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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2016 - 17 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: approximately 10-11 weeks old at the beginning of the treatment period
- Mean body weight at at the beginning of the treatment period: 308 g (range: 268 g to 365 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- 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: 24 October 2016 to 17 November 2016.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATION:
- Emulsion in the vehicle.
- Concentration in vehicle: 10, 50 and 150 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 (Gas Chromatography with FID detection)
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: The dose formulations containing the test item in corn oil at 2 and 200 mg/mL were found to be homogeneous. They are therefore considered to be suitable for routine administration in GLP Toxicological studies, based on a daily preparation.
Duration of treatment / exposure:
Day 6 to Day 20 p.c.
Frequency of treatment:
Daily
Duration of test:
up to day 21 p.c.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females (for groups 2 to 4).
For group 1, 23 females (because one female was prematurely sacrificed before administration on Day 1. The data of this female are not presented in the study but kept in the raw data).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
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 100, 500 or 1000 mg/kg/day. An additional group of eight pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

At 1000 mg/kg/day, there were:
- mortality: one female was found dead on Day 19 p.c. Prior to death, ptyalism, piloerection, emaciated appearance and dacryhorrhea were observed, as well as a body weight loss and reduced food consumption. There were no macroscopic post-mortem findings at necropsy,
- ptyalism in all females,
- increases in the absolute and relative mean liver weights,
- thickening of forestomach mucosa in 5/8 females together with depressed area in the stomach or forestomach mucosa in two of them,
- no effects on pre- and post-implantation losses, number of viable fetuses and fetal body weight,
- no findings at external (including oral cavity) examination of the fetuses.

At 500 mg/kg/day, there were ptyalism in all females and increases in the absolute and relative mean liver weights.

At 100 mg/kg/day, there were no findings.

Therefore, 750 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing approximately a 3- to 5-fold interval (i.e. 50 and 250 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure
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.

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 once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT (GAIN):
- 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 weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy.
The body weight of each animal was recorded before sacrifice at the end of the treatment period. Liver from all study animals was weighed as soon as possible after dissection and preserved.
The ratio of organ weight to "net body weight" was therefore calculated.

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

MICROSCOPIC EXAMINATION:
A microscopic examination was performed on the liver from the first ten control and high-dose females (groups 1 and 4) sacrificed at the end of the treatment period.
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- 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.

For the apparently non-pregnant female, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).
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).
PathData software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 2.
Test item treatment-related clinical signs of minor toxicological importance consisted of ptyalism observed in most of females given 250 mg/kg/day and in all females given 750 mg/kg/day for a 1 to 15-day duration.
Reflux at administration noted on one occasion in 1/24 females given 250 mg/kg/day resulted from the gavage procedure.
Cutaneous lesions on the tail, associated with scabs or mass in the urogenital region, were noted in 2/24 females given 50 mg/kg/day. Reddish/brownish vaginal discharge was transiently observed in 1/24 females given 250 or 750 mg/kg/day, together with chromodacryorrhea at 750 mg/kg/day. These findings were considered to be unrelated to the test item treatment, as they were reported on a limited number of occasions and/or are findings commonly observed in rats of this strain and age.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test item treatment-related deaths.
At 0 mg/kg/day, one female was sacrificed prematurely on Day 6 p.c. (before administration) due to poor clinical condition (marked ataxia for both hind limbs, together with markedly reduced proprioception). At macroscopic post-mortem examination, no relevant findings were observed. This female was pregnant. Data for this female are kept in the raw data but are not presented in the study report.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See Table 3.
There were no effects on mean body weight or mean body weight change.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See Table 4.
There were no effects of treatment with the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Table 5.
Statistically significant increases in absolute and relative-to-body liver weights were observed in animals treated with the test item at 750 mg/kg/day. A relationship with the test item was considered to be likely.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related macroscopic findings.

Macroscopic observations were not attributed to the test item treatment as they were reported at isolated incidences and/or are findings commonly observed in rats of this strain and age.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 6.
The livers from the first ten females of the high-dose and control groups were submitted to microscopic examination.
Increased mitosis (minimal to moderate) was observed in 3/10 high-dose females, but not in control animals. Of notice, the absolute and relative to body liver weights in these three females were above the average liver weight values observed in the control group. Since this finding was not observed in controls, a relationship with the test item could not be excluded. Increased hepatocyte mitoses has occasionally been described in rodent livers. Causes vary from physiological responses such as during early growth, during pregnancy, and following partial hepatectomy to post-necrotic repair. In the present case, the pathogenesis of this finding remained unclear. However, given its low incidence and in absence of associated findings (such as necrosis/degeneration), this finding was considered to be non-adverse.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 7.
At 250 and 750 mg/kg/day, when compared with controls, females had a low mean gravid uterus weight (-12% and -10%, p < 0.05, respectively). This correlated with the lower mean number of fetuses at these dose-levels. These findings were considered to be non-adverse as they were not dose-related and of low magnitude.
There were no dose-related effects on mean carcass weight or net body weight change at any dose-level.
Number of abortions:
no effects observed
Description (incidence and severity):
See Table 8.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Table 8.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Tables 1 and 8.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 8.
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 8.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 8.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Localisation:
other: Non-adverse effects on liver
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See Table 9.
There were no effects on mean fetal body weight.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no effects on mean fetal body weight.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 9.
There were no effects on mean sex ratio (percentage of male fetuses).
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 10.
Variations
There was no test item treatment-related increase of the frequency of external variations.
In the 750 mg/kg/day group, one fetus had limb hyperflexion. This is a common finding in this species and strain, therefore any relationship to the test item treatment was considered unlikely.

Malformations
See Table 11.
There was no test item treatment-related increase in the frequency of external malformations.
Cleft palate was noted in one malformed fetus from one litter at 50 mg/kg/day (incomplete ossification of the palate at skeletal observation) and in three fetuses from one litter at 750 mg/kg/day (split palate at skeletal examination).
As palate malformation was observed without a dose-relationship with a similar litter incidence in the low and the high-dose groups, a test item treatment-related effect was considered to be unlikely.
In the control group, one fetus had an ombilical hernia; this finding is commonly observed in this species and strain.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See Table 12.
Cartilage
When compared with controls, there was a dose-related increase in ossification delay (cartilage of the thoracic vertebra were present, but there was incomplete ossification of the centrum).

Variations
See Table 13.
When compared with controls, there was an apparent dose-related increase (statistically significant at 750 mg/kg/day) in ossification delay (incomplete ossification of the thoracic vertebra centrum). As the fetal and litter incidences were of low magnitude and within the range of the Historical Control Data, these effects were considered to be of no toxicologically relevance in absence of test item treatment-related effects on mean fetal body weight.

Malformations
See Table 14.
There was no test item treatment-related increase in the frequency of skeletal malformations.
At 750 mg/kg/day, one litter had one malformed fetus with split palate (cleft palate at external examination). As the malformation was of isolated occurrence and as fetal and litter incidences were within the Historical Control Data, this was considered to be unrelated to the test item treatment.
At 50 mg/kg/day, one litter had one malformed fetus with an absent lumbar vertebra. As lumbar malformation was observed without a dose-relationship and was within the range of the Historical Control Data, this was considered to be unrelated to the test item treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Variations
See Table 15.
There was no test item treatment-related increase of the frequency of soft tissue variations.
Tissue variations (i.e. short or absent innominate artery, dilated renal pelvis, dilated ureter and reddish foci on the thymus) were observed in control and test item-treated groups, without a dose relationship and with an incidence similar to the Historical Control Data, were considered to be unrelated to the test item treatment.

Malformations
See Table 16.
There was no test item treatment-related increase in the frequency of soft tissue malformations.
At 250 mg/kg/day, the right kidney and right ureter were absent in one fetus. As these malformations were observed without a dose relationship and/or with an incidence similar to Historical Control Data (absent kidney), they were considered to be unrelated to the test item treatment.

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Pregnancy status

 

Dose-level(mg/kg/day)

0

50

250

750

Number of females

24

24

24

24

Found dead females

1

0

0

0

Non-pregnant females

0

0

1

0

Females with total resorption

0

0

0

0

Females with live fetuses at term

23

24

23

24

 

Table 2: Clinical signs

 

Dose-level (mg/kg/day)

0

50

250

750

Ptyalism

 

 

 20
(usually from the first week of treatment)

 24
(usually from Day 7p.c.)

Reflux at administration

 

 

1

 

Number of affected animals

0/23

0/24

20/24

24/24

( ): in brackets: daysp.c.of occurrences.

 

Table 3: Mean body weight and body weight changes (g)

 

Dose-level (mg/kg/day)

          0

50

250

750

Body weight (g)

 

 

 

 

Day 6p.c.

        312

308

306

307

 

-

(-1)

(-2)

(-2)

Day 21p.c.

        473

477

455

458

 

-

(+1)

(-4)

(-3)

Body weight change (g)

 

 

 

 

Days 6 - 21p.c.

      +161

+168

+149

+151

 

-

(+4)

(-7)

(-6)

p.c.         : post-coitum.

-              : not applicable.

( )           : in brackets, percentage differencevs.controls.

 

Table 4: Mean food consumption (g/animal/day)

 

Dose-level (mg/kg/day)

0

50

250

750

. Days 6 - 9p.c.

23

23

22

21

. Days 9 - 12p.c.

25

25

25

25

. Days 12 - 15p.c.

27

27

26

28

. Days 15 - 18p.c.

30

30

29

32

. Days 18 - 21p.c.

30

31

30

33

p.c.:post-coitum.

 

Table 5: Maternal organ weight

 

Sex

Female

Group

2

3

4

Dose-level (mg/kg/day)

50

250

750

Exam. animals / Num. of animals

23/23

24/24

24/24

- Final body weight

0

-2

-1

- Liver  

.absolute

-2

0

+15**

.relative

-3

+1

+16**

Statistical significance: **: p<0.01 (the significance concerned the organ weights values and not the percentages).

 

Table 6: Incidence and severity of the main microscopic finding in the liver of control and high dose females

 

Sex

Female

Group

1

4

Dose-level (mg/kg/day)

0

750

Exam. Animals/ Num. of animals

10/23

10/24

Liver

 

 

- Increased mitoses; hepatocyte

 

3

Minimal (grade 1)

-

1

Slight (grade 2)

-

1

Moderate (grade 3)

-

1

-: no findings.

 

Table 7: Mean carcass, net change and gravid uterus weights (g)(a)

 

Dose-level (mg/kg/day)

0

50

250

750

Gravid uterus weight

110

112
(+2)

97*
(-12)

99*
(-10)

Carcass weight

363

365
(+1)

359
(-1)

359
(-1)

Net weight change from Day 6p.c.

+51

+57

+52

+53

( ): in brackets, percentage differencevs.controls.

p.c.         :post-coitum.

(a)                  : weights are rounded values.

Statistical significance*: p<0.05.

 

Table 8: Hysterectomy data

 

Dose-level (mg/kg/day)

     0

   50

 250

 750

HCD

Number of pregnant females
at hysterectomy

   23

   24

   23

   24

      407

Number of females with live fetuses
at termination

   23

   24

   23

   24

      388

Number of females with total resorption

     0

     0

     0

     0

          0

Mean number ofcorpora lutea

   15.9

   15.8

   15.3

   14.9

[13.8; 16.0]

Mean number of implantation sites

   14.5

   14.6

   13.2

   13.4

[12.5; 14.5]

Mean pre-implantation loss (%)

     8.3

     6.9

   13.8

     9.9

[6.2; 14.0]

Mean number of live fetuses

   13.8

   13.8

   12.0*

   12.3*

[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

     0.6

     0.9

     1.1

     0.9

/

Mean number of late resorptions

     0.1

     0.0

     0.0

     0.2

/

Mean post-implantation loss (%)

     5.0

     6.0

     7.8

     8.2

[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.

Statistically significant*: p<0.05.

 

Table 9: Fetal body weight and sex-ratio

 

Dose-level (mg/kg/day)

0

50

250

750

HCD

Mean fetal body weight (g)

5.77
-

5.89
(+2)

5.73
(-1)

5.82
(+1)

[5.5; 5.9]

Mean fetal body weight
of males (g)

5.93
-

6.05
(+2)

5.90
(-1)

5.99
(+1)

[5.7; 6.1]

Mean fetal body weight
of females (g)

5.59
-

5.76
(+3)

5.53
(-1)

5.66
(+1)

[5.4; 5.7]

Mean percentage
of male fetuses (%)

51.3

47.0

51.7

45.5

[44.0; 55.4]

( ): in brackets, percentage differencevs.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.

 

Table10: Fetal (litter) incidences (%) of external variations

 

Dose-level (mg/kg/day)

       0

     50

    250

    750

HCD

Dams with live fetuses

     23

     24

     23

      24

      388

Number of live fetuses

    317

   330

    277

    295

    5000

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

1 (4.2)

6 (1.5)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

0 (0)

1 (0.3)

7 (0.1)(b)

Limb hyperflexion, F (L)

0 (0)

0 (0)

0 (0)

0.3 (4.2)

0.4 (4.8)(a)

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a): maximum incidence.

 

Table 11: Fetal (litter) incidences (%) of external malformations

 

Dose-level (mg/kg/day)

          0

      50

      250

      750

HCD

Dams with live fetuses

        23

      24

        23

        24

      388

Number of live fetuses

      317

    330

      277

      295

    5000

Litters affected, n (%)

1 (4.3)

1 (4.2)

0 (0)

1 (4.2)

11 (2.8)(b)

Fetuses affected, n (%)

1 (0.3)

1 (0.3)

0 (0)

3 (1.0)

13 (0.3)(b)

Cleft palate, F (L) (%)

0 (0)

0.3 (4.2)

0 (0)

1.0 (4.2)

0.4 (5.0)(a)

Ombilical hernia,           F (L) (%)

0.3 (4.3)

0 (0)

0 (0)

0 (0)

0.4 (4.8)(a)

F             : fetal incidence.

L             : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)            : maximum incidence.

(b)            : mean incidence.

 

Table 12 Fetal (litter) incidences (%) of cartilage findings

 

Dose-level (mg/kg/day)

           0

       50

       250

    750

HCD

Dams with live fetuses

         23

       24

         23

      24

         388

Number of live fetuses

       164

      175

       145

    153

       2596

Litters affected, n (%)

23 (100.0)

22 (91.7)

22 (95.7)

20 (83.3)

236 (60.8)(b)

Fetuses affected, n (%)

130 (79.3)

121 (69.1)

98 (67.6)

67 (43.8)

990 (38.1)(b)

Cartilage of thoracic vertebra (e) present, F(L)

4.9 (30.4)

5.7 (33.3)

9.0 (43.5)

9.8 (50.0)

31.5 (87.5)(a)

F             : fetal incidence.

L             : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)            : maximum incidence.

(b)            : mean incidence.

 

Table 13: Fetal (litter) incidences (%) ofskeletal variations

 

Dose-level (mg/kg/day)

           0

      50

     250

     750

HCD

Dams with live fetuses

         23

      24

      23

      24

         388

Number of live fetuses

       164

    175

     145

     153

        2596

Litters affected, n (%)

23 (100.0)

22 (91.7)

22 (95.7)

20 (83.3)

345 (88.9)(b)

Fetuses affected, n (%)

133 (81.1)

125 (71.4)

101 (69.7)

69 (45.1)

1267 (48.8)(b)

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

3.0 (17.4)

5.1 (29.2)

6.2 (34.8)

8.5(50.0)*

29.6 (87.5)(a)

F             : fetal incidence.

L             : litter incidence.

Statistically significant*: p<0.05 for the number of litters affected.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)            : maximum incidence.

(b)            : mean incidence.

 

Table 14: Fetal (litter) incidences (%) of fetal skeletalmalformations

 

Dose-level (mg/kg/day)

      0

     50

  250

         750

HCD

Dams with live fetuses

    23

     24

   23

          24

      388

Number of live fetuses

  164

    175

  145

         153

    2596

Litters affected, n (%)

0 (0)

1 (4.2)

0 (0)

1 (4.2)

15 (3.9)(b)

Fetuses affected, n (%)

0 (0)

1 (0.6)

0 (0)

1 (0.7)

18 (0.7)(b)

Palate: split, F(L)

0 (0)

0 (0)

0 (0)

0.7 (4.2)

0.7 (5.0)(a)

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

0 (0)

0.6 (4.2)

0 (0)

0 (0)

1.3 (8.7)(a)

F             : fetal incidence.

L             : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

(a)            : maximum incidence.

(b)            : mean incidence.

 

Table 15: Fetal (litter) incidences (%) of soft tissue variations

 

Dose-level (mg/kg/day)

       0

     50

    250

    750

HCD

Dams with live fetuses

      23

     24

      23

      24

      387

Number of live fetuses

    153

   155

    132

    142

    2404

Litters affected, n (%)

6 (26.1)

11 (45.8)

6 (26.1)

6 (25.0)

86 (22.2)(b)

Fetuses affected, n (%)

9 (5.9)

12 (7.7)

11 (8.3)

11 (7.7)

119 (5.0)(b)

Dilated renal pelvis, F (L)

2.0 (8.7)

3.9 (25.0)

4.5 (13.0)

4.2 (20.8)

9.5 (28.6)(a)

Short innominate artery, F (L)

0 (0)

0.6 (4.2)

0 (0)

2.8 (12.5)

3.7 (22.7)(a)

Absent innominate artery, F (L)

0 (0)

2.6 (16.7)

1.5 (8.7)

0.7 (4.2)

5.1 (25.0)(a)

Dilated ureter, F (L)

3.3 (13.0)

2.6 (16.7)

3.0 (13.0)

2.8 (16.7)

7.1 (28.0)(a)

Thymus: reddish foci, F (L)

0.7 (4.3)

0 (0)

0.8 (4.3)

0 (0.0)

/

F             : fetal incidence.

L             : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in HCD.

(a)            : maximum incidence.

(b)            : mean incidence.

 

Table 16: Fetal (litter) incidences (%) of soft tissue malformations

 

Dose-level (mg/kg/day)

       0

      50

    250

    750

HCD

Dams with live fetuses

      23

      24

      23

      24

         387

Number of live fetuses

    153

     155

    132

    142

       2404

Litters affected, n (%)

0 (0)

0 (0)

1 (4.3)

0 (0)

7 (1.8)(b)

Fetuses affected, n (%)

0 (0)

0 (0)

1 (0.8)

0 (0)

13 (0.5)(b)

Absent kidney, F (L)

0 (0)

0 (0)

0.8 (4.3)

0 (0)

0.7 (4.5)(a)

Absent ureter, F(L)

0 (0)

0 (0)

0.8 (4.3)

0 (0)

/

F             : fetal incidence.

L             : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in HCD.

(a)            : maximum incidence.

(b)            : mean incidence.

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 50, 250 and 750 mg/kg/day. The 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 was considered to be 750 mg/kg/day,
- the NOAEL for embryo-fetal development was considered to be 750 mg/kg/day.
Executive summary:

The potential toxic effects of Luperox DH on the pregnant female and on embryonic and fetal development, was evaluated 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 a dose-level of 50, 250 or 750 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 sacrificed 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 of each dam was weighed and samples of liver and stomach were collected and preserved in 10% buffered formalin.

The concentrations of the test item in the dose formulations (+1.4% to +8.5%) remained within an acceptable range of variations (± 15%) when compared to the nominal concentrations.

No test item was observed in the control dose formulation. All females were pregnant with the exception of one female given 250 mg/kg/day. There were no test item treatment-related unscheduled deaths in any group. Ptyalism was noted in 20/24 females given 250 mg/kg/day and in 24/24 dams given 750 mg/kg/day. There were no effects on body weight or body weight change at any dose-level. There were no effects on food consumption at any dose-level. Test-item related effects consisted of increased liver weights at 750 mg/kg/day. Increased mitosis was observed in 3/10 high-dose females; a relationship with the test item could not be excluded. Although the pathogenesis of both findings remained unclear, in the absence of other histopathological findings (such as necrosis/degeneration), they were considered to be non-adverse. No treatment-related and/or adverse changes were observed in the net body weight change and in the hysterectomy data. There were no effects on fetal body weight or sex-ratio at any dose-level. There were no test item treatment-related variations or malformations at externa, soft tissue and skeletal examination.   

On the basis of the results obtained in this study:

.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 750 mg/kg/day,

.            the NOAEL for embryo-fetal development was considered to be 750 mg/kg/day.

Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopropylbenzene hydroperoxide
EC Number:
247-988-1
EC Name:
Diisopropylbenzene hydroperoxide
Cas Number:
26762-93-6
Molecular formula:
C12H18O2
IUPAC Name:
reaction mass of 1-(3-isopropylphenyl)-1-methylethyl hydroperoxide and 1-(4-isopropylphenyl)-1-methylethyl hydroperoxide
Test material form:
liquid
Details on test material:
SMILES : [*]OO.[*]C(C)C.CC(C)c1ccccc1
Molecular formula: C12H18O2
Molecular Weight: 196.288

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion