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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
in its original version (1981)
Deviations:
yes
Remarks:
Deviations to current OECD 414 (2001): - Treatment was terminated after gestation day 19. - Mid-dose group was inappropriate with only 16 animals and a maternal mortality over 10%. - Light sequence with 14 h light/10 h dark disagreed with recommendations
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
in its original version (1981)
Deviations:
yes
Remarks:
- Light sequence with 14 h light/10 h dark disagreed with recommendations of the guideline. - Relative humidity in the animal facility exceeded on several occasions the recommended range.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Test material: Ziram
- Lot/Batch number: P15/62
- Description: White powder
- Purity: 98%
- Stability: Stable

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ranch Rabbits, Crawley Down, Sussex, UK.
- Age at study initiation: 16-28 weeks
- Weight at study initiation: 3.00 – 4.38 kg

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose
Details on mating procedure:
Each female with one male. To ensure ovulation chorionic gonadotrophin was injected.
Duration of treatment / exposure:
Day 7-19 post mating
Frequency of treatment:
Daily
Duration of test:
Day 28 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
3, 7.5, 15 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
16 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CLINICAL SIGNS
- Daily

MORTALITY
- Twice daily

BODY WEIGHT
- Days 0, 7, 8, 9, 10, 13, 16, 19, 23 and 28 of gestation.

FOOD CONSUMPTION
- Days 0-3, 3-7, 7-10, 10-13, 13-16, 16-19, 19-23, 23-25, 25-28 of gestation

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 28
- Organs examined: Uterus

Ovaries and uterine content:
Examination of uterine content
Examination included:
- Gravid uterus weight
- Number of corpora lutea
- Number and position of of live and death foetuses
- Number of early and late resorptions
- Pregnancy status

Fetal examinations:
- General: Each foetus was individually identified, weighed (to calculate the litter weight), sexed externally (gonadal inspection) and given a gross examination for external malformations, anomalies and variations.
- Soft tissue examinations: All foetuses were evaluated for visceral malformations/ variations. Evaluations were performed on the fresh foetal specimens shortly after removal from the uterus.
- Skeletal examinations: After the visceral inspection, the foetuses were eviscerated and processed for staining of the ossified skeletal structures using the Alizarin Red S staining procedure.
Statistics:
Continuous and semi-continuous responses and some discrete responses:
- Statistical evaluation was made using an analysis of variance technique for normally distributed errors or by non-parametric techniques for non-normally distributed errors.
Analysis of variance established the significance of the variability between all the groups to determine a treatment-related response. The standard deviation obtained from this analysis was used for ‘t’ tests between the control and treatment groups. Where necessary the data were suitably transformed before analysis.
Non-parametric testing was carried out using the Kruskal-Wallis test to determine a treatment-related response. Significant differences between control and treatment groups were determined using the Wilcoxon rank sum test.
All tests were carried out at 1 and 5% significance levels for a two-sided risk.

Discrete responses:
- Statistical analysis was carried out using Fisher’s two-sum randomisation (permutation) test with a Monte Carlo simulation for computation of significance levels. The litter was the experimental unit and a square root transformation was used for weighting the number of incidences and adjusting for different litter sizes.
Each treatment group was tested against the control at 1 and 5% significance for a one-sided risk.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS
- No effects.

MORTALITY
- One high-dose animal died on day 23 of gestation and one mid-dose on day 13. In addition one control animal and one mid-dose animal were killed (day 14 and 15) after showing weight loss and noisy respiration.
No treatment-related abnormalities were observed in these animals.

BODY WEIGHT
- High-dose animals showed body weight loss from day 9 to 13 of gestation, followed by partial recovery.
In mid-dose animals a slight reduction in weight gain was observed from day 8 to 16 of gestation, including a slight weight loss from days 10-13 with compensatory weight gain from day 16 onwards.

FOOD CONSUMPTION
- Food intake of high-dose animals was lower during the treatment period, most marked during day 10-16 of gestation. Mid-dose animals showed also slightly reduced food intake for days 10-16 but without statistically significance.

UTERUS WEIGHTS
- Mean gravid uterus weight was decreased in animals dosed at 15 mg/kg bw/day and reflected the lower litter weight observed in this group.
The mean uterus weight in the mid-dose group was also decreased but this was primarily due to the higher mean pre-implantation loss in this group, discussed under ceasarean data.

CAESAREAN DATA
- Implantation
In mid-dose animals the pre-implantation loss was increased. This was considered to be not treatment-related as implantation would be expected to occur before start of dosing at day 7 of gestation.
- Post-implantation loss
The post-implantation loss was increased in dams dosed at 15 mg/kg bw/day.

OTHER
- Necropsy findings were unaffected by ziram treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
ca. 7.5 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 3 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In the high- and mid-dose group the litter size was decreased. This was considered a result of high post-implantation loss respectively high pre-implantation loss.
Additionally in the high-dose group litter weight, mean foetal weight and crown/rump length were lower than controls.
The observed incidence in major skeletal anomalies was increased but considered to be not treatment-related as these defects are known to occur spontaneously in this strain of rabbit.

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
ca. 7.5 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 7.8.2-B1                    Toxic effects in dams and foetuses

Parameter

0 mg/kg

3 mg/kg

7.5 mg/kg

15 mg/kg

Dose-response

+/–

No. of dams:

16

16

16

16

Mortality

1

1

2

Body weight gain

↓ day 8-16

↓ entire gestation period

+

Food intake

day 7-19 **

Post-implantation loss (%) *

8.6

4.1

7.2

16.9

Foetuses (no.) and litter:

128

140

103

107

Mean litter weight

↓ **

Mean foetal weight

Litter size

Mean crown/rump length (mm)

98.7

98.5

99.2

96.5

*   Post implantation loss was higher in top dose than controls but equals the highest value seen in control groups during the six most recent studies.The increase is partially due to a high incidence of late intrauterine deaths in one animal.

** Statistically significantly different from control

Applicant's summary and conclusion