Climbazole

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-05-08 to 2001-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 414 with GLP regulations.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

no data

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

Test animals

Species:

rabbit

Strain:

Chinchilla

Details on test animals or test system and environmental conditions:

Acclimatization: 7 days minimum (prior to pairing) under test conditions with an evaluation of the health status;
Number of animals: 96 mated females, 24 per group;
Age at pairing: 17 to 23 weeks;
Body weights (day 0 post coitum): 2510-4297 g;
Conditions: Animals were housed under standard laboratory conditions: air-conditioned with 10-15 air changes per hour; temperature range 20 ± 3°C and relative humidity range 30-70%), 12 h artificial fluorescent light / 12 h dark with background music played at a centrally defined low volume for at least 8 h during the light period. Accommodation individually in stainless steel cages
Diet: Pelleted standard Kliba 3418 rabbit maintenance diet was available ad libitum
Water: Tap water, provided in water bowls, was available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

water

Remarks:

Bi-distilled water with 4% CMC.

Details on exposure:

Mated female were assigned to the four groups containing each 24 rabbits. The dose levels selected are 0, 15, 30, and 60 mg/kg body weight/ day (based from dose range-finding study in rabbit) during the gestation from day 6 through 27 post coitum.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test item/vehicle mixtures were determined from samples drawn during the first and last week of the treatment period using HPLC technique.

Details on mating procedure:

After acclimatization, females were placed in cages with sexually mature males (1:1) until copulation had been observed. After mating, the females were removed and caged individually. The day of mating was designated day 0 post coitum. Mated females were assigned to the four groups using a randomization procedure based on body weight adjusted so that a similar number of rabbits was allocated to each group on each day of mating and ensuring an acceptable distribution of males to which the females were mated.

Duration of treatment / exposure:

day 6 to 27 of pregnancy

Frequency of treatment:

once daily

Duration of test:

not applicable

No. of animals per sex per dose:

24 female pregnant rabbits per dose level

Control animals:

yes

Details on study design:

not applicable

Examinations

Maternal examinations:

Bahaviour and appearance; mortality; food consumption; body weight-increase were recorded

Ovaries and uterine content:

no data

Fetal examinations:

Caesarean and investigation of the foetuses:
The caesarean was carried out on the female rabbits after injecting sodium pentobarbital (1 mL/kg body weight) anaesthetic on the 28th day of pregnancy.

Post mortem examination, including grass macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea in each ovary, were performed and the data recorded. When considered appropriate, samples of macroscopic changes in the dams were fixed in neutral phosphate-buffered 4% formaldehyde solution for possible macroscopic examination. The uteri (and contents) of all females with live fetuses were weighed at necropsy to enable the calculation of the corrected body weight gain. Additionally, placental weights were determined.

If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites. The number and distribution of implantations in uterine horns, classified as empty implantation sites, embryonic resorptions, fetal resorptions, dead fetuses or live fetuses were recorded. Fetuses were removed from the uterus, killed by subcutaneous injection of sodium pentobarbital (0.1 mL per fetus), and weighed individually, examined for grass external abnormalities and prepared for examination as follows:

1) The fetuses were dissected; the organs examined and any abnormal findings were recorded. The sex of each fetus was noted.
2) The thoracic organs were fixed in neutral phosphate buffered 4% formaldehyde solution (without separation) and examined for malformations of the heart (septum defects) and major blood vessels. The lungs were also examined for abnormal findings.
3) After the skin had been removed, the cranium was examined for the degree of ossification.
4) From half of the fetuses the heads were separated from the trunks and fixed in a solution of trichloroacetic acid and formaldehyde. They were serially sectioned and examined (evaluation of the internal structures of the heads, including the eyes, brain, nasal passages and tongue). Descriptions of any abnormal findings were recorded.
5) From all fetuses the skin with the exception of over the paws and the dorsal cervical fat pads was removed and discarded. The trunks of the fetuses without the heads and the fetuses with heads were then processed through solutions of ethanol, glacial acetic with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. Fetuses with external abnormalities were photographed.

Statistics:

Mean body weight gain (%), mean corrected body weight gain (corrected for uterus weight) and mean daily food consumption were calculated by a computer program based on online recorded data.
Calculations for evaluation of the reproduction data were performed by a computer program based on online recorded data. The following data were calculated: Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, sex ratios of fetuses and fetal body weights.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean fetal weights were calculated from the individual weights both on a per group and on a per litter basis.
To test the statistical significance, the following procedures were used:
1) Means and standard deviations of various data were calculated and included in the report.
2) If the variables could be assumed to follow a normal distribution, the Dunnett many-one test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
3) The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
4) Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Indices:

no data

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: 30 mg/kg body weight

Details on maternal toxic effects:
General tolerability:
At 60 mg/kg/day, one female was found dead on day 27 post coitum and one female was killed in extremis on day 25 post coitum. At 30 mg/kg/day, one female was found dead on day 28 post coitum. No mortalities were noted at 15 mg/kg/day or in vehicle control. One female was killed after abortion at 30 and 15 mg/kg/day and in vehicle control, each. These abortions were noted on post coital days 26, 28 and 26, respectively. At 60 mg/kg/day, four females were noted with local alopecia (affecting cervical regions, abdomen or legs), which was in one case combined with bitten-off claws at the left hind leg. Alopecia was noted from post coital days 13, 12, 23 and 10, respectively, until necropsy. From one female, which was killed in extremis on that day, bloody vaginal discharge was noted on day 25 post coitum. At 30 mg/kg/day, local alopecia on the left foreleg and the cervical region was noted for one female from post coital day 21 until necropsy.

Food consumption and body weights:
At 60 mg/kg/day, food consumption of dams with live fetuses at caesarean section was markedly reduced during the first four recording intervals after the start of treatment (i.e. post coital days 6-18). From post coital day 18 until caesarean section food consumption was similar to vehicle controls. At 30 mg/kg/day, food consumption was clearly reduced following start of treatment until day 18 post coitum although to a lesser extent than at 60 mg/kg/day. Food consumption from day 18 post coitum until caesarean section was similar to vehicle control. Food consumption of at 15 mg/kg/day was unaffected by treatment with the test item. Body weight gain of dams with live fetuses at Caesarean section was statistically significantly decreased from post coital days 7 to 23 at 60 mg/kg/day and from days 7 to 22 at 30 mg/kg/day when compared to vehicle controls. As for food consumption body weight gain was similar to that of vehicle controls towards the end of the treatment period. Body weight gain at 15 mg/kg/day was similar to vehicle controls during the entire study.

Reproduction data:
At 60 mg/kg/day, a test item-related increase in the incidence of total post-implantation loss (i.e. implantation sites only at caesarean section) was noted (7 dams versus 1 dam in vehicle control). When calculated for all females surviving until scheduled necropsy (including females with total post-implantations loss), the mean number of fetuses per dam was statistically significantly decreased at 60 mg/kg/day (5.6 versus 8.6 in vehicle control). Also at 30 mg/kg/day the number of fetuses per dam was decreased (7.5) but to a lesser extent. When calculated only for dams with live fetuses at caesarean section (excluding females with total post-implantation loss), there was a slight increase in post-implantation loss at 60 and 30 mg/kg/day (9.9 and 9.7% of implantation sites versus 6.9% in vehicle control).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: clinical signs, reduced food consumption, reduced body weight gain, mortality

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Body weights and sex ratios
No test item-related effects on mean fetal body weights or sex ratios were noted.

External and fresh visceral examination
No fetal abnormalities that were considered to be test item-related were noted.

Examination of fixed fetal heads (including brains) and fixed fetal thoracic organs
No fetal abnormalities that were considered to be test item-related were noted.

Skeletal and cartilage examination
No fetal abnormalities that were considered to be test item-related were noted.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of performance of mated females

Group Dose (mg/kg)	1 (0)	2 (15)	3 (30)	4 (60)
Number of mated females	24	24	24	24
Number of pregnant females	23	23	23	23
Non pregnant	1	1	1	1
Implantation sites only	1	0	1	7
Killed after abortion	1	1	1	0
Killed in extremis or died spontaneously	0	0	1	2
Number of females with live fetuses at termination	21	22	20	14

 

Applicant's summary and conclusion

Conclusions:

In a GLP, OECD 414 prenatal toxicity study in rabbits treatment with the test item caused mortalities and clinical symptoms, reduced food consumption and decreased body weight gain from 30 mg/kg body weight/day onwards. Based on these results, the NOEL (no observed effect level) for maternal organisms was considered to be 15 mg/kg body weight/day.
Treatment with the test item up to and including 60 mg/kg body weight/day caused no effects on any fetal data recorded. Based on these results, the NOEL for fetal organisms was considered to be 60 mg/kg body weight/day.

Executive summary:

In order to detect effects on embryonic and fetal development in pregnant rabbits, test item (HR 00/600306) was administered orally (gavage) once daily, from day 6 through to day 27 post coitum at dose levels of 15, 30 or 60 mg/kg body weight/day. The dose levels were selected based from previous dose range-finding study in rabbits. There were 24 mated female rabbits in each treatment group.

Treatment with the test item caused mortalities and clinical symptoms, reduced food consumption, decreased body weight gain, and altered reproductive data e.g. pre- and post-implantation loss, etc from 30 mg/kg body weight/day onwards. Based on these results, the NOEL (no observed effect level) for maternal organisms was considered to be 15 mg/kg body weight/day.

Treatment with the test item up to and including 60 mg/kg body weight/day caused no effects on any fetal data recorded e.g. body weight, sex ratio, external, skeletal or visceral abnormalities, etc. Based on these results, the NOEL for fetal organisms was considered to be 60 mg/kg body weight/day.