Climbazole

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-04-27 to 1978-09-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was conducted according to a method which is generally similar to the recommendations given in ICH guideline S5(R2), but it did not follow the principles of GLP. However, the study is relatively well documented and appears to be scientifically sound.

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:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH segment I-III guideline S5(R2)

Principles of method if other than guideline:

The effects of the test item, compound No. 34054 (crinipan) on fertility and general reproductive performance were evaluated in a study in Charles River CD rats. This study consisted of two phases: In phase I, both male and female rats were treated orally with the test item at dose levels of 7.2, 36, and 100 mg/kg/d from prior to mating throughout the mating, gestation, and lactation periods. In phase II, only the males were treated, while the females remained untreated. The control group received the vehicle, 3% carboxymethyl cellulose (CMC) on a comparable regimen.
The rats were observed for signs of toxicity, mortality as well as for changes in appearance, behaviour, and body weights. Fertility, gestation length, the day of parturition and the number of live and dead pups were recorded. All pups were weighed at designated intervals during lactation, examined for abnormalities, and counted daily for survival. Uterine examinations were performed on approximately one half of the females in each group on gestation day 13. Additionally, the phase I females were vaginally smeared to determine the effects of the test compound on the estrous cycle.

GLP compliance:

no

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:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc. (Portage, Michigan, USA)
- Age at study initiation: Males: approx. 70 d; females: approx. 110 d
- Weight at study initiation: Males: 322-388 g; females: 200-277 g
- Housing: Prior to mating, the rats were individually housed in hanging wire mesh cages. During mating, the rats were housed in units of 1 male and 2 females in plastic cages on ground corn cob bedding. Following mating, the females were housed individually in plastic cages on ground corn cob bedding.
- Diet: Purina Laboratory Chow, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 10 d at minimum

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 h darkness / 12 h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

3% in distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Amount of vehicle: 10 mL/kg/d

The test item was pulverized with a mortar and a pestle prior to mixing. Using a tissue homogenizer, the test material was suspended daily in 3% aqueous (distilled water) CMC.

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 15 d
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear, referred to as day 0 of pregnancy
- After 10 d of unsuccessful pairing, the unmated female was house with another male of the same group for a further 5 d.
- Further matings after 2 unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in plastic cages

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

In phase I, males and females were treated daily starting 10 weeks or 2 weeks prior to mating, respectively. Both males and females received the test item during the whole mating, gestation, and lactation period or until sacrifice (i.e., after weannig for females, no time point specified for males).
In phase II, males were treated daily starting 86 d prior to mating, females were left untreated. The males received the test item throughout the study period (no time point of termination specified)

Frequency of treatment:

Animals were treated daily.

Details on study schedule:

not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
7.2, 36, and 100 mg/kg/d
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 20 females were used per dose level.

Control animals:

yes, concurrent vehicle

Details on study design:

Due to signs of toxicity observed in females at the dose level of 100 mg/kg/d during the regular (phase I) study, this study was extended by a second phase (phase II) to evaluate the effects of the test compound on the health and reproductive performance of male rats.
In this phase II part of the study, 30 male rats were treated daily with the test compound using the same dose regimen as for phase I of the study, but this time, treatment was started 86 days before mating, and the treatment was continued for the whole study period (no time point of termination specified). The treated males were mated with 60 female rats (divided into three groups of 20 females per group), which were left untreated.

Positive control:

No positive control was used.

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Parameter checked: Mortality, signs of toxicity, and changes in appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly until sacrifice; Females: Weekly until evidence of copulation, and on gesation days 0, 7, 13, and 20 and on lactation days 0, 7, 14, and 21. Females selected for 13-day uterine examinations were weighed on gestation days 0, 7, and 13.

OTHER:
Male and female fertility and length of the gestation period were calculated.

Oestrous cyclicity (parental animals):

Duration of the estrous cycle was recorded for the phase I treated females.

Sperm parameters (parental animals):

No sperm parameters were recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
After birth, the pups were counted, sexed, weighed, and observed at designated intervals during lactation. At weaning, the pups were examined for external abnormalities and then sacrificed.

Postmortem examinations (parental animals):

SACRIFICE
Approximately half of the female animals from each group (phase I) were sacrificed on gestation day 13 to examine their uterine contents. The remaining females were allowed to deliver.
The females from the phase I and phase II segments which failed to deliver by the 24th day after mating were sacrificed by an overdose of carbon dioxide.
The male rats were sacrificed at the end of phase II, examined for external abnormalities, and discarded.

GROSS NECROPSY
The abdominal and thoracic cavities of the sacrified females were examined, and the uterus and ovaries were evaluated for any condition that could prevent pregnancy.
The females that did deliver were similarly sacrificed and examined. In addition, implantation scars were counted and recorded.
Adults that died during the study were necropsied to determine the cause of death.

13-DAY UTERINE EXAMINATIONS
The pups from both phases of this study were observed daily for signs of toxicity and changes in appearance and behaviour.
Survival of the pups was recorded daily throughout lactation. Individual body weights were recorded on days 0, 4, and 21 of lactation. On lactation day 21, the number of male and female pups in each litter was determined. Pups found dead on lactation day 0 were examined for skeletal abnormalities. All normal appearing pups were sacrificed and discarded.

Postmortem examinations (offspring):

SACRIFICE
- The offspring was sacrificed at weaning.

GROSS NECROPSY
- The offspring was only examined for external abnormalities (at weaning).

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

All statistical analyses compared the treatment groups with the phase I control group with the level of significance at p < 0.05.

Gestation, 4- and 21-d survival indices and the number of post implantation losses/number of implantations per dam were compared and differences were tested for significance using the Mann-Whitney U-test as described by Siegel (1) and Weil (2).

The number of liveborn pups, corpora lutea, and implantation sites were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variance, and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie (3) using Dunnett's multiple comparison tables (4) to judge significance of differences.

The mean live pup weights per litter were compared by analysis of variance (hierarchal classification) and t-test as described by Steel and Torrie (see citation above) using Dunnett's multiple comparison tables (4) to judge significance of differences.

References:
1.Siegel S (1956) Nonparametric statistics for the behavioral sciences. McGraw-Hill, New York, USA
2. Weil CS (1970) Selections of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis, or carcinogenesis. Food Cosmet Toxicol 8:177-82.
3. Steel RGD, Torrie JR (1960) Principles and procedures of statistics. McGraw-Hill. New York, USA
4. Dunnett C (1964) New tabIes for multiple comparisons with a control. Biometrics 20:482-91

Reproductive indices:

The following two reproductive indices were calculated from the results of this study:
Fertility index for males = (number of fertile males / total number of males mated) x 100
Fertility index for females = (number of pregnant females / total number of females mated) x 100

Offspring viability indices:

The following three offspring viability indices were calculated from the results of this study:
Gestation index = (number of live pups at birth / total number of pups born) x 100
Viability index = (number of pups surviving 4 d / total number of live pups at birth) x 100
Lactation index = (number of pups surviving 21 d / total number of pups surviving 4 d) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (parental animals)"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (parental animals)"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (parental animals)"

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (parental animals)"

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (parental animals)"

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Phase II parental animals:
The general behaviour and appearance of the male and female rats in the 7.2 mg/kg/d treatment group and the males in the 36 mg/kg/d group were similar to the control rats. Increased activity, hair loss, and a slight incidence of salivation were observed in male rats of the 100 mg/kg/d group and in females of the 36 and 100 mg/kg/d groups. In addition, red ocular and nasal discharge, a slight incidence of dark yellow urine abdominal stains, and self-mutilation of the extremities and abdomen were observed in females of the 100 mg/kg/d. The severity and frequency of these observations decreased as the treatment progressed, except for the increase in activity of the male rats receiving 100 mg/kg/d. This increase in activity was evident until sacrifice, with the exception of study week 8.
Survival of the rats was 100% for the males at all dose levels and for the females of the control and 7.2 mg/kg/d dose level. At the dose levels of 36 and 100 mg/kg/d, one female each died during delivery.

Phase II parental females:
No overt effects on general behaviour, appearance, or survival were observed.

BODY WEIGHT (PARENTAL ANIMALS)
Phase I parental animals:
Mean body weights of the males at the 7.2 and 36 mg/kg/d treatment levels and of the females at all three treatment levels were similar to the control rats. Mean body weights of the 100 mg/kg/d males were slightly lower than the control males during the treatment (see table 1 of the section "Any other information on results incl. tables").

Phase II parental females:
No effects on body weight were observed.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Phase I parental females:
The estrous cycles of the females in the control group and at the 7.2 and 36 mg/kg/d dose levels were within normal ranges.
At 100 mg/kg/d, 9 of the 20 females were in the diestrus stage of estrous varying from 5-16 d in length.

Phase II parental females:
not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Phase I parental animals:
Gestation lengths were comparable to the control for the females receiving 7.2 and 36 mg/kg/d. At 100 mg/kg/d, two females did not complete delivery until gestation day 24. At completion of delivery, neither female had surviving pups. This resulted in an increased gestation length at this treatment level in comparison to the control group (see table 2 of the section "Any other information on results incl. tables").
Male and female fertility indices of the 7.2 and 36 mg/kg/d groups and the males in the 100 mg/kg/d group were comparable to the control. At 100 mg/kg/d, female treatment level, fertility was reduced when compared to the respective control index (see table 3 of the section "Any other information on results incl. tables").

Phase II parental females:
No effects on reproductive performance were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects found

2

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (offspring)"

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

See section "Details on results (offspring)"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results (offspring)"

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Phase I pups:
No differences were seen in the pups in the treated groups with regard to general behaviour, and appearance when compared to the control litters. Survival of the pups was not affected by treatment with the test item at dose levels of 7.2 and 36 mg/kg/d.
The number of pups born alive/total pups born showed a statistically significant decrease at the 100 mg/kg/d dose level when compared to controls. The number of live pups per litter at birth was similar in the control and the 7.2 mg/kg/d groups. In the 36 and 100 mg/kg/d groups, the number of live pups per litter was lower when compared to controls, with the difference being statistically significant for the 100 mg/kg/d dose level (see table 4 of the section "Any other information on results incl. tables").

Phase II pups:
No overt effects on offspring viability were reported.

CLINICAL SIGNS (OFFSPRING)
Phase I pups:
No effects on general behaviour and appearance were reported.

Phase II pups:
No effects on general behaviour and appearance were reported.

BODY WEIGHT (OFFSPRING)
Phase I pups:
Mean pup body weights were not affected by the treatment. Lactation day 21 body weights were significantly increased in the 100 mg/kg/d group when compared to the controls (see table 5 of the section "Any other information on results incl. tables"). However, this difference was attributed to the significantly fewer number of pups in each litter.

Phase II pups:
The body welghts of the pups in the 7.2 and 36 mg/kg/d groups were significantly decreased at lactation day 4 and 21 when compared to the control pups of phase I (see table 6 of the section "Any other information on results incl. tables"). However, these differences were not considered biologically meaningful due to the lack of a dose-related trend.

13-DAY UTERINE EXAMINATIONS
Phase I:
No meaningful or statistically significant differences in the number of viable and nonviable implantations, total implantations, resorptions, or corpora lutea were observed between the 7.2 and the 36 mg/kg/d treatment groups and the control group. At 100 mg/kg/d, the mean numbers of viable and total implantations were decreased, mean resorptions were increased, and the ratio of implantation sites to corpora lutea was moderately decreased as compared to the respective control values but these differences were not statistically significant (see table 7 of the section "Any other information on results incl. tables").
The ratio of implantation sites to corpora lutea were as follows for the control and the 7.2, 36, and 100 mg/kg/d group, respectively: 90%, 91%, 87%, and 78%. The ratio difference in the 100 mg/kg/d group was primarily attributed to 2 females.

Phase II:
There were no remarkable differences between the 3 untreated groups and the phase I control group with respect to the number of viable and nonviable implantations, total implantations, resorptions, or corpora lutea. In the female group mated with the 7.2 mg/kg/d males, the mean number of corpora lutea was significantly decreased when compared to the phase I control group (see table 8 of the section "Any other information on results incl. tables"). Due to the lack of a dose-related trend, this difference was attributed to random occurrence.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Mean body weights of phase I males (100 mg/kg/d and control group)

Week of study	0 mg/kg/d (control)		100 mg/kg/d
	Mean body weight	Weight ranges	Mean body weight	Weight ranges
0	357	326-388	357	322-380
1	398	372-440	382	348-408
2	428	395-465	410	367-432
3	455	420-500	436	392-465
4	451	412-525	423	375-457
5	469	430-545	453	393-484
6	491	442-575	475	415-505
7	493	440-566	478	415-497
8	517	455-594	494	415-522
9	532	469-608	509	440-538
10	549	484-620	524	445-565
11	541	471-618	513	429-538
12	554	482-627	521	440-550
13	564	482-634	530	440-569
14	572	484-650	537	440-600
15	577	489-648	536	439-592

Table 2: Mean lenght of gestation in phase I animals

Dose level (mg/kg/d)	Mean length of gestation (d)
0	22.0
7.2	22.1
36	22.1
100	22.8

Table 3: Fertility indices of male and female phase I animals

Dose level (mg/kg/d)	Pregnant females / total females mated	Index (%)	Fertile males / total males mated	Index (%)
0	19/20	95	9/10	90
7.2	19/20	95	9/10	90
36	19/20	95	9/10	90
100	15/20	75	8/10	80

Table 4: Viability of phase I pups

Dose level (mg/kg/d)	Live pups at birth / total born	Index (%)	Pups surviving 4 d / live pups at birth	Index (%)	Pups surviving 21 d / live pups retained at 4 d	Index (%)
0	118/119	99	113/118	96	113/113	100
7.2	113/119	95	112/113	99	112/112	100
36	74/80	93	70/74	95	70/70	100
100	50/87	57*	42/50	84	41/42	98

* = significantly lower than control group (p<0.05)

Table 5: Mean body weights of phase I pups on lactation day 21

Dose level (mg/kg/d)	Mean body weight of male pups (g)	Mean body weight of female pups (g)
0	43.1	41.7
7.2	45.1	42.9
36	45.1	42.2
100	47.8	46.3

** = significantly higher than control group (p<0.01)

Table 6: Mean body weights of phase II pups on lactation days 4 and 21

Dose level (mg/kg/d)	Lactation day 4	Lactation day 21
	Mean body weight of male and female pups (g)	Mean body weight of male pups (g)	Mean body weight of female pups (g)
0	10.7	43.1	41.7
7.2	9.9*	38.0**	36.1**
36	9.8*	38.4**	37.4**
100	10.2	41.7	40.4

Pups of the phase I control group were referred to as control

* = significantly lower than control group (p<0.05)

** = significantly lower than control group (p<0.01)

Table 7: Results of the day-13 uterine examinations in phase I animals

Dose level (mg/kg/d)	Implantations		Resorptions		Post implantation loss	Total implantations	Corpora lutea
	Live	Dead	Late	Early
0	13.8	0.0	0.0	1.1	1.1	14.9	16.6
7.2	14.0	0.0	0.0	0.8	0.8	14.8	16.3
36	12.7	0.0	0.0	1.4	1.4	14.1	16.3
100	11.5	0.0	0.0	2.3	2.3	13.8	17.7

Table 8: Results of the day-13 uterine examinations in phase II animals

Dose level (mg/kg/d)	Implantations		Resorptions		Post implantation loss	Total implantations	Corpora lutea
	Live	Dead	Late	Early
0	13.8	0.0	0.0	1.1	1.1	14.9	16.6
7.2	12.8	0.0	0.0	0.4	0.4	13.2	13.8**
36	13.3	0.0	0.0	0.8	0.8	14.1	15.2
100	13.4	0.0	0.0	0.6	0.6	14.0	15.0

Pups of the phase I control group were referred to as control

** = significantly lower than control group (p<0.01)

Applicant's summary and conclusion

Conclusions:

From the results of this study, it was concluded that the daily oral administration of the test item to male rats at dose levels of 7.2, 36, and 100 mg/kg starting 86 d prior to mating and during mating did not adversely affect the fertiliy and reproductive performance of the males.
In contrast, the daily oral administration of the test item to female rats at 100 mg/kg/d, starting 2 weeks prior to mating and throughout the whole mating, gestation and lactation period elicited severely toxic effects in the parental females, especially in the beginning of the experimental period, resulting in disruptions of the estrus cycle and an increased number of stillbirths. No overt effects on reproduction were observed when the female rats were treated with dose levels of 7.2 and 36 mg/kg/d. Thus, no observable adverse effect levels (NOAELs) for the reproductive effects of the test compound of 100 and 36 mg/kg/d can be derived for males and females, respectively.
Concerning parental toxicity, NOAELs of 36 mg/kg/d and 7.2 mg/kg/d can be derived for males and females, respectively.

Executive summary:

The effects of the test item, compound No. 34054 (crinipan), on fertility and general reproductive performance were evaluated in a study in Charles River CD rats.

This study consisted of two phases: In phase I, both male and female rats were treated with the test item from prior to mating throughout the mating, gestation, and lactation period. In phase II, only the males were treated, while the females remained untreated. Dosage levels of 7.2, 36, and 100 mg/kg/d were administered orally by gavage. The control group received the vehicle, 3% CMC, on a comparable regimen.

The rats were observed for signs of toxicity and mortality as well as for changes in appearance, behaviour, and body weights. Fertility, gestation length, the day of parturition, and the numbers of live and dead pups were recorded. All pups were weighed at designated intervals during lactation, examined for abnormalities, and counted daily for survival. Uterine examinations were performed in approximately one half of the females in each group on gestation day 13.

Additionally, the phase I females were vaginally smeared to determine the effects of the test compound on the estrous cycle.

In this study, it was found that treatment with the test compound induced slight behavioural changes in parental males at the dose level of 100 mg/kg/d. However, the compound did not adversely affect the reproductive performance of the male test animals, as supported by the findings made in phase II of this study (i.e., with females left untreated).

In contrast, parental females treated with the test item at a dose level of 100 mg/kg/d exhibited severe toxicity during the initial stages of treatment which caused disruptions of the estrous cycle and an increased number of stillbirths.

At the 36 mg/kg/treatment level, the female rats exhibited slight-to-moderate toxicity during the initial stages of treatment with no observable effects on estrous cycle or stillbirths. Thus, no observable adverse effect levels (NOAELs) for the reproductive effects of the test compound of 100 and 36 mg/kg/d can be derived for males and females, respectively.

Concerning parental toxicity, NOAELs of 36 mg/kg/d and 7.2 mg/kg/d can be derived for males and females, respectively.