Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 January 2017 to 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on test animals and environmental conditions:
- Number: 88 virgin mated females. The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on Day 0 of gestation (G 0).
- Strain and sanitary status: Wistar rats: Crl: WI (Han). All animals received a clinical inspection for ill-health on arrival.
- Breeder: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age/Weight:
Age at mating: 10 to 13 weeks.
Body weight range at mating: 189 to 238 g
- Housing: One air-conditioned room in a barrier protected unit (building K1), Caging: Females were individually housed in plastic cages (dimensions 375 x 215 x 180 mm) meeting European directive 2010/63/EU requirements.
- Food and water: Rat powdered/pelleted commercial complete rodent diet ad libitum (Diet reference SAFE A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian. The rats had free access to the food throughout the study. Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation: no information
- Allocation to group: Performed by random assignment after arrival (computer-generated random numbers).
- Identification: Animals were identified using transponder implants (Biolog-id).

ENVIRONMENTAL CONDITIONS
- temperature: 22 + 3 °C (target range),
- relative humidity: > 35 % (target),
- light/dark cycle: 12 hours light (artificial)/12 hours dark,
- ventilation: At least 10 air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
DOSE FORMULATION PREPARATION
Preparation: The test item was prepared as a suspension in the vehicle at concentrations of 50, 100 and 200 mg/mL, according to Standard Operating Procedures of the Test Facility. No correction factor was taken into account for dose calculations.
Frequency of preparation: Weekly.
Storage of formulations (including vehicle): Refrigerated (between +2 and +8 °C).
Stability of the test item in the vehicle: Formulations, protected from light and in the range 5-200 mg/mL, were demonstrated to be stable for 24 hours at room temperature (between +15 and +25 °C), for 8 days refrigerated (between +2 and +8 °C) and for 3 weeks under frozen conditions (between -15 and 25 °C) (Test Facility study no. AB21611).



VEHICLE
The vehicle was propylene glycol..
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on Day 0 of gestation (G 0).

Duration of treatment / exposure:
The dose formulations were administered daily from day 6 to day 20 p.c., inclusive. Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.
Frequency of treatment:
Daily
Duration of test:
One month
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
Dose / conc.:
500 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
dose volume : 5 ml/kg/day
No. of animals per sex per dose:
22 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the DRF phase.




Examinations

Maternal examinations:
MORBIDITY AND MORTALITY:
All animals were observed at least twice daily.

CLINICAL SIGNS:
All animals were observed daily for clinical signs during the study.
During the treatment period, the animals were observed before and at least twice after dosing to detect any abnormalities in appearance, behaviour or other signs of reaction to treatment. A full clinical examination was performed on each weighing day.

BODY WEIGHT:
All animals were weighed individually on Days 0, 6, 9, 12, 15, 18 and 21 of gestation .

FOOD CONSUMPTION:
Individual food consumption was measured for the periods (Days) 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 during gestation .

POST-MORTEM EXAMINATIONS: Yes
All females were weighed before necropsy and killed by carbon dioxide inhalation followed by exsanguination on Day 21 of gestation, on the day after the last treatment. They were dissected and examined for macroscopic pathological changes.
Any abnormal organs/tissues were sampled and preserved in 10 % neutral formalin. No further examinations were performed.

Ovaries and uterine content:
The ovaries and uterus of each female were removed and examined.
The placentae were also examined. The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of implantations
- number and distribution of live foetuses
- number and distribution of intrauterine implantations, classified as follows:
- live foetuses
- dead foetuses
- early resorptions
- late resorptions
- gravid uterus weight
- individual foetal weights
- foetal sex.

Resorptions were classified on the basis of the presence (late) or absence (early) of foetal or decidual tissue in addition to placental tissue.
The uterus of all females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.




Fetal examinations:
Each foetus was examined for external defects. All foetuses were killed by an oral administration of sodium pentobarbitone (CEVA Santé Animale).
Approximately one half of each litter was submitted to fresh visceral examination of the body (abdominal and thoracic cavities) and then processed for skeletal examination. The ossified skeleton was stained with Alizarin red following maceration of the soft tissues in aqueous potassium hydroxide solution. The stained specimens were preserved in glycerol.
The remaining foetuses were preserved in Harrisson's fluid for subsequent examination of the head only by the modified Wilson-Barrow technique. The remaining carcass was retained fixed but not examined further.
Soft tissue and skeletal examinations were performed using a binocular microscope.
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the normality of the data distribution tested by the Shapiro-Wilk's test
- the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were 10 out 22 females given 1000 mg/kg/day that showed transient but adverse clinical signs including irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour. These clinical signs were observed mainly shortly after the first or second dosing (G 6 and G 7).
There was a higher incidence and persistence of hypersalivation associated or not with abnormal foraging and/or pedalling in all treated groups (21/22, 22/22 and 22/22 females in the 250, 500 and 1000 mg/kg/day groups, respectively) when compared with the control group (6/22 females). Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.
There were no other treatment-related clinical signs in any group.
Wheezing was noted for control female no. 101 on G 17. This isolated finding in the control group was considered incidental. Other incidental clinical signs included hairloss and scab(s).
Mortality:
no mortality observed
Description (incidence):
There is no unscheduled deaths in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although the mean body weight gain was slightly lower from G 6 to G 9 for females given 1000 mg/kg/day (9.7 g) than for other groups (13.0, 13.0 and 11.1 g in the control, 250 and 500 mg/kg/day groups, respectively), the mean final body weight was comparable in all groups. This slight decrease is considered as non-adverse.
There were no treatment-related effects on mean body weight gain during the dosing period (G 6 to G 21) in other groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slightly lower mean food consumption between G 6 and G 12 for females given 1000 mg/kg/day (19.6 g/day) than in other groups (20.8, 21.8 and 21.4 g/day in the control, 250 and 500 mg/kg/day groups, respectively). This was no longer observed thereafter.
There was no treatment-related effect on food consumption during the dosing period (G 6 to G 21) in other groups.
This slight and transient decrease is not considered as adverse.

Food efficiency:
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:
no effects observed
Description (incidence and severity):
There was no treatment-related effect on gravid uterus weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings in any group.
A cyst on the left ovary was noted for female no. 139 given 500 mg/kg/day and a dark focus on the liver of female no. 185 given 1000 mg/kg/day. These isolated findings were considered incidental.
Localized or diffuse alopecia was occasionally observed with a sporadic incidence.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
There were no unscheduled deaths in any group.
Transient but marked test item-related clinical changes were observed in 10 out of 22 females in the 1000 mg/kg/day group on G 6 or G 7 shortly after dosing. These included irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour.
Hypersalivation noted for essentially all females in the vehicle, 250, 500 and 1000 mg/kg/day groups, with or without abnormal foraging and/or pedalling were clearly associated with the vehicle (Polyethylene glycol 400) but might possibly be slightly exacerbated by the test item. Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.
There was a transient slightly lower mean body weight gain and mean daily food consumption for females given 1000 mg/kg/day compared with the concurrent control towards the initiation of treatment. The mean body weight at the end of the dosing period was, however, comparable in all groups.
There were no treatment-related macroscopic findings or effects on mean gravid uterus weight in any group.
There were 22 pregnant females at terminal caesarean in each of the control, 250, 500 and 1000 mg/kg/day groups, all of which had viable foetuses.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups, all of which had viable foetuses.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean numbers of corpora lutea and implantation sites were comparable in all groups. The pre-implantation loss was slightly higher for females given 250 and 500 mg/kg/day (12.14 and 12.92%, respectively) when compared with other groups (7.03 and 7.44% for group 1 and 4, respectively). However mean values were within the historical control range and these differences were considered incidental.
There was no test item-related effect on embryo-foetal survival in any group. Mean live litter size was consequently comparable in all groups.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups, all of which had viable foetus. There was no-test item effect on embryo-foetal survival in any group. Mean live litter size was consequently comparable in all groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 22 pregnant females at the terminal caesarean sections in the control and in each of the treated groups.
Details on maternal toxic effects:
Under the experimental conditions of the study, oral (gavage) administration of 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was associated with transient and non-adverse slightly lower body weight gain and food consumption at 1000 mg/kg/day. Transient but adverse clinical changes at the start of treatment in the 1000 mg/kg/day group included irregular and/or rapid breathing, partly closed eye(s), decreased activity, dark eyes, recumbency and /or subdued behaviour.
In view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean foetal weight in any group.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of treatment on sex ratio in any group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter size was comparable in all groups.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 3 foetuses from as many litters with external malformation in the 1000 mg/kg/day group compared with none in other groups.
One foetus had meningoencephalocele (foetus no.4 from dam no. 167), one had anasarca (foetus no 4 dam no. 174) and the other had anal atresia and thread-like tail (foetus no. 11 from dam no. 175).
The incidence of each external malformation was equal or lower to the maximal incidence per study in the historical control data. None of these observations were therefore clearly attributed to the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 5 foetuses from as many litters with skeletal malformations in the 1000 mg/kg/day group compared with none in other groups.
Consistent with the meningoencephalocele observed externally, the foetus no.4 from dam no.167 had interparietal and parietal misshapen associated with hole in cranium. This foetus also showed exoccipital fused with right first vertebral cervical arch.
Correlated with the anal atresia and thread-like tail noted at the external examination, the foetus no.11 from dam no.175 showed multiple skeletal abnormalities including sacral arch vertebra misshapen, 2nd sacral centrum absent, absence of the 3rd and 4th sacral vertebrae and the absence of all caudal vertebrae.
Foetus no.4 from dam no.174 with anasarca also had mandible fused and palatine split and one foetus from each of dams nos. 182 (foetus no. 7) and 188 (foetus no. 3) had fused sternebra or fused mandible, respectively.
All skeletal malformations were observed in the historical control data (2014-2016) with a maximal incidence comparable or higher to that of the 1000 mg/kg/day treated group. Therefore, these skeletal malformations can not be clearly related to an effect of the test item.
The incidence of other less severe skeletal anomalies and variations did not suggest any association with the test item.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In addition to the foetus no.4 (dam no.174) with multiple abnormalities of the heart, likely associated with the anasarca noted externally, 2 foetuses from two litters had visceral malformations in the 1000 mg/kg/day group, compared with none in other groups.
One foetus had retroesophageal subclavian artery (foetus no. 6 from dam no. 168) and the other one had a situs inversus (foetus no.13 from dam no. 174).
In view of the lower incidence of each visceral malformation when compared with the maximal incidence per study of the historical control data (2014-2016) , none of these malformations were considered clearly related to an effect of the test item.
The incidence of other less severe visceral anomalies and variations such as absent innominate artery, transposed umbilical artery and convoluted or dilated ureters did not suggest any association with the test item.

Details on embryotoxic / teratogenic effects:
There was no test item-related effect on embryo-foetal survival, foetal weight or foetal sex ratio in any group.
In total, the number of foetuses (litters) with external, skeletal and/or visceral malformations in the 1000 mg/kg/day group were 7/240 (6/22) leading to a foetal (litter) incidence of 2.9% (27%) compared with none in other groups. All malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the historical control data (2014-2016). In addition, in view of broad spectrum of these malformations (including general, head, caudal malformations and other visceral or skeletal malformations such as situs inversus, retroesophagial subclavian artery and sternebra or mandible fused), a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on a slight increase of the foetal and litter incidences of malformations at 1000 mg/kg/day which was more likely attributed to the maternal clinical signs.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of malformations – Individual descriptions:

 

Dose level (mg/kg/day)

Female identification number

Foetus identification number(s)

Malformation(s)#

0

/

/

No malformations observed

250

/

/

No malformations observed

500

/

/

No malformations observed

1000

167

4

Meningoencephalocele

Interparietal and parietal misshapen associated with hole in cranium

Exoccipital fused with right 1stvertebral cervical arch

168

6

Retroesophageal right subclavian artery (arising from descending aorta)

174

4

Anasarca

Multiple abnormalities of the heart (interventricular septum defect with aorta arising from right ventricle)

Mandible, fused

Palatine, split

13

Situs inversus (abdominal and thoracic cavities)

175

11

Anal atresia

Thread‑like tail

2ndsacral arch vertebra misshapen; 2ndsacral centrum absent; absence of the 3rdand 4thsacral vertebrae; absence of all caudal vertebrae

182

7

Sternebra, fused (3rdand 4th)

188

3

Mandible, fused

#: including external, visceral and skeletal examinations.

maximal foetal incidence per study or total incidence of external malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD (mean)

Head malformation

0.4 (meningoencephalocele)

0.4 (exencephaly)

0.04

Anasarca

0.4

0.5

0.12

Anal atresia

0.4

1

0.04

maximal foetal incidence per study or total incidence of visceral malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of

 malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD (mean)

Retroesophageal subclavian artery

0.8

1.8

0.05

Situs inversus

0.8

1.4

0.23

maximal foetal incidence per study or total incidence of skeletal malformations in the historical control data (2014-2016) compared with study no.AB21616 (in percent):

Type of malformation

Incidence in study AB21616

Maximal incidence per study in HCD

Total incidence in HCD

Mandible fused

1.6

1.8

0.1

Sternebra fused

0.8

0.8

0.05

Palatine split

0.8

0.8

0.1

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, oral (gavage) administration of 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin) to the pregnant Wistar rat from implantation through to the day before caesarean section at 250, 500 or 1000 mg/kg/day was associated with transient and non-adverse slightly lower body weight gain and food consumption at 1000 mg/kg/day. Transient but adverse clinical changes at the start of treatment in the 1000 mg/kg/day group included irregular and/or rapid breathing, partly closed eye(s), decreased activity, dark eyes, recumbency and /or subdued behaviour.
In view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat.
Post-implantation data were not affected by test item in any group.
A slight increase of the foetal and litter incidences of malformations was observed at 1000 mg/kg/day compared to the control. However, all these malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. Therefore a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect .
Based on these findings, the NOAEL for embryo-fetal development was considered to be 500 mg/kg/day.

Executive summary:

The objectives of the study were to determine the potential of the test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine the NOAEL (no observed-adverse effect level) for maternal toxicity and developmental toxicity.

The test item, 3-ethoxy-4-hydroxybenzaldehyde (Ethylvanillin), was administered by daily gavage at dose levels of 250, 500 and 1000 mg/kg/day to groups of 22 mated female Wistar rats from days 6 to 20 of gestation inclusive. A control group received a similar volume (5 mL/kg) of the vehicle Propylene Glycol. Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on day 21 of gestation and litter parameters were recorded. At necropsy, the females were examined macroscopically and the gravid uterus was weighed.

All foetuses were weighed, sexed and examined for external abnormalities. Half of the foetuses were submitted to fresh visceral examination of the body (abdominal and thoracic cavities) prior to processing for skeletal examination. The remaining foetuses were preserved for subsequent examination of the head only by the modified Wilson-Barrow technique.

All formulations at 50, 100 and 200 mg/mL of Test item: 3-ethoxy-4-hydroxybenzaldehyde in vehicle (Propylene Glycol) including the vehicle, used on the first day of treatment were in agreement with acceptance criteria. No significant amount of test item was detected in the vehicle sample. There were no unscheduled deaths in any group. Transient but marked test item-related clinical changes were observed in 10 out of 22 females in the 1000 mg/kg/day group on G 6 or G 7 shortly after dosing. These included irregular and/or rapid breathing associated or not with partly closed eye(s), wheezing, decreased activity or dark eyes. Among these animals, six displayed recumbency and /or subdued behaviour. Hypersalivation noted for essentially all females in the vehicle, 250, 500 and 1000 mg/kg/day groups, with or without abnormal foraging and/or pedalling were clearly associated with the vehicle (Polyethylene glycol 400) but might possibly be slightly exacerbated by the test item. Hypersalivation, with associated observations of abnormal foraging, pedalling and/or Straub tail, is commonly observed in rodents given oral formulations with poor palatability and is, therefore, considered of no toxicological significance.

There was a transient slightly lower mean body weight gain and mean daily food consumption for females given 1000 mg/kg/day compared with the concurrent control towards the initiation of treatment. The mean body weight at the end of the dosing period was, however, comparable in all groups. There were no treatment-related macroscopic findings or effects on mean gravid uterus weight in any group.

There were 22 pregnant females at terminal caesarean in each of the control, 250, 500 and 1000 mg/kg/day groups, all of which had viable foetuses. Post-implantation data were not affected by test item in any group. There was no test item-related effect on embryo-foetal survival, foetal weight or foetal sex ratio in any group. In total, the number of foetuses (litters) with external, skeletal and/or visceral malformations in the 1000 mg/kg/day group were 7/240 (6/22) leading to a foetal (litter) incidence of 2.9 % (27 %) compared with none in other groups. All malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. In addition, in view of broad spectrum of these malformations (including general, head, caudal malformations and other visceral or skeletal malformations such as situs inversus, retroesophagial subclavian artery and sternebra or mandible fused), a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect in view of the marked clinical signs observed on the first days of dosing at the high dose of 1000 mg/kg/day.

In conclusion, in view of the nature of the clinical signs, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was established as 500 mg/kg/day in the female rat. Some malformations were observed at 1000 mg/kg/day compared with none in the control group. However, all these malformations were already noted in our more recent historical control data with an incidence comparable or lower to the maximal incidence per study in the HCD. Therefore a test item-related effect was doubtful and the malformations were more likely attributed to a maternal mediated effect. Based on these findings, the NOAEL for embryo-fetal development was considered to be 500 mg/kg/day.