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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:
weight of evidence
Study period:
07 Aug 1995 to 14 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
1981
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
1984
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
1987
Qualifier:
according to
Guideline:
other: Japanese MAFF 59 Nohsan No. 4200
Version / remarks:
1985
Qualifier:
according to
Guideline:
other: European Communities Commission Directive 87/302/EEC, OJ No. L133/24
Version / remarks:
1988
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rabbit
Strain:
other: Russian Chbb:HM
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: at least 3 months
- Weight at study initiation: 2424-3668 g
- Fasting period before study: not specified
- Housing: Individually in battery cages with steel slatted floors
- Diet: Pelleted certified standard feed (Nafag No. 814), ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 7 days prior to insemination

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 2°C
- Humidity: 50 ± 20%
- Air changes: Approximately 16/hour
- Photoperiod: 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %(w/w) aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered in an aqueous solution of carboxymethylcellulose (0.5% w/w). Suspensions at the appropriate concentrations were freshly prepared every day using a high-speed homogeniser. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Verification analyses of the actual test material concentrations, stability and homogeneity in the prepared suspensions were performed on two dates during the administration. All samples were analysed by HPLC.
- Concentration analysis results: The mean concentrations of the test substance in suspensions were 98.3%, 97.2 %, 99.0% and 101.4 % of the nominal concentrations (1.25, 3.75, 12.5 and 37.5 mg/mL respectively).
- Homogeneity results: The homogeneity of the test substance in suspension was considered to be in an acceptable range (-2% to +2%).
- Stability results: The test substance was found to be stable in suspension for the period of dosing.
Details on mating procedure:
- Impregnation procedure: artificial insemination with diluted semen from bucks of the same strain
- The day of insemination was designated day 0 of pregnancy
Duration of treatment / exposure:
day 7 to day 19 of gestation
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
low
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low-mid
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
high-mid
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high
No. of animals per sex per dose:
19
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of the results of a dose range finding study.
- Justification species selection: The rabbit is one of the species recommended for teratology studies required by regulatory authorities. Background data for this strain were available at the test facility.
- Justification route selection: Administration by gavage was selected as the oral route is a possible route for human exposure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
Animals were examined twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS
- Time schedule for examinations: daily

BODY WEIGHT
- Time schedule for examinations: daily

FOOD CONSUMPTION
Food consumption was recorded on days 4, 7, 12, 16, 20, 24 and 29.

POST-MORTEM EXAMINATIONS
- The animals were killed on day 29 by intravenous injection of a fast-acting barbiturate anaesthetic and foetuses were removed by hysterectomy.
- The following were recorded: Macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals
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:
The foetuses were numbered, sexed (on the basis of anogenital distance), externally examined and weighed. They were then killed and processed for visceral or skeletal examination. Foetal heads were assigned to either visceral or skeletal examination at an approximate 1:1 ratio within each litter, independent of sex and starting with skeletal. In the case of gross external anomaly or malformation, foetuses were allocated to one technique depending on the type and incidence of finding.
Statistics:
Analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-test in case of a significant result in the ANOVA. Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher’s Exact test in case of a significant result in the Chi-Square test. Non-parametric data (e.g. mean percent affected foetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One high dose group animal (150 mg/kg bw/day) presented with vaginal bloody discharge on day 19. A second high dose animal had vaginal bloody discharge on days 18 & 19. Bloody discharge in the perineal area was noticed for a third high dose animal on day 22 and was terminated on the same day, due to severe weight loss. Bloody discharge in the perineal area was detected in a total of 13/19 dams in the high dose group between days 14 and 23. These observations were considered treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One high dose group animal (150 mg/kg bw/day) presented with vaginal bloody discharge on day 19 and was found dead on day 20. A second high dose animal had vaginal bloody discharge on days 18 & 19 and was killed moribund on day 19. A third high dose animal was terminated due to severe weight loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The absolute mean body weight of high dose group dams (150 mg/kg bw/day) was slightly reduced from gestation day 16 to day 29 as compared to controls but without statistical significance. A marked body weight loss was noted between days 7 and 12 and during the whole treatment period. A reduced weight gain resulted from day 7 to 29 (not statistically significant). The reduction was attributed to treatment. A similar non-significant reduction was noted at 50 mg/kg bw/day for days 7-19.
See Table 1 in "Any other information on results incl. tables" for more information.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean maternal food consumption was significantly and dose-dependently reduced in the 50 and 150 mg/kg bw/day groups during the treatment period (days 7 to 12 and days 12 to 16) and from day 16 to day 20 in the 150 mg/kg bw/day group. A significant, compensatory increase in food consumption in the high dose group was recorded in the post-treatment period.
See Table 2 in "Any other information on results incl. tables" for more information.
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:
effects observed, treatment-related
Description (incidence and severity):
In the high dose females found dead or killed prior to day 29, haemorrhagic contents of the uterus were observed and considered treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Pre-implantation loss and the number of implantation sites were comparable for all groups. There was a higher post-implantation loss at the high dose due to an increase in early resorptions. Increased numbers of post-implantation losses in 3 high dose dams resulted from total resorption and were considered treatment-related.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Number of total resorption at term at highest doses are increased.
See Table 3 in "Any other information on results incl. tables" for more information.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Mean number of early resorptions was increased at the highest dose.
See Table 3 in "Any other information on results incl. tables" for more information.
Dead fetuses:
no effects observed
Description (incidence and severity):
Values of mean number of live foetuses remains almost the same with the dose range.
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Increased "number pregnant, premature deaths" at the highest dose.
See Table 3 in "Any other information on results incl. tables" for more information.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
pre and post implantation loss
early or late resorptions
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
vagina
Description (incidence and severity):
bloody vaginal discharge in most animals in high dose group, haemorrhagic contents of the uterus found in three animals found dead or killed before scheduled termination.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the highest dose, mean foetal body weights were significantly lower than controls.
See Table 3 in "Any other information on results incl. tables" for more information.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related malformations, anomalies or variations
See Table 4 in "Any other information on results incl. tables" for more information.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related malformations. Significantly increased foetal (but not litter) incidence of fused sternebrae 3 and 4 in the high dose group was correlated with reductions in mean foetal body weight and considered related to maternal toxicity due to treatment. Other skeletal anomalies were unaffected by treatment. The significantly increased foetal (but not litter) incidence of absent ossification of the medial phalanx of anterior digit 5 in four high dose foetuses may indicate slight maternal toxicity. Other skeletal variations were considered unrelated to treatment.
See Table 4 in "Any other information on results incl. tables" for more information.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related malformations, anomalies or variations
See Table 4 in "Any other information on results incl. tables" for more information.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced mean body weight in high dose group
Description (incidence and severity):
Mean foetal body weights were significantly lower than controls in high dose groups. No teratogenicity observed.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1: Intergroup comparison of body weight gain(g) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

15

50

150

7-12

-26

-23

-17

-30

-105**

12-16

57

69

73

47

38

16-20

21

-2

-17**

3

-6

7-19

51

61

40

17

-67**

7-29

114

144

167

134

38

Net change

-112

-159

-150

-160

-172

** Statistically significant difference from control group mean, p<0.01

Net change = carcass weight minus day 7 body weight

 

Table 2: Intergroup comparison of food consumption (g/animal/day) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

15

50

150

7-12

88.7

92.4

88.5

69.2**

21.4**

12-16

86.7

91.2

79.1

64.2**

38.5**

16-20

96.3

95.9

88.5

80.6

56.8**

20-24

96.3

99.2

93.1

100.9

125.1*

24-29

93.4

96.8

100.3

103.3

121.6*

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 3: Ceasarean section observations

Observation

Dose level (mg/kg bw/day)

0 (control)

5

15

50

150

Number of females inseminated

19

19

19

19

19

Number not pregnant

4

0

0

0

1

Number pregnant, premature deaths

0

0

0

0

3

Number pregnant at term

15

19

19

19

15

Number with total resorption at term

0

0

0

1

3

Number with live foetuses at term

15

19

19

18

12

Gravid uterus weight (g)

226

303

316

294

210

Mean number of corpora lutea

6.7

7.2

7.2

6.9

7.1

Mean number of implantation sites

4.7

5.4

5.9

5.4

5.4

% Pre-implantation loss

32.6

23.1

17.5

23.7

25.1

Mean number live foetuses

3.7

5.1

5.4

4.6

3.0

Mean number early resorptions

1.0

0.3

0.5

0.6

2.4

Mean number late resorptions

0.0

0.0

0.1

0.2

0.0

% post implantation loss

21.0

6.3

9.9

16.3

45.6

Total number viable foetuses - males

31

48

46

46

22

Total number viable foetuses - females

24

49

56

40

23

% Males

56.4

49.5

45.1

53.5

48.9

Mean foetal body weight (g)

44.0

41.5

41.7

42.1

37.5**

Mean male foetal body weight (g)

44.4

41.7

43.0

42.2

38.8**

Mean female foetal body weight (g)

41.8

40.9

40.8

41.1

36.6*

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 4: Incidence of selected foetal skeletal anomalies and variations (% foetuses affected per litter)

Observation

Dose level (mg/kg bw/day)

0 (control)

5

15

50

150

Fused sternebra 3 and sternebra 4

0/55

(0.0)

0/97

(0.0)

2/102

(1.8)

1/88

(0.8)

5/45*

(14.6)

Anterior digit 5, medial phalanx, absent ossification

0/55

(0.0)

1/97

(1.3)

0/102

(0.0)

0/88

(0.0)

4/45*

(5.9)

* Statistically significant difference from control group mean, p<0.05

Applicant's summary and conclusion

Conclusions:
- Maternal toxicity occurred at 150 mg/kg bw/day (3 premature deaths, bloody discharge, reduced food consumption, body weight and body weight gain) resulting in foetal toxicity (increased post-implantation loss, reduced foetal weight and increased incidence of two skeletal observations).
- Maternal toxicity also occurred at 50 mg/kg bw/day (reduced food consumption) but there was no foetal toxicity.
- On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 15 mg/kg bw/day and for developmental toxicity was 50 mg/kg bw/day.
- There was no evidence of teratogenicity.
Executive summary:

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rabbits. The test material was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 5, 15, 50 and 150 mg/kg body weight to 19 inseminated Russian Chbb:HM rabbits per group from day 7 to 19 of pregnancy inclusive, using a dose volume of 4 mL/kg body weight. Dams were killed on day 29 of pregnancy, just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent examination. In the 150 mg/kg bw/day, there were a number of treatment-related findings. Bloody discharge in the perineal area or from the vagina was detected in 15 dams. Three of those dams were found dead, killed in moribund condition or terminated prematurely due to severe weight loss and bloody discharge. Mean body weights were reduced from day 16 to 29 and there was a mean body weight loss during the treatment period (days 7-19). A slight non-significant mean body weight gain reduction occurred in the 50 mg/kg bw/day dose group. Food consumption was dose-dependently reduced during treatment in the 150 and 50 mg/kg bw/day groups. A compensatory increase in food consumption was noticed during post-treatment in the high dose group.Increased treatment-related post implantation losses resulted from total resorption in 3 dams of the high dose group. At necropsy, haemorrhagic contents were noted in the uteri of the 3 dams not surviving to scheduled termination. Foetal weights in the 150 mg/kg bw/day group were significantly reduced compared to controls and this finding was attributed to maternal toxicity. The incidence and type of external, visceral and skeletal findings was generally not affected by the treatment but a few isolated skeletal findings may indicate a treatment-related delay in ossification. Maternal toxicity occurred at 150 mg/kg bw/day (3 premature deaths, bloody discharge, reduced food consumption, body weight and body weight gain) resulting in foetal toxicity (increased post-implantation loss, reduced foetal weight and increased incidence of two skeletal observations). Maternal toxicity also occurred at 50 mg/kg bw/day (reduced food consumption) but there was no foetal toxicity. On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 15 mg/kg bw/day and for developmental toxicity was 50 mg/kg bw/day. There was no evidence of teratogenicity.