Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2017 - 06 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age at the beginning of the treatment period: approximately 18-20 weeks old on the day of treatment
- Mean body weight at the beginning of the treatment period: 3681 g (range: 3215 g to 4170 g)
- Fasting period before study: no
- Housing: The animals were individually housed in noryl cages (Tecniplast, 4200 cm2).
- Diet: (SAFE, Augy, France) pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8h/16h

IN-LIFE DATES: 29 May 2017 to 06 July 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose (1%) in drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 33.3, 100 and 333 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with UV detection (HPLC-UV)
Test item concentrations: within an acceptable range of variation (-10.4% to -2.7%) when compared to the nominal values (± 15% required).
Homogeneity: Dose formulations ranging from 2 mg/mL to 333.3 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies within the day of the preparation.
Stability: not assessed, dose formulation prepared daily.
Details on mating procedure:
The females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.
Duration of treatment / exposure:
The dose formulations were administered daily from Days 6 to 28 p.c., inclusive.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a preliminary study where time-mated New Zealand White female rabbits were given the test item by gavage from Days 6 to 28 p.c. at 100, 300 or 1000 mg/kg/day.
. In this study, there were no apparent test item-related effects at 100 and 300 mg/kg/day. At 1000 mg/kg/day, there were no mortality, no test item-related clinical signs and no obvious effects at necropsy. One pregnant female had a food consumption = 10 g/day from Day 15 p.c. and lost 18% of body weight between Days 12 and 29 p.c. Two other pregnant females had a food consumption lower than 50 g/day at the end of the study and lost concomitantly 4 or 7% of body weight. Mean fetal body weight was lower in their litter. There were no such effects on food consumption, body weight and fetal body weight in the fourth pregnant female.
In absence of death and severe suffering and in order to induce some maternal toxicity at the high-dose at least, as requested by the guideline, the same dose levels were selected for the present study.

- Rationale for animal assignment: stratified procedure.

Examinations

Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during treatment period, including weekends and public holidays.
One female from group 4, showing signs of poor clinical condition, was weighed and humanely euthanized.

CLINICAL SIGNS:
- Time schedule: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c. and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each animal was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 29 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 and distribution of dead and live fetuses,
- Number of implantation sites,
- Number of early resorptions,
- Number of late resorptions,
- Number of uterine scars and evaluation of placentas.
The placenta weight was recorded for each live fetus and the fetal weight/placental weight ratio was calculated.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: body weight, sex.
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s 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
Historical control data:
Cf attached document

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Se table 1.
The clinical signs recorded during the treatment period in pregnant animals were recorded in isolated animals and/or without dose-relationship. They were therefore considered to be not test item-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female treated at 300 mg/kg/day was found dead on Day 12 p.c. This female was difficult to administer during the 3 days before, and blood was found on the gavage tube on the eve. At necropsy, reddish color of the left lung was observed and the female had only late resorptions in the uterus. This death was considered to be related to the gavage procedure.
Another female treated at 1000 mg/kg/day was prematurely euthanized on Day 23 p.c. following abortion (placentas and fetus in the bedding); emaciated appearance and absence of feces were recorded for this female for 6 or 8 days before abortion. From treatment initiation, its food consumption was near or at 0 g/day and concomitant to body weight losses (-16% between Days 6 and 19 p.c.). At necropsy, thickened pyloric mucosa, reddish content in the vagina and dilatation of the gall bladder were observed.
A relationship between this abortion and the test item treatment could not be excluded as it happened at the high-dose.
There were no other unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
At 1000 mg/kg/day, there were no test item treatment-related effects on mean body weights. However, on Days 6 to 12 p.c. females had a mean body weight loss followed by a low mean body weight gain. Mean body weight change was then comparable with controls from Day 12 to 24, but at the end of the treatment period there were more females losing weight (<10%; generally associated with reduced food consumption) than in controls.
These effects on mean body weight change were considered to be test item-related and non-adverse (slight and/or reversible effect, no toxicological impact on mean body weights).
At 100 and 300 mg/kg/day, there were no test item treatment-related effects.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 1000 mg/kg/day and when compared with controls, mean food consumption was statistically significantly lower on Days 6 to 15 p.c. At the end of the treatment, food consumption was strongly reduced (associated with body weight loss) in more females when compared with controls.
These effects were considered to be test item-related and non-adverse (no severe differences from controls; no toxicological impact on mean body weights).
At 100 and 300 mg/kg/day, there were no test item treatment-related effects.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the findings recorded in surviving pregnant females were considered to be test item-related (no dose-relationship).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See table 4.
Gravid uterus weight and net body weight change
There were no test item-related effects on mean carcass weight and mean net body weight change.

At 100 and 1000 mg/kg/day when compared with controls, mean uterus weight was statistically significantly lower. This finding was considered to be due mainly to the lower number of fetuses in both groups (not test item-related).

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
See table 5.
One female treated at 1000 mg/kg/day was prematurely euthanized on Day 23 p.c. following abortion (placentas and fetus in the bedding); emaciated appearance and absence of feces were recorded for this female for 6 or 8 days before abortion. From treatment initiation, its food consumption was near or at 0 g/day and concomitant to body weight losses (-16% between Days 6 and 19 p.c.). At necropsy, thickened pyloric mucosa, reddish content in the vagina and dilatation of the gall bladder were observed.
A relationship between this abortion and the test item treatment could not be excluded as it happened at the high-dose.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
There were no effects on mean hysterectomy data considered to be test item-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
There were no effects on mean hysterectomy data considered to be test item-related.
At 100 mg/kg/day when compared with controls and historical control data, mean number of live fetuses per litter was lower, reaching statistical level. This was due to high mean rate of pre-implantation loss (therefore not test item-related as treatment started at implantation), as well as to high mean post-implantation loss percentage and mean number of resorptions (considered to be incidental in absence of dose-relationship and as within historical control data).
At 1000 mg/kg/day when compared with controls and historical control data, mean number of live fetuses per litter was lower also. It was considered to be due to the high mean rate of pre-implantation loss (therefore also not test item-related).
Dead fetuses:
effects observed, non-treatment-related
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
On Day 29 p.c., there were 23/24, 22/24, 19/23 and 23/23 pregnant females with live fetuses in control, 100, 300 and 1000 mg/kg/day groups, respectively.
At 300 and 1000 mg/kg/day, the two prematurely euthanized/dead females were pregnant.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
See table 6.
There were no toxicologically significant differences in mean data between groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no test item-related effects on mean percentage of male fetuses per litter (mean data comparable between groups and close to 50%).
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related external variations and malformations in litters.
The only fetal external variations (paw or limb hyperflexion) were mostly recorded in the control group, and the only fetal external malformations (bent tail or short + narrowed tail) were recorded in one fetus of the control group (associated with fused caudal vertebrae) or in one fetus of the 100 mg/kg/day group (associated with fused and absent caudal vertebrae) only.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 8 to 10.
In test item groups, all cartilages of bones were present or with a correct shape, with a few exceptions: fused, bipartite and misshapen cartilages of sternebrae. For both latter findings, the incidences were isolated and/or similar with controls (no test item-related effects therefore). For the fused cartilages of sternebrae, see discussion on the malformation fused sternebrae below.
At 1000 mg/kg/day and when compared with controls, there were a few skeletal variations (incomplete ossification of interparietal, 1st to 4th sternebrae and 1st metacarpal; thickened ribs) with higher incidences. However, the differences from controls were slight, and/or the incidences were either within or close to the upper limit of the Historical Control Data.
At 300 mg/kg/day, there were 3 fetuses from 3 different litters with unossified pubis, while no control fetus and rare historical control data fetuses had the same findings. Nevertheless this finding was not observed in the other test item groups and the incidence was slight. The incidences of incompletely ossified interparietal were lower/close to the historical control data maximum.
A test item treatment effect was therefore uncertain (especially for thickened ribs at the high-dose whose incidence was slightly higher than Historical Control Data) for the above mentioned variations, but considered of to be non-adverse as they are variations (not malformations), were noted in absence of toxicologically significant effects on mean fetal weight and as litter incidence were generally within Historical Control Data.
The other fetal skeletal variations in test item groups (including extra sternebral ossification site, ossification point on 13th thoracic vertebra and dumbbell ossification of centrum at 1000 mg/kg/day) were noted with incidences isolated or comparable/lower to controls and/or historical control data, and/or with no dose-relationship and statistical significance. They were considered to be incidental or of no toxicological significance.
At 300 and 1000 mg/kg/day, fetal and litter incidences of interparietal or parietal split were higher than in controls or Historical Control Data; a test item treatment effect cannot be excluded. However, these findings were not considered to be adverse in view of the slight incidences and slight differences from controls (no statistical differences), since they do not impact the global shape of the skull and since they are probably due to ossification delay.
Fused sternebrae was present in test item-treated groups at higher fetal and litter incidences than in controls. However, the incidences were generally within historical control data and there were no dose-relationship. A test item treatment effect was considered to be unlikely.
The other fetal skeletal malformations in test item groups were noted with incidences isolated or comparable/lower to controls and/or historical control data and/or with no dose-relationship or statistical significance. They were considered to be incidental.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
At 1000 mg/kg/day when compared with controls, there were higher litter and fetal incidences of findings on the gall bladder. Indeed, 8 fetuses among 6 litters (vs. 2 fetuses among 2 litters in controls) had a morphology finding on their gall bladder (small, bilobed, enlarged, with colored nodule). These findings were considered to be test item-related and non-adverse in view of the slight incidence and as these findings are variations.
At 300 and 100 mg/kg/day, there were higher litter incidences of absent innominate artery than control data and historical control data maximum. In absence of clear dose-relationship, and taking into account that fetal incidences were within historical control data and that the litter incidence at 1000 mg/kg/day was close to the historical control data maximum, an effect of the test item treatment was considered to be unlikely.
The statistical significantly higher fetal incidence of short innominate artery was not attributed to the test item treatment (close to historical control data maximum, no dose-relationship).
The other fetal visceral variations in test item groups, including the ovary serous cyst and the liver colored nodule at 1000 mg/kg/day, were noted with incidences isolated and/or comparable to controls and/or historical control data and/or with no dose-relationship or statistical significance. They were considered to be incidental.
There were no fetal visceral malformations ascribed to the test item treatment.
The only fetal visceral malformations (marked dilated cerebral ventricle, absent aortic arch) were observed in 1 or 2 fetuses of isolated litters from the 100 and 300 mg/kg/day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs

 

Dose level (mg/kg/day)

0

100

300

1000

Emaciated appearance

1

Absence of feces

1

White coloured feces

1

Soiled urogenital area

1

Cutaneous lesions on head

1

Reddish vaginal discharge

1

Blood in the bedding

1

1

1

1

Abnormal growth of teeth

1

Number of affected animals

1/24

2/24

2/23

4/23

Table 2: Body weight

 

Dose level (mg/kg/day)

0

100

300

1000

Mean body weight

 

 

 

 

Day 6p.c

3693

3659

3657

3694

 

 

-1

-1

0

Day 9p.c

3725

3701

3687

3677

 

 

-1

-1

-1

Day 12p.c

3795

3754

3753

3700

 

 

-1

-1

-3

Day 15p.c

3889

3839

3828

3783

 

 

-1

-2

-3

Day 19p.c

3951

3881

3886

3856

 

 

-2

-2

-2

Day 24p.c

4049

3937

4016

3989

 

 

-3

-1

-1

Day 29p.c

4080

3991

4060

3970

 

 

-2

0

-3

Mean body weight change

 

 

 

 

Days 6 - 9p.c.

+32

+41

+30

-16*

Days 9 - 12p.c.

+71

+54

+63

+23*

Days 12 - 15p.c.

+93

+85

+75

+83

Days 15 - 19p.c.

+62

+42

+57

+74

Days 19 - 24p.c.

+98

+55

+130

+105

Days 24 - 29p.c.

+31

+54

+44

-18

Days 6 - 29p.c.

+387

+331

+399

+283

In italic, percentage difference (%)vs. controls;Statistical significancevs. controls:*: p<0.05.

Table 3: Food consumption

 

Dose level (mg/kg/day)

0

100

300

1000

Days 6 - 9p.c.

164

168

+2

156

-7

119#

-27

Days 9 - 12p.c.

175

185

+6

172

-2

131#

-25

Days 12 - 15p.c.

165

171

+4

160

-3

131*

-21

Days 15 - 19p.c.

188

181

-4

177

-6

163

-13

Days 19 - 24p.c.

160

168

+5

163

+2

160

0

Days 24 - 29p.c.

107

118

113

90

 

 

+10

+6

-16

In italic, percentage difference (%)vs. controls; Statistical significancevs. controls:*: p < 0.05, #: p < 0.001.

Table 4: Gravid uterus

 

Dose level (mg/kg/day)

0

100

300

1000

Gravid uterus weight

526

430*

-18

487

-7

425**

-19

Carcass weight

3555

3561

3573

3545

Net weight change from Day 6p.c.

-139

-98.3

-87.5

-142

Statistical significance:*: p < 0.05, **: p < 0.01; data rounded to 3 significant figures when applicable;in italic, percentage difference (%)vs.controls.

Table 5: Hysterectomy data

Dose level (mg/kg/day)

0

100

300

1000

HCD

Number of females with live fetuses

23

22

19

23

159

Mean number ofcorpora lutea

11.8

11.3

11.5

11.3

[11.4-12.8]

Mean number of implantation sites

10.8

9.3

10.3

9.1

[9.5-11.4]

Mean pre-implantation loss (%)

8.8

18.1

10.5

20.1

[10.5-17.9]

Mean number of live fetuses

10.0

7.5**

9.4

8.2

[8.5-10.6]

Mean number of early resorptions

0.3

1.0

0.3

0.7

Late + early [0.36-1.50]

Mean number of late resorptions

0.5

0.8

0.5

0.3

Mean post-implantation loss (%)

6.2

20.1*

7.9

9.2

[3.9-20.5]

Statistical significance:*: p < 0.05 **: p < 0.01.

HCD: historical control data (2015-2016; New Zealand White rabbits): study means [minimum-maximum].

 

Table 6: Fetal body weight and placental weight

 

Dose level (mg/kg/day)

0

100

300

1000

HCD

Mean fetal body weight

35.7

38.2

+7

34.7

-3

34.4

-4

[34.6-39.3]

Mean placental weight

5.26

5.80

+10

5.27

0

5.40

+3

-

Mean ratios of fetal weight/placental weighta

 

6.89

 

6.68

 

6.75

 

6.47

 

-

In italic, percentage difference (%)vs. controls.

a: calculated with Excel, no statistics performed.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): study means [minimum-maximum].
-: not presented in HCD.

Table 7: Soft tissue variations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

23

22

19

23

159

Number of fetuses

231

164

179

188

1558

Gall bladder

 

 

 

 

 

Small gall bladder, F (L)

0.4 (4.3)

1.8 (9.1)

1.1 (8.7)

0.6 (5.6)

Bilobed gall bladder, F (L)

1.1 (8.7)

0.6 (5.6)

Colored nodule on gall bladder, F (L)

0.4 (4.3)

1.1 (8.7)

0.4 (4.0)

Enlarged gall bladder, F (L)

1.1 (4.3)

0.5 (5.3)a

Liver

 

 

 

 

 

Colored nodule

 

 

 

0.5 (4.3)

0.4 (4.0)

Ovary

 

 

 

 

 

Serous cyst

 

 

 

0.5 (4.3)

0.6 (5.6)

Vessels

 

 

 

 

 

Short innominate artery, F (L)

19.0 (87.0)

28.7* (86.4)

21.8 (73.7)

26.1 (82.6)

26.8 (94.4)

Absent innominate artery, F (L)

9.5 (56.5)

14.0 (63.6)

14.5 (84.2)

16.0 (73.9)

17.9 (72.0)

Litters with visceral variations, n (%)

21 (91.3)

21 (95.5)

18 (94.7)

22 (95.7)

143 (89.9)

Fetus with visceral variations, n (%)

71 (30.7)

75** (45.7)

74* (41.3)

86** (45.7)

481 (30.9)

F: fetal incidence; L: litter incidence,n: number; Statistical significance:*: p < 0.05, **: p < 0.01.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 8: Cartilage examinations

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

23

22

19

23

159

Number of fetuses

231

164

179

188

1558

Sternebrae

 

 

 

 

 

Fused cartilages

1.3 (4.3)

1.2 (9.1)

2.2 (10.5)

1.1 (8.7)

1.8 (16.7)

Bipartite cartilage

 

 

0.6 (5.3)

 

-

Misshapen

0.4 (4.3)

0.6 (4.5)

0.5 (4.3)

-

Litters with cartilage findings, n (%)

22 (95.7)

20 (90.9)

19 (100)

23 (100)

 

Fetuses with cartilage findings, n (%)

104 (45.0)

86 (52.4)

86 (48.0)

102 (54.3)

 

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 9: Fetal skeletal variations

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

23

22

19

23

159

Number of fetuses

231

164

179

188

1558

Skull

 

 

 

 

 

Interparietal: incomplete ossification

0.9 (8.7)

2.4 (13.6)

5.0* (31.6)

4.8* (21.1)

4.1 (36.8)

Sternebra

 

 

 

 

 

Incomplete ossification of 1stto 4thsternebrae, F (L)

0.0 (0.0)

1.2 (9.1)

1.1 (10.5)

3.2** (17.4)

4.4 (26.3)

Extra sternebral ossification site

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.3)

1.2 (8.3)

Rib

 

 

 

 

 

Thickened rib(s), F (L)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

1.6 (13.0)

1.0 (10.5)

Ossification point on 13ththoracic vertebra

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.3)

0.6 (5.6)

Metacarpal bone

 

 

 

 

 

Incomplete ossification of 1stmetacarpal, F (L)

5.6 (30.4)

3.7 (22.7)

6.7 (31.6)

11.7* (47.8)

8.4 (61.1)

Pelvis

 

 

 

 

 

Pubis: unossified, F (L)

0.0 (0.0)

0.0 (0.0)

1.1 (10.5)

0.0 (0.0)

0.5 (5.6)

Thoracic vertebrae

 

Dumbbell ossification of centrum

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.5 (4.3)

-

Litters with skeletal variations, n (%)

23 (100.0)

22 (100.0)

19 (100.0)

23 (100.0)

159 (100)

Fetuses with skeletal variations, n (%)

202 (87.4)

139 (84.8)

151 (84.4)

170 (90.4)

1382 (88.7)

F: fetal incidence; L: litter incidence,n: number; Statistical significance (on number of fetuses affected not on the percentages presented in the table):*: p < 0.05, **: p < 0.01.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 10: Fetal skeletal malformations

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

23

22

19

23

159

Number of fetuses

231

164

179

188

1558

Head skull

 

 

 

 

 

Interparietal: split, F(L)

2.2 (21.7)

3.0 (22.7)

4.5 (31.6)

1.6 (13.0)

1.2 (11.1)

Interparietal: absent

3.9 (21.7)

1.2 (4.5)

3.4 (21.1)

0.5 (4.3)

3.4 (31.6)

Parietal: split

0.4 (4.3)

0.0 (0.0)

0.0 (0.0)

1.6 (13.0)

0.4 (4.2)

Skullcap: hole

0.9 (8.7)

0.6 (5.3)

0.3 (2.7)

Caudal vertebrae

 

Fused

0.4 (4.3)

0.6 (4.5)

0.5 (4.3)

0.9 (8.3)

Absent

0.6 (4.5)

0.4 (4.2)

Misaligned

0.5 (4.3)

-

Sternebrae

 

 

 

 

 

Fused

2.2 (13.0)

3.7 (18.2)

6.7* (26.3)

4.3 (17.4)

2.2 (20.8) [or 5.1 (31.3)a]

Rib

 

Fused

0.5 (4.3)

0.9 (8.3)

Litters with skeletal malformations, n (%)

10 (43.5)

8 (36.4)

12 (63.2)

10 (43.5)

46 (28.9)

Fetuses with skeletal malformations, n (%)

20 (8.7)

13 (7.9)

27 (15.1)

17 (9.0)

56 (3.6)

F: fetal incidence; L: litter incidence;n: number; Statistical significance (on number of fetuses affected not on the percentages presented in the table):*: p < 0.05.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

a: HCD from 2013-2014.

Applicant's summary and conclusion

Conclusions:
On the basis of the experimental conditions and results obtained in this study, the dose of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development, based on the non-adverse effects on dam body weight change and food consumption and on non-adverse increased incidences of the fetal gall bladder and skeletal variations at this dose.
Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage), to pregnant female rabbits from implantation to the day prior the scheduled hysterectomy [Days 6 to 28 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female New Zealand White rabbits received the test item by oral route (gavage) at doses of 100, 300 and 1000 mg/kg/day once daily from Day 6 post-coitum (p.c.) until Day 28 p.c. inclusive. One additional group of 24 time-mated females received the vehicle [methylcellulose (1%) in drinking water treated by reverse osmosis] under the same experimental conditions and acted as a control group. A constant dose-volume of 3 mL/kg/day was used.

Formulation concentrations were checked twice (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 1 to 5 days from their arrival.

On Day 29 p.c., females were euthanized and submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for external, soft tissue and/or skeletal (bones and cartilages) abnormalities. Placentas were grossly observed and weighed.

 

Results

Test item concentrations in the dose formulations analyzed during the study were within an acceptable range of variations when compared to the nominal values (± 15% accepted) and there was no test item in the control dose formulations.

On Day 29 p.c., there were 23/24, 22/24, 19/23 and 23/23 pregnant females with live fetuses in control, 100, 300 and 1000 mg/kg/day groups, respectively.

At 1000 mg/kg/day, there was a premature euthanasia following abortion; clinical signs, body weight loss and poor food consumption were observed beforehand. A relationship between the test item treatment and the abortion could not be excluded.

There were no other clinical signs or necropsy findings considered to be test item-related.

There were no test item effects on mean body weight and mean food consumption at 100 and 300 mg/kg/day. At 1000 mg/kg/day, there were slight mean body weight loss on Days 6 to p.c.(-16 g vs. +32 g in controls, p<0.05) and on Days 24 to 29 p.c.(-18 g vs.+31 g) and a low mean body weight gain on Days 9 to 12 p.c.(+23 g vs.+71 g, p<0.05). Concomitant low mean food consumption was noted (-21 to -27% on Days 6 to 15 p.c. vs. controls, p<0.05 or p<0.001). These effects were considered to be test item-related and non-adverse (no toxicological impact on mean body weight, no strong differences from controls).

There were no test item-related effects on mean uterus weight, mean carcass weight and mean net body weight change in any groups.

 

There were no test item-related effects on mean hysterectomy data and mean fetal sex ratio in any groups.

There were no toxicologically significant differences in mean fetal and placental weights data between groups in any groups.

There were no test item-related external variations and no external and visceral malformations in litters in any groups. A test item relationship with increased skeletal variation incidences at 300 and 1000 mg/kg/day (mainly incomplete ossification of 1st to 4thsternebrae, interparietal and 1stmetacarpal bone; thickened ribs) were considered uncertain but non-adverse.

At 1000 mg/kg/day when compared with controls, there were higher litter and fetal incidences of variations on the gall bladder (8 fetuses among 6 litters affected vs. 2 fetuses among 2 litters in controls). These findings were considered to be test item-related and non-adverse.

At 300 and 1000 mg/kg/day, a test item treatment effect on slightly increased incidences of split interparietal (4.5% of fetuses and 31.6% of litters affected at 300 mg/kg/day vs. 2.2% and 21.7% in controls, respectively) or parietal (1.6% of fetuses and 13.0% of litters affected vs .0.4% and 4.3% in controls, respectively) could not be excluded but considered as non-adverse since the differences from control incidences were low (no statistical differences), since the findings were of low incidences and do not impact the global shape of the skull and since they are probably due to ossification delay.

 

Conclusion

On the basis of the experimental conditions and results obtained in this study, the dose of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for maternal parameters and forembryo-fetal development, based on the non-adverse effects on dam body weight change and food consumption and on non-adverse increased incidences of fetal gall bladder and skeletal variations at this dose.