Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 June 2016 to 21 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Age at study initiation: at least 11 weeks old at mating
- Weight at study initiation: 193-252 g (the variation did not exceed ± 20% of the mean weight)
- Fasting period before study: No
- Housing: Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-24.8°C
- Humidity (%): 31-63%
- Air changes (per hr): 15-20 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light / 12 hrs dark

IN-LIFE DATES: From: 21 June 2016 To: 21 July 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) at the appropriate concentrations according to the dose level and volume selected. The test item was formulated considering the density of the test item and then it was corrected with the purity of the test item in the applied water solution. Formulations were prepared fresh prior to administration to animals.

VEHICLE
- Amount of vehicle (if gavage): 1.79 mL/kg body weight
- Lot/batch no. (if required): 809 0416
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of test item formulations for concentration and/or homogeneity was performed by an UV-spectrophotometric method in the Analytical Laboratory of CiToxLAB Hungary Ltd.
Control, top, middle and bottom duplicate samples were taken from test item formulations two times during the study (approximately during the first and last week of treatment): one set to analyse and one set as a back-up.
All formulations were found to be in the range of 91-101% of the nominal concentrations and were homogenous. No test item was detected in the control (vehicle) formulation.
These results were considered suitable for the study purposes.
Similarly, duplicate samples were taken on each occasion from the control (vehicle) formulation for concentration measurement.
Stability of the NOPCOTE 1661 in the vehicle (distilled water) was assessed during the method validation study (CiToxLAB study code: 12/331-316AN). According to the results, the test item is stable in the vehicle in the concentration range of 2.894 (w/w)% - 46.300 (w/w)% on the day of preparation.
Details on mating procedure:
After acclimation (5 days), the females were paired according to their oestrus cycle (examined shortly before start of pairing) with males in the morning for approximately 2 hours (1 male : 1 female) until at least 24 sperm positive females/group are attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
6-14 days pc.
Frequency of treatment:
Daily, 7 days per week.
Duration of test:
Approximately 1 month, from first mating of animals to last necropsy on gestation day 20.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 (control, low and high dose), 25 (mid dose).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were set based on available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats (CiToxLAB study code: 12/331-220PE) and another combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats (CiToxLAB study code: 12/331-220P), with the aim of inducing toxic effect(s), but no death or suffering at the highest dose and a NOAEL at the lowest dose.
In those studies, doses of 62.5, 250 and 1000 mg/kg bw/day were administered by oral gavage to wistar rats. Administration at high dose level for at least 28 consecutive days was associated with minor changes in clinical chemistry and urinalysis parameters in males and females. In addition, ulcers of the non-glandular mucosa of the stomach were noted in 4/12 males and 4/12 females. No adverse effect was found on reproduction/developmental parameters at this high dose level. No adverse effects or test item related histopathology findings were observed at the low or mid dose levels.
Based on these results, the doses selected for this study were 100, 300 and 1000 mg/kg bw/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side clinical observations were made at twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At GD6 and then weekly.
- Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
In addition, on GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT: Yes
- Time schedule for examinations: GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
Food was measured on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
Food consumption was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
Dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed.
Stomach of all dams was retained in 10% buffered formalin solution.



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
- Other: Placentas were examined macroscopically
Fetal examinations:
- External examinations (including great arteries): Yes: all per litter.
- Soft tissue examinations: Yes: half per litter. The abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture then after fixation the body was micro-dissected by means of a dissecting microscope. Skeletal examinations: Yes: half per litter. After fixation the skeletons were stained nd examined by means of a dissecting microscope.
- Head examinations: Yes: half per litter. The heads were examined by Wilson's free-hand razor blade method.
- Others: Individual weight.
Statistics:
The statistical evaluation of data was performed using the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).
The heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out.
If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied.
If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.
The Chi squared test was used for comparing group incidences against the controls.
Indices:
Maternal Data:
-Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
-Clinical signs (by gestation day)
-Mortality (by gestation day)
-Body weight and body weight gain: mean ± S.D.
-Corrected body weight on GD20 (body weight-gravid uterine weight), corrected body weight gain and corrected net body weight gain: mean ± S.D.
-Net body weight change (Body weight on GD20 minus body weight on GD6 minus gravid uterine weight): mean ± S.D.
-Food consumption: mean ± S.D.
-Gross pathology findings
-Gravid uterine weight: mean ± S.D.

Caesarean Section and Necropsy Data:
-Number of corpora lutea: mean ± S.D.
-Number of implantations: mean ± S.D.
-Number and percentage of live foetuses: mean ± S.D.
-Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation mortality, Early and late embryonic, as well as foetal death
-Preimplantation loss: %, group mean
-Postimplantation loss: %, group mean

Foetal Data:
-Sex distribution: %, group mean
-Foetal body weight (accuracy 0.01 g): mean * S.D.
-External abnormalities/litter: %, group mean
-Visceral abnormalities/litter: %, group mean
-Skeletal abnormalities/litter: %, group mean

Historical control data:
See Section “Any other information on results incl. tables”

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects or systemic clinical signs were noted in any dose groups.
Fur thin and/or alopecia observed in some control and test item treated animals were considered as irrelevant findings.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled mortality during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes compared to the control were observed in the mean body weights or body weight gain values in any dose group.
No statistical significance in any of the dose groups when compared to the control group was observed for corrected body weight gain and corrected net body weight gain values (when results were adjusted by the uterus weight), although the high dose group had lower values (by approximately 8-9%).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No statistical significant differences compared to the control in the mean daily food consumption of dams were observed in any period for any of the dose groups, although in the last observation period (GD18-20) lowed food consumption (by approximately 21%) was recorded in the High dose group.
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):
No observations were recorded for any animals in the study except of recording the fur thin.
The stomach of all animals was carefully examined, but no findings were observed in any control or test item treated animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The study makes no reference of any abortion.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups.
No test item effect was observed in preimplantation loss of the test item treated groups when compared to the control.
There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The total intrauterine mortality was comparable with the control, no statistically significant differences were observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There was no statistically significant difference in foetal death in any group compared to the control.
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:
no effects observed
Description (incidence and severity):
Ninety-seven females (24 animals in the Control, Low and High dose groups and 25 animals in the Mid dose group) were mated in the study. The number of confirmed pregnant, evaluated dams was 24, 24, 24 and 21 in the Control, Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups, respectively.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: No adverse effect observed up to the highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For the mean body weight/foetus significant difference was observed in the Low and High dose groups when compared to the control. However the slightly higher mean foetal body weight values showed no dose response and all values were within the historical range, hence the statistical differences were not considered as a test item related effect.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The total number of retarded foetuses (runts) as well as the number of affected litters in the test item treated groups was comparable with the control value.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The weight of foetuses per litter in any of the dose groups did not differ significantly from the control mean value.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No external malformations were recorded in the study. External variation was recorded for one foetus in the High dose group, but based on the isolated occurrence it was considered incidental, ascribed to individual variability and not related to treatment (although concurrent control data did not contain this observation but it was included in the historical control database).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the skeletal findings were consistent in general nature and the observed incidence was within the study concurrent control data or the existing historical control data, therefore they were considered as incidental findings.
No skeletal malformations were recorded in the study.
In one case (wavy ribs), an increased incidence of changes was observed in the absolute number of findings in the Mid and High dose groups with a frequency of 8.8% and 11.6% for foetuses, and 29.2% and 33.3% for litters, respectively. The difference gained statistical significance (p<0.05) in the High dose group when compared to control (3.6% for foetuses and 16.7% for litters). However, this observation is a common background finding and occurred with a comparable incidence in the historical control (9.9% for foetuses and 30.8% for litters). Furthermore, the incidence in the individual control groups of the historical control database showed this variation in a 4.1-18.6% range for foetuses and 8.7-56.5% for litters. Therefore, this finding was not considered as a test item related adverse effect.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the visceral findings were consistent in general nature and the observed incidence was within the study concurrent control data or the existing historical control data or were considered as incidental background finding, therefore they were considered not related to the test item treatment.
There were no malformations in the Mid or High dose groups. A low incidence malformation (one foetus) in the Low dose group was ascribed to incidental background findings, unrelated to treatment.
The number of foetuses with variation was statistically higher (p<0.05) in the Low dose group, consequently the number of intact foetuses was significantly lower (p<0.05) in the same group when compared to the control. This fact was caused by the significantly (p<0.05) increased number of foetuses with thymic cord variation in the Low dose group. In case of this visceral variation, although the occurrence (5.7% for foetuses, 16.7% for litters) in the Low dose group was higher rate than in the actual control group (0.7% for foetuses, 7.1% for litters), but it was comparable to the rate of the HC database (2.7% for foetuses, 11.3% for litters). Furthermore, there was no dose response, so this fact was not considered as a test item related effect.
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities were observed on the placentas of any animals in the Control, Low, Mid or High dose groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: No treatment related adverse effect observed in any of the parameters at the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
There were no effects of the test item on external, visceral and/or skeletal development of foetuses in the study which could be related to the test item administration. Increased incidence of the skeletal variation of wavy ribs in the Mid and High dose groups was comparable with the relevant historical control data.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of the dose formulation analysis

Nominal
concentration
(mg/mL)

Measured concentration
(mg/mL)

Percentage of the nominal concentration
(%)

Analytical sampling #1(27 June 2016)

Control

not detectable

-

4.54% w/w

4.44 ± 0.105

98

13.63% w/w

12.4 ± 0.293

91

45.4% w/w

43.8 ± 1.04

97

Analytical sampling #2(12 July 2016)

Control

not detectable

-

4.54% w/w

4.54 ± 0.319

100

13.63% w/w

13.8 ± 0.449

101

45.4% w/w

45.8 ± 1.395

101

Table 2: Summary of pregnancy data

Parameters

Dose (mg/kg bw/day)

0

100

300

1000

Number of mated females

24

24

25

24

Number of non-pregnant females

0

0

1

2

Number of females with ≤ 5 implantation sites

0

0

0

1

Number of evaluated females
on GD20 (Caesarean section)

24

24

24

21

Table 3: Summary of the intrauterine evaluation

Parameters

Dose (mg/kg bw/day)

0

100

300

1000

Number of evaluated dams

24

24

24

21

Mean number of corpora lutea

12.21

11.13

11.67

11.57

Mean number of implantations

11.67

10.71

10.71

10.81

Preimplantation loss, mean

0.54

0.42

0.96

0.76

Preimplantation loss (%), mean

4.71

3.58

8.21

6.43

Early embryonic loss, mean

0.21

0.25

0.08

0.10

Early embryonic loss (%), mean

2.21

2.33

0.79

0.76

Late embryonic loss, mean

0.00

0.04

0.17

0.00

Late embryonic loss (%), mean

0.00

0.33

1.50

0.00

Dead foetuses, mean

0.00

0.00

0.00

0.00

Dead foetuses (%), mean

0.00

0.00

0.00

0.00

Postimplantation loss, mean

0.21

0.33

0.25

0.10

Postimplantation loss (%), mean

2.21

3.04

2.29

0.76

Total intrauterine mortality, mean

0.75

0.75

1.21

0.86

Total intrauterine mortality (%), mean

6.46

6.54

10.17

7.14

Viable foetuses, mean

11.46

10.42

10.46

10.71

Table 4: Examination of viable foetuses

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of examined litters

24

24

24

21

 

Viable foetuses, mean

11.46

10.42

10.46

10.71

 

Male foetuses, mean

5.88

5.42

5.13

5.76

 

Female foetuses, mean

5.58

5.00

5.33

4.95

 

Total number of foetuses

275

250

251

225

 

Total number of male foetuses

141

130

123

121

 

Total number of female foetuses

134

120

128

104

 

Sex ratio (males), %

50.96

52.00

49.33

53.48

 

Mean foetal weight / litter (g)

3.406

3.509

3.422

3.483

 

Mean body weight / foetus (g)

3.400

3.509**DN

3.418

3.486**DN

 

Number of foetuses with retarded body weight

12

6

11

10

 

Number of affected litters (Runts)

10

4

9

8

 

DN:’s multiple range test, *: p<0.05, **: p<0.01

Table 5: Summary table of the external abnormalities

 

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

24

24

21

485

Total number of examined foetuses

275

250

251

225

4955

Total number of intact (normal) foetuses

275

250

251

224

--

Number of foetuses with malformation

0

0

0

0

--

Number of foetuses with variation

0

0

0

1

--

External malformations

No external malformations were observed

External variations

Pale foetus

--

--

--

1 / 1

1 / 1

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control

Table 6: Summary table of the visceral abnormalities

 

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

24

24

21

485

Total number of examined foetuses

138

123

126

113

2484

Total number of intact (normal) foetuses

135

113*CH

118

108

--

Number of foetuses with malformation

0

1

0

0

--

Number of foetuses with variation

3

9*CH

8

5

--

Visceral malformations

Liver lobe (caudate process), absent

--

1 / 1

--

--

--

Visceral variations

Thymic cord

1 / 1

7 / 4*CH

3 / 3

3 / 3

66 / 55

Renal papilla, small

--

--

1 / 1

1 / 1

18 / 16

Ureter, convoluted

--

1 / 1

1 / 1

--

3 / 3

Brachiocephalic trunk, short

2 / 2

1 / 1

3 / 3

1 / 1

43 / 35

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control. CH: Chi2test, *: p<0.05, **: p<0.01

Table 7: Summary table of the skeletal abnormalities

 

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

24

24

21

484

Total number of examined foetuses

137

127

125

112

2467

Total number of intact (normal) foetuses

123

116

104

92

--

Number of foetuses with malformation

0

0

0

0

--

Number of foetuses with variation

14

11

21

20

--

Skeletal malformations

No skeletal malformations were observed

Skeletal variations

Skull, 2 or more bones incomplete ossification

6 / 5

3 / 3

12 / 5

7 /4

474 / 326

Ossified sternebra (3 or less)

1 / 1

4 / 2

3 / 3

2 / 1

46 / 37

Sternebra(s), misaligned

--

1 / 1

--

--

6 / 6

Ribs, wavy

5 / 4

5 / 4

11 / 7

13 / 7*CH

243 / 149

Vertebra, dumbbell or asymmetric or unilateral ossification (2 or more)

--

--

2 / 1

--

79 / 73

Vertebra, unossified

--

1 / 1

--

--

2 / 1

Vertebra, bipartite ossification

1 / 1

1 / 1

1 / 1

--

24 / 24

Pubis, unossified

--

1 / 1

--

--

6 / 6

Carpal ossified ≤ 2.5

--

1 / 1

--

--

4 / 4

Tarsal ossified ≤ 3.5

1 / 1

2 / 2

--

1 / 1

75 / 53

Notes: Numbers represent the number of abnormalities / number of affected litters.HC: historical control.CH: Chi2test, *: p<0.05, **: p<0.01

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study performed on the test item, no significant toxicological changes on dams, embryos or foetuses were observed and NOAEL for maternal toxicity, NOAEL for embryotoxicity, NOAEL for foetotoxicity and NOAEL for teratogenecity were determined to be 1000 mg/kg bw/day.
Executive summary:

A prenatal developmental toxicity study was performed in accordance with OECD 414 guideline (GLP study) in order to assessthe effects of the test item on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. After mating, the pregnant dams were treated daily by oral (gavage) administration at 3 dose levels, 100, 300 and 1000 mg/kg bw/day, from gestation day GD6 up to and including GD19. Caesarean section and necropsy with macroscopic examination was performed on GD20. Control dams were treated with the vehicle (distilled water) only. The number of confirmed pregnant, evaluated dams was 24 in the Control, 24 in the Low, 24 in the Mid and 21 in the High dose groups, respectively.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain, individual food consumption and maternal reproductive parameters associated with uterine, for dams, and weight, external, visceral and skeletal examinations, for foetuses. Placentas were examined macroscopically.

There were no unscheduled mortality or treatment related clinical signs in the study. No significant differences in the body weight, body weight gain and daily food consumption of dams in any treated group were observed. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day. There were no effects on the early or late embryonic loss in the test item-treated evaluated dams. The mean number of viable foetuses, the mean foetal body weight per litter and the sex ratio did not differ significantly from the control mean value in any of the treated groups. There were no effects of the test item on external, visceral and/or skeletal development of foetuses in the study which could be related to the test item administration. Increased incidence of the skeletal variation of wavy ribs in the Mid and High dose groups was comparable with the relevant historical control data.

In conclusion, from the observations made in the dams and their foetuses, there were no significant toxicological changes on embryos or foetuses, and therefore, the NOAEL for maternal toxicity, NOAEL for embryotoxicity, NOAEL for foetotoxicity and NOAEL for teratogenecity were determined to be 1000 mg/kg bw/day.