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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:
17 Oct 1995 - 09 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
yes
Remarks:
treatment started on Day 7 of pregnancy when implantation has already taken place
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1987
Deviations:
yes
Remarks:
treatment started on Day 7 of pregnancy when implantation has already taken place
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1984
Qualifier:
according to
Guideline:
other: JMAFF (Prenatal Developmental Toxicity Study) adopted 1985
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain (as specified by the author): Hoe: WISKf(SPF71) Wistar
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: Approx. 8-10 weeks
- Housing: Individually in Makrolon cages (type III) on soft wood granulate
- Diet: Ssniff R-Z (V1324), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 45-68
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% w/v aqueous methylcellulose
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each concentration, samples were taken towards the start, middle and end of the dosing period (Day 7 to 16). Only the samples from the start were analysed. The archieved concentration, stability and the homogeneous distribution of the test material in the vehicle over 4 hours were acceptable.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: The day of sperm detection was defined as day 1 of gestation, and the day of mating was defined as day 0 of pregnancy. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.
Duration of treatment / exposure:
Day 7-16 of pregnancy
Frequency of treatment:
Daily
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 120 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
23 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a dose-range finding study groups of mated female Wistar rats received the test compound orally at the dose levels of 500 mg/kg bw (2 females) or 1000 mg/kg bw (4 females) daily from day 7 to 16 of pregnancy and were killed at day 21.
One animal at 1000 mg/kg bw/d was found dead on day 17. No clinical signs were observed in the other animals of the 1000 mg/kg body weight group or in the animals treated with 500 mg/kg bw/d. Body weight change, food consumption and litter data did not reveal any compound-related effect. Based on the results of this study and on request of the sponsor, the dose levels of 0, 15, 120 and 1000 mg/kg bw/d were selected. 1000 mg/kg bw/d: limit dose; 15 mg/kg bw/d lowest NOEL (in the rat 90-day study) and was considered likely to be a certain no effect level. The intermediate dose level, 120 mg/kg bw/d, was approximately the log mid-point between the high and low dose levels.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Several times daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 1, 4, 7, 10, 14, 17, 19 and 21 of pregnancy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between Days 1-7, 7-10, 10-14, 14-19 and 19-21 of pregnancy.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes, externally and internally
- Sacrifice on Gestation day 21
- Organs examined: thoracic and abdominal contents, Uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes
- Other: The fetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
- Other: crown-rump length was recorded
Statistics:

All data were recorded on-line and compiled by a data processing system (ARTEMIS). The statistical evaluation is based on the assumption of a monotone dose-response relationship.
Statistical comparisons of the low dose groups with the simultaneous control group were only carried out if significant effects were detectable in the high dose group. Two-sided questions were generally tested as follows: a two-sided comparison with the high dose group was followed by a one-sided test for the low-dose group. In case of the caesarean section data of the multivariate statistics were first of all calculated and used in selecting relevant dose groups. For the individual parameters, sequential comparisons with the high dose group and sequential tests at the 5% level for the low dose were then conducted.

For the Wilcoxon test the exact distribution of the meaned ranks was calculated.

Food consumption: mean consumption/100 g bw was always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon.
Body weight gain: the change in weight was determined in comparison to the initial weight. The univariate evaluation was carried out using t-tests.

The caesarean section data of the fetuses were used to calculate litter mean values. Multivariate evaluation was carried out using the test statistics of Wilks. In the univariate analysis,t-tests were used.

The number of corpora lutea, implantation sites and live foetuses, and quotas of dead embryonic primordia undergoing resorption in the animals were likewise analysed using one-sided Wilcoxon tests.

The findings obtained at autopsy, during visceral examination and skeletal examination of the fetuses were evaluated separately for the foetuses and for the litters by exact Fisher test at significance levels of 5% and 1% and compared with actual and previous control group data.
Indices:
% of pre- and post-implantation loss, % of implantation
Historical control data:
Historical data used for comparison are not given in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: At 1000 mg/kg bw/day: Mortality, decreased body weight gain and food consumption

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY:
One high dose group animal died on Day 21 of pregnancy. The mortality was considered to be treatment-related since 1000 mg/kg bw/day was about half of the acute oral LD50 value for rats and one mortality occurred on Day 17 in the range-finding study.
No clinical signs were observed in any of the animals.

BODY WEIGHT: (see Table "Group mean body weights")
At 1000 mg/kg bw/day body weight gain was slightly but statistically significantly reduced from Day 10 on. Body weight changes before beginning of treatment (Days 4 and 7) were not considered to be treatment-related.

FOOD CONSUMPTION: (see Table "Group mean food consumption")
At 1000 mg/kg bw/day food consumption was slightly but statistically significantly decreased between Days 7-19 of pregnancy. Decreases in food consumption before beginning of treatment (Days 1 and 7) were not considered to be treatment-related.

POST-MORTEM EXAMINATIONS:
At 1000 mg/kg bw/day one female exhibited whitish fluid in the renal pelvis at necropsy. The animal found dead on Day 21 showed autolysis. The uterus contained 12 well-developed foetuses.

CESARIAN SECTION DATA: (see Table "Developmental toxic effects")
With the exception of one female from the control group and four females dosed at 120 mg/kg bw/day, respectively, all animals became pregnant.
Gravid uterus weights were comparable in all groups.
Number of early and late conceptuses undergoing resorption based on per cent of implantations ('early intrauterine deaths') was slightly but significantly increased in the animals dosed at 1000 mg/kg bw/day. As a consequence, the number of live fetuses based on per cent of implantations was slightly, but significantly decreased. Incidence of dead fetuses ('late intrauterine deaths') was not increased.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
= NOEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Remarks:
= NOEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Slightly increased incidence of embryonic deaths (see maternal data).

Details on embryotoxic / teratogenic effects:
One dead fetus occurred in the control group, and two dead fetuses were observed in the intermediate dose group.
Fetal body weights, crown-rump lengths, litter size, placental weights and sex ratios remained unaffected by test substance administration. In all external, skeletal and visceral examinations no compound related effects were observed, no statistical differences of the findings between the groups combined with dose-dependency were found and the incidences were within the historical range of the strain used.
Variations: No compound-related effects observed
Retardations: No compound-related effects observed
Dead fetuses: External examination did not reveal any abnormalities.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Group mean body weights (g)

Day

Dose level (mg/kg bw/day)

0

15

120

1000

1

221.0

223.6

220.7

220.8

4

239.9

239.8

240.0

236.7*

7

252.9

249.8

250.4

247.4*

10

264.2

260.0

262.7

254.1*

14

280.6

275.7

280.5

269.2*

17

299.4

296.3

303.5

286.5*

19

322.3

318.8

328.0

306.5*

21

348.8

344.5

354.4

328.7*

* Significantly less than control

 

Table 2: Group mean food consumption (g/100 g bw)

Day

Dose level (mg/kg bw/day)

0

15

120

1000

1-7

8.3

7.9

8.0

7.7*

7-10

7.8

7.5

7.7

6.9*

10-14

7.8

7.5

7.7

7.2*

14-19

8.0

7.6

8.0

7.5*

19-21

7.4

7.1

7.2

7.3

* Significantly less than control

 

Table 3: Developmental toxic effects (Group means)

 

Dose level (mg/kg bw/day)

0

15

120

1000

Early intrauterine deaths

 

      Total

14

14

13

30

      % of implantations

5.03

4.69

5.60

12.00#

Late intrauterine deaths

 

      Total

1

0

2

0

      % of implantations

0.35

0.00

1.16

0.00

Number of foetuses

 

      Total

256

275

233

230

      % of implantations

94.62

95.31

93.24

88.0*

#Significantly higher than control

* Significantly less than control

In conclusion daily administration to rats of Hoe 122006 at the regulatory limit dose level of 1000 mg/kg bw/day from Day 7-16 of pregnancy caused maternal toxicity (as evidenced by mortality, decreased body weight gain and decreased food consumption) and a slightly increased incidence of embryonic deaths. However, there was no evidence of any effect on foetal morphology and no teratogenic effect.

Neither maternal nor embryofoetal toxicity were observed after repeated administration of 120 mg/kg bw/day. The 'No Observed Effect Level' (NOEL) for maternal toxicity and embryofetal effects was 120 mg/kg body weight per day.

Applicant's summary and conclusion