Climbazole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

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

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.

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

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: 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)"

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.

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

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

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

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

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.

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

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

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)

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

36 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The effects of crinipan (compound No. 34054) 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 prior to mating,and 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/day were administered orally by gavage. The control group received the vehicle, 3% carboxymethylcellulose (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, day of parturition, and 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. It was found that treatment with the test compound induced slight behavioural changes in parental males at the dose level of 100 mg/kg/day. 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/day 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/day 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, NOAELs for the reproductive effects of the test compound of 100 and 36 mg/kg/day can be derived for males and females, respectively. Concerning parental general toxicity, NOAELs of 36 and 7.2 mg/kg/day could be derived for males and females, respectively.

Short description of key information:
Daily oral administration of crinipan, to male rats at dose levels of 7.2, 36, and 100 mg/kg/d starting 86 day prior to mating and during mating did not adversely affect the fertility and reproductive performance of the males. In contrast, 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 estrous 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, 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 general toxicity, NOAELs of 36 and 7.2 mg/kg/d can be derived for males and females, respectively.

According to REACh Annex VIII, section 8.7.1., column 2, the screening test for reproductive and developmental toxicity in a single species is not required if a prenatal developmental toxicity study is available.
Since results of an ICH segment I-III as well as from three developmental toxicity studies in rats and rabbits (an embryotoxicity/teratogenicity study in rats, a perinatal and postnatal development study in rats, and a prenatal developmental toxicity study in rabbits) are available for crinipan (see IUCLID section 7.8.1 and 7.8.2), the screening test was waived.

Justification for selection of Effect on fertility via oral route:
The study was conducted according to a method which is generally similar to the recommendations given in ICH guideline S5(R2), but did not follow the principles of GLP. However, the study is relatively well documented and appears to be scientifically sound.

Effects on developmental toxicity

Description of key information

Based on the results from prenatal developmental toxicity studies with climbazole in rats and rabbits, 15 and 30 mg/kg/day could be considered as NOAEL for maternal and prenatal developmental toxicity, respectively.
Investigations for embryotoxic and teratogenic effects of climbazole after oral administration to the rats (key study):
Climbazole caused neither embryotoxic nor teratogenic effects in doses up to 30 mg/kg/day. However, 100 mg/kg/day resorption rate was increased indication for embryotoxic effects. This effect can be evaluated as secondary resulting from the strong toxic effects on the mother in this dose group. From this study, NOAELs for maternal toxicity and for prenatal developmental toxicity could be considered each as 30 mg/kg/day.
Perinatal and postnatal study in rats (supportive study):
Maternal toxic effects were observed at dose level of 36 mg/kg/day and/or higher doses. The embryo-feto-toxic effects observed at these dose levels were possibly secondary to maternal toxic effects. However, at 7.2 mg/kg/day neither maternal nor fetal toxic effects were significantly noticeable. Hence 7.2 and 36 mg/kg/day could be considered as NOAEL for maternal and pre- or post-natal developmental toxicity, respectively.
Dose range finding study to a prenatal developmental toxicity study in rabbits:
Based on the results of this dose range-finding study, dosages of 15, 30, and 60 mg/kg/day are proposed for the main study in this animal species.
Prenatal developmental toxicity study in the rabbit:
Treatment with climbazole caused mortalities and clinical symptoms, reduced food consumption and decreased body weight gain from 30 mg/kg/day onwards. Treatment with the test item up to and including 60 mg/kg/day caused no effects on any fetal data recorded. Based on these results, the NOAEL for maternal and fetal organisms were considered to be 15 and 60 mg/kg/day, respectively.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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.

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

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

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).

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

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

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

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.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The following prenatal developmental toxicity studies were conducted with climbazole in rats and rabbits. Based on the results from these studies, 15 and 30 mg/kg/day could be considered as NOAEL for maternal and prenatal developmental toxicity, respectively.

 

Investigations for embryotoxic and teratogenic effects of climbazole after oral administration to the rats (key study):

BAY e 6975 (climbazole) was investigated for maternal tolerability and embryotoxic and/or teratogenic effects in rats. Each of 4 groups of 25 female rats (FB30-strain) was treated daily with one of the following oral doses of BAY e 6975 from the 6th to the 15th day of pregnancy. Control = 0 mg/kg (vehicle material only); Group I = 10 mg/kg BAY e 6975; Group II = 30 mg/kg BAY e 6975; and Group III = 100 mg/kg BAY e 6975. The substance was held in a 0.5% aqueous tylose suspension; the standard volume applied was 10 mL/kg bodyweight. The following observations were made:

l) Doses of up to 30 mg/kg were tolerated by the mother animals without symptoms. Following administration of 100 mg/kg, the majority of the mother animals were mutilated, with deep lacerations on their extremities or stomach. Furthermore, weight-gain during the treatment period was significantly lower in the middle and upper dose groups than in the control group. Therefore, 30 or 100 mg/kg should be regarded as toxic to them, whose self-mutilation must have caused by a specific mechanism.

2) Doses of up to 30 mg/kg had neither embryotoxic nor specific teratogenic effects. In the higher dose group, the number of resorptions was significantly increased, and as a result the number of foetuses was significantly reduced.

It was observed that BAY e 6975 in all doses up to 10 mg/kg was tolerated by the mother animals without causing damage. 30 mg/kg and 100 mg/kg can be considered as toxic to the mother. The embryotoxic effects, in the sense of an increased rate of resorption observed after administration of the 100 mg/kg dose, can be considered as secondary caused by the clear toxicity of this dose group to the mother. From this study, each NOAELs for maternal toxicity and for prenatal developmental toxicity could be considered as 30 mg/kg/day.

 

Perinatal and postnatal study in rats (supportive study):

A perinatal and postnatal study was conducted with climbazole (test item no. 34054) in 80 mated Charles River CD rats. Dosage levels of 7.2, 36 and 100 mg/kg/day were administered orally by gavage beginning on day 15 of gestation and continuing throughout gestation and lactation. A control group received the vehicle, 3% carboxymethyl cellulose (CMC) on a comparable regimen at 10 mL/kg/day. During the treatment period the females were observed for clinical signs of toxicity, mortality and changes in appearance, behavior and body weight. The day of parturition, numbers of live pups and stillbirths were recorded. All pups were weighted at birth, day 4 and 21, examined for abnormalities and counted daily for survival. A slight increase in hair loss of the extremities and red nasal discharge were seen in the rats in the 7.2 mg/kg/day group. Increased activity, red ocular and nasal discharge, hair loss and self mutilation of the extremities and abdomen were seen in many rats in the 36 mg/kg/day group. The severity and frequency of these observations decreased as treatment continued. However, the severity and frequency of these observations were increased in the 100 mg/kg/day group. For the first five days of treatment increased activity was noted for the remainder of the day after dosing. However, prior to the next day's dosing, this increase returned to a level comparable to the controls. One rat in the 36 mg/kg/day group, and 16/20 rats in the 100 mg/kg/day group died by lactation day 18. The deaths of all rats on this study were attributable to the test material. Accentuated lobulation of the liver was the most consistent finding at necropsy. Yellowish liver and congested lungs were noted in many of the animals that died. Mean maternal body weights throughout gestation and lactation were similar in the rats from the both control and the 7.2 mg/kg/day groups. The body weights on gestation day 29 and throughout lactation were slightly less for the dams in the 36 mg/kg/day group when compared to the controls. There were only 2 surviving pregnant rats on gestation day 20 in the 100 mg/kg/day group and severe body weight loss was observed in these animals. There were no biologically meaningful or statistically significant differences in the mean number of stillborn pups mean number of live pups at birth, pup survival indices through weaning, mean pup weight at birth or mean pup weight gains through weaning for the pups of the 7.2 or 36 mg/kg/day groups when compared to the controls. The mean number of live pups at birth in the 36 mg/kg/day group was slightly less when compared to the control group; there was no meaningful associated increase in stillbirths. The dam in the 100 mg/kg/day group that delivered on gestation day 24 produced only stillborn pups. The other dam in this group had 4 stillborn pups out of 13 total delivered and all pups died by lactation day 2. These 9 pups at birth were considerably lighter than the pups in the other groups. One stillborn pup in the control group was observed to have an open eye lid. This pup was placed in Bouin's fixative and found to have a diaphragmatic hernia and absent kidney, ureters, pancreas, spleen and urinary bladder. The small intestine was malformed and not connected to the stomach or colon. In conclusion: Maternal toxic effects were observed ≥ 36 mg/kg/day. The embryo-feto-toxic effects observed at these dose levels were possibly secondary to maternal toxic effects. However, at 7.2 mg/kg/day dose levels neither maternal or nor fetal toxic effects were significantly noticeable, and hence 7.2 mg/kg/day could be considered as NOAEL for maternal toxicity while 36 mg/kg/day could be considered as NOAEL for pre- and post-natal developmental toxicity, under the present experimental conditions.

 

Dose range finding study to a prenatal developmental toxicity study in rabbits:

In this dose range-finding study for a prenatal developmental toxicity study in female Chinchilla rabbits, animals treated with oral doses of 60 and 90 mg/kg/dayclimbazole (crinipan) showed test item-related clinical symptoms as well as a marked transient reduction in food consumption and body weight. An initial reduction in food consumption was also noted in females treated with 30 mg/kg/day. The total post-implantation losses in two females at 90 mg/kg/day and increased pre-implantation loss in the single dam with live foetuses at caesarean section were considered to be the consequence of a high maternal toxicity. Treatment with the test item up to and including 60 mg/kg/day did not cause any effects on the foetal data recorded. Abnormal skeletal and cartilage findings were observed in 2 foetuses from the single litter at 90 mg/kg/day. As these malformations occurred in association with marked maternal toxicity, it cannot be ruled out that these foetal abnormalities were due to the maternal toxicity rather than to direct embryo-foetal toxicity. Based on the results of this dose range-finding study, dosages of 15, 30, and 60 mg/kg/day are proposed for the main study in this animal species.

 

Prenatal developmental toxicity study in the rabbit:

The study was conducted as per OECD 414 guidelines under GLP conditions. In order to detect effects on embryonic and fetal development in pregnant rabbits climbazole (HR 00/600306) was administered orally by gavage once daily, from day 6 through to day 27 post coitum at dose levels of 15, 30 or 60 mg/kg/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 from 30 mg/kg body weight/day onwards. Based on these results, the NOAEL for maternal organisms was considered to be 15 mg/kg/day. Treatment with the test item up to 60 mg/kg/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 NOAEL for fetal organisms was considered to be 60 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Tihs was a well conducted study to OECD 414 guidelines and under GLP.

Justification for classification or non-classification

The substance does not have to be classified regarding toxicity to reproduction according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC), because of the following:

 

In developmental toxicity studies in rats and rabbits oral treatment with the substance caused no toxicologically relevant and substance treatment related effects on any fetal data recorded e.g. body weight, sex ratio, external, skeletal or visceral abnormalities. At high doses, at which pronounced maternal toxicity occurred, an increased number of resorptions were observed. These were considered to be secondary effects from the toxicity on the pregnant animals.

 

Rabbits were treated on gestational days 6-27 at dose levels of 15, 30 or 60 mg/kg bw/day (Becker, 2001). Treatment with the test item caused mortalities in the pregnant rabbits (1/24 and 2/24 at 30 and 60 mg/kg/day, respectively) and clinical symptoms, reduced food consumption (severely reduced down to approx.. 50% of that of controls during gd 6-18 at 60 mg/kg/day), prominently decreased body weight gain at 30 and 60 mg/kg/day.

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). No increases in malformations or other effects on fetuses were found.

The resorptions were considered to be a reaction of the maternal organism to the general bad state of the animals, i.e. weight loss at the beginning of pregnancy.

 

Rats were treated orally (gavage) on gestational day 6-15 at dose levels of 10, 30 or 100 mg/kg bw/day (Schlüter, 1981). Treatment with the test item caused no mortality, but 21/25 animals of the high dose group gave the impression of being ill. The weight gain during the treatment period was only 63% that of the control group. The number of resorptions was significantly increased at the high dose group, while the number of malformations was not increased. No teratogenic or embryotoxic effects were observed at 30 and 10 mg/kg/day. The resorptions were considered to be a secondary effect of maternal toxicity.

 

Rats were treated orally (gavage) from gestational day 15 until the end of lactation at dose levels of 7.2, 36 and 100 mg/kg bw/day (Goldenthal, 1979). One rat in the mid dose group, and 16/20 rats of the high dose group died by lactation day 18. Yellowish liver and congested lungs were noted in many of the animals that died. The two surviving high dose females showed severe body weight loss and a high rate of stillbirths. Body weights of the mid dose animals were slightly less than that of controls. The number of stillborn pups was increased in 2/18 pregnant females of the mid dose group. As this occurred in two animals only in this study and was not observed at a similar dose level in the other study in rats (Schlüter, 1981), this effect was not considered to meet the classification threshold. The stillbirths in the high dose group were clearly related to maternal toxicity and thus were considered to be a secondary effect. No increases in malformations or other effects on fetuses were found.

 

 

In a one-generation study in rats, males were treated from 10 weeks prior to mating and females were treated two weeks prior to mating, throughout mating, gestation and lactation periods until sacrifice at dose levels of 7.2, 36, and 100 mg/kg (Goldenthal, 1979).. Increased activity, hair loss and salivation were seen in females at the mid and high doses. High dose females showed signs of red ocular and nasal discharge, self mutilation. One pregnant rat of the mid and high dose groups died. There was no significant effect on body weights in females. High dose females showed an elongated diestrus stage, an increased gestation length. The number of live pups at birth was slightly, but not statistically significantly lower in the mid dose group, while it was markedly reduced at the high dose. The high dose group showed a decreased number of viable and total implantations and an increased number of resorptions. No effect on pup survival or other offspring parameters was noted. The effects on estrous cycle, gestation length and resorptions were considered to be secondary of maternal stress and toxicity by the study authors.

No adverse effects were noted from treatment of lactating rats on offspring parameters.