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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
testing lab.
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
purity: > 99 .9 %

Test animals

Details on test animals or test system and environmental conditions:
Sexually mature, virgin Himalayan rabbits, which were free from clinical signs of disease, were used for the investigations. This strain was selected since extensive experience is available on Himalayan rabbits and this strain has been proved to be sensitive to substances with a teratogenic potential. Unique identification of the rabbits by ear tattoo had already been carried out by the breeder.
During the acclimatization and the study periods, the rabbits were housed singly. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
The animals were accommodated in fully airconditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%. The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6 .00 hours). The food used was pelleted Kliba maintenance diet, which was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was drinking water of tap water quality from water bottles.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 7 to day 19 p.i.) always at approx. the same time of day (in the morning). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.). The control group was dosed with the vehicle only (doubly distilled water). Each day the test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of test substance was weighed in a volumetric flask, subsequently topped up with doubly distilled water and intensively shaken.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in doubly distilled water for a period of 4 hours at room temperature were carried out in a range-finding maternal toxicity study in Himalayan rabbits.
Details on mating procedure:
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. This implied that 0.2 ml of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
During the acclimatization period the animals were assigned to the different test groups according to a randomization plan and on the basis of their body weights. The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.). At the beginning of the study (day 0) the does were between 22 and 37 weeks old. Their mean body weight was approx. 2,641 g.
On day 29 p.i., all surviving females were sacrificed in randomized order and examined macroscopically. The fetuses were removed from the uterus and further investigated with different methods.
Duration of treatment / exposure:
day 7 through day 19 post insemination (p.i.)
Frequency of treatment:
Duration of test:
until day 29 p.i.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
The selection of doses for the present examination was based on the results of a preceding range-finding study in Himalayan rabbits. From the results of this preliminary study it could be seen, that cyclohexanone caused slight signs of maternal toxicity at 450 mg/kg body weight, but induced no substance-related effects on the does of the low and the intermediate dose groups (50 and 150 mg/kg body weight/day). There were no substance-related effects on the gestational parameters in any of the groups and the fetuses showed no malformations.


Maternal examinations:
Food consumption, body weight data, corrected body weight gain (net maternal body weight change), clinical symptoms, mortality
Ovaries and uterine content:
Weight of uterus before it was opened, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations, conception rate, preimplantation loss, postimplantation loss.
Fetal examinations:
Examination of the fetuses after dissection from the uterus, soft tissue examination of the fetuses, skeletal examination of the fetuses, malformations, variations, retardations, unclassified observations.
The DUNNETT-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight.
FISHER's Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter.
If the results of these tests were significant, labels (* for p < 0.05, ** for p < 0.01) were printed in summary tables.
Historical control data:

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg body weight/day: unsteady gait in all animals about 2 - 4 hours after the daily gavaging during the first treatment days
mortality observed, non-treatment-related
Description (incidence):
One control animal died due to gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg - body weight/day: body weight loss at the beginning of the treatment period; weight gain during the treatment period was only 25% of the control animals. This corresponds to the reduced food intake of the high dose dams.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg - body weight/day: statistically significantly reduced food consumption (about 15% less than the controls) during the treatment period
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

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:
no effects observed
Changes in pregnancy duration:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
Effect level:
250 mg/kg bw/day
Basis for effect level:
body weight and weight gain
clinical signs

Maternal abnormalities

no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
One control and one intermediate dose fetus each showed external malformations. For the control fetus cleft palate and kinky tail were observed, while the
intermediate dose fetus showed cheiloschisis. Only one type of external variation (pseudoankylosis) was found and it was seen in 4 out of 75 control fetuses (in 2 out of 13 litters), in one out of 105 fetuses from the 50 mg/kg group (in one out of 15 litters) and in one out of 81 fetuses of the 250 mg/kg group (in one out of 15 litters).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations of the fetal skeletons were noted for 2 out of 75 control fetuses (= 2 .7%) (in 2 out of 13 litters (= 15%)) and in 2 out of 81 intermediate dose fetuses (= 2 .5%) (in 2 out of 15 litters (= 13%)). These malformations were related to the vertebral column (cervical/lumbar vertebrae fused and/or of irregular shape), the scapula(e) (deformed), the sternum (sternebrae severely fused (bony plate)) and the hindlimbs (bent with involvement of the bones). None of the fetuses of the low and the high dose group showed skeletal malformations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The examination of the organs of the fetuses revealed several types of soft tissue malformations in fetuses of the control and the intermediate dose group . In the control group one out of 75 examined fetuses 1.3%) from one out of 13 litters (= 7 .7%) showed hydrocephaly . In the 250 mg/kg group in total 3 out
of 81 examined fetuses (= 3 .7%) from 3 out of 15 litters (= 20%) were malformed . All of them had a septal defect ; one of these fetuses (No . 7 of doe No .
38) had in addition a hypoplastic thymus and another one (No . 3 of doe No . 43) showed additionally malformations of the great vessels, agenesia of gallbladder and hypoplastic kidneys (associated with other kidney findings) (see also Tab . B 034 , Volume II). No soft tissue malformations occurred at 50 or 500 mg/kg body weight/day.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
No substance-induced signs of embryo-/fetotoxicity, especially no teratogenic effects were observed in the present full-scale prenatal toxicity study up to
and including 500 mg/kg body weight/day.

Effect levels (fetuses)

Dose descriptor:
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: no teratogenic effects up to and including the high dose

Overall developmental toxicity

Developmental effects observed:

Applicant's summary and conclusion

Under the conditions of this study, the test substance caused some overt signs of maternal toxicity at 500 mg/kg body weight/day (reduced food consumption, impaired body weight gain and unsteady gait), but was not toxic to the does at 50 and 250 mg/kg body weight/day.
There occurred no substance-related adverse effects on the gestational parameters and on the fetuses up to and including the highest dose level (500 mg/kg body weight/day); at all dose levels no indications for substance-induced teratogenic effects were observed.