Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13. Apr. - 30. Jul. 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guidelines for the Testing of Chemicals Section 4: Health effects (ministry of environmental protection of People's Republic of China
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adapted 2001
Deviations:
yes
Remarks:
dosing started on Day 6 instead Day 0, only 12 instead of 20 animals/dose were used, no detailed clinical signs description, no food consumption evaluated, not all fetuses observed for soft tissue or skeletal malformations, no data on individual animals
GLP compliance:
not specified
Remarks:
study was performed in China and it is not clear whether it was performed according to GLP
Limit test:
no

Test material

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

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jingling Seed Rabbit Farm
- Age at study initiation: 6 month
- Weight at study initiation: day 6 of gestation: 3.23±0.58 kg
- Fasting period before study: no
- Housing: not specified
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 20
- Humidity (%): 55 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with corn oil. Animals were gavaged based on 1 mL/kg bw/day.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: artificial insemination; the day when the rabbit was artificially fertilized was set as Day 0
- Proof of pregnancy: rabbits were palpated every day to make sure that they were pregnant
Duration of treatment / exposure:
Day 6 to Day 18 of gestation
Frequency of treatment:
daily
Duration of test:
up to Day 29 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
25.3 mg/kg bw/day (actual dose received)
Dose / conc.:
126.4 mg/kg bw/day (actual dose received)
Dose / conc.:
632 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to the results of an acute oral toxicity test of female rabbits (LD50 = 3160 mg/kg bw), three dose groups of 25.3, 126.4 and 632.0 mg/kg bw/day were used along with a vehicle control (0 mg/kg bw/day).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: checked for general health condition (no further details given)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily during dosing until Day 18, after that on Day 21, 24, 27 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes (macroscopic examination)
- Sacrifice on gestation day # 29
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 resorptions: Yes, but not differentiated between early or late resorptions
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No data
Statistics:
SPSS10.0 was used to perform statistics. All ratios were analyzed by Chi-square test. Study measurments were analyzed by ANOVA or nonparametric statistics. Fetus body length, tail length and body weight were compared by t-test. The data of fetuses was analyzed in the unit of litter.
Indices:
None
Historical control data:
Not reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At Day 3 of dosing one rabbit in the high dose group showed cachexia (body becoming thin) and died on Day 9 of dosing.
No abnormalities were detected in the other dosing groups.
These results were described in the summary only. No individual datawas reported.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal in the high dose group died on the day 9 of dosing.
This result was described in the summary only. No individual data was reported.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The dead rabbit in the high dose group did not show any abnormalities in macroscopic examination.
No abnormalities were detected in the other groups.
These results were described in the summary only. No individual data was reported.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group in 6 of 11 rabbits all the fetuses died in utero. For 5 of 11 animals in the high dose group all the fetuses were alive at sacrifice of dams. This corresponds to the observation that live fetuses in the high dose group were significantly lower and dead fetuses were significantly higher compared with the control group.
No significant differences were observed in the mid dose and low dose group compared with the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
The number of corpus luteum and blastocyst implantation and the uterus and fetus weight in all treatment groups showed no significant difference when compared with the control group.
The number of absorptions and sexual ratio in the all treatment groups showed no significant changes when compared to the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
126.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group in 6 of 11 rabbits all the fetuses died in utero. For 5 of 11 animals in the high dose group all the fetuses were alive at sacrifice of dams. This corresponds to the observation that live fetuses in the high dose group were significantly lower and dead fetuses were significantly higher compared with the control group.
No significant differences were observed in the mid dose and low dose group compared with the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the low dose group 1 of 119 fetuses had cephalocele and 1 of 119 fetuses umbilical hernia. This was considered to be an incidental occurence since in all other treatment groups no abnormalities were observed and no significant difference compared with the control group was reported.
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
In the low dose group 1 of 44 (2.3%) fetuses had a brain cavity. In the high dose group 2 of 14 (14.3%) fetuses had a cleft palate. The external malformations in the low-, mid- and high dose group had no significant difference when compared with the control group. However, in the high dose group a treatment-related effect cannot be ruled out.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
31

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
126.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: cleft palate
Description (incidence and severity):
2/14 (14.3%): no data if these findings were found in one dam or two dams

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
632 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Pregnant rabbits of implantation (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

No. of corpus luteum

No. of blastocyst implantation

Uterus & fetus weight (g)

0

12

9.2±3.0

7.9±3.1

512.9±179.4

25.3

12

11.0±1.3

10.5±1.3

596.8±115.6

126.4

12

9.3±2.0

8.3±2.3

500.6±139.5

632

11

9.3±2.5

7.8±2.6

380.6±202.0

Table 2: Fetus (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

live fetus

dead fetus

no of absorptions

sexual ratio (male:female)

0

12

7.6±2.7

0.3±0.6

0.1±0.3

100:113

25.3

12

9.9±1.4

0.4±0.7

0.2±0.6

100:143

126.4

12

7.9±2.2

0.4±0.9

0.0±0.0

100:94

632

11

3.9±5.0**

3.9±3.8**

0.0±0.0

100:96

** p<0.01 compared to controls

Table 3: Growth and development (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

Fetus body length (cm)

Fetus tail length (cm)

Fetus bodyweight (g)

0

12

10.504±0.542

1.535±0.070

47.47±7.65

25.3

12

10.236±0.551

1.476±0.083

42.44±7.36

126.4

12

10.525±0.570

1.526±0.130

44.52±7.10

632

5

10.231±0.597

1.445±0.114

41.54±6.97

Table 4: Skeletal malformations in the fetuses

Type                                                                                                         Dose (mg/kg bw/day)

                                                                                   0                     25.3                     126.4           632

                                                                                 (n=60)              (n=75)             (n=62)             (n=29)

Rib malformation                                  9 (15.0%)       9 (12.0%)       16 (25.8%)       3 (10.3%)

Sternum ossification rudimentary or not ossified       12 (20.0%)       12 (16.0%)       5 (8.1%)       1 (3.4%)

Note: number in front of () was number of malformed fetuses, number insed () was ratio of abnormality.

Table 5:  Splanchnic malformations in the fetuses

Type                                                        Dose (mg/kg bw/day)

                                         0              25.3        126.4               632

                                         (n=31)       (n=44)       (n=33)        (n=14)

Cleft tongue, cleft palate       0              0                   0              2 (14.3%)

Brain cavity                          0              1 (2.3%)       0              0

Note: number in front of () was number of malformed fetuses, number insed () was ratio of abnormality.

Applicant's summary and conclusion

Conclusions:
In a tetratogenicity study with rabbits performed using a protocol similar to OECD 414 one of 12 females died in the high dose group. In the high dose group 6/11 rabbits showed dead fetuses whereas 5/11 had alive fetuses. Skeletal malformations had no significant difference in the treated groups compared with the control group. In the low dose group 2.3% fetuses had a brain cavity. In the high dose group 14.3% fetuses had a cleft palate. The external malformations in the low-, mid- and high dose group had no significant difference when compared with the control group. However, in the high dose group a treatment-related effect cannot be ruled out. Thus, a NOAEL of 126.4 mg/kg bw/day was established for maternal toxicity and developmental toxicity.