Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Age : 7 weeks old for males and females, strains were obtained from Biogenomics Limited
- Weight at study initiation : 323.38 - 356.63 g for males, 212.21 - 237.18 g for females
- Temperature: 18.25-25.69 °C
- Humidity: 51.09 - 73.75 %
- 12-hour dark cycle
- 15-15 room air change per hour
- Water and diet ad libitum
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
- M/F ratios per cage ; 1/1
- Proof of pregnancy: sperm detection in vagina
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Dosing schedule :
- males: premating period of 2 weeks, then mating period for 15 days, total of 29 days
- females: premating period of 2 weeks then mating, pregnant and 3 days lactation period , total of 41 ~ 51 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 250, 500, 1,000 mg/kg b.w/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose (5 animals/sex/positive control)
Control animals:
yes, concurrent vehicle
Details on study design:
- Parameters assessed during study P and F1 : copulation index (matings/pairs x 100), fertility index (Pregnancies/matings x 100), gestation index (live litters/pregnancies x 100), viability index (No. of live offspring at day1 or day3/No. of live offspring at birth × 100), body weight of live pups (on day 0 and 4), No. of corpora lutea, No. of female mated, abortion, premature birth, gestation period, sex ratio (Total No. of male pups/Total No. of female pups)
Positive control:
Yes: Cyclophosphamide (4.5 mg/kg/day)
Parental animals: Observations and examinations:

MORTALITY: observation once daily

DETAILED CLINICAL OBSERVATIONS AND BODY WEIGHT: Yes
Clinical observations performed and frequency : Clinical signs were observed twice a day over the administration period, and once daily during the weekend. Changes in skin, hair, eyes, mucous membrane, respiratory system, autonomic nervous system, central nervous system, mobility and behavioral patterns were recorded with careful attention to tremor, seizure, ptyalorrhea, diarrhea, drowsiness, stupor or coma. Body weight were observed once a week respectively.

Moreover, haematology and biochemistry for males only at the time of necropsy after 28 days of chemical exposure (detailed in 7.5.1)
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
- Sperm examination : concentration, motility, morphology was observed at necropsy.
Litter observations:
Sex ratio (Total No. of male pups/Total No. of female pups) was assessed, body weight of live pups (on day 0 and 4)
Body weight of neonates was recorded on day 1 pp and on day 3 pp.
All living neonates were subjected to an external examination within 24 hours after birth.
Postmortem examinations (parental animals):
- Organs examined at necropsy (macroscopic and microscopic) :
Organ weight : liver, kidney, adrenal gland, testis, epididymis, thymus, spleen, brain, heart, ovary, uterus, thyroid gland, spermary, prostrate gland. - Microscopic : brain, spinal cord, stomach, pancreas, jejunum, ileum, cecum, colon, rectum, liver, kidney, adrenal gland, spleen, heart, thymus, thyroid, bronchus, lung, pituitary gland, ovary, uterus, vagina, testis, epididymis, spermary, prostrate gland, mammary gland, bladder, nodi lymphatici mesenterici, nodi lymphatici, nervus ischiadicus, femoral marrow and all gross lesion.
Postmortem examinations (offspring):
Grossly visible abnormalities; external, soft tissu and skelettal abnormalities
Statistics:
Parametric data were analyzed using One-way Analysis of variance. If significance was detected, Dunnett¿s multiple comparison test was used to detect significant difference between the negative controls and treatment groups. One-sided Student¿s t-test was used to detect significant difference between the negative controls and the positive controls.
Non-parametric data were analyzed using Kruskal-Wallis analysis, and Dunn¿s multiple comparison test was conducted using ranked data.
Categorized data were analyzed using descriptive analysis or Two-sided Fisher¿s exact test.
All differences were considered significant at 5%.
Reproductive indices:
Parameters assessed during study P and F1 : copulation index (matings/pairs x 100), fertility index (Pregnancies/matings x 100), gestation index (live litters/pregnancies x 100), No. of corpora lutea, No. of female mated, abortion, premature birth, gestation period,
Offspring viability indices:
Viability index (No. of live offspring at day1 or day3/No. of live offspring at birth × 100),
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
minimal testes degeneration in 5 of 9 rats, seen on only 1/5 rat on the satellite group, and not associated with gross necropsy findings, testicular atrophy and decrease of fertility.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: increase of food consumption during the 4 first weeks only in the highest dose group
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no significant difference between experimental and control groups in the precoital time, copulation, fertility and gestation rate.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No deaths were found in all animals including control groups. Body weight : Body weight gains were slightly increased (approximately 7 % more than controls) in female recovery group exposed to 1,000 mg/kg only after 29 days.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No significant body weight changes were observed in all treatment animals except highest dose recovery group. Low body weight gain of pups showed in 1,000 mg/kg dose.
Food/water consumption : The mean food consumption of 1,000 mg/kg was significantly increased at first and fourth weeks of chemical exposure in male. No other treatment groups were showed abnormal food consumption.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant decrease in absolute and relative weights of only left testes and relative wt of epididymis was observed in ONE male exposed to 1000 mg/kg (p < 0.05) and is considered as sporadic effect as only seen on left testis.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Severe testis atrophy was seen in one male of the highest exposure male. In the 1,000 mg/kg treatment group,minimal teste degeneration was observed in several animals of this high dose-group but these changes were not seen in recovery animals. Moreover there is no effect on fertility. No graduation of the severity of this effect is given. this effect has to be noted but is not considered as adverse. In the female reproductive organ, slight affects were observed in one uterus of female exposed to 1,000 mg/kg is not considered as tratement-related. See table 4

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): none


GROSS PATHOLOGY (PARENTAL ANIMALS): no effects


Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
>= 1 000 mg/kg bw/day
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decrease of body weight gain at day 3 post-partum
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
BODY WEIGHT (OFFSPRING)
The pups exposed to highest levels of benzoyl peroxide showed significant decrease in body weight gain (p < 0.01). The body weight gain of pups on the day 3 after birth decreased at 1,000 mg/kg. Benzoyl peroxide has adverse effects on development of pups with high birthrate of runts at 1000 mg/kg dose level. No significant differences appeared between the treatment and control group in any other observation. See tables 2 and 3.


Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table 1

DOSE : (mg/kg)        0     250    500    1000    Cyclophos
phamide
No. of mated males 10 10 10 10 10
Copulation index (%) 100 100 100 100 100 (5/5)
Fertility index (%) 100 100 100 90 100 (5/5)
                                         (9/10)
No. of mated females 10 10 10 10 10
Copulation index (%) 100 100 100 100 100 (5/5)
Fertility index (%) 100 100 100 90 100 (5/5)
                                         (9/10)
Gestation index (%) 100 100 100 100 40 (2/5)
No. of dams 10 10 10 9 2
No. of corpora lutea 169 182 171 157 90
Mean 16.9 18.2 17.1 17.4 18
+-S.D +-4.1 +-2.3 +-1.9 +-2.5 +-1.9
No. of implantations 150 150 161 136 81
Mean 15.0 15.0 16.1 15.1 16.2
+-S.D +-1.8 +-2.9 +-1.9 +-2.1 +-1.6
Mean percent 9 16.1 5.8 12.9 9.9
preimplantation loss
No. of embryo/fetal 10/3 9/3 7/3 3/0 64/9
death
No. of live pups born
Mean 13.7 13.8 15.1 14.8 1.6***
+-S.D +-2.7 +-2.5 +-2.1 +-2.2 +-2.3
Mean pregnancy 20.4 20.8 20.6 20.8 22
period (day)
Viability index 97.5 98.1 100 100 53.3***
on day 1 pp
Viability index 100 100 100 100 90*
on day 3 pp
Body weights
of pups (g)
Male 1 DAY mean 6.88 7.22 6.91 6.72 4.59
+-S.D +-0.53 +-0.81 +-0.64 +-0.79 +-0.77
Male 3 DAY mean 8.93 9.10 8.69 8.12*** 7.16
+-S.D +-0.91 +-1.03 +-0.79 +-1.01 +-0.88
Female 1 DAY mean 6.51 6.93***6.73 6.21 4.33
+-S.D +-0.53 +-0.76 +-0.59 +-0.79b +-0.74
Female 3 DAY mean 8.60 8.79 8.48 7.49*** 5.48
+-S.D +-0.99 +-1.03 +-0.89 +-1.03 +-0.89
Precoital time
Mean (day) 2.2 3.2 2.4 2.3 5.3
+-S.D +-1.2 +-1.3 +-1.2 +-1.2 +-5.3
Number of females showing evidence of copulations was decreased at 1000 mg/kg but this effect has not to be taken into account, since a severe atrophy of left teste appeared in only one male at 1000 mg/kg (male 80) and since this male was
mated with female 35 that was not later pregnant. The atrophy of left teste can not be related to BPO treatment since absolute no effect is observed on right teste, including no histhopatological effect.

Table 2:
Grossly visible abnormalities, external, soft tissue and skeletal abnormalities:
In the 1000 mg/kg dose group, the incidence of runts was significantly higher then control group.

Treatment      0     250    500    1000   Cyclophosphamide
(mg/kg/day)
No. with major 0 1 0 0 0
abnormalities
Mean % of pups 0 0.6 0 0 0
examined
No. of females 0 1 0 0 0
affected
No. with minor 20 20 18 60 8
abnormalities
Mean % of pups 14.2 12.4 11.5 41.4*** 100.0***
examined
No. of females 6 4 5 6 2
affected
No. with 0 0 0 0 0
variants
*** Statistically significant difference from control group (p
< 0.001)
Conclusions:
The NOAEL for reproduction toxicity in male rats is 500 mg/kg based on reduced weight of testes and epididymis at 1,000 mg/kg. NOAEL for developmental toxicity is also 500 mg/kg based on significant decreased weight gain of pups with high birthrate of runt at 1,000 mg/kg.
Executive summary:

A combined repeated dose and reproduction/developmental toxicity screening test (OECD TG 422) was conducted using Sprague-Dawley rats (NIER, Korea, 2001). Benzoyl peroxide was administered to rats by gavage at doses of 0, 250, 500 and 1,000 mg/kg/day. Males were dosed for 29 days and females were dosed for 41 - 51 days from 14 days before mating to day 3 of lactation

throughout the mating and pregnancy period. No treatment-related changes in precoital time and rate of copulation, fertility and gestation were noted in any benzoyl peroxide treated groups. Minimal symptoms such as vacuolation or hyperplasia, were seen in 1000 mg/kg group, but this is not considered to have been related to benzoyl peroxide treatment. Adverse effects on reproduction were shown at the highest dose of 1,000 mg/kg/day in male rats with the reduction of reproductive organ¿s weight and slight testes degenerations. In female rats, no adverse effects were observed during the test period. The NOAEL for reproduction toxicity in male rats was 500 mg/kg/day. No variants were found. High birthrate of runt was seen and body weight gain of pups was significantly decreased (male 9 % ; female 12.9 % of control wt) at dose of 1,000 mg/kg/day. The study concluded that Benzoyl peroxide has adverse effects on development of pups with high birthrate of runt at 1000 mg/kg dose level. The NOAEL for developmental toxicity was determined as 500 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The NOAEL for the reproduction was 500 mg/kg bw in a well conducted combined repeated dose and reproduction/developmental toxicity study [OECD TG 422].

OECD 422 study in rats with BPO

A combined repeated dose and reproduction/developmental toxicity screening test (OECD TG 422) was conducted using Sprague-Dawley rats (Park, 2002). Benzoyl peroxide was administered to rats by gavage at doses of 0, 250, 500 and 1,000 mg/kg/day. Males were dosed for 29 days and females were dosed for 41 - 51 days from 14 days before mating to day 3 of lactation throughout the mating and pregnancy period. No treatment-related changes in precoital time and rate of copulation, fertility and gestation were noted in any benzoyl peroxide treated groups. Minimal symptoms such as vacuolation or hyperplasia, were seen in 1000 mg/kg group, but this is not considered to have been related to benzoyl peroxide treatment. Adverse effects on reproduction were shown at the highest dose of 1,000 mg/kg/day in male rats with the reduction of reproductive organ’s weight and slight testes degenerations. In female rats, no adverse effects were observed during the test period. The NOAEL for reproduction toxicity in male rats was 500 mg/kg/day. No variants were found. High birthrate of runt was seen and body weight gain of pups was significantly decreased (male 9 % ; female 12.9 % of control wt) at dose of 1,000 mg/kg/day. The study concluded that Benzoyl peroxide has adverse effects on development of pups with high birthrate of runt at 1000 mg/kg dose level. The NOAEL for developmental toxicity was determined as 500 mg/kg/day.

Effects on developmental toxicity

Description of key information
In a developmental toxicity study in rats (OECD 414) with benzoyl peroxide, 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) and 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.
Link to relevant study records
Reference
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.
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
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
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
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
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
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.
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
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.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP guideline study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 study in rats

The potential toxic effects of dibenzoyl peroxide on the pregnant female and on embryonic and fetal development was evaluated 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) (Chevalier, 2014a). This GLP study was carried out according to OECD guideline No. 414. Three groups of 24 time-mated female Sprague-Dawley rats received dibenzoyl 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 21p. 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 21p. 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 dibenzoyl 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.

Range-finding developmental toxicity study in rats

The potential toxic effects of dibenzoyl peroxide on the pregnant female and on embryonic and fetal development was evaluated 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), in order to select dose-levels for a further main study (Chevalier, 2014b). Three groups of eight time-mated female Sprague-Dawley rats received the test item once daily from days 6 to 20 p. c., by the oral route, at dose-levels of 100, 300 or 1000 mg/kg/day (dose-levels expressed as dibenzoyl peroxide using a corrective factor of 1.34 to take into account a concentration of 74.8% in the test item). Another group of eight 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 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 abnormalities.

All females were pregnant, except one at 100 mg/kg/day and another at 1000 mg/kg/day. No unscheduled mortality occurred and no test item-related clinical signs were observed during the study. At 1000 mg/kg/day, the body weight gain was transiently lower than that of the control group between days 6-9p. c. (-31%, not significant). This effect coincided with a lower food consumption during this period (-12%, p<0.05) without impacting the group mean body weight recorded throughout the dosing period. At necropsy, there were no macroscopic post-mortem findings in any of the dams examined. Compared to control mean values, the gravid uterus weight was 15% lower at 1000 mg/kg/day (but not significant) and correlated with the lower fetal weight recorded in this group (-10%, p<0.01). No effects on gestation parameters or on the sex ratio were noted. At external examination of the fetuses, there were no variations or malformations at any dose-level.

Dibenzoyl peroxide was administered to female Sprague-Dawley rats by oral gavage from Days 6 to 20 p. c. at dose-levels of 100, 300 or 1000 mg/kg/day. Slight maternal toxicity was recorded at 1000 mg/kg/day (transient and initial low body weight gain and food consumption). At this dose-level, the mean fetal body weight was 10% lower than that of the control group. None of the gestational parameters were affected, and there were no variations/malformations at the external examinations of fetuses. Based on the results of this study, 1000 mg/kg/day was considered to be lower than the Maximum Tolerated Dose for a developmental toxicity study.

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP) criteria, the substance is not classified for reproduction toxicity or for developmental toxicity.

Additional information