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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:
13 October 2017 - 30 November 2017
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 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 material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age: at the beginning of the treatment period, the animals were 10-11 weeks old
- Mean body weight: at the beginning of the treatment period, the animals had a mean body weight of 291 g (range: 241 g to 334 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H 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): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 06 November 2017 to 30 November 2017.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 40, 120 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: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS MS).
Test item concentrations: a sample was taken from control and test item dose formulations and analyzed using the validated method.
Homogeneity: dose formulation is a solution
Stability: the stability of dose formulation preparations was demonstrated for up to 23 days when stored in plastic vial at room temperature and protected from light.
Dose formulations ranging from 5 mg/mL to 200 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies within the stability period validated.
Details on mating procedure:
The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated Day 0 p.c.
Duration of treatment / exposure:
From Day 6 to Day 20 p.c., inclusive.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 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:
- Rationale for dose selection:
The dose levels were selected by the Sponsor based on a preliminary study for effects on embryo-fetal development by the oral route in rats.
In this study, three groups of eight pregnant female Sprague-Dawley rats received the test item, daily by oral administration (gavage) throughout gestation (Day 6 to Day 20 p.c.) at dose levels of 200, 600 or 1000 mg/kg/day. An additional group of eight pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosage volume was 5 mL/kg/day.
At 1000 mg/kg/day, there was:
- ptyalism in all females,
- slight increases in the absolute and relative mean liver weights,
- no effects on pre- or post-implantation losses, number of viable fetuses and fetal body weight,
- higher litter and fetal incidences of fetuses with variations (i.e. protruding tongue and reddish color of amniotic fluid) and/or malformations (i.e. short snout, misshapen genital tubercle, exencephaly and cleft palate).

At 600 mg/kg/day, there was ptyalism in all females and slight increases in the absolute and relative mean liver weights.
At 200 mg/kg/day, there were no findings.

Based on the above data, the dose levels selected for the present study were: 200, 600 and 1000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

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

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

- ORGAN WEIGHTS:
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to net body weight was calculated.


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 and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites,
- gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half 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, treatment-related
Description (incidence and severity):
See table 1.
Test item-related clinical signs of minor toxicological importance consisted of ptyalism, observed for 2 to 12 days in most females given 600 mg/kg/day and in all females given 1000 mg/kg/day.

Reflux at administration, noted on one occasion in 1/24 females given 1000 mg/kg/day, resulted from the gavage procedure.

The other clinical signs consisted of cutaneous lesions on the head (1/24 control females), areas of hair loss on forelimbs (2/24 females given 600 or 1000 mg/kg/day) and/or scab on the neck (1/24 control females and 1/24 females given 1000 mg/kg/day). These findings were considered to be unrelated to the test item as they were also reported in control animals, in a limited number of test item-treated animals and/or are findings commonly observed in rats of this strain and age.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See table 2.
There were no effects on mean body weight.

At 1000 mg/kg/day and when compared with controls, a slightly higher mean body weight gain was noted between Days 9 and 12 p.c. (+24 g vs. +19 g in controls). As this difference was transient, of minimal amplitude and mainly due to one female that gained 40 g, a test item relationship was considered to be unlikely.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 1000 mg/kg/day, when compared with controls, a lower mean food consumption (-13%, p<0.05) was noted between Days 6 and 9 p.c., with a return towards control values thereafter. This finding was considered to be test-item treatment related but non-adverse.

At 200 and 600 mg/kg/day, there were no effects on mean food consumption.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in liver or kidney weights.

Differences in organ weights were minor and reflected the usual range of individual variations.
When compared with controls, the mean absolute and relative liver weights were minimally higher (up to +8%) in females at 1000 mg/kg/day, reaching statistical significance for the relative weight only. These findings were not considered to be related to the test item given the low magnitude of the changes and as individual relative liver weights in this group were all below the highest control value.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Brownish foci were observed on the stomach mucosa only in 1/24 females given the high dose level. Given the isolated occurrence, a relationship to the test item was considered to be doubtful.

All other macroscopic observations (i.e. reddish discolored area on the lungs, adhesions between the liver and one kidney, marked dilatation of the renal pelvis, dilatation of ureters and/or enlarged placenta) were not attributed to the test item as they were of isolated occurrence, reported both in control and test item treated animals with comparable incidences, not dose-related and/or are findings commonly observed in rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
See table 4.
There were no effects on mean gravid uterus weight, mean carcass weight or net body weight change.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See table 5.
There were no effects.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See table 5.
There were no effects.

Early or late resorptions:
no effects observed
Description (incidence and severity):
See table 5.
There were no effects.
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 5.
There were no effects.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See tables 5 and 13.
There were no effects.

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:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
In controls, mean fetal body weights and mean fetal body weights of males and females were higher than the upper limit of the Historical Control Data (HCD). Therefore, from 200 mg/kg/day, lower mean body weight (-4% to -5% vs. controls) was noted. This was mainly due to female fetuses rather than male fetuses at 200 mg/kg/day (males: -3% and females: -5% with p<0.01 vs. controls), 600 mg/kg/day (males: -3% and females: -4% with p<0.05 vs. controls) and 1000 mg/kg/day (males: -5% with p<0.05 and females: -5% with p<0.01 vs. controls).
As the differences from controls in mean fetal body weights were poorly dose-related, minimal (less than 10% differences in all test-item treated groups) and within the range of the HCD, a relationship with the test item is questionable.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 6.
There were no effects on sex ratio (percentage of male fetuses).
External malformations:
no effects observed
Description (incidence and severity):
Variations
There were no external variations in the test item-treated groups.

Malformations
See table 7.
There were no external malformations in the test item-treated groups.
In the control group, 4 fetuses had cleft palate, together with bent tail for one of them; these findings are recorded at comparable incidence in the HCD and/or are common observations in this species and strain.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilage
See table 10.
When compared with controls, there was a dose-related increase of delayed ossification. Cartilage of hyoid, sternebra(e), forepaw proximal phalanx and metatarsal bone were present, but there was incomplete ossification of the interparietal, hyoid and/or 6th sternebra and/or unossification of forepaw proximal phalanx and/or 1st metatarsal. As the litter and fetal incidences of controls were below the lower limit or in the low range of the HCD, and as the litter and fetal incidences of the test item-treated groups were within the range of the HCD, a relationship with the test item is questionable.
Other cartilage observations [i.e. cartilage of cervical vertebra(e) present and/or cartilage of rib present] were observed at 200 and/or at 1000 mg/kg/day. These findings were not dose-related and were noted at lower or similar incidences in the HCD, therefore a test item relationship was considered as unlikely.

Variations
See table 11.
When compared with controls, there was a dose-related increase in incomplete ossification [interparietal, hyoid and/or 6th sternebra (statistically significant at 1000 mg/kg/day)] and/or unossification [forepaw proximal phalanx (statistically significant from 600 mg/kg/day) and/or 1st metatarsal (statistically significant from 200 mg/kg/day)]. As the litter and fetal incidences of controls were below the lower limit or in the low range of the HCD, and as the litter and fetal incidences of the test item-treated groups were within the range of the HCD, a relationship with the test item is questionable.
Other skeletal variations [i.e. incomplete ossification of cervical vertebra(e) centrum, thickened rib and/or wavy rib] were observed at 200 mg/kg/day. As these findings were not dose-related and were noted with a lower incidence than that observed in the HCD, they were therefore considered to be unrelated to the test item.

Malformations
See table 12.
There were no skeletal malformations in the test item-treated groups.
In the control group, one litter had three malformed fetuses: L28862-01, -07 and -11 with split palate (cleft palate at external examination). These findings are observed with an incidence similar to the HCD and/or are commonly noted in this species and strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See table 8.
There were no test item treatment-related soft tissue variations.

Tissue variations (i.e. short or absent innominate artery and reddish foci on thymus) were observed in control and/or test item-treated groups, with an isolated occurrence and/or with a lower incidence than that observed in the HCD. These findings were therefore considered to be unrelated to the test item treatment.
Lower, statistically significant incidences of dilated ureter were noted at 600 and 1000 mg/kg/day. This was due to higher fetal and litter control incidences and was therefore considered to be of no relevance.

Malformations
See table 9.
There were no test item-related increases in the frequency of soft tissue malformations.

At 1000 mg/kg/day, the left ureter was markedly dilated in one fetus . This correlated with the dilatation of the left renal pelvis. As this malformation was observed with an incidence similar to HCD, it was considered to be unrelated to the test item treatment.

In the control group, one fetus had cleft palate (also observed at external examination); this finding is commonly noted in this species and strain.

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

200

600

1000

Ptyalism

 

 

14

24

Reflux at administration

 

 

 

1

Number of affected animals

0/24

0/24

14/24

24/24

 

Table 2: Body weight

Dose level (mg/kg/day)

             0

200

600

1000

Body weight (g)

 

 

 

 

Day 6 p.c.

           290

294

291

291

 

-

(+1)

(0)

(0)

Day 9 p.c.

304

310

304

303

 

-

(+2)

(0)

(0)

Day 12 p.c.

323

328

326

327

 

-

(+2)

(+1)

(+1)

Day 15 p.c.

347

349

347

349

 

-

(+1)

(0)

(+1)

Day 18 p.c.

391

396

387

394

 

-

(+1)

(-1)

(+1)

Day 21 p.c.

446

447

437

453

 

-

(0)

(-2)

(+2)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+14

+15

+13

+12

Days 9 - 12 p.c.

+19

+18

+22

+24*

Days 12 - 15 p.c.

+24

+21

+20

+22

Days 15- 18 p.c.

+44

+47

+40

+44

Days 18 - 21 p.c.

+54

+51

+50

+60

Days 6 - 21 p.c.

+155

+152

+146

+162

 

-

(-2)

(-6)

(+5)

p.c.   : post-coitum.

-       : not applicable.

( )     : in brackets, percentage difference vs. controls. Statistical significance *: p<0.05.

 

Table 3: Food consumption

Dose level (mg/kg/day)

0

200

600

1000

. Days 6 - 9 p.c.

23

23

21

20*

. Days 9 - 12 p.c.

25

25

25

26

. Days 12 - 15 p.c.

28

26

26

28

. Days 15 - 18 p.c.

32

31

30

32

. Days 18 - 21 p.c.

31

29

28

31

p.c.   : post-coitum.

Statistical significance *: p<0.05.

Table 4: Net body weight change

Dose level (mg/kg/day)

0

200

600

1000

Gravid uterus weight

105

104
(-1)

96
(-9)

110
(+5)

Carcass weight

341

343
(+1)

341
(0)

344
(+1)

Net body weight change from Day 6 p.c.

+50

+48

+49

+52

( )     : in brackets, percentage difference vs. controls.

p.c.         : post-coitum.

(a)         : weights are rounded values.

Table 5: Hysterectomy data

Dose level (mg/kg/day)

       0

  200

  600

1000

HCD

Number of pregnant females
at hysterectomy

   24

   22

   24

   23

      407

Number of females with live fetuses
at termination

   24

   22

   24

   23

      388

Number of females with total resorption

     0

     0

     0

     0

          0

Mean number of corpora lutea

   14.0

   14.0

   14.3

   14.8

[13.8; 16.0]

Mean number of implantation sites

   13.2

   13.5

   13.0

   14.0

[12.5; 14.5]

Mean pre-implantation loss (%)

     5.5

     3.8

     9.0

     4.9

[6.2; 14.0]

Mean number of live fetuses

   12.5

   12.9

   11.9

   13.5

[11.6; 13.8]

Dead fetuses (%)

     0.0

     0.0

     0.0

     0.0

[0.00; 0.50]

Mean number of implantation scars

     0.0

     0.0

     0.0

     0.0

/

Mean number of early resorptions

     0.7

     0.6

     1.1

     0.5

/

Mean number of late resorptions

     0.0

     0.0

     0.0

     0.0

/

Mean post-implantation loss (%)

     5.4

     4.3

     9.0

     3.7

[3.5; 11.1]

HCD     : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014
             to July 2016), [min.; max.].

/          : not reported in HCD.

 

Table 6: Fetal body weight and sex ratio

Dose level (mg/kg/day)

0

200

600

1000

HCD

Mean fetal body weight (g)

5.98
-

5.75*
(-4)

5.73*
(-4)

5.69**
(-5)

[5.5; 5.9]

Mean fetal body weight
of males (g)

6.11
-

5.92
(-3)

5.90
(-3)

5.83*
(-5)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.85
-

5.55**
(-5)

5.59*
(-4)

5.53**
(-5)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

52.3

50.9

46.3

54.1

[44.0; 55.4]

( )        : in brackets, percentage difference vs. controls.

HCD     : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014
             to July 2016), [min.; max.].

-          : not applicable.

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

 

Table 7: Fetal external malformations

Dose level (mg/kg/day)

0

200

600

1000

HCD

Dams with live fetuses, n

24

22

24

23

388

Live fetuses, n

299

283

286

311

5000

. cleft palate,L (F)

4.2 (1.3)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

5.0 (0.4)(a)

. bent tail,L (F)

4.2 (0.3)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

5.0 (0.4)(a)

Litters affected, n (%)(c)

1 (4.2)

0 (0.0)

0 (0.0)

0 (0.0)

11 (2.8)(b)

Fetus affected, n (%)(c)

4 (1.3)

0 (0.0)

0 (0.0)

0 (0.0)

13 (0.3)(b)

n: number.

HCD:Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016);(a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all malformations combined.


 

Table 8: Fetal soft tissue variations

 

Dose level (mg/kg/day)

          0

    200

     600

   1000

HCD

Dams with live fetuses, n

        24

       22

        24

        23

        387

Live fetuses, n

     145

    137

     137

     151

      2404

. short innominate artery, L (F)

0 (0)

0 (0)

0 (0)

4.3 (0.7)

22.7 (3.7)(a)

. absent innominate artery, L (F)

4.2 (0.7)

4.5 (0.7)

0 (0)

13.0 (2.0)

25.0 (5.1)(a)

. dilated ureter, L (F)

25.0 (5.5)

9.1 (1.5)

0** (0)

4.3* (0.7)

28.0 (7.1)(a)

. thymus: reddish foci, L (F)

0 (0)

0 (0)

0 (0)

4.3 (0.7)

0 (0)

Litters affected, n (%)(c)

6 (25.0)

3 (13.6)

1 (4.2)

6 (26.1)

86 (22.2)(b)

Fetus affected, n (%)(c)

8 (5.5)

3 (2.2)

1 (0.7)

7 (4.6)

119 (5.0)(b)

n: number.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016); (a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all variations combined.

Statistical significance for affected fetuses/litter: *: p < 0.05 and **: p<0.01

 

Table 9: Fetal soft tissue malformations

Dose level (mg/kg/day)

          0

      200

     600

1000

HCD

Dams with live fetuses, n

        24

         22

        24

        23

            387

Live fetuses, n

     145

      137

     137

     151

         2404

. marked dilated ureter, L(F)

0 (0)

0 (0)

0 (0)

4.3 (0.7)

4.8 (4.8)(a)

. cleft palate, L(F)

4.2 (0.7)

0 (0)

0 (0)

0 (0)

0 (0)

Litters affected, n (%)(c)

1 (4.2)

0 (0)

0 (0)

1 (4.3)

7 (1.8)(b)

Fetus affected, n (%)(c)

1 (0.7)

0 (0)

0 (0)

1 (0.7)

13 (0.5)(b)

n: number.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016); (a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all malformations combined


 

Table 10: Fetal skeletal examinations

Cartilage

Dose level (mg/kg/day)

              0

        200

          600

   1000

HCD

Dams with live foetuses, n

            24

          22

            24

        23

            388

Live fetuses, n

          154

        146

          149

      160

          2596

. cartilage of hyoid present, L(F)

4.2 (0.6)

18.2 (2.7)

12.5 (3.4)

26.1*(5.0)*

30.4 (7.4)(a)

. cartilage of sternebra(e) present, L(F)

0 (0)

4.5 (0.7)

12.5 (2.0)

17.4*(2.5)

21.7 (3.8)(a)

. forepaw: cartilage of proximal phalanx present, L(F)

33.3 (14.3)

59.1 (21.9)

70.8*(26.8)**

69.6*(33.8)#

85.0 (50.0)(a)

. cartilage of metatarsal bone present, L(F)

20.8 (5.2)

50.0 (13.7*)

54.2*(20.8)#

73.9#(28.1)#

72.7 (36.8)(a)

Litters affected, n (%)(c)

21 (87.5)

18 (81.8)

23 (95.8)

23 (100.0)

236 (60.8)(b)

Fetus affected, n (%)(c)

65 (42.2)

79* (54.1)

75 (50.3)

104# (65.0)

990 (38.1)(b)

n: number.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016); (a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all cartilage findings combined.

Statistically significant *: p<0.05, **: p<0.01 and # :p<0.001 for the number of fetuses or litters affected.

 

Table 11: Fetal skeletal variations

Dose level (mg/kg/day)

              0

      200

      600

    1000

HCD

Dams with live fetuses, n

            24

        22

         24

         23

             388

Live fetuses, n

          154

      146

      149

      160

          2596

. interparietal: incomplete ossification, L(F)

4.2 (0.6)

22.7 (4.8*)

12.5 (2.0)

30.4*(7.5)**

45.0 (9.2)(a)

. hyoid: incomplete ossification, L(F)

4.2 (0.6)

18.2 (2.7)

12.5 (3.4)

26.1*(4.4)

55.0 (14.8)(a)

. 6thsternebra: incomplete ossification, L(F)

0 (0)

0 (0)

0 (0)

17.4*(2.5)

16.7 (2.9)(a)

. forepaw: unossified proximal phalanx, L(F)

33.3 (14.3)

59.1 (21.9)

70.8* (26.8)**

69.6*(33.8)#

85.0 (50.0)(a)

. 1stmetatarsal: unossified, L(F)

20.8 (5.2)

50.0 (13.7)*

54.2*(20.8)#

73.9#(28.1)#

72.7 (36.8)(a)

Litters affected, n (%)(c)

21 (87.5)

19 (86.4)

23 (95.8)

23 (100.0)

345 (88.9)(b)

Fetus affected, n (%)(c)

68 (44.2)

82* (56.2)

76 (51.0)

104# (65.0)

1267 (48.8)(b)

n: number.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016); (a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all variations combined.

Statistically significant *: p<0.05, **: p<0.01 and #:p<0.001 for the number of fetuses or litters affected.

Table 12: Fetal skeletal malformations

Dose level (mg/kg/day)

        0

     200

  600

         1000

HCD

Dams with live fetuses, n

     24

       22

     24

              23

        388

Live fetuses, n

   154

     146

  149

            160

      2596

. palate: split, L(F)

4.2 (1.9)

0 (0)

0 (0)

0 (0)

5.0 (0.7)(a)

Litters affected, n (%)(c)

1 (4.2)

0 (0)

0 (0)

0 (0)

15 (3.9)(b)

Fetus affected, n (%)(c)

3 (1.9)

0 (0)

0 (0)

0 (0)

18 (0.7)(b)

n: number.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016); (a): upper litter incidence by study; (b): mean number (percentage) of litters or fetuses affected; (c): all malformations combined. 

Table 13: Pregnancy status

Dose level(mg/kg/day)

0

200

600

1000

Number of females

24

24

24

24

Non-pregnant females

0

2

0

1

Females with live fetuses at term

24

22

24

23

 

Applicant's summary and conclusion

Conclusions:
The test item was administered to pregnant Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 200, 600 and 1000 mg/kg/day. A control group received the vehicle, corn oil, under the same experimental conditions.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters is considered to be 1000 mg/kg/day,
- the NOAEL for embryo-fetal development is considered to be 1000 mg/kg/day.
Executive summary:

The potential toxic effects of OO-tert-butyl O-(2-ethylhexyl) peroxycarbonate (TBEC) on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive]. This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). Three groups of 24 time-mated female Sprague-Dawley rats received the test item by the oral route (gavage), at dose levels of 200, 600 or 1000 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated female rats received the vehicle only, corn oil, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. 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 weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). The liver and the kidneys of each dam were weighed and samples of liver, kidney and stomach were collected and preserved in 10% buffered formalin.

The concentrations of the test item in the dose formulations (-2.6% to +9.0%) remained within an acceptable range of variations (± 10%) when compared to the nominal concentrations. No test item was found in the control dose formulation. All females were pregnant with the exception of two females given 200 mg/kg/day and one female given 1000 mg/kg/day. There were no unscheduled deaths. Ptyalism was noted in 14/24 females given 600 mg/kg/day and in 24/24 females given 1000 mg/kg/day. This finding was considered to be test item treatment-related but non-adverse. There were no effects on body weight or body weight change at any dose level.  At 1000 mg/kg/day, lower food consumption (-13% vs. controls, p < 0.05) was observed between Days 6 and 9 p.c., with a return to control values thereafter. This finding was considered to be test item treatment-related but non-adverse. Brownish colored foci were observed on the stomach mucosa at the high-dose level in 1/24 females. This finding was considered to be non-adverse, and a test item treatment relationship was considered to be doubtful. There were no test item-related changes in liver or kidney weights. There were no effects on mean carcass weight, net body weight gain or gravid uterus weight. There were no effects on hysterectomy parameters (mean numbers of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss).

From 200 mg/kg/day and when compared with controls, statistically significant lower mean fetal body weights were noted. They were mainly due to the lower body weight of female fetuses at 200 mg/kg/day (males: -3% and females: -5% with p<0.01 vs. controls), 600 mg/kg/day (males: -3% and females: -4% with p<0.05 vs. controls) and 1000 mg/kg/day (males: -5% with p<0.05 and females: -5% with p<0.01 vs. controls). As these minimal changes were within the range of the Historical Control Data and mainly resulted from control values higher than the upper limit of the Historical Control Data, a relationship with the test item is questionable. There were no toxicological effects on sex-ratio at any dose level. There were no variations and no test item treatment-related malformations at external examination. There were no test item treatment-related variations or malformations at soft tissue examination. At 600 and/or 1000 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses within complete ossification of interparietal, hyoid and/or 6th sternebra and/or unossification of forepaw proximal phalanx and/or 1st metatarsal. As incidences of these non-adverse findings (the cartilage/bone structure still present) were lower than that observed in the Historical Control Data and as control incidences were below the lower limit or in the low range of the Historical Control Data, a relationship with the test item is questionable. There were no test item-related malformations at skeletal examination.

TBEC was administered to pregnant Sprague-Dawley rats by gavage, once daily, from Day 6 to Day 20 p.c., inclusive, at the dose levels of 200, 600 and 1000 mg/kg/day. A control group received the vehicle, corn oil, under the same experimental conditions.

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters is considered to be 1000 mg/kg/day,

. the NOAEL for embryo-fetal development is considered to be 1000 mg/kg/day.