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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 1993 to 05 July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Supplier: BASF.

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, Federal Republic of Germany
- Strain: Chbb:HM (outbred strain)
- Age at study initiation: At Day 0, the does were between 22 and 37 weeks old (sexually mature).- Weight at study initiation: At Day 0 mean body weight was approximately 2641 g.
- Housing: Singly in type 12.2395.C stainless steel wire mesh cages (Draht Bremer GmbH, Marktheidenfeld, Federal Republic of Germany) with floor area of about 3000 cm². The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: pelleted Kliba maintenance diet type 23-341-4 for rabbits, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland, ad libitum throughout the study from the day of supply to the day of necropsy.
- Water: Water of tap water quality was provided in water bottles available ad libitum throughout the study from the day of supply to the day of necropsy.- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: Central air conditioning guaranteed a range of temperature of 20 - 24 °C. There were no deviations from these limits.
- Humidity: Relative humidity of 30 - 70%. There were no deviations from these limits.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours of light from 06:00 to 18:00 hours and 12 hours of darkness from 18:00 to 06:00 hours).

IN-LIFE DATES
- From: 22 March 1993
- To: 01 June 1993

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(doubly distilled)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Each day the test material solutions were freshly prepared shortly before the test material was administered. For the preparation of the solutions, an appropriate amount of test material was weighed in a volumetric flask, subsequently topped up with doubly distilled water and intensively shaken.

VEHICLE
- Concentration in vehicle: 0, 500, 2500 and 5000 mg/100 mL for the 0, 50, 250 and 500 mg/kg dose levels, respectively.
- Amount of vehicle: The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 post insemination (p.i.)).
- Dose volume: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSES
- All analyses were carried out at the analytical department of the testing facility.

ANALYSES OF THE TEST MATERIAL
- Analytical determinations of the purity and the homogeneity of the test material itself were carried out before the beginning of the study by GC. The stability of the test material over the study period has been proven by reanalysis.The homogeneity of the test material was proven by visual inspection.

ANALYSES OF THE SOLUTIONS OF TEST MATERIAL
- Analytical verifications of the stability of the test material 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. The test material solutions were analysed by GC. As the test material preparations were true solutions, the homogeneity did not need to be proven analytically.
Details on mating procedure:
- Impregnation procedure: Artificial insemination after an acclimatisation period of at least 5 days, whereby 0.2 mL of a synthetic hormone (Receptal®) 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.The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 p.i.
Duration of treatment / exposure:
Day 7 to Day 19 post insemination (p.i.)
Frequency of treatment:
Once daily during the period of major organogenesis
Duration of test:
29 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control receiving distilled water.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The selection of doses for the present examination was based on the results of a preceding range-finding study in Himalayan rabbits in which the test material was administered as an aqueous solution to 4 - 5 pregnant female rabbits/group by stomach tube in doses of 50, 150 and 450 mg/kg body weight on day 7 through day 19 post insemination. A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water). In this preliminary study, the test material caused slight signs of maternal toxicity at 450 mg/kg body weight, but induced no material-related effects on the does of the low and the intermediate dose groups (50 and 150 mg/kg body weight/day). There were no test material-related effects on the gestational parameters in any of the groups and the foetuses showed no malformations. Taking this into consideration, the doses were chosen for the full-scale prenatal toxicity study in Himalayan rabbits.- Rationale for animal assignment: Animals were assigned to the different test groups according to a randomisation plan and on the basis of their body weights.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice daily on working days or once daily (Saturday, Sunday or on public holidays) on days 0 - 29 p.i.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0- 29 p.i.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results.Corrected body weight gain (net maternal body weight change) was calculated after terminal sacrifice (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.).

FOOD CONSUMPTION: Yes
-The consumption of food was determined daily during the entire study period.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 p.i.- Organs examined: The surviving dams were sacrificed in randomised order by intravenous injection of a pentobarbital and the foetuses were removed from the uterus.Dams that died intercurrently as well as the contents of the uterus from these animals were examined, if possible, in the same way as at terminal sacrifice (with the exception of uterus weight). After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
- Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes; only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy.
- Number of late resorptions: Yes; embryonic or foetal tissue in addition to placental tissue visible.
- Other: Dead foetuses; hypoxemic foetuses which did not breathe spontaneously after the uterus had been opened.
Fetal examinations:
- External examinations: Yes: Each foetus was weighed and examined macroscopically for any external findings. The viability of the foetuses and the condition of the placentae, umbilical cords, foetal membranes and fluids were examined. Individual placental weights were recorded.
- Soft tissue examinations: Yes: After the foetuses had been sacrificed by CO2, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the foetuses was determined by internal examination of the gonads.
- Skeletal examinations: Yes: After fixation in ethyl alcohol the skeletons, with the possible exception of the skulls, were stained according to a modified method of Dawson. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.
- Head examinations: Yes: After skinning, all foetuses (if required without heads) were fixed in ethyl alcohol. After fixation for approx. 1 - 5 days, the foetuses were removed from the fixative for a short while and a cross section of the heads from all intact foetuses was made in the parietal bone area using a scalpel. The two halves of the heads were carefully bent to allow a thorough examination of the brain. Subsequently, the foetuses were placed back into the fixative for further fixation.If heads of foetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus or cleft palate), the heads of these foetuses were severed from the trunk, fixed in Bouin's solution and later processed and assessed according to Wilson's method.About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.
Statistics:
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 various parameters.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 foetal 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 foetuses with malformations, variations, retardations and/or unclassified observations in each litter.For the parameter “food consumption”, the “mean of means” was calculated. The “mean of means” values allow a rough estimation of the total food consumption during the different time intervals (pre-treatment, treatment and post-treatment period); they are not exactly precise values because the size of the intervals taken for calculation differs. For the “mean of means” values no statistical analysis was performed.
Indices:
Calculation of conception rate and pre- and post-implantation losses were carried out.Conception rate (%) = (number of pregnant animals / number of fertilised animals) x 100Pre-implantation loss (%) = ((number of corpora lutea – number of implantations) / number of corpora lutea) x 100Post-implantation loss (%) = ((number of implantations – number of live foetuses) / number of implantations) x 100
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high dose animals showed unsteady gait about 2 - 4 hours after the daily gavaging during the first 3 days of the treatment period. On the following days (days 10 and 11 p.i.) unsteady gait occurred only in some of the high dose females. This finding has to be related to the test material administration. No defecation was recorded for high dose female No. 55 on days 19 and 20 p.i.; this doe consumed only minimal amounts of food the days before.
Mortality:
no mortality observed
Description (incidence):
One mis-gavaged doe of the control group (No. 10) was found dead on day 21 p.i.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant differences between the controls and the test material-treated does concerning mean body weights. If the mean body weight gain over the whole treatment period (Days 7 - 19 p.i.) is calculated, the high dose females reached only about 25% of the body weight gain of the control group. This corresponds to the reduced food intake of the high dose dams and is therefore assessed as a test material-induced effect. During the post-treatment period (Days 19 - 29 p.i.) the increased food consumption of the 500 mg/kg dams resulted in a statistically significantly increased weight gain.The weight gains of the dams of test groups 1 and 2 (50 or 250 mg/kg body weight/day) did not show any statistically significant differences in comparison to the controls. In the absence of test material-related effects on the food consumption of these dams, all differences in respect to body weight change are considered to be spontaneous in nature.The results of the corrected body weight gain (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.) do not show any differences of biological relevance between the groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the 500 mg/kg dams was statistically significantly reduced during the first days of the treatment period. The consumption of approximately 15% less of the diet in the high dose group in comparison to the controls during the treatment period is assessed as a clear substance-related effect.Food consumption of dams of test groups 1 and 2 (50 and 250 mg/kg body weight/day) was uninfluenced by the test substance administration.
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Description (incidence and severity):
There were no substantial differences concerning the uterus weights between the controls and test groups 1, 2 or 3 (50, 250 and 500 mg/kg body weight/day). All these values lie within the range of biological variation. No other organ weights were reported.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy none of the does exposed to the test substance showed any substance-induced lesions. Only some spontaneous necropsy findings were recorded for single animals of all dose groups, including the controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

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:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Details on maternal toxic effects: Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (day 29 p.i.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.In this study, the following females were partially or totally excluded from the above mentioned calculations:
- Test group 0 (Control):
- Female No. 11790 (6) - not pregnant
- Female No. 11886 (10) - died intercurrently

- Test group 3 (500 mg/kg body weight/day):
- Female No. 11300 (49) - not pregnant

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
One control and one intermediate dose foetus each showed external malformations. The control foetus showed kleft palate and kinky tail; the intermediate dose foetus showed cheiloschisis. These external malformations occur sporadically in control foetuses. Furthermore, no dose-response relationship was given. The findings in this study are considered to be spontaneous in nature.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Malformations of the foetal skeletons were noted for 2.7% of control foetuses (occurring in 15% of the litters) and 2.5% of the foetuses of the intermediate dose group (occurring in 13% of the litters). Skeletal malformations were related to the vertebral column (cervical and lumbar vertebrae fused and/or of irregular shape), deformed scapulae, severely fused sternebrae (bony plate) and the hindlimbs (bent with involvement of bones). None of the low and the high dose groupss showed skeletal malformations. Skeletal variations were observed, which included splitting of skull bones, epactal bone between nasal and frontal bones, accessory 13th ribs, shortened or absent 12th ribs, rudimentary cervical ribs, accessory lumbar vertebra and sternebrae of irregular shape, fused or accessory sternebra or bipartite sternebra. All of the described skeletal variations appeared without any relation to dosing and/or without statistically significant differences between the control group and the substance-treated groups.Skeletal retardations (incomplete or missing ossification of skull bones, vertebral column, sternebrae and talus) occurred in all groups, including the controls, at a comparable frequency. All differences between the groups concerning foetal skeletal retardations are withouth biological relevance. This includes the statistically significantly higher litter incidence of incomplete ossified or smaller sternebrae at 50 and 500 mg/kg body weight/day. For this finding, which appears in the historical control data at even higher litter incidence, no clear dose-response relationship was found.
Visceral malformations:
no effects observed
Description (incidence and severity):
The examination of the organs of the foetuses revealed several types of soft tissue malformations in foetuses of the control and the intermediate dose group. Malformations consisted of hydrocephaly, septal defect, hypoplastic thymus, malformations of the great vessels, agenesia of the gallbladder and hypoplastic kidneys. No soft tissue malformations occurred in the 50 or 500 mg/kg body weight/day dose groups. Because no relation to dosing is given and because most soft tissue malformations were also present at low incidence in the historical control data, the recorded visceral findings were considered to be spontaneous in nature.
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
There were no dose-related foetal abnormalities.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Mean Maternal Food Consumption During Gestation (g/animal/day)

Days

Parameter

Test Group 0

Control

Test Group 1

50 mg/kg bw/day

Test Group 2

250 mg/kg bw/day

Test Group 3

500 mg/kg bw/day

0 to 1

Mean

118.4D

124.9

122.2

118.9

1 to 2

Mean

119.2D

131.8

124.9

125.1

2 to 3

Mean

121.8D

133.3

126.9

122.7

3 to 4

Mean

123.7D

135.1

129.0

126.3

4 to 5

Mean

127.2D

135.9

132.6

127.0

5 to 6

Mean

120.7D

126.3

128.3

122.2

6 to 7

Mean

121.4D

131.6

121.4

120.9

7 to 8

Mean

112.3D

131.6*

105.2

60.1**

8 to 9

Mean

110.8D

129.2*

105.9

75.0**

9 to 10

Mean

110.5D

122.8

111.6

89.1**

10 to 11

Mean

111.2D

119.4

109.6

97.6

11 to 12

Mean

109.5D

118.4

108.4

95.8

12 to 13

Mean

107.4D

117.6

106.9

94.2

13 to 14

Mean

103.0D

112.4

105.6

92.5

14 to 15

Mean

96.4D

115.4

96.1

86.1

15 to 16

Mean

98.3D

113.5

94.8

89.7

16 to 17

Mean

93.0D

120.2

98.8

91.2

17 to 18

Mean

102.9D

124.5

105.4

102.0

18 to 19

Mean

98.8D

122.2

110.0

97.5

19 to 20

Mean

93.2D

126.9*

101.0

101.3

20 to 21

Mean

98.9D

118.5

113.1

112.0

21 to 22

Mean

99.4D

118.9

106.5

112.0

22 to 23

Mean

100.8D

117.5

104.2

118.0

23 to 24

Mean

102.9D

117.4

105.6

119.3

24 to 25

Mean

113.4D

119.5

112.0

121.8

25 to 26

Mean

117.0D

120.2

114.2

122.3

26 to 27

Mean

118.9D

123.6

115.7

127.5

27 to 28

Mean

113.7D

111.2

113.8

121.1

28 to 29

Mean

118.7D

124.4

107.9

130.6

D = Dunnett-test (two-sided)

*p0.05

**p0.01

Table 2: Mean Maternal Body Weight Change During Gestation (g)

Days

Parameter

Test Group 0

Control

Test Group 1

50 mg/kg bw/day

Test Group 2

250 mg/kg bw/day

Test Group 3

500 mg/kg bw/day

0 to 7

Mean

63.4D

68.2

68.7

56.5

7 to 19

Mean

70.9D

99.6

33.1

18.0

19 to 29

Mean

132.8D

190.3

147.5

198.9*

0 to 29

Mean

268.9D

358.1

249.2

273.4

D = Dunnett-test (two-sided)

*p0.05

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects and the NOAEL for teratogenicity/developmental toxicity was 500 mg/kg/day as no test material-related effects were observed.
Executive summary:

A study was conducted to investigate the potential developmental toxicity of cyclohexanone to rabbits via the oral route. The study was conducted in accordance with the standardised guidelines OECD 414 and EU Method B.31 under GLP conditions.

The test material was administered as an aqueous solution to 15 artificially inseminated female Himalayan rabbits/group (Chbb:HM (outbred strain)) by oral gavage at doses of 0, 50, 250 and 500 mg/kg body weight on Day 7 through to Day 19 post-insemination (p.i.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (doubly distilled water).

The state of health of the animals was checked each day. Food consumption and body weights of the animals were recorded regularly throughout the study period. On Day 29 p.i., all surviving females were sacrificed and assessed by gross pathology. The foetuses were dissected from the uterus, sexed, weighed and investigated for any external, soft tissue and skeletal findings.

At 50 and 250 mg/kg bw/day there were no test material-related effects on does, gestational parameters or foetuses. At 500 mg/kg bw/day statistically significantly reduced food consumption (about 15% less than the controls) during the treatment period was observed. Body weight loss at the beginning of the treatment period was also observed; weight gain during the treatment period was only 25% of the control animals. All the animals in this group were also observed to have an unsteady gait 2 – 4 hours after daily gavaging during the first treatment days.

Thus, the test material caused some overt signs of maternal toxicity at 500 mg/kg body weight/day, but was not toxic to the does at 50 and 250 mg/kg body weight/day.

There were no test material-related adverse effects on the gestational parameters and on the foetuses up to and including the highest dose level (500 mg/kg body weight/day); at all dose levels no indications for test material-induced teratogenic effects were observed.

Under the conditions of this study, the NOAEL for maternal toxicity was determined to be 250 mg/kg/day based on systemic effects. The NOAEL for teratogenicity and developmental toxicity was 500 mg/kg/day as no test material-related effects were observed. The read-across from cyclohexanone to dyclohexanol is considered to be valid for the characterisation of the developmental/teratogenicity toxicity endpoints. The results of this study with cyclohexanone are considered to be valid for classification and labelling purposes.