Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to internationally accepted guideline. No deviations from guideline protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EC Directive 88/302/EEC B.31 Teratogenicity Test - Rodent and Non-Rodent
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): PAC
Lot/Batch number: 5A7990
Description: White crystalline powder
Purity: 99.48% w/w
Stability: Analytical confirmation of the stability of the substance in the vehicle was performed by the Sponsor and communicated to the Study Laboratory: Stability of the test material in the vehicle resulted to be about 20 days.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia
- Age at study initiation: 16-17 weeks old
- Weight at study initiation: 2700-3200 g

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose 400 cps aqueous solution
Details on exposure:
Concentration in vehicle: 0, 12.5, 25, 75 mg/ml
Total volume applied: 4 ml/kg bw/day
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Mating period: 14 days
Duration of treatment / exposure:
12 days: pregnant dams were dosed by gavage during days 6-18 of gestation.
Frequency of treatment:
Dosing regime: 7 days/week
Duration of test:
Dams were sacrificed on day 29 of gestation, i.e. 10 days after last dosage
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Number of dams and doses
20 at 0 mg/kg
20 at 50 mg/kg
20 at 100 mg/kg
20 at 300 mg/kg
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were selected on the ground of a preliminary teratogenicity study, performed using pregnant rabbits dosed orally (on days 6 – 18 of gestation) with PAC at 0, 100, 500 or 1000 mg/kg/day. Test design was based on OECD guideline 414. A number of treatment-related deaths were seen: at 1000 mg/kg/day, 4/6 decedents showed gastro-intestinal tract ulceration and at 500 mg/kg day 2/4 decedents showed oedema of the gastric submucosa. Surviving animals of these test groups showed reduced bodyweight gain. These maternally toxic treatments were associated with embryotoxicity (increased resorptions), reduced foetal weights and some skeletal anomalies (most retarded ossification of head or sternum). However no treatment-related malformed foetuses were seen. At 100 mg/kg/day, only slight reduction of bodyweight gain in treated does was seen.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: not specified
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:
- Litter examinations: litter size, number of dead foetuses, foetal weight, sex ratio.
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Five animals (one from the control group (day 17), one from the low dose group (day 19), two from the mid-dose group (day 12 and day 24 and one from the high-dose group (day 12)) died during the study. All deaths were considered as incidental, although it could be possible that treatment induced a worsening of effects.

No clinical signs or behavioural changes were found in any experimental group. A dose-related decrease in maternal absolute body weight was recorded for all the treatment groups during PAC administration, achieving statistical significance at 300 mg/kg/day. Decreased body weight gain achieved statistical significance at 100 and 300 mg/kg/day during the treatment period. A slight recovery in body weight gain was observed at the end of exposure (day 19-29 of gestation) in the 300 mg/kg/day group. Changes in body weight were consistent with the decreased food intake recorded for all treatment groups during the administration period. A similar trend was observed also for water intake.

No pathological changes were found during the autopsy and no abortions were found in any group.

2 animals with only resorptions were present, one in the low-dose group and another in the mid-dose group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
50 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:
Effects on fetus - litter examinations:
- An increase in the incidence of does with early resorptions was observed in all treated groups (30-35%) compared to controls (8.33%). This included two does, one in the low-dose group and another in high-dose group with only resorptions present. In addition, a statistically significant (p < 0.01) increase in the percentage of early resorptions (group total number of early resorptions/group total number of implantations) was observed in the mid- and high dose groups (12% and 11%, respectively), even with the exclusion of the 2 cases of only resorptions from the mean calculations.
- The mean viable foetal weight of the pups from the hig-dose group was significantly lower (16%, p < 0.01) than the respective controls. No other effects were observed in litter parameters. No treatment-related malformations, anomalies and variants were observed at any tested dose.

Effects on fetus - Gross:
- No treatment-related effects in any groups.

Effects on fetus - Soft tissue:
- No visceral malformations were found at any dosages.

Effects on fetus - Skeletal:
- No important differences were found between the skeletal anomalies and variants of the treated groups and dose of the control group.
- The head's examination did not show any malformation, anomaly or variant in any group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 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

Maternal effects

Parameter

control

 

low dose

medium dose

high dose

dose-response
+ / -

Number of dams examined

20

20

20

20

 

Clinical findings during application of test substance

-

-

-

-

-

Mortality of dams

state %

5%

5%

10%

5%

-

Abortions

-

-

-

-

-

Body weight gain (g) - day 0-6

27.5

52.3

22.5

20.7

 

day 6-19

318.3

154.6*

94.2**

-93.6**

+

day 19-29

151.7

171.5

196.7

342.9**

+

day 0-29

497.5

378.5

313.3

270.0

+

Pregnancies

12/20

14/20

13/20

14/20

-

Necropsy findings in dams dead before end of test

Not examined

Acute pneumonia

Kidney - tubular dilatation atrophy

 

Liver - centrilobular necrosis and degeneration

Kidney - tubular dilatation atrophy

 

* p< 0.05; ** p< 0.01

Litter response

Parameter

Mean#

control

low dose

medium dose

high dose

dose-response
+ / -

Corpora lutea

 

A

115/12

140/14

132/13

150/14

-

B

115/12

130/13

121/12

150/14

-

Implantations

 

A

105/115

91.30%

128/140

91.43%

102/132*

77.27%

139/150

92.67%

-

B

105/115

91.30%

118/140

90.77%

95/132**

78.51%

139/150

92.67%

-

Early resorptions

 

A

2/105

1.90%

18/128***

14.06%

18/102***

17.65%

15/139**

10.79%

-

B

2/105

1.90%

8/118

6.78%

11/95**

11.58%

15/139**

10.79%

-

Early + late resorptions

 

A

4/105

3.80%

23/128***

17.97%

18/102**

17.65%

20/139**

14.39%

-

B

4/105

3.80%

13/128*

11.02%

11/95*

11.58%

20/139**

14.39%

 

total number of fetuses

A

105

128

102

139

-

B

105

118

92

139

-

pre-implantation loss, %

 

A

9.3

7.9

22.7*

7.21

-

B

9.3

8.5

21.6*

7.21

-

post-implantation loss, %

A

5.0

17.5

20.2

13.9

-

B

5.0

11.1

13.5

13.9

-

total number of litters

A

12

14

13

14

-

B

12

13

12

14

-

live fetuses / litter

A

8.42

7.50

6.31

8.43

-

B

8.42

8.08

6.83

8.43

 

dead fetuses / litter

A

0.00

0.00

0.15

0.07

 

B

0.00

0.00

0.17

0.07

-

fetus weight (mean), g

B

47.64

45.62

48.03

40.53**

-

placenta weight (mean), g

B

7.17

7.44

7.85

7.09

-

Fetal sex ratio, m/f

 

 

0.83

0.91

0.86

0.90

-

*p< 0.05; ** p< 0.01; *** p< 0.001

 

#Mean A: all dams were included in the mean

 Mean B: the 2 cases of only resorptions were excluded

 

Examination of the fetuses

Parameter

control

 

low dose

medium dose

high dose

dose-response
+ / -

External malformations*

[%]

0.00

0.00

0.00

0.85

-

External anomalies*

[%]

0.00

0.00

0.00

0.00

-

Skeletal malformations*

[%]

0.00

0.00

0.00

0.00

-

Skeletal anomalies*

[%]

15.84

21.90

18.29

10.17

-

Skeletal variants*

[%]

29.70

41.90*

37.80

22.03

-

Visceral malformations*

[%]

0.00

0.00

0.00

0.00

-

Visceral anomalies*

[%]

0.00

0.00

0.00

0.00

-

Visceral variants*

[%]

0.00

0.00

0.00

0.00

-

* p< 0.05

 

Applicant's summary and conclusion

Conclusions:
LO(A)EL maternal toxic effects 100 mg/kg bw/day. General toxicity, decreased body weight and body weight gain.
NO(A)EL maternal toxic effects 50 mg/kg bw/day
LO(A)EL embryotoxic / teratogenic effects 300 mg/kg bw/day. Statistically significant decrease in the mean viable foetal weight of the pups.
NO(A)EL embryotoxic / teratogenic effects 100 mg/kg bw/day
Executive summary:

20 pregnant female New Zealand white rabbits/group were exposed to 0, 50, 100 and 300 mg/kg/day by gavage. Pregnant dams were dosed by gavage during days 6-18 of gestation. On day 29 of gestation, animals were sacrificed and the potential effects of the treatment on pregnancy and foetuses were analysed.

Maternal toxicity (decreased body weight and body weight gain) was observed at ≥ 100 mg/kg bw/day.

An increased incidence of early resorptions was observed in all the treatment groups and 2 dams with only resorptions were observed, one in the 50 mg/kg/day and another in the 100 mg/kg/day groups. However,the incidences of these findings were in the range of normal variability seen in this strain. The group mean number of early resorptions in the 100 and 300 mg/kg/day groups was also in the range of normal variability and the increase in the percentage of early resorption observed in these treatment groups was considered to be due to a high individual value.

A statistically significant decrease in the mean viable foetal weight of the pups was observed at 300 mg/kg/day. No treatment-related malformations, anomalies and variants were observed at any tested dose.