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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:
From 8 APR 1997 to 5 MAY 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: approximately 8 - 10 weeks
- Housing: in fully air-conditioned rooms in makrolon cages (type III) on soft wood granulate
- Diet: Ssniff R- (V1324), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least five days under study conditions (guaranteed by practically identical breeding conditions)
- Animal identification: ear tags and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.0
- Humidity (%): 35-62
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 APR 1997 (= day 1) To: 5 MAY 1997

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
, deionised
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test compound was prepared daily, immediately before dosing. Each concentration was prepared from a separate weighed quantity of the test compound. Formulations were stirred continuously in the animal room during dosing. Homogeneity and stability of the test compound in the vehicle were assessed prior to the start of the study.
Dose volume: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
In the laboratory's own breeding facility, virgin female animals in the pre-oestrus or oestrus phase were mated overnight with sexually mature males in the ratio 1 male : 1 female and were caged individually after detection of sperm in vaginal smears. 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:
from day 7 to 16 of pregnancy
Frequency of treatment:
once daily
Duration of test:
animals were sacrificed on day 21 of pregnancy and necropsied
No. of animals per sex per dose:
23 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the available toxicological data of the test compound, the dose levels of 0, 250, 500 and 1000 mg/kg body weight per day were selected for the present study (i.e. 0, 160, 320, and 640 mg surrogate substance/kg bw/day). Testing of dose levels > 1000 mg/kg bw is not necessary with regard to the present testing guideline (OECD TG 414, cf. Limit Test).
- route of exposure selection rationale: The oral route is considered to be a potential exposure route in man.
- species selection rationale: The rat has proved to be a suitable species for developmental toxicity (teratogenicity) testing with many chemical substances and is the species of choice (rodent) according to international testing guidelines.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined before the start of the study and were shown to be in good general health condition.
The behaviour and general health condition of the animals were observed several times daily (on weekends and public holidays once daily).
- Cage side observations checked were included in the study report.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal was recorded between days 1-4, 4-7, 7-10, 10-14, 14-17, 17-19 and 19-21 of pregnancy
- mean daily diet consumption calculated as g food/100 g body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: thoracic and abdominal contents (emphasis on uterus); placentae were weighed/measured and examined for gross external abnormalities
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
-Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); number of intra-uterine deaths;
Statistics:
All data were recorded online and compiled by a data processing system (ARTEMIS).
The statistical evaluation is based an 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. In the univariate analysis, two-sided questions (body weight of dams, relative food consumption, crown-rump length, foetal weight and placental weight) 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 foetuses, 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.
The t-tests and the test statistics of Wilks are based an common variance estimations for all study groups. For the Wilcoxon test the exact distribution of the meaned ranks was calculated.
In the case of the daily food consumption of the dams, the mean consumption per 100 g bw was always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon. In examining the bw of the dams, 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 foetuses 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects: no effects

Details on maternal toxic effects:
There were no deaths during the study and no clinical signs were observed in any of the animals. Body weight gain and food consumption were not affected by the administration of the test compound. No compound-related effects were observed at necropsy of the animals. Dilation of the kidney pelvis was observed more frequently in the high dose group, but this finding occurs spontane¬ously in the rat strain used. Gravid uterus weights were comparable in all groups. One female from the low dose group, three females from the intermediate dose group and two females from the high dose group did not become pregnant. Statistical evaluation did not reveal an increase in the incidence of the numbers of early and late conceptuses undergoing resorption (designated 'early intrauterine deaths' in the printouts) and dead foetuses (designated 'late intrauterine deaths' in the printouts). Most of the resorptions were early conceptuses undergoing resorption.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
640 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
640 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
One female of the intermediate dose group had one dead foetus. There was no dose-dependency, and statistical evaluation did not reveal differences between the groups. Crown-rump lengths, litter size, number of live foetuses, foetal and placental weights remained unaffected by the administration of the test compound. Sex ratio of the foetuses was not altered by the administration of the test compound.
For details on external, skeletal and visceral examinations see fiel below.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
640 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: no adverse toxic effects at highest dose group

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

External, skeletal and viscerall examination

Major defects:

One markedly stunted foetus occurred in the low dose group. Bilateral dysplasia and partial aplasia of the fibula were observed in one foetus from the intermediate dose group, which also exhibited shortened femur and shortened and bent tibia. Another foetus from this dose group was edematous and showed complete fusion of lung lobes. As there was no dose­dependency, a compound-related effect is unlikely.

Minor defects:

The foetuses scheduled for skeletal examination showed blood in the abdominal cavity (1/133, 0/137, 0/117, 0/131) and distension of the kidney pelvis (1/133, 1/137, 6/117, 4/131) at autopsy, the latter finding being statistically significant in the intermediate dose group. All incidences were within the historical range of the rat strain used, and there was no dose-dependency. Therefore, these findings are considered not to be treatment-related.

Minor skeletal defects consisted of splitting of bone an interparietal bone (0/133, 0/137, 0/117,1/131), fragmented thoracic vertebral centres (3/133, 0/137, 1/117, 0/131), fused or longitudinally displaced sternebrae (6/133, 1/137, 0/117, 0/131), wavy and/or thickened ribs (19/133,18/137, 6/117, 17/131) and costad bent scapulae (1/133, 4/137, 0/117, 0/131). In these cases, statistical evaluation did not reveal differences between the groups, and - except bent scapulae in the low dose group, which showed no dose-dependency - the values were within the historical range of the rat strain used. Therefore, a compound-related effect is not evident.

Examination of body cross-sections revealed a statistically significantly increased incidence of distended kidney pelvis in the intermediate dose group based an absolute numbers (0/122, 3/123, 4/106, 4/120) and in the intermediate and high dose group based an the number of affected litters (0/23, 3/22, 4/20, 4/21). The incidence was within the historical range of the rat strain used, and there was no dose-dependency. Therefore, a compound-related effect is not evident. Blood in the thoracic cavity was observed in one control foetus and two foetuses from the high dose group. The latter incidence was above the historical range of the rat strain used. However, only two foetuses were affected, and statistical evalution did not reveal significant differences. Therefore, a compound-related effect is not evident. Other minor defects observed at body cross-section consisted of blood in the abdominal cavity (3/122, 4/123,1/106, 4/120), haematoma in the liver (0/122, 0/123, 1/106, 1/120), blood in the vicinity of the kidney (0/122, 0/123, 1/106, 0/120) and distended ureter (1/122, 2/123, 2/106, 0/120). In all cases, there was no dose-dependency or the values were within the historical range of the rat strain used. Furthermore, statistical evaluation did not reveal differences between the groups.Therefore, these findings are considered not to be treatment-related.

Variations:

Statistical evaluation revealed an increased incidence of an extra rib at the 7th cervical vertebra (0/133, 5/137, 2/117, 2/131) in the low dose group. This incidence was above the historical range of the rat strain used. However, there was no dose-dependency, and the incidences of the higher dose groups were within the historical range of the rat strain used. Therefore, a compound- related effect is unlikely. One foetus of the intermediate dose group showed anlage of 7 sternebrae, and an extra rib at the 1st lumbar vertebra was observed in all groups(32/133, 24/137, 19/117, 31/131). In these cases, statistical evaluation did not reveal differences between the groups, and the incidences were within the historical range of the rat strain used. Therefore, a compound-related effect can be ruled out.

Retardations:

Statistical evaluation revealed increased incidences of non-ossified metacarpale 5 based on the number of affected litters (2/23, 6/22, 7/20, 5/21). Absolute incidences (4/133, 8/137, 7/117, 10/131) were not statistically significant compared to the control group. All values were within the historical range of the rat strain used. Therefore, a compound-related effect is not

evident. Further retardations consisted of slight- or non-ossification of individual skull bones(69/133, 58/137, 46/117, 69/131), not visible incisors (0/133, 2/137, 0/117, 0/131), weakly ossified cervical vertebral arch (1/133, 0/137, 0/117, 0/131), weakly ossified lumbar vertebral arch (2/133, 0/137, 0/117, 0/131), non ossified sacral vertebral arch or centres (0/133, 2/137, 1/117, 0/131), ossification of less than two caudal vertebral centres (35/133, 42/137, 25/117,36/131), non or weakly ossified sternebrae (27/133, 14/137, 11/117, 17/131), weakly ossified ribs (1/133, 0/137, 1/117, 0/131), weakly or non ossified metacarpale 2 and/or 4 (0/133, 3/137, 0/117, 1/131), non ossified phalanx III of the 1st to 5th toe of the forepaw (0/133, 2/137, 0/117,0/131), non or weakly ossified ischium/pubis (0/133, 1/137, 0/117, 2/131), non ossified meta­tarsale 2, 3 and 4 (0/133, 1/137, 0/117, 0/131), non ossified metatarsale 5 (0/133, 2/137,1/117, 0/131) and non ossified phalanx III of the 1st to 5th toe of the hindpaw (5/133, 4/137,3/117, 7/131). In all these cases, statistical evaluation did not reveal differences between the groups. A compound-related effect is not evident.

Morpholooical findinos in dead foetuses:

Autopsy of the dead foetus could not be performed due to the small size. All bones were non or weakly ossified.


Applicant's summary and conclusion

Conclusions:
Oral administration of the test item (DMDHEU, a surrogate substance to submission substance) to the rat at doses of 250, 500 or 1000 mg/kg body weight (corresponds to 160, 320, or 640 mg surrogate substance/kg bw/day ) from day 7 - 16 of pregnancy did not cause any maternal toxicity or embryotoxicity. With regard to the present study the No Observed Adverse Effect Level (NOAEL) is 640 mg/kg/day.
Executive summary:

Groups of 23 mated female Wistar rats received the test item (DMDHEU, a surrogate substance to submission substance)
by oral gavage once daily at the dose levels of 0, 250, 500 or 1000 mg/kg body weight (corresponding to 160, 320 or 640 mg surrogate substance/kg bw/day) from day 7 to 16 of pregnancy (day 0 = day of mating; day 1 = day of sperm detection) and were sacrificed an day 21 of pregnancy.
There were no deaths during the study and no clinical signs were observed in any of the animals. Body weights and food consumption were not affected by the administration of the test compound. No compound-related effects were observed at necropsy of the animals.
Gravid uterus weights, crown-rump lengths, litter size, sex ratios, foetal and placental weights remained unaffected by the administration of the test compound. There was no increase in the number of early or late conceptuses undergoing resorption. Morphological examination of the foetuses did not reveal any compound-related effect.
With regard to the present study the No Observed Adverse Effect Level (NOAEL) is 640 mg/kg bw/day.