandula parathyroidea, adrenal, thymus, spleen, mesenteric lymph node, cervical lymph node, kidney, urinary bladder, testis, epididymis, seminal vesicle, prostate, mammary gland, skin, bone marrow including sternum and femur.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
Statistics:
- Multiple Test
Homogeneity of variance among groups was tested by the Bartlett’s method. As a result, in a case where variance was homogeneous, the one-way analysis of variance was carried out. In a case where a significant difference was present among the groups, the paired comparison test between the control group and each treated group was carried out using the Dunnett’s method. In a case where variance was not homogeneous, the Kruskal-Wallis rank test was carried out. If the result was significant, the Dunnett’s test was carried out for a mean rank difference between the control group and each treated group.
Body weight, food consumption, quantitative items of urinalysis, count of estrus, estrous cycle, number of living-together days, gestation length, hematological examination values, blood chemical examination values, organ weights, number of corpora lutea, number of implantation sites, number of live born pups, implantation index, sex ratio, delivery index, live birth index, and viability index.
- x2 Test: Copulation index, fertility index, and birth index
- Mann-Whitney’s U Test, Fisher’s Exact Test: The Mann-Whitney’s U test was conducted for expression frequency and increase in severity of histopathological findings. The Fisher’s exact test was conducted for pancreas acinus basophilic focus and lung osseous metaplasia.
Reproductive indices:
- Male mating index
- Female mating index
- Female fertility index
- Gestation index
Offspring viability indices:
- Live birth index
- Postimplantation loss
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Male and female rates: no changes in clinical signs related to administration of the test article were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Male and female rats: the body weights of the treated groups changed almost similarly to those of the control group through out the administration period. No notable abnormalities were observed n food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The estrous cycle in each of the treated groups, that is, the count of estrus and the mean duration of cycles, was almost similar to that in the control group, and no significant differences were recognized between the control group and the respective treated groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation Index and Fertility Index:
The copulation index of each group was 100 %. There were also no significant differences in the average number of days until copulation between the control group and the treated groups. There were no significant differences in the fertility index between the control group and the treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Male rats: a tendency toward a high value for an absolute weight of the liver, a significant high value for a relative weight of the liver, and significant high values for absolute and relative weights of the kidney in the 500 mg/kg bw/d group. A significant high value for a relative weight of the kidney in the 150 mg/kg bw/d group was observed as well as in the 50 mg/kg bw/d group.
Female rats: no substance related abnormalities.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No substance related abnormalities

HISTOPATHOLOGY (PARENTAL ANIMALS)
Male and female rats: no substance related abnormalities

OTHER FINDINGS (PARENTAL ANIMALS)
Number of Females with Live Pups, Gestation Length, Delivery, and Nurture States:
there were no significant differences in the gestation length between the control group and the treated groups. No abnormal delivery was observed in the treated groups, and the birth index was 100 % in each group.
As for the nurture states, no abnormalities were recognized in nurture behaviors such as nesting, pups collection, and lactation in any of the control group and the treated groups.
Number of Corpora Lutea, Number of Implantation Sites, Implantation Index, and Number of Pups Born:
The number of corpora lutea, number of implantation sites, and implantation index in the treated groups were almost similar values to those in the control group, and no significant differences were recognized between the control group and the treated groups. There were no significant differences in the live birth index between the control group and the treated groups. However, in the 500 mg/kg bw/d group, a tendency toward a low value for the number of pups born was recognized, and a significant low value for the delivery index was recognized. No single animal data are given in the report, only average numbers therefore an interpretation of this finding is not possible.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Results of hematological examinations/ blood chemistry tests (hemoglobin and urea nitrogen) in male rats.
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adversed effects observed.
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: In the 500 mg/kg treated group, a tendency toward a low value for a number of pups born and a low value for a delivery index were recognized.
Remarks on result:
other: Generation: P generation; reproductive performance and fertility (migrated information)
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adversed effcets observed.
Remarks on result:
other: Generation: P generation; reproductive performance and fertility (migrated information)
VIABILITY (OFFSPRING)
There were no significant differences in the viability index on day 4 after birth between the control group and the respective treated groups.

BODY WEIGHT (OFFSPRING)
The body weights at birth and on day 4 after birth in the treated groups showed almost similar values to those in the control group, and there were no significant differences between the control group and the respective treated groups.

GROSS PATHOLOGY (OFFSPRING)
In the autopsy on day 4 after birth, no abnormalities were recognized in the control group and the treated groups.

OTHER FINDINGS (OFFSPRING)
Sex Ratio, External Malformations:
There were no significant differences in the sex ratio between the control group and the treated groups. In external observations of live born pups, a vestigial tail was recognized in 1 pup in the 500 mg/kg bw/d group.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
An OECD guideline study.
Additional information

Toxicity to reproduction (oral):

A combined repeated dose and reproductive/developmental toxicity screening test was performed according to OECD guideline 422, with the test substance in the following concentrations: 0 (olive oil – vehicle), 50, 150, and 500 mg/kg bw/d. The test substance was administered orally to male rats for 49 days and to females for 54 days.

Effects on Reproductive/Developmental Toxicity of Parent Animals (P):

In a 500 mg/kg bw/d treated group, a tendency toward a low value for a number of pups born and a low value for a delivery index were recognized. However, there were no effects of the test article on the estrous cycle and count of estrus of females, number of days until male and female copulation, copulation index, fertility index, and gestation length, birth index, delivery, nurture states, number of corpora lutea, number of implantation sites, and implantation index of females.

Effects on Live Born Pups (F1):

There were no effects of the administered test substance in any dose-group on the number of still born pups, live birth index, sex ratio, and no pups with external malformations were observed. There were no effects of the test substance on the viability index and weights on day 4 after birth, and no abnormalities were recognized in autopsy.

Therefore, under the conditions of the present test, the no observed effect levels for reproductive/developmental toxicity of parent animals were considered to be 500 mg/kg bw/d for males, 150 mg/kg bw/d for females, and 500 mg/kg bw/d for live born pups.


Short description of key information:
In a combined repeated dose and reproductive/developmental toxicity screening test, performed according to OECD guideline 422 NOELs values for reproductive/developmental toxicity of parent animals of 500 mg/kg bw/d for males, 150 mg/kg bw/d for females, and 500 mg/kg bw/d for live born pups were determined.

Justification for selection of Effect on fertility via oral route:
A reliable OECD guideline study.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for toxicity to reproduction/ development under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction/ developmentunder Regulation (EC) No. 1272/2008, as amended for the sixth time in Regulation EC 605/2014.