Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 December 2014 -- 03 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

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:
other: turbid liquid
Details on test material:
- Name of test material: Luperox 610
- Active material:
. Chemical name : 3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate
. CAS number : 95718-78-8
- Physical state: turbid liquid
- Lot/batch No.: 99676 E12-401
- Analytical purity: 89.9% of active material
- Expiry date: 07 June 2015
- Storage conditions: at -20°C and protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation/Mean body weight at study initiation: the animals were 10-11 weeks old and had a mean body weight of 264 g (range: 226 g to 320 g).
- Housing: polycarbonate cages, individually
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 4 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 29 December 2014 to 03 February 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Care was taken to not use glass bottles but HDPE (high-density polyethylene) bottles (for reconditioning) and PP (polypropylene) bottles (for formulations).

The test item was administered as an emulsion in the vehicle.
The test item was thawed at +2-8°C and homogenized before sampling. Then, it was mixed with the required quantity of vehicle (considering a test item density of 1). The entire formulation preparation process was performed on ice (test item, vehicle and formulation bottles kept on ice; formulation homogenization and aliquoting into daily flasks performed on ice) with the exception of the weighing. The test item and formulations were never at room temperature except for test item weighing during formulation preparation.

Dose formulations were prepared for use up to 9 days, stored at +2-8°C and protected from light and delivered to the study room on ice and protected from light.

VEHICLE
- Concentration in vehicle: 10, 50, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Homogeneity/Stability: the homogeneity and stability was checked on a range of dose formulations prepared at levels which cover the lowest and highest concentrations used in this study.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: detection of a vaginal plug
Duration of treatment / exposure:
day 6 to day 20 post-coitum
Frequency of treatment:
Daily
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg/day
Basis:
actual ingested
the dose-levels are expressed in active material (3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate) and correspond to the doses of 55.6, 278 and 1112 mg Luperox 610/kg/day, taking into account the correction factor of 1.112.
No. of animals per sex per dose:
24 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of two studies:
- a preliminary embryo-foetal development toxicity study, where four groups of eight time-mated Sprague-Dawley females were given the test item in 0.5% carboxymethylcellulose at +2-8°C at 0, 100, 300 or 1000 mg/kg/day from Days 6 to 20 p.c.. There was no mortality. Ptyalism was recorded in all females treated at 300 and 1000 mg/kg/day. Two females treated at 1000 mg/kg/day also had transient hypoactivity. There were no toxicological significant effects in mean body weight and mean food consumption, except lower food consumption in the first week of treatment at 1000 mg/kg/day. Mean liver weight was increased at 1000 mg/kg/day with one liver noted as enlarged at necropsy. However, absence of homogeneity of the formulations used in this study was demonstrated in concomitant assays,
- a preliminary 2-week toxicity study, where the test item was administered daily by gavage to Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day for 2 weeks in corn oil. At 1000 mg/kg/day, findings were limited to ptyalism in both sexes and lower body weight gain and food consumption at treatment initiation in males. Non-adverse higher liver weights were recorded in females. At 100 and 300 mg/kg/day, findings were limited to ptyalism in isolated animals.

- Rationale for animal assignment: computerized stratification procedure

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION:
- Time schedule: on Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs.


Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight,
- Number of corpora lutea,
- Number of implantations,
- Number and distribution of early resorptions,
- Number and distribution of late resorptions,
- Number and distribution of dead and live fetuses,
- Number and distribution of uterine scars,
- Evaluation of placenta.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : number dead and live, body weight, sex
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at 1000 mg/kg/d

Details on maternal toxic effects:
There were no effects of test item treatment on the pregnancy status (Table 6).

There were no unscheduled deaths.

Ptyalism recorded in most females at 250 mg/kg/day and in all females at 1000 mg/kg/day for several days (from 2 to 15 days) during the treatment period was considered to be of minor toxicological significance.
At 1000 mg/kg/day, there were 2/24 females with pallor of extremities together with round back or piloerection, reddish vaginal discharge and hypoactivity in the last 2 or 5 days of treatment, respectively. There were also two other females with piloerection from Day 19 or 20 p.c.. These clinical signs were considered to be toxicologically relevant.
At 250 mg/kg/day, one female had loud breathing from Day 16 p.c. and piloerection, hypoactivity and emaciated appearance from Day 18 p.c.. At the same period, its food consumption was close to zero and the female lost -20% of body weight. This bad clinical condition was not considered to be related to the test item treatment but to the perforation of oesophagus seen at necropsy.
The other clinical signs noted in test item-treated groups (reddish vaginal discharge on Days 15-16 p.c., reflux at dosing, areas of hair loss and cutaneous lesion) are commonly observed findings in rats of this strain when housed in laboratory conditions and were of isolated incidence (Table 1).

At 1000 mg/kg/day and when compared with controls, mean body weights were statistically significantly lower throughout the entire treatment period (down to -15% at termination). Mean body weight gain over the treatment period was at -40% from controls. These effects were attributed to the test item treatment and considered to be adverse.
There were no effects on mean body weight or mean body weight change at 50 and 250 mg/kg/day (Table 2).

At 1000 mg/kg/day and when compared with controls, there were statistically significant lower mean food consumptions throughout the entire treatment period (down to -45% on Days 6 to 9 p.c., p<0.001). This effect was attributed to the test item treatment and considered as adverse.
At 50 and 250 mg/kg/day, there were no effects on mean food consumption (Table 3).

At 1000 mg/kg/day and when compared with controls, females had a lower mean gravid uterus weight (-16%, p<0.05) and mean carcass weight (-15%, p<0.001) and, a mean net body weight loss (-2.8 g vs. +39.8 g, p<0.001), correlating with the effects on mean body weight and mean fetal body weight. These findings were considered to be test item treatment-related and adverse (Table 4).
At 50 and 250 mg/kg/day, there were no test item treatment-related effects on mean gravid uterus weight, mean carcass weight and mean net body weight change from Day 6 p.c.. The slightly lower mean net body weight at 250 mg/kg/day when compared with controls was due to one female with the oesophagus perforation.

At 1000 mg/kg/day, more than half of the females had thickened mucosa of the duodenum (rarely jejunum) and dilatation of the duodenum (rarely jejunum). Three of them also had whitish mucosa of the duodenum. These effects on intestines were considered to be test item treatment-related. More than half of the females had also reduced-in-size thymus.
Blackish mandibular lymph node(s) was also recorded in three of these females and enlarged adrenals in two of these females. A relationship to the test item treatment cannot be excluded but was considered as non-adverse.
At 250 mg/kg/day, one female had the same necropsy findings as at 1000 mg/kg/day. This was thus ascribed to the test item treatment but considered as non-adverse in view of the isolated incidence. The necropsy findings of one female were considered to be related to a gavage error and not to the test item treatment.
There were no test item treatment-related necropsy findings at 50 mg/kg/day (Table 5).

There were no effects on mean hysterectomy data (Table 6).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 other: mg/kg/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
250 other: mg/kg/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at 1000 mg/kg/d

Details on embryotoxic / teratogenic effects:
There were no effects on sex ratio (percentage of male fetuses).

At 1000 mg/kg/day and when compared with controls, there were lower mean fetal body weights in both sexes which were considered to be test item treatment-related and adverse.
At 50 and 250 mg/kg/day, there were no toxicologically relevant effects on mean fetal body weights. The very low mean body weight of one litter (2.85 g) was considered to be a consequence of the bad health condition of the mother following oesophagus perforation (Table 7).

There were no fetal external variations.
The only fetal external malformation was narrowed tail noted in one fetus at 250 mg/kg/day and in one fetus at 1000 mg/kg/day. The significance of this finding is discussed below.

No fetal soft tissue variations (dilated cerebral ventricle, whitish nodule on liver, dilated renal pelvis and reddish foci on thymus) were considered to be test item-related in view of the absence of dose-relationship and/or of the isolated litter incidence.
The only fetal soft tissue malformation was hydropericardium noted in one fetus at 1000 mg/kg/day. The significance of this findings is discussed below.

At 1000 mg/kg/day and when compared with controls, statistically significantly higher incidences of fetuses with skeletal variations in presence of effects on mean fetal body weights indicated the beginning of ossification delays at this dose which was considered to be secondary to the maternal toxicity and of limited toxicological significance.
At 250 mg/kg/day, most of the skeletal variations indicating ossification delays concerned litter of female with oesophagus perforation correlating with its low mean body weight and were considered to be a consequence of the bad health condition of the mother following oesophagus perforation.
The higher fetal incidence of ossification point on 14th thoracic vertebra was considered to be of no toxicologically relevance in absence of statistical significance on litter incidence and of test item treatment related effects on mean fetal body weight at this dose (Table 8).
At 50 mg/kg/day, there were no test item treatment-related fetal skeletal variations.

At 250 mg/kg/day, litter of female with oesophagus perforation had one fetus with unossified interparietal and unossified centrum of thoracic vertebrae and another fetus with unossified centrum of thoracic vertebrae (Table 9).
Absent lumbar vertebra was recorded in one fetus of each group treated at 50 or 250 mg/kg/day.
The significance of these findings is discussed below.

There were no dose-related increases in the incidences of litters with malformed fetuses (Table 10).
Moreover, a relationship of skeletal malformations in litter of female with oesophagus perforation with the test item treatment was considered to be unlikely. The mother was found with oesophagus perforation and consequent body weight loss of -20% at the end of the gestation period. The bone non-ossifications were not observed in any other litters or doses.
Therefore, there were no test item treatment-related malformations in the study.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 other: mg/kg/day
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1. Most relevant clinical signs observed in females

Dose-level (mg/kg/day)

0

50

250

1000

Ptyalism

 

 

18

24

Round back

 

 

 

1c

Pallor of extremities

 

 

 

2b,c

Reddish vaginal discharge

 

 

 

2b

Piloerection

 

 

1a

3b

Hypoactivity

 

 

1a

1b

Emaciated appearance

 

 

1a

 

Loud breathing

 

 

1a

 

a: one female; b: including one female, c: including one female.

Table 2 . Mean body weights and body weight changes (g)

Dose-level (mg/kg/day)

0

50

250

1000

Body weight (g)

 

 

 

 

Day 6 p.c.

267

263

264

262

 

 

(-1)

(-1)

(-2)

Day 9 p.c.

279

277

276

264*

Day 12 p.c.

301

300

297

283*

Day 15 p.c.

321

319

317

297**

Day 18 p.c.

360

356

352

328**

Day 21 p.c.

411

410

394

348#

 

 

(0)

(-4)

(-15)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+13

+14

+12

+2#

Days 12 - 15 p.c.

+21

+19

+20

+14*

Days 18 - 21 p.c.

+51

+54

+42
(+46 excluding one female with oesophagus perforation)

+20#

Days 6 - 21 p.c.

+144

+147

+130
(+136 excluding one female with oesophagus perforation)

+86#

Statistically significantvs.controls:*: p<0.05, **: p<0.01, #: p<0.001;in brackets and italic, percentage differences vs. controls.

Table 3. Mean food consumptions (g/female/day) 

Dose-level (mg/kg/day)

0

50

250

1000

. Days 6 - 9 p.c.

22

22

21

12#

. Days 9 - 12 p.c.

25

24

23

20#

. Days 12 - 15 p.c.

26

25

25

21#

. Days 15 - 18 p.c.

26

27

25

21*

. Days 18 - 21 p.c.

26

24

23

17#

Statistically significant vs.controls:*: p<0.05, #: p<0.001.

Table 4. Mean gravid uterus, carcass and net body weights (g)

Dose-level (mg/kg/day)

0

50

250

1000

Gravid uterus weight

105

107

97.1

88.7*

Carcass weight

306

304

297
(301
excluding female with oesophagus perforation)

259#

Net body weight change from Day 6 p.c.

39.8

40.0

33.0
(37.5 excluding female with o
esophagus perforation)

-2.8#

Statistically significant vs.controls:*: p<0.05, #: p<0.001. 

Table 5. Macroscopic post mortem examination 

Dose-level (mg/kg/day)

0

50

250

1000

Thymus: reduced in size

 

 

 

14#

Intestines (jejunum or mainly duodenum): thickened mucosa

 

 

 

13#

Intestines (jejunum or mainly duodenum): dilated

 

 

1

12#

Intestines (duodenum): whitish mucosa

 

 

1

3

Mandibular lymph node(s): blackish

 

 

 

3

Adrenals: enlarged

 

 

1a

2

Oesophagus: perforation

 

 

1a

 

Heart: pericardium: whitish deposit

 

 

1a

 

Statistically significantvs.controls: #: p<0.001; a: one female.

Table 6. Mean hysterectomy data

Dose-level (mg/kg/day)

0

50

250

1000

Number of females with live fetuses at term

23

24

24

23

Number ofcorpora luteaper female

15.2

15.9

14.5

15.9

Number of implantations per female

13.9

14.4

13.1

13.9

Pre-implantation loss per female (%)

9.0

8.7

10.0

11.7

Number of fetuses per female

13.5

13.5

12.5

12.9

Percentage of dead fetuses (%)

0

0

0

0

Number of uterine scars + resorptions per female

0.4

0.9

0.6

1.0

Number of early resorptions per female

0.4

0.8

0.6

0.9

Post-implantation loss per female (%)

3.5

6.3

5.5

8.2

 

Table 7. Mean fetal body weights per litter (g)

Dose-level (mg/kg/day)

0

50

250

1000

Fetal body weight (males + females)

5.70

 

5.78

(+1)

5.64

(-1)

(5.76 excluding litter D25228)

4.99#

(-12)

In brackets and italic, percentage differences form controls; statistically significantvs.controls: #: p<0.001.

Table 8. Most relevant skeletal variations

Dose-level (mg/kg/day)

0

50

250

1000

Litters evaluated

23

24

24

23

Fetuses evaluated

163

169

155

154

Ossification point on 14ththoracic vertebra, F(L)

1 (1)

7 (2)

8* (5)

7* (3)

Cervical vertebra(e): unossified centrum, F(L)

3 (3)

4 (4)

10* (4)

14** (8)

Caudal vertebra(e): incomplete ossification of centrum, F(L)

2 (2)

1 (1)

4 (2)

11** (8)

Incomplete ossification of 6thsternebra, F(L)

1 (1)

 

4 (2)

7* (3)

Unossified 1stmetatarsal,F(L)

12 (8)

10 (5)

19 (7)

32# (10)

Statistically significant vs.controls: *: p<0.05, **: p<0.01, #: p<0.001.

F(L): Fetal (Litter) incidences.

 

Table 9. Fetal skeletal malformations

Dose-level (mg/kg/day)

0

50

250

1000

Litters evaluated

23

24

24

23

Fetuses evaluated

163

169

155

154

Interparietal: unossified, F(L)

 

 

1 (1)a

 

Thoracic vertebra(e): unossified centrum, F(L)

 

 

2 (1)a

 

Lumbar vertebra(e): absent, F(L)

 

1 (1)

1 (1)

 

a: litter D25228.

Table 10. Distribution of malformations across litters and fetuses  

Dose-level (mg/kg/day)

0

50

250

1000

Number of females with live fetuses at term

23

24

24

23

Number of fetuses with external examination

310

324

300

296

Number of fetuses with soft tissues examination

147

155

145

142

Number of fetuses with skeletal examination

163

169

155

154

Narrowed tail(a)

 

 

D25230-10

D25254-2

Hydropericardium(b)

 

 

 

D25250-2

Lumbar vertebra(e): absent(c)

 

D25195-1

D25222-1

 

Interparietal: unossified(c)

 

 

D25228-7

 

Thoracic vertebra(e): unossified centrum(c)

 

 

D25228-7

D25228-5

 

Number of litters affected

0 (0%)

1 (4.2%)

3 (12.5%)

2 (8.7%)

Number of fetus affected

0 (0%)

1 (0.6%)

4 (2.9%)

2 (1.0%)

(a): external examination; (b): soft tissues examination; (c): skeletal examination.

 

Applicant's summary and conclusion

Conclusions:
The test item was administered by oral gavage daily from Day 6 to Day 20 p.c. inclusive to time-mated female Sprague-Dawley rats at doses of 50, 250 and 1000 mg/kg/day.
Under the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day, based on the absence of adverse effects in the dams in this group and on the adverse effects seen at 1000 mg/kg/day (body weight, food consumption and/or necropsy findings),
- the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day, based on the absence of adverse effects in litters in this group and on a lower mean fetal body weight at 1000 mg/kg/day, in the context of maternal toxicity,
- no teratogenic effect was observed.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of Luperox 601 on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.) inclusive). The study was performed based on the OECD guideline No. 414 and in compliance with the Good Laboratory Practices. Three groups of twenty-four time-mated Sprague-Dawley rats were administered with the test item, daily during the period of organogenesis (from Day 6 to Day 20 p.c. inclusive), by gavage at doses of 50, 250 or 1000 mg/kg/day (expressed in active material: 3‑hydroxy-1,1-dimethylbutyl peroxyneodecanoate). One additional group of 24 time-mated females received the vehicle (corn oil) under the same experimental conditions. A dose-volume of 5 mL/kg/day was used. Formulation concentrations were checked at the beginning and at the end of the study.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 or 3 days during the study. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal/cartilage abnormalities.

The test item concentrations in the administered dose formulations analyzed were within an acceptable range of variation when compared to the nominal concentrations(± 15%). No test item was observed in the control dose formulation. There were no effects on pregnancy status. At termination on Day 21 p.c., there were 23/24, 24/24, 24/24 and 23/24 females with live fetuses at 0, 50, 250 and 1000 mg/kg/day, respectively. There were no unscheduled deaths. Ptyalism noted at 250 and 1000 mg/kg/day was considered to be of minor toxicological significance. At 1000 mg/kg/day and towards the end of the treatment period, there were 3/24 females with piloerection and 2 had pallor of extremities, round back, reddish vaginal discharge and/or hypoactivity. These clinical signs were considered to be toxicologically relevant. There were no test item treatment-related clinical signs at 50 mg/kg/day. At 1000 mg/kg/day, mean body weights were lower throughout the entire treatment period (-15% vs. controls for terminal mean body weight, p<0.001). This correlated with lower mean food consumptions throughout the entire treatment period (down to -45% on Days 6 to 9 p.c., p<0.001) and, with lower mean gravid uterus weight and mean carcass weight and with a mean net body weight loss.These effects were considered to be adverse. At 50 and 250 mg/kg/day, there were no effects on mean food consumption, mean body weight, mean body weight change, mean gravid uterus weight, mean carcass weight or mean net body weight change. At necropsy at 1000 mg/kg/day, more than half of the females had thickened mucosa and dilatation of the duodenum (rarely of jejunum). Three of them also had whitish mucosa of the duodenum. More than half of the females had also reduced-in-size thymus. These effects were considered to be test item treatment‑related.

Blackish mandibular lymph node(s) and enlarged adrenals were also noted in a few of these females; a relationship to the test item treatment cannot be excluded but was considered as non-adverse. At 250 mg/kg/day, similar necropsy findings were observed in one female and ascribed to the test item treatment but considered as non-adverse in view of the isolated incidence. There were no test item treatment-related necropsy findings at 50 mg/kg/day. At hysterectomy, there were no test item treatment-related effects in any dose-level.

There were no effects on mean percent of male fetuses (sex ratios). There were no fetal external variations and no test item treatment-related fetal soft tissue variations or external, soft tissues and skeletal malformations. There were also no test item treatment-related effects on cartilages.

At 1000 mg/kg/day, there were lower mean fetal body weights in both sexes (-12% vs. controls, p<0.001), associated with ossification delays (of limited toxicological significance), most probably secondary to the lower mean body weight gain and mean food consumption of the dams.

At 50 and 250 mg/kg/day, there were no relevant effects on mean fetal body weights and no relevant skeletal variations.

Under the experimental conditions of this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day, based on the absence of adverse effects in the dams in this group and on the adverse effects seen at 1000 mg/kg/day (body weight, food consumption and/or necropsy findings),

- the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day, based on the absence of adverse effects in litters in this group and on a lower mean fetal body weight at 1000 mg/kg/day, in the context of maternal toxicity,

- no teratogenic effect was observed.