Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
The prenatal developmental toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 August 2016 to 05 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 12 - Nousan No. 8147
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
452-330-3
EC Name:
-
Cas Number:
314020-40-1
Molecular formula:
C14H20N2O2
IUPAC Name:
2-(2,6-diethyl-4-methyl-phenyl)propanediamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, UK
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: 177 to 246 g
- Fasting period before study: No
- Housing: Animals were housed individually in solid-floor cages with appropriate bedding provided.
- Diet (e.g. ad libitum): VRF1 manufactured by SDS available ad libitum
- Water (e.g. ad libitum): Mains tap water available ad libitum
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) aqueous CMC with 0.1% (v/v) Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The required amount of test item was added to a container and tared on a balance. The test item was then wetted with a small quantity of vehicle and initially made into a smooth paste. After further addition of vehicle and mixing, the suspension was made up to final weight with vehicle and stirred until visibly homogeneous. Formulations were then placed in an ultrasonic bath for a minimum of 15 minutes (in short bursts, as necessary, to prevent heating of the formulation) to break up any remaining large particulate. Formulations were then divided into daily aliquots for dosing and stored refrigerated (2 °C to 8 °C). Test item formulations were stirred for at least 15 minutes before the start of dosing and until completion of dosing, to ensure thorough re-suspension and homogeneity.

- Concentration in vehicle: 0, 1.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from each test item formulation prepared for the first day of dosing and were analysed for test item using a validated method to confirm homogeneity and also achieved concentrations. Having confirmed satisfactory homogeneity for the first day of dosing, triplicate samples were taken from all test item formulations prepared towards the end of the dosing period and analysed to determine achieved concentrations only. Triplicate samples were taken from formulations prepared for the first day of dosing and towards the end of the dosing period from the vehicle used to dose Controls and were analysed to confirm absence of test item.
All remaining samples were retained and were discarded once the final formulation analysis
results were accepted.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant from Charles River (UK) Limited.
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Animals were dosed from Day 6 to Day 19 of gestation.
Frequency of treatment:
Animals were dosed once daily.
Duration of test:
Dosing started on Day 6 of gestation and animals were killed on Day 20 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor after examining existing toxicity data. The top dose level of 1000 mg/kg/day was selected on the basis of the results from a 90-day study and a reproduction and developmental toxicity screening study. In these studies, 1000 mg/kg/day was well tolerated with no detrimental effects on body weight or food intake and no adverse clinical observations. Therefore, 1000 mg/kg/day was considered to be an appropriate high dose level for use in this study. This is also the limit dose recommended in the OECD guideline for this study type.

- Randomisation: Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded at the supplier on Day 0 of gestation (making sure that females mated with the same male were spread across the groups). The cages were positioned in the battery using a randomised cage allocation procedure, starting at the top left-hand corner of the rack and then working from left to right, top to bottom. All groups were allocated to each rack. Each animal was uniquely identified by a subcutaneously implanted micro-identification device.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 of gestation by the supplier then daily from Day 5 to 20 of gestation, inclusive

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Organs or tissues showing any macroscopic abnormalities were removed and retained in fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained.
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]
- Decaptiation: Yes [half per litter]
- Soft tissue examinations: Yes: [all - intact foetuses and the bodies of the decapitated foetuses using a combined sectioning/dissection technique ]
- Skeletal examinations: Yes: [all - intact foetuses and the bodies of the decapitated foetuses]. Carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.
- Head examinations: Yes: [ all - half per litter serial section of Bouins fixed heads, remaining half per litter heads of intact foetuses - coronal section made through the head along the frontal parietal suture and brain examined.
Statistics:
Test were two-sided with significance levels of 5% and 1%. The litter, was considered as the experimental unit. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).
For Quantitative Data: Body weight, cumulative body weight gain from the start of dosing, food intake, terminal body weight, numbers of corpora lutea, implants, live foetuses, dead foetuses, early intrauterine deaths, late intrauterine deaths, gravid uterus weight, total litter weight, placental weight and mean foetal weight (sexes separately and combined) were analysed using a parametric ANOVA.
For Percentages: Pre-implantation loss, post-implantation loss, sex ratios (% male foetuses) and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation (Freeman and Tukey).
Maternal Performance: (e.g. the proportion of females with live foetuses at termination, abortions, total resorptions) were analysed by a two-tailed Fisher’s Exact Text comparing each treated group to the control group.
Foetal Morphology Data: The incidence of foetal malformations and developmental variations (external, visceral and skeletal) were summarised as the proportion of foetuses affected, the proportion of litters affected and the proportion of foetuses affected within each litter. The proportions of litters affected were analysed by the exact version of the Cochran-Armitage Test. The percentages of foetuses affected within each litter was analysed by the exact version of the Jonckheere Trend Test. In both cases the tests were performed in a step-wise manner where, when a test was significant at the 5 % level, the test was repeated after removing the then top dose, until only the Control group was left. Tests were one-sided looking for increase in treated groups versus the Control group.
Indices:
Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites) / no. of corpora lutea (x 100)
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses) / no. of implantation sites (x 100)
Mean pre- and post-implantation losses were calculated on a proportional litter basis.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Yellow stained bedding was observed in the cages of females given 1000 mg/kg/day. This was considered to be test item-related but non-adverse.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females in the group given 1000 mg/kg/day ate statistically significantly less (p<0.01) than the Controls from Day 6 to Day 15 of gestation so that their overall mean food intake for the entire dosing period was 13 % lower than the Controls.
At 15 or 150 mg/kg/day mean food intake was similar to Control values.
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
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, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the mean incidence of post-implantation loss was slightly higher than that of the Controls; however, this was due to one female with total resorption, this female had only one implantation that was an early intrauterine death. Given the absence of an effect of the test substance on post-implantation loss in the litters with live foetuses at 1000 mg/kg/day, the single occurrence of total resorption with only a single implantation is considered to be an incidental event unrelated to administration of the test item. For this reason, the dam and litter have been excluded from calculation of the group mean data.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the mean incidence of post-implantation loss was slightly higher than that of the Controls; however, this was due to one female with total resorption, this female had only one implantation that was an early intrauterine death. Given the absence of an effect of the test substance on post-implantation loss in the litters with live foetuses at 1000 mg/kg/day, the single occurrence of total resorption with only a single implantation is considered to be an incidental event unrelated to administration of the test item. For this reason, the dam and litter have been excluded from calculation of the group mean data.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day group mean foetal weight (3.35 g) was slightly, but statistically significantly lower than Control (-9 %, p<0.01) and below the historical Control ranges for this laboratory (3.50 g to 3.91 g). There was no effect on foetal weight at 15 mg/kg/day or 150 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the overall incidence of minor foetal abnormalities was significantly higher than the Controls (p<0.001). This included increases in the number of foetuses showing skeletal and cartilage abnormalities of the cervical vertebrae and ribs together with the presence of cervical ribs; the majority of these incidences were outside the historical Control range.
In addition, at 1000 mg/kg/day, there were significantly higher (p<0.001) incidences of related variant findings of the cervical vertebrae and non - or incomplete ossification of the 5th and 6th sternebrae and 5th metacarpal (p<0.05, p<0.01, p<0.001), the majority of these findings were outside the historical Control range.
The minor defect of partially fused jugal and maxilla was noted in 5 foetuses from 3 litters in the group given 1000 mg/kg/day (p<0.05). Although the incidence was slightly higher than the historical Control range, this abnormality is seen in this strain of rats in this laboratory and as such the incidence was considered likely to be spontaneous and not an adverse effect of test substance administration.
At 1000 mg/kg/day, there were significantly higher incidences of the minor defects, irregular ridging of the palate (p<0.01) and incomplete ossification of the nasal bones (p<0.001), compared with Control, both of which were outside the historical Control range.
At 150 mg/kg/day, there were marginal increases in the incidences of minor abnormalities of the cervical vertebrae and incomplete ossification of the nasal bones compared with the Controls which correlated with increases seen also at 1000 mg/kg/day. These incidences were also above the historical Control range.
The findings at 150 or 1000 mg/kg/day related to ossification are considered to be transient in nature and/or of no developmental consequence. The increased incidences of other skeletal and cartilage minor foetal abnormalities and variants were clearly test item-related and considered to represent an adverse effect of the test item on foetal development.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
Description (incidence and severity):
At 1000 mg/kg/day, the overall incidence of minor foetal abnormalities was significantly higher than the Controls (p<0.001). This included increases in the number of foetuses showing skeletal and cartilage abnormalities of the cervical vertebrae and ribs together with the presence of cervical ribs; the majority of these incidences were outside the historical Control range.
In addition, at 1000 mg/kg/day, there were significantly higher (p<0.001) incidences of related variant findings of the cervical vertebrae and non - or incomplete ossification of the 5th and 6th sternebrae and 5th metacarpal (p<0.05, p<0.01, p<0.001), the majority of these findings were outside the historical Control range.
The minor defect of partially fused jugal and maxilla was noted in 5 foetuses from 3 litters in the group given 1000 mg/kg/day (p<0.05). Although the incidence was slightly higher than the historical Control range, this abnormality is seen in this strain of rats in this laboratory and as such the incidence was considered likely to be spontaneous and not an adverse effect of test substance administration.
At 1000 mg/kg/day, there were significantly higher incidences of the minor defects, irregular ridging of the palate (p<0.01) and incomplete ossification of the nasal bones (p<0.001), compared with Control, both of which were outside the historical Control range.
At 150 mg/kg/day, there were marginal increases in the incidences of minor abnormalities of the cervical vertebrae and incomplete ossification of the nasal bones compared with the Controls which correlated with increases seen also at 1000 mg/kg/day. These incidences were also above the historical Control range.
The findings at 150 or 1000 mg/kg/day related to ossification are considered to be transient in nature and/or of no developmental consequence. The increased incidences of other skeletal and cartilage minor foetal abnormalities and variants were clearly test item-related and considered to represent an adverse effect of the test item on foetal development.

Overall developmental toxicity

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

Any other information on results incl. tables

Table 1: Foetal findings with statistical significance

 

 

 

Dose level (mg/kg/day)

 

 

 

Background data range

Finding

 

0

15

150

1000

 

Oral cavity – palate: irregular ridging

No Foetuses

No Litters

5 (4.8)

5

5 (5.1)

4

7 (6.0)

6

20 (20.2)**

13***

1.7-8.5

2-5

Skull- nasal- uni- or bilateral: incomplete ossification

No Foetuses

No Litters

3 (2.5)

2

5 (5.4)

3

7 (7.1)

4

73 (71.8)***

19***

2.1-6.3

1-6

Skull- jugal and maxilla- uni or bilateral: partial fusion

No Foetuses

No Litters

0 (0.0)

0

0 (0.0)

0

0 (0.0)

0

5 (5.1)*

3*

0.6-3.1

1-2

Cervical vertebra – one

or more neural arch:

misshapen

No Foetuses

No Litters

2 (1.0)

2

7 (3.5)

5

12 (6.5)*

7

63 (31.8)***

18***

3.6-3.6

4-4

Cervical vertebra –

cartilaginous ventral

plate - uni- or bilateral:

fused

No Foetuses

No Litters

1 (0.4)

0

0 (0.0)

0

3 (1.5)

3

10 (4.8)***

2

0.3-1.0

1-2

Cervical vertebra –

additional cartilaginous

ventral plate – uni- or

bilateral: fused

No Foetuses

No Litters

1 (0.4)

0

0 (0.0)

0

3 (1.5)

3

10 (4.6)***

8***

0.7-0.7

1-1

Rib – uni – or bilateral:

cervical

No Foetuses

No Litters

6 (3.0)

5

11 (7.8)

6

12 (6.4)

7

71 (33.4)***

16***

1.2-12.6

2-9

Rib – one or more: costal

cartilage fused severe

No Foetuses

No Litters

0 (0.0)

0

1 (0.5)

1

0 (0.0)

0

13 (6.0)***

6***

0.9-0.9

1-1

Rib – cervical rib – left:

costal cartilage fused

severe

No Foetuses

No Litters

0 (0.0)

0

0 (0.0)

0

0 (0.0)

0

8 (3.9)***

6***

0.0-0.0

0

Rib – cervical rib – uni- or

bilateral: with costal

cartilage

No Foetuses

No Litters

2 (0.9)

1

2 (1.1)

1

1 (0.6)

1

26 (11.9)***

9***

0.8-3.4

1-3

Sternum – 2nd sternebra:

incomplete ossification

No Foetuses

No Litters

2 (0.7)

2

3 (1.4)

3

2 (0.9)

1

8 (3.7)

6*

0.8-8.9

1-7

Cervical vertebra –

cartilaginous ventral

plate – uni- or bilateral:

incomplete

No Foetuses

No Litters

1 (0.4)

1

0 (0.0)

0

2 (0.9)

2

11 (5.5)***

9***

0.8-1.7

1-3

Cervical vertebra –

cartilaginous ventral

plate – uni- or bilateral:

on 5th cervical vertebra

No Foetuses

No Litters

1 (0.4)

1

5 (2.5)

4

10 (5.7)

7*

57 (29.1)***

16***

0.9-5.9

2-5

Cervical vertebra –

cartilaginous ventral

plate – uni- or bilateral:

misshapen

No Foetuses

No Litters

0 (0.0)

0

2 (1.0)

2

2 (1.2)

1

12 (6.5)***

8***

0.9-0.9

1-1

Cervical vertebra –

additional cartilaginous

ventral plate – uni- or

bilateral: on 5th cervical

vertebra

No Foetuses

No Litters

1 (0.4)

1

0 (0.0)

0

3 (1.5)

3

19 (9.2)***

14***

0.3-2.7

1-2

Sternum – 5th sternebra:

not ossified

No Foetuses

No Litters

1 (0.4)

1

8 (6.5)*

7

12 (6.5)*

7

97 (45.8)***

17***

1.1-10.7

1-7

Sternum – 5th sternebra:

incomplete ossification

No Foetuses

No Litters

6 (2.9)

5

6 (3.7)

4

19 (9.7)

8

24 (12.7)***

14***

1.7-15.3

2-11

Sternum – 6th sternebra:

not ossified

No Foetuses

No Litters

1 (0.4)

1

5 (2.6)

1

1 (0.5)

1

10 (4.6)**

7**

0.3-4.7

1-4

Sternum – 6th sternebra:

incomplete ossification

No Foetuses

No Litters

5 (2.4)

3

8 (4.9)

4

2 (1.2)

2

18 (7.8)

8*

2.1-21.5

2-12

Forelimb – 5th

metacarpal – uni- or

bilateral: not ossified

No Foetuses

No Litters

33 (15.9)

12

34 (21.5)

12

21 (10.5)

12

80(39.1)**

17*

2.5-36.0

3-16

Figures in brackets are group mean percent values

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

Applicant's summary and conclusion

Conclusions:
Administration of the test substance to the pregnant Crl:WI(Han) rat at 15, 150 or 1000 mg/kg/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, elicited slight maternal toxicity at 1000 mg/kg/day in terms of reduced food intake. A slightly lower mean foetal weight was observed at a dose level that induced maternal toxicity. There were no test item related major foetal abnormalities, at any dose level, however, an increased incidence of minor foetal abnormalities and variants was noted at both 150 and 1000 mg/kg/day above both the concurrent and historical Control ranges. Based on the above findings, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was considered to be 1000 mg/kg/day and NOAEL for embryo-foetal development was considered to be 15 mg/kg/day.
Executive summary:

The purpose of this study was to investigate the effects of the test item on the pregnant rat and developing organism when administered by oral gavage daily, from Day 6 to Day 19 of gestation, inclusive.

Four groups of 22 sexually mature, timed-mated female Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg/day test substance daily, by oral gavage, from Day 6 to Day 19 of gestation, inclusive, at a dose volume of 10 mL/kg body weight. Body weights, food intake and clinical observations were recorded. All animals were killed on Day 20 of gestation, a necropsy was performed and the internal organs examined for gross abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral, skeletal and cartilage abnormalities.

There were no deaths or adverse clinical observations during the study. Yellow stained bedding was observed in the cages of females given 1000 mg/kg/day. Group mean body weights and body weight gains were similar to Controls at all dose levels. There was no adverse effect of the test substance on terminal body weight when adjusted for the weight of the gravid uterus. Females in the group given 1000 mg/kg/day ate less than the Controls from Day 6 to 15 of gestation (the difference was statistically significant) so that their overall mean food intake for the entire dosing period was 13% lower than the Controls. At 15 or 150 mg/kg/day mean food intake was similar to Control values. There were no test item-related maternal necropsy findings.

There were 21, 19, 21 and 20 females in the groups given 0, 15, 150 or 1000 mg/kg/day, respectively, with live foetuses on Day 20 of gestation. The mean number of live foetuses per female was similar in all groups. Uterine/implantation parameters at 15 or 150 mg/kg/day were comparable with Controls. At 1000 mg/kg/day group mean foetal weight was slightly, but statistically significantly, lower than Control values (-9 %). There was no effect on foetal weight at 15 or 150 mg/kg/day. Mean placental weight and sex ratio were similar in all groups. There were no major foetal abnormalities considered to be test item-related. At 150 and 1000 mg/kg/day, there were higher incidences of minor and variant foetal abnormalities compared with Control that affected the cervical vertebrae, ribs, sternebrae, skull and palate. These significantly higher incidences of several skeletal minor abnormalities and/or variants (affecting skull, cervical vertebrae, ribs and sternebrae) were outside of the historical Control range seen within this laboratory and were considered to be related to the test item. Increases in the minor or variant foetal abnormalities associated with skeletal ossification were considered to be transient in nature and/or of no developmental consequence.

Administration of the test substance to the pregnant Crl:WI(Han) rat at 15, 150 or 1000 mg/kg/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, elicited slight maternal toxicity at 1000 mg/kg/day in terms of reduced food intake. A slightly lower mean foetal weight was observed at a dose level that induced maternal toxicity. There were no test item related major foetal abnormalities at any dose level, however, an increased incidence of minor foetal abnormalities and variants was noted at both 150 and 1000 mg/kg/day above both the concurrent and historical Control ranges.

Based on the above findings, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was considered to be 1000 mg/kg/day and NOAEL for embryo-foetal development was considered to be 15 mg/kg/day.