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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3043N9Z

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 76-83 days
- Weight at study initiation: (P) Males: 281.5 - 310.2 g; Females: 179.3 - 202.5 g
- Housing: individually except over night mating period; pregnant females were housed with their litter
- Diet: ground Kliba Iaboratory diet rat/mouse/hamster, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: from bottles; ad libitum
- Acclimatisation period: 6 days
- Animal identification: ear (P) and skin (F1) tattoo

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70 %
- Air changes: fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h/12 h



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of the solutions the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with doubly distilled water and subsequently thoroughly mixed. For the mid and high dose groups (200 and 1,000 mg/kg body weight/day), the test substance was dissolved in doubly distilled water using an ultrasonic bath.
The test substance solutions were prepared at the beginning of the administration period and thereafter at 4 day intervals.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Mating period: 14 days
- Further matings after one unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually and after birth with litter
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSES OF THE TEST SUBSTANCE PREPARATIONS
Stability analyses
The stability of the aqueous test substance solutions for a period up to 4 days at room temperature was demonstrated.

Concentration control analyses
The concentration control analyses of the aqueous test substance solutions revealed that the values were in the expected range of the target concentrations. Thus, the correctness of the prepared concentrations was confirmed.

Food analyses
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.

Drinking water analyses
On the basis of the analytical findings, the drinking water was found to be suitable.

Bedding analyses
On the basis of the findings of analysis the bedding was found to be suitable.
Duration of treatment / exposure:
Daily administration starts at least 14 days before mating in both sexes. Male animals are killed on day 28-29 of administration. Females and pups were sacrificed 4 days post partum.
Frequency of treatment:
once daily
Details on study schedule:
- Details of mating:
After 14 days of administration, mating period starts. Mating is carried out over-night for 14 days. The paired animals belong to the same dose group.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 femals per dose and control group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
50 mg/kg body weightlday: as the expected no observed adverse effect level
200 mg/kg body weightlday: as the intermediate dose level and eventually as higher no observed adverse effect level
1,000 mg/kg body weightlday: as the dose level where toxic effects were expected
Positive control:
A positive control with a known reprotoxic agent was not included in the study protocol.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and daily on week-ends/holidays for mortality; daily for clinical signs of toxicity
- Cage side observations include: clinical evident signs of toxicity; nesting, littering and lactational behavour

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly (males); females weekly until the day 0 post coitum; on the day 0 post coitum, weekly thereafter and n the day of parturition

FOOD CONSUMPTION: Yes
- Food consumption was determined once weekly except during pairing period.

OTHER:
- For mating pairs, the number of mating days until vaginal sperm could be detected and gestational status were recorded.
Sperm parameters (parental animals):
lmmediately after necropsy and organ weight determination the right testis and cauda epididymides were taken from the F0 males of all dose groups.
The following parameters were determined:
- sperm motility
- sperm morphology
- sperm head count (cauda epididymides)
- sperm head count (testis)
Sperm motility examinations were carried out in a randomized sequence.
Sperm head count (cauda epididymides and testis) were evaluated for the control and the high dose group, only.
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, weight gain, physical and behavioural abnormalities

GROSS EXAMINATION OF PUPS:
yes, liveborn and stillborn pups were examined for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed following the mating period
- Maternal animals: All surviving animals ca. 9 days p.p.

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

HISTOPATHOLOGY
Ovaries, left testis, left epididymes, kidneys, all gross lesions were prepared for microscopic examination, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Only stillborn pups, pups suffering premature death and those displaying notable abnormalities were subjected to gross necropsy
- Gross necropsy consisted of external and internal examinations
Statistics:
STATISTICS OF CLINICAL EXAMINATIONS:
Food consumption (parental animals) body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litte: two-sided Dunnett test
Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy, males with >4% abnormal spermmaturation: Fisher's exact test
Proportions of affected pups per litter with necropsy observations: one-sided Wilcoxon-test
Total spermatids/g testis, Total sperm/g cauda epididymides, % motility: one-sided Wilcoxon test
Weight parameters: two-sided Kruskal-Wallis test and Wilcoxon test
Reproductive indices:
For the males, mating and fertility indices were calculated according to the following formulas:
 Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females*) x 100
*defined by a female with vaginal sperm or that gave birth to a litter or with pups/implantations in utero
 
Male fertility index: (number of males proving their fertility)/(number of males placed with females *) x100
 *defined by a female giving birth to a litter or with pups/implantations in utero

For the females, mating, fertility and gestation indices were calculated according to the following formulas: 
Female mating index= (number of females mated*)/(number of females placed with males) x100
 *defined as the number of females with vaginal sperm or that gave birth to a litter or with pups/implantations in utero
 
Female fertility index (%) = (number of females pregnant*)/(number of females mated**) x 100
*defined as the number of females that gave birth to a litter or with pups/implantations in utero
**defined as the number of females with vaginal sperm or that gave birth to a litter or with
pups/implantations in utero
 
Gestation Index (%)= (number of females with live pups on the day of birth)/(number of females pregnant *) x 100
 *defined as the number of females that gave birth to a litter or with- pups/implantations in utero
 
The total number of pups delivered and the number of liveborn and stillborn pups were noted and the live birth index was calculated according to the following formulas:
Live birth index (%) = (number of liveborn pups at birth)/ (total number of pups born) x 100
 
Moreover, after sacrifice of the female animals, the implantation sites were counted and the postimplantation loss was calculated for each individual pregnant animal according to the following formula:
Post implantation loss (%) = (number of implantations - number of pups delivered) /(number of implantations)x 100
 

Offspring viability indices:
The viability index was calculated according to the following formula:
Viability index= (number of live pups on day 4 after birth)/(number of liveborn pups on the day of birth) x100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
1000 mg/kg bw: transient salivation after gavaging (induced by bad taste, substance induced but not considered as adverse). No further clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the male and female F0 parental animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and mean body weight gains of the F0 parental rats of all substance-treated groups were comparable to the values of the concurrent controls during the different study phases.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the male and female rats of test groups 1, 2 and 3 (50, 200 and 1,000 mg/kg body weight/day) did not show any test substance related changes. Furthermore, the food consumption of the F0 females during gestation and lactation periods for F1 Iitters was not affected by the test substance administration; again the mean values were fully comparable to that of the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Weight parameters
Absolute weights: The mean kidney weight was significantly increased in rats of the high dose group. This was regarded treatment related.

Relative weights (related to terminal body weight): The mean kidney weight was significantly increased in rats of the high dose group. This was regarded treatment related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few gross lesions were noted which were regarded to have developed fortuitously and unrelated to treatment. Four females (from different dose groups) had neither pups nor implantation sites. Histopathological examination of testes/epididymides of the respective males revealed no correlate for the non-pregnancies of female mating partners.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: focal vacuolization of tubular epithelia in the renal cortex of two male and of two female rats

Histopathology of testes, epididymides and ovaries did not reveal any treatment related microscopic finding.

Four female animals were not pregnant. No microscopic findings were recorded in these females that may contribute to this observation. In the respective male mating partners also no microscopic findings were noted that were indicative for an impaired fertility.
No correlate was obtained, histopathologically, that may account for the significantly increased mean absolute and relative kidney weights in male and female rats of the high dose group. However, two male and two female rats of the high dose group revealed focal or multifocal vacuolization of tubular epithelia in the renal cortex.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There occurred no treatment-related effects in the different sperm parameters examined at or after the sacrifice of the F0 parental males. The number of homogenization resistant testicular spermatids or caudal epididymal sperm, the percentages of abnormal and normal sperm and sperm motility data were similar between the examined dose groups and the concurrent control group and showed no biologically significant differences.
Reproductive performance:
no effects observed
Description (incidence and severity):
For nearly all parental males of test groups 0, 50, 200 and1000 mg/kg bw/day which were placed with females to generate F1 pups, mating was confirmed within the scheduled mating interval. The male mating index was 90 % - 100 %.
The male fertility index was 80 % - 100 %.

The female mating index and the mean cohabitation time (duration until sperm was detected) reflect the normal range of biological variation inherent in the strain used in this study. Consequently, the differences between the groups are assessed as spontaneous in nature and without any biological relevance.
With the exception of one control female and one high dose female all mated females of all test groups became pregnant. Therefore, the fertility indices varied between 89 % and 100 %. The sporadic occurrence of infertility in one female each in the control and the high dose group is assessed as being incidental.
There occurred no gross and/or histopathological findings in these females or its mating partners, which could explain the observed infertility of these pairs. The mean duration of gestation was very similar in all groups (between 21.6 and 21.8 days).
The gestation index was 88 % in the control group, but reached 100 % in all other test groups (50, 200 and 1,000 mg/kg body weight/day). Thus, there were no indications for substance-induced intrauterine embryo-/fetolethality.

Implantation was not affected by treatment since the mean number of implantation sites was comparable between all test groups taking normal biological variation into account. The mean number of F1 pups delivered/dam was not affected by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups and the live birth index reached 100 % in all test groups.
Thus, the administration of the test item did not adversely affect reproduction and delivery data of the F0 generation parental females.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOAEL for reproductive performance and fertility
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOAEL for general systemic toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
None of the F1 pups showed any clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index as indicator for pup mortality between days 0-4 p.p. was 99 - 100 % for all test groups including the controls. Thus, there were no substance-related differences concerning pup viability and mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights/pup body weight gains did not show any statistically significant differences between the substance-treated groups and the concurrent control group. Furthermore, the number of “runts" (i.e. stunted pups) did not show any differences between the groups for which a substance-induced origin could be assumed.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination did not reveal any differences between the controls and the substance-treated groups.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Pup number and status at delivery
The mean number of delivered F1 pups/dam and the percentage of liveborn pups were not affected by the administration of the test substance.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 4 p.p. did not show any substantial differences between controls and treated groups; all observable differences are regarded to be spontaneous in nature.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOAEL for developmental toxicity for the F1 progeny

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion