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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:
26 February 2014 -- 27 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzoyl peroxide
EC Number:
202-327-6
EC Name:
Dibenzoyl peroxide
Cas Number:
94-36-0
Molecular formula:
C14H10O4
IUPAC Name:
benzoyl benzenecarboperoxoate
Test material form:
other: moistened white powder
Details on test material:
- Name of test material: Luperox A75
- Physical state: moistened white powder
- Lot/batch No.: 11628 201D10-041
- Analytical purity: 74.8% dibenzoyle peroxide in water
- Expiry date: June 2014
- Storage conditions: protected from light, at room temperature and tightly closed in a dry, well ventilated and insulated area.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: approximately 10-11 weeks old on the day of treatment
- Mean body weight at study initiation: 272 g (range: 214 g to 326 g)
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study

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

IN-LIFE DATES: 27 February 2014 to 27 March 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder using a mortar and pestle, and then mixed with the required quantity of vehicle.
The frequency of test item dose formulations, storage and delivery conditions were performed according to homogeneity and stability study (suspensions stable and homogeneous when stored 10 days at room temperature and protected from light).

VEHICLE
- 0.5% methylcellulose
- Concentration in vehicle: 20, 60 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: HPLC/UV
Before the start of treatment the suitability of the dose formulation process was confirmed. The homogeneity and stability was also determined on a range of dose formulations prepared at levels which cover the lowest and highest concentrations proposed for use in this study.
Precise details concerning the validation process, the homogeneity and stability study are documented in the homogeneity and stability study.

Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: detection of a vaginal plug
Duration of treatment / exposure:
Day 6 to day 20 post-coitum
Frequency of treatment:
Daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
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 time-mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a preliminary prenatal development study in which seven or eight pregnant females per group received the test item at dose-levels 100, 300 or 1000 mg/kg/day. At 1000 mg/kg/day, signs of slight maternotoxicity were evidenced (transient effects on body weight gain and food consumption) and the fetal body weight was 10% lower than that of controls.
At external examinations of the fetuses, no variations or malformations were noted at any dose-level.
Based on these data, the dose-levels of 100, 300 and 1000 mg/kg/day were selected as 1000 mg/kg/day was expected to produce slight signs of maternal toxicity and as it is the limit dose recommended by the OECD test guideline.

- Rationale for animal assignment: computerized stratification procedure

Examinations

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

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

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

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

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantation sites
- Number of early resorptions
- Number of late resorptions
- Number of uterine scars, evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : number dead and live, body weight, sex
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy status:
At study termination on Day 21 p.c., there were 22, 20, 22 and 19 females with live fetuses in the groups given 0, 100, 300 or 1000 mg/kg/day, respectively. The number of pregnant females at term (19) was considered not to have an impact on the overall interpretation of the study in view of the total number of fetuses evaluated per group.

Mortality:
At 1000 mg/kg/day, two females (C22573 and C22575) were prematurely sacrificed on Day 21 p.c. because of delivery (pups found in the bedding). These unscheduled deaths were not considered to be related to treatment with Luperox A75 in absence of clinical signs preceding the sacrifice, but most probably to have occurred by chance.

Clinical signs:
There were no test item-related clinical signs during the study.
The only clinical signs consisted of reddish vaginal discharge in 1/24, 1/24 and 3/24 females given
0, 300 or 1000 mg/kg/day, respectively, on Day 13 or 15 p.c., and area of hair loss observed on different parts of the body in 1/24 females given 1000 mg/kg/day between Days 16 and 18 p.c.. As these signs were reported both in control and test item-treated animals, on limited occasions and/or are findings commonly observed in females of this strain and age, they were considered to be unrelated to the test item treatment.

Body weight and body weight change:
At 1000 mg/kg/day, the mean body weight gain was lower than controls (+127 g vs. +153 g). This difference was due to the lower body weight gain recorded between Days 18 and 21 p.c. (+38 g vs. +57 g in controls, p<0.05). This effect on mean body weight gain was attributed to the test item treatment and correlated with reduced mean food consumption during the period of Days 18 to 21 p.c. (see below).
However, the terminal body weight recorded on Day 21 p.c. was not statistically significantly different from the mean control value.
At 100 and 300 mg/kg/day, there were no effects on mean body weight or mean body weight change (see Table 1).

Food consumption :
At 1000 mg/kg/day, the mean food consumption was 15% lower than controls between Days 18 and 21 p.c. (p<0.001). This effect on mean food consumption was attributed to the test item treatment and correlated with that noted on mean body weight gain during the same period (see Table 2).

Maternal necropsy findings:
There were no test item-related macroscopic findings at necropsy.
All macroscopic observations (i.e. renal pelvis dilation, ureter dilation, uterine horns with colored contents, scars of implantation sites in two females who have delivered and fused placenta with one implantation site for two fetuses), were not attributed to the test item treatment as they were reported both in control and test
item-treated animals and/or are findings routinely observed in females of this strain and age.

Gravid uterus weight :
At 1000 mg/kg/day and when compared with controls, there was a lower mean gravid uterus weight (-10%) and a lower mean net body weight change between Days 6 and 21 p.c. (+39.0 g vs. +43.9 g) with no effects on the mean carcass weight.
These observations confirmed a slight effect of the test item both on the maternal body weight and the gravid uterus weight.
At 100 and 300 mg/kg/day, there was no effect on mean gravid uterus weight or on mean net body weight change (see Table 3).

Hysterectomy data :
On Day 21 p.c., all principal pregnant females had live concepti.
There were no effects on gestation parameters at any dose-level.
The higher mean post-implantation loss recorded at 300 and 1000 mg/kg/day was considered to be physiological variations unrelated to the test item treatment since the mean values were within the historical control data (mean% = 6.5, min-max = 2.0-8.7%) (Table 4).

Organ weights
There were no differences in the weights of kidneys recorded at the time of hysterectomy of the dams between the control and the test-item treated groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Body weight and sex:
At 1000 mg/kg/day and when compared with controls, there was a lower mean fetal body weight (-16%, p<0.001). This finding was attributed to the test item treatment, mean value was below HCD and correlated with the lower mean gravid uterus weight recorded in this group (see Table 5).
At 100 and 300 mg/kg/day, there was no effect on mean fetal body weight.

The sex ratio was unaffected by the test item treatment at any dose-level.

Fetal external examination :
There were no test item treatment-related effects on the incidences of external variations (autolysed fetuses was the unique variation observed without any dose-relationship).
There were no fetal external malformations in any of the fetuses examined.

Fetal soft tissue examination:
- Malformations
There were no fetal soft tissue malformations in the study.
- Variations
There were no test item treatment-related soft tissue variations and no malformations in any of the fetuses observed in any groups. The dilated renal pelvis litter and fetal incidences of all group were higher than the upper limit of the historical control data (controls including) but with no dose-relationship. Therefore, this finding was considered not to be unrelated to the test item treatment (Table 7).

Fetal skeletal examination (Table 8):
- Malformations
There were no skeletal malformations at examination of fetuses from any groups that could be ascribed to the test item treatment. The unossifed centrum of thoracic vertebrae concerned 2 fetuses in 1 litter among the high dose group and was considered as an isolated phenomenon reflecting a developmental delays rather than an abnormality (although fetal and litter incidences were slightly above the HCD).
- Variations
At 1000 mg/kg/day, there was an increased number of litters with fetuses with incomplete ossification of various part of the skeleton, as summarized below (only statistical difference were reported):

All these development variations were considered to be test item treatment related since they were often recorded at higher incidence when compared with historical control data and in fetuses with the lower mean fetal body weights.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weight and body weight changes (g)

 

Dose-level (mg/kg/day)

0

100

300

1000

Body weight

 

 

 

 

. Day 6 p.c.

273

269

275

276

. Day 18 p.c.
% from controls

369
-

369

0

367

-1

364

-1

. Day 21 p.c.
% from controls

426
-

424
0

421
-1

403
-5

Body weight change

 

 

 

 

. Days 18 - 21 p.c.

+57

+55
-

+54
-

+38*
-

. Days 6 - 21 p.c.

+153
-

+155
1

+146
-

+127**
-

Statistically significant vs. controls: *: p<0.05, **: p<0.01. In bold type: relevant differences from controls: -: not applicable.

Table 2 : Mean food consumption (g/day)

 

Dose-level (mg/kg/day)

0

100

300

1000

Days 18 to 21p.c.

33

32

32

28#

% from controls

-

-3

-3

-15

Statistically significantvs.controls: #: p<0.001. In bold: relevant differences from controls; -: not applicable.

Table 3: Gravid uterus weight

 

Dose-level (mg/kg/day)

0

100

300

1000

Gravid uterus weight
% from controls

109.2
-

107.6
-1

101.9
-7

98.5
-10

Carcass weight

317.1

316.0

319.5

315.9

% from controls

-

0

+1

0

Net body weight change
from Day 6 p.c.

+43.9

+47.1

+44.3

+39.0

-: not applicable; In bold: relevant differences from controls.

Table 4: Hysterectomy data

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of females with live fetuses at term

22

20

22

19

Mean number ofcorpora luteaper female

15.5

15.3

14.8

16.1

Mean number of implantations per female

14.3

14.6

13.7

15.1

Mean pre-implantation loss per female (%)

7.6

6.4

8.0

5.8

Mean number of fetuses per female

14.0

13.8

12.8

14.1

Dead fetuses (%)

0.3

0.0

0.3

0.0

Mean number of resorptions per female

0.3

0.8

0.9

0.9

Mean post-implantation loss per female (%)

2.6

5.0

6.7

6.3

Table 5: Mean fetal body weight (g)

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Mean fetal body weight (g)

5.7

5.7

5.7

4.8#

5.7[5.5-5.8]

% from controls

-

+1

+0

-16

-

Statistically significantvs.controls: #: p<0.001. In bold: relevant differences from controls, -: not applicable.

Values were rounded, HCD: Historical Control Data; HCD with mean [min-max].

Table 7:Litter (L) and Fetal (F) incidences of soft tissue variations

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

22

19

-

Live fetuses, n

147

132

134

129

-

Kidney

 

 

 

 

 

. dilated renal pelvis, L (F), %

9.1 (1.4)

15.0 (2.3)

13.6 (2.2)

21.1 (5.4)

7.1 (1.3)

Adrenal gland

 

 

 

 

 

. colored focus, L (F), %

0 (0)

0 (0)

4.5 (1.5)

0 (0)

na

Vessels

 

 

 

 

 

. absent innominate artery

0 (0)

0 (0)

4.5 (0.7)

5.3 (0.8)

na

. short innominate artery

0 (0)

0 (0)

4.5 (0.7)

0 (0)

na

Ureter

 

 

 

 

 

. dilated, L (F), %

0 (0)

15.0 (3.8*)

0 (0)

10.5 (1.6)

5.7 (1.4)

Statistically significancevs.controls: *: p<0.05,

HCD: historical control data ; na: not applicable as not seen in HCD; -: not applicable.

Table 8: Litter (Fetal) incidences (%) of skeletal incomplete (I) or unossified (U) ossifications

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

22

20

22

19

-

Live fetuses, n

159

144

147

139

-

parietal (I)

22.7(3.1)

10.0(1.4)

13.6(2.0)

47.4(16.5#)

15.6(3.7)

interparietal (I)

18.2(2.5)

15.0(2.1)

22.7(4.1)

63.2**(23.0#)

31.9(7.5)

supraoccipital (I)

0(0)

0(0)

0(0)

47.4#(13.7#)

5.0(1.2)

frontal (I)

0(0)

0(0)

4.5(0.7)

21.1*(6.5#)

2.8(0.4)

hyoid (I)

18.2(2.5)

30.0(7.6)

36.4(7.5)

52.6*(18.0#)

16.3(4.2)

hyoid (U)

9.1(2.5)

5.0(0.7)

4.5(0.7)

52.6**(11.5**)

0.7(0.1)

cervical vertebrae – centrum (U)

9.1(1.9)

35.0(7.6*)

22.7(6.1)

47.4*(16.5#)

40.4(11.0)

caudal vertebrae – (U)

4.5(0.6)

5.0(1.4)

9.1(1.4)

52.6#(18.7#)

na

caudal vertebrae – arch (I)

0(0)

10.0(1.4)

18.2(4.1*)

42.1#(9.4#)

5.7(1.2)

caudal vertebrae – centrum (I)

9.1(1.3)

0(0)

9.1(1.4)

31.6(7.2*)

5.0(1.0)

caudal vertebrae – arch (U)

4.5(0.6)

0(0)

9.1(1.4)

52.6#(23.0#)

3.5(0.8)

sternebrae 6th (I)

4.5(0.6)

5.0(0.7)

9.1(1.4)

47.4**(10.8#)

na

sternebrae 5th (U)

4.5(0.6)

5.0(0.7)

0(0)

57.9#(12.9#)

na

sternebrae 6th (U)

0(0)

0(0)

0(0)

15.8(5.0**)

na

metacarpal (I)

0(0)

0(0)

0(0)

42.1#(7.9#)

5.7(1.5)

metacarpal (U)

0(0)

5.0(0.7)

0(0)

15.8(7.2#)

0.7(0.2)

forepaw – proximal (U)

45.5(12.6)

55.0(20.1)

50.0(16.3)

94.7(71.9#)

13.5(3.7)

forepaw – distal (U)

45.5(18.9)

70.0(31.9*)

72.7(25.2)

84.2*(50.4#)

7.8(5.2)

metatarsal 1st (U)

31.8(8.2)

40.0(8.3)

31.8(13.6)

94.7#(71.2#)

13.5(3.7)

hindpaw – distal (U)

40.9(14.5)

50.0(19.4)

63.6(20.4)

84.2**(48.9#)

6.4(3.5)

Total fetal variations (fetuses/litter)

54.2

71.0

59.8

92.2#

na

Statistically significance vs. controls: *: p<0.05; **:p<0.01; #: p<0.001;

HCD: historical control data; na: not applicable as not seen in HCD; -: not applicable.

Applicant's summary and conclusion

Conclusions:
When dibenzoyle peroxide was administered to female Sprague-Dawley rats by gavage from Days 6 to 20 p.c. at 100, 300 or 1000 mg/kg/day, signs of maternal toxicity were recorded at the highest dose-level (lower body weight gain and food consumption, lower uterus weight). Fetal findings were recorded at 1000 mg/kg/day in a context of maternal toxicity. In this group, there was evidence of fetal growth retardation (body weight and incomplete ossification of the skeleton).
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day (effects on body weight change and on food consumption at 1000 mg/kg/day),
- the NOAEL for embryo-fetal development was considered to be 300 mg/kg/day (based on higher incidence of fetuses with incomplete ossification at 1000 mg/kg/day),
- no teratogenic effect was observed up to 1000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of dibenzoyle peroxide on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.)inclusive). This GLP study was carried out according to OECD guideline No. 414. Three groups of 24 time-mated female Sprague-Dawley rats received dibenzoyle peroxide once daily from Day 6 to Day 20 p.c., by the oral route, at dose-levels of 100, 300 or 1000 mg/kg/day. Another group of 24 time-mated rats received the vehicle only, 0.5% methylcellulose, 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 21p.c., the females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissues and skeletal abnormalities (including cartilage). The kidneys of dams were weighed.

The concentrations of the test item in the dose formulations remained within the acceptable range of variations (-9% to -5%) when compared to the nominal concentrations (± 15%). At study termination on Day 21p.c., there were 22, 20, 22 and 19 females with live fetuses in the groups given 0, 100, 300 or 1000 mg/kg/day, respectively. Two pregnant females at 1000 mg/kg/day were prematurely sacrificed on Day 21 p.c. because of delivery. No test item-related clinical signs were observed during the study. Low body weight gain and food consumption were recorded at 1000 mg/kg/day between Days 18 and 21 p.c. and these observations were associated with a low net body weight change. At necropsy of the dams, there were no test item-related macroscopic post-mortem findings and no effects on kidneys weights. At hysterectomy, the gravid uterus weight was 10% lower at 1000 mg/kg/day and correlated with the 16% lower fetal body weight when compared with controls. No effects on gestation parameters or on sex ratio were noted.There were no effects on mean numbers of pre- and post-implantation losses in any groups. At external fetal examination, there were no effects on the incidence of variations, and there were no external malformations. At soft tissue examination, there were no variations and no malformations considered to be related to treatment with the test item. At skeletal examination, there was an increased number of litters with fetuses with incomplete ossification of the skeleton at 1000 mg/kg/day.

When dibenzoyle peroxide was administered to female Sprague-Dawley rats by gavage from Days 6 to 20 p.c. at 100, 300 or 1000 mg/kg/day, signs of maternal toxicity were recorded at the highest dose-level (lower body weight gain and food consumption, lower uterus weight). Fetal findings were recorded at 1000 mg/kg/day in a context of maternal toxicity. In this group, there was evidence of fetal growth retardation (body weight and incomplete ossification of the skeleton).

On the basis of the results obtained in this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day (effects on body weight change and on food consumption at 1000 mg/kg/day),

- the NOAEL for embryo-fetal development was considered to be 300 mg/kg/day (based on higher incidence of fetuses with incomplete ossification at 1000 mg/kg/day),

- no teratogenic effect was observed up to 1000 mg/kg/day.