Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The oral (gavage) administration of Bis(2,4 -dichlorobenzoyl) peroxide (CAS# 133 -14 -2), paste, 50% in silicone oil, for ninety consecutive days, to Wistar rats of either sex at dose levels of 100, 300 or 1000 mg/kg bw/day resulted in treatment-related effects sperm analysis,

reduced testes, epididymides and cauda epididymis weight and microscopic changes in the testes / epididymides (tubular atrophy with an accompanying aspermia in the epididymis) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day. These changes were considered to represent adverse effects of treatment on the male reproductive system. No toxicologically significant effects were evident in females treated with 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day.

The 'No Observed Adverse Effect Level (NOAEL) was therefore considered to be 300 mg/kg bw/day for females and 100 mg/kg bw/day for males.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
no further testing on fertility is necessary because the substance is known to have an adverse effect on fertility, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage fertility (H360F), and the available data are adequate to support a robust risk assessment and classification and labelling
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Excellent (GLP and OECD)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Available information from the 90 -day and 28 -day repeated dose toxicity studies revealed toxicologically significant findings in the male reproductive system and are considered toxicologically significant findings on fertility. It was therefore concluded, based mostly on the 90 -day study results, that these toxicologically significant findings in males would require the substance to be classified as reproductive toxicant Category 1B. Available data are complete and no further animal testing is deemed necessary


Short description of key information:

An oral (gavage) study of CAS# 133 -14 -2, paste, 50% in silicone oil, for 90 days, administered to Wistar strain rats at dose levels of 100, 300 or 1000 mg/kg bw/day was conducted (OECD 408). A vehicle alone (Arachis oil BP) group was included. Recovery was assessed after 4 -weeks in two recovery groups (males and females) which were previously treated with the high dose (1000 mg/kg bw/ day) or the vehicle alone for ninety consecutive days. Conclusion: Treatment-related effects in body weight development, hematology parameters (anemia), sperm analysis (reduced sperm concentration, motility and increased sperm morphological abnormalities), reduced testes, epididymides and cauda epididymis weight and microscopic changes in the bone marrow (fatty vacuolation and subsequent decreased cellularity), testes/epididymides (tubular atrophy with an accompanying aspermia in the epididymis) and thymus (atrophy) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day were noted. These changes were considered as adverse effects of treatment. After recovery period, microscopic changes in thymus had completely resolved, while the reversibility of bone marrow changes were not complete. In testes and epididymis, the microscopic changes and aspermia persisted. No toxicologically significant effects were evident in females treated with 300 mg/kg bw/day orin animals of either sex treated with 100 mg/kg bw/day. The 'No Observed Adverse Effect Level (NOAEL) was therefore considered to be 300 mg/kg bw/day for females and 100 mg/kg bw/day for males. The latter, being the lowest NOAEL for this substance, was used as a starting point in DNEL calculations.

Results from a previously conducted 28 -day repeated oral dose study support the adverse findings from the 90 -day study. In that study, for some males at 1000 mg/kg bw/day, increased Sertoli-cell vacuolation with focal, segmental tubular degeneration was observed in the testes accompanied, in some cases, by spermatid retention in stage X tubule. In addition, interstitial edema with inflammatory cell or mononuclear cell infiltration as well as oligospermia and/or increased intraductal cellular debris were observed in the epididymis of the males showing testicular change. The NOAEL from the 28 -day study was 300 mg/kg bw/d. It was therefore concluded that the toxicologically significant findings in males from the 90 -day study (NOAEL: 100 mg/kg bw/day) would require the substance to be classified as reproductive toxicant Category 1B. Data are complete and no further animal testing is deemed necessary.

Justification for selection of Effect on fertility via oral route:
Two studies (OECD 408 with additional reproductive parameters and OECD 414) were conducted. The results from the OECD 408 (repeated oral dosing study that included an evaluation of additional reproductive parameters, viz., estrus cycle, sperm analysis) revealed adverse findings leading to classifying the substance as a reproductive toxicant Category 1B based on gross necropy and microscopic findings in male reproductive organs, and male sexual function (sperm analysis data: concentration, motility and morphology) data. These results are in line with the adverse histopathologic findings noted the male reproductive system in a previous 28 -day repeated oral dose study in rats. For these reasons, and to save animals, no further animal testing is deemed necessary for this endpoint.

Effects on developmental toxicity

Description of key information

In the OECD 414 study, the oral (gavage) administration of Bis(2,4 -dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil to pregnant Sprague-Dawley rats from gestation Days 5 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation (-23% cumulative gain at Day 20 of gestation) that was preceded by an initial reduction (-19%) in food consumption at 1000 mg/kg bw/day.  No similar effects were apparent at 300 or 100 mg/kg bw/day.  Consequently, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

In the same study, in-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day, although reduced fetal and placental weights and an increased incidence of visceral (kinked/dilated ureters, increased renal pelvic cavitation and left sided umbilical artery) and skeletal findings (reduced ossification) indicated an adverse effect on fetal growth.  No adverse treatment-related changes in the measured fetal parameters or embryofoetal development were detected at 300 or 100 mg/kg bw/day.  The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 27 January 2016 and 01 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 20001
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley Crl:CD (SD) IGS BR strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: not reported
- Weight at study initiation: 183 - 273g
- Fasting period before study: None
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3”C
- Humidity (%): 50 ± 20%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 January 2016 To: 17 February 2016
Route of administration:
oral: gavage
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks.

Preparation of Calibration Standards
Stock solutions of test item in extract solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with extract solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL. The standard solutions contained the equivalent amount of vehicle to that of the relevant samples.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters below.

Preparation of Test Samples
The formulations received were diluted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extract solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and Precision Samples
This was performed under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.

Instrumentation Parameters

HPLC : Agilent Technologies 1200, incorporating autosampler and workstation
Column : Prodigy 5µ C8 (250 x 4.6 mm id) at 40°C
Mobile phase : Methanol: water (90:10 v/v)
Flow-rate : 1 mL/min
UV detector wavelength: 250 nm
Injection volume: 25 µL
Retention time : ~ 3.8 and 4.8 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
This was performed under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.

Homogeneity and Stability in Vehicle Formulations
This was performed under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.

Refrigerated storage (nominally +4ºC)
This was performed under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Concentration of Dose Formulations

The mean concentrations of Test Item in test formulations were within applied limits ±10%, (excluding Analysis 2; 250 mg/mL Rep 2 and Analysis 3; 250 mg/mL Rep 2) confirming accurate formulation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.
The homogeneity and stability was confirmed for Test Item in Arachis Oil BP formulations at nominal concentrations of 25 mg/mL and 250 mg/mL when stored refrigerated for 21 days under Envigo Study Number 41502366: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil: Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat with Recovery Groups.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±11% of nominal concentrations, confirming accurate formulation

Details on mating procedure:
Not reported for the following reason:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
14 Days - Day 5 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
18 Days (Day 3 to Day 20 of Gestation)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Guideline requirement
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.
- Other:
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes. All animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes.Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION AND COMPOUND INTAKE: Water intake was observed daily by visual inspection of the water bottles for any overt changes

POST-MORTEM EXAMINATIONS: Yes:
Terminal Investigation
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible.
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus
Fetal examinations:
The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation.

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
((No. corpora lutes - No. implantations)/No. corpora lutea) x 100

Percentage post-implantation loss was calculated as:
((No. implantations - No. live fetuses)/No. implantations)) x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = (No. male fetuses/Total No. fetuses) x 100

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were detected.
One female treated with 1000 mg/kg bw/day had noisy respiration on Day 15. One control female also had noisy respiration on Day 17 and a further control had Chromodacryorrhoea (Right Eye only) between Days 17 and 20. Generalized fur loss was also noted in one female animal treated with 300 mg/kg bw day on Days 19 and 20. Due to the isolated nature of these observations, these observations were considered to be incidental
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.001) in body weight gain between Days 5 and 6 of gestation, with actual body weight losses being evident in the majority of females. A slight recovery was evident between Days 6 and 7 however body weight gain for the remainder of gestation was lower when compared to controls, attaining statistical significance (p<0.05) between Days 11 and 14. Cumulative body weight gain throughout the treatment period for these females was statistically significantly reduced (-23% at Day 20). Body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (-43%) in females treated with 1000 mg/kg bw/day.
A slight, but statistically not significant reduction in body weight gain was evident in females treated with 300 mg/kg bw/day between Days 5 and 6 and Days 7 and 8 of gestation. This resulted in a statistically significant reduction in overall body weight gain between Days 5 and 8. Recovery was evident thereafter and by Day 20, overall body weight gain was comparable to controls. There was also no effect on body weight gain after the adjustment for the contribution of the gravid uterus.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.001) in body weight gain between Days 5 and 6 of gestation, with actual body weight losses being evident in the majority of females. A slight recovery was evident between Days 6 and 7 however body weight gain for the remainder of gestation was lower when compared to controls, attaining statistical significance (p<0.05) between Days 11 and 14. Cumulative body weight gain throughout the treatment period for these females was statistically significantly reduced (-23% at Day 20). Body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (-43%) in females treated with 1000 mg/kg bw/day.
A slight, but statistically not significant reduction in body weight gain was evident in females treated with 300 mg/kg bw/day between Days 5 and 6 and Days 7 and 8 of gestation. This resulted in a statistically significant reduction in overall body weight gain between Days 5 and 8. Recovery was evident thereafter and by Day 20, overall body weight gain was comparable to controls. There was also no effect on body weight gain after the adjustment for the contribution of the gravid uterus.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected in treated females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
No clinical signs of toxicity were detected.
One female treated with 1000 mg/kg bw/day had noisy respiration on Day 15. One control female also had noisy respiration on Day 17 and a further control had Chromodacryorrhoea (Right Eye only) between Days 17 and 20. Generalized fur loss was also noted in one female animal treated with 300 mg/kg bw day on Days 19 and 20. Due to the isolated nature of these observations, these observations were considered to be incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
gross pathology
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in the number of fetuses reported as small was evident in litters from females treated with 1000 mg/kg bw/day. Consequently, the overall number of fetuses/litters affected was also statistically significantly increased at the treatment level.
Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 indicated any adverse effect of maternal treatment at 300 or 100 mg/kg bw/day on fetal development.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean fetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance in both fetal weights and litter weights. Placental weights were also statistically significantly reduced (-11%) in these females.
No similar effects on fetal, litter or placental weights were detected in females treated with 300 or 100 mg/kg bw/day.
Changes in postnatal survival:
not examined
External malformations:
not specified
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of affected fetuses/litters increased compared with control and differences frequently attaining statistical significance. These parameters included incomplete ossification of the frontal, nasal and premaxilla bones of the skull, incomplete/no ossification of one thoracic vertebral centrum, incomplete ossification of the cervical (neural) arch, incomplete/no ossification of the sacral (neural) arch, less than 4 caudal vertebrae ossified, incomplete/no ossification of one sternebra, costal cartilage not fused to sternebra, ossification centre associated with 7th cervical vertebra, no ossification of the metacarpals, incomplete ossification of the metatarsals and incomplete ossification of the femur. There was also a lower incidence of fetuses showing ossification of the ventral arch of vertebra 1 and ossification of one or more forepaw phalanges. The group mean incidences of these findings were higher than observed for the historical control ranges.
At 300 mg/kg bw/day, an increased incidence of fetuses/litters showing incomplete ossification of the femur was evident. Although group mean values were above historical control range, the group mean values for control litters were also higher than historical control range. This variation was seen in isolation of other developmental changes at this dose level and in the absence of an effect on mean fetal weight. Consequently, this was considered to be an isolated finding which was not regarded as evidence of adverse developmental toxicity
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral examination of fetuses at 1000 mg/kg bw/day, revealed an increased incidence of fetuses/litters showing kinked ureters, dilated ureters and increased renal pelvic cavitation. An increased incidence of fetuses/litters showing left sided umbilical artery was also evident at this dose level. The group mean incidences of these findings were higher than observed for the historical control ranges.
No such effects were evident in fetuses from females treated with 300 or 100 mg/kg bw/day.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Maternal exposure to 1000 mg/kg bw/day of the test item was associated with actual body weight losses between Days 5 and 6 of gestation and continued lower maternal body weight gain during the remainder of gestation and an initial reduction (-19%) on food consumption. While part of the lower overall weight gain observed was attributable to reduced fetal weight, an underlying effect on the pregnant dam was still present when body weight gain was adjusted for the contribution of the gravid uterus.

In-utero survival of the developing conceptus appeared unaffected by maternal treatment at 1000 mg/kg bw/day with both pre and post-implantation losses being comparable to control. This was despite a clear treatment-related reduction in fetal weight which resulted in lower litter weight at this treatment level and which attained statistical significance.  A reduction in placental weights were also observed for these fetuses.  The lower fetal weight at 1000 mg/kg bw/day is suggestive of a retardation of fetal growth at this treatment level and this was supported by subsequent findings observed at fetal examination.  External examination of the fetuses at necropsy revealed an increased incidence of small fetuses from females treated with 1000 mg/kg bw/day.  Visceral examinations also revealed a small number of findings (kinked ureters, dilated ureters and increased renal pelvic cavitation) that indicated a treatment-related disturbance of the normal development of the kidneys and ureters and an increased incidence of left sided umbilical artery.

Skeletal evaluation of fetuses from females treated with 1000 mg/kg bw/day showed significant differences compared to controls. The number of individual sites with reduced ossification and the difference in their incidence compared to controls was particularly higher, with a wide range of structures affected.  Generalised delays have a common ‘finger print’, characterised by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation (e.g. supra occipital bone and metacarpals) and denotes generalized growth delays with subsequent catch-up postnatally (Carney and Kimmel, 2007).  

For females treated with 300 or 100 mg/kg bw/day, clinical signs, body weight performance, food consumption and macroscopic necropsy examinations did not indicate any obvious effect of treatment, therefore 300 mg/kg bw/day is considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female. Litter data, fetal, litter and placental weights, external fetal appearance and detailed visceral and skeletal fetal evaluation at 300 or 100 mg/kg bw/day did not indicate any adverse effect of maternal treatment on the developing conceptus.  A dose level of 300 mg/kg bw/day is therefore considered to be the No Observed Adverse Effect Level (NOAEL) for the developing conceptus.  

Conclusions:
The oral (gavage) administration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil to pregnant Sprague-Dawley rats from gestation Days 5 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation (-23% cumulative gain at Day 20 of gestation) and an initial reduction (-19%) on food consumption at 1000 mg/kg bw/day. No similar effects were apparent at 300 or 100 mg/kg bw/day. Consequently, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day, although reduced fetal and placental weights and an increased incidence of visceral (kinked/dilated ureters, increased renal pelvic cavitation and left sided umbilical artery) and skeletal findings (reduced ossification) indicated an adverse effect on fetal growth. No adverse treatment-related changes in the measured fetal parameters or embryofoetal development were detected at 300 or 100 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.
Executive summary:

The study was performed according to the study plan and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

•       US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

•       Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

•       OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

•       Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.  

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents.  The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded.  Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

The stability and homogeneity of the test item formulations were performed under Envigo Study Number 41502366 and were shown to be stable for up to 21 days.  The test item formulations were analysed for concentration and results showed the formulations were within acceptable limits.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

There were no clinical signs of toxicity detected.

Body Weight

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain between Days 5 and 6 of gestation, with actual body weight losses being evident in the majority of females.  Body weight gain for the remainder of gestation was lower when compared to controls and statistical significance was achieved between Days 11 and 14.  Body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (-43%) in females treated with 1000 mg/kg bw/day.  No toxicologically significant effects were detected in females treated with 300 or 100 mg/kg bw/day.

Food Consumption

A statistically significant reduction (-19%) in food consumption was evident in females treated with 1000 mg/kg bw/day between Days 5 and 8.  Recovery was evident thereafter.  No treatment-related effects were detected in females treated with 300 or 100 mg/kg bw/day.

Water Consumption

No adverse effect on water consumption was detected.

Post Mortem Studies

No macroscopic abnormalities were detected in treated females.

Litter Data and Litter Placental and Fetal Weights

The number of implantations and subsequent embryofoetal survival were unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day.  At 1000 mg/kg bw/day, mean fetal weights for both sexes were statistically significantly lower than control, resulting in statistically significantly lower mean litter weights.  Placental weights were also statistically significantly reduced at this dose level.  No toxicologically significant effects were detected at 300 or 100 mg/kg bw/day.

Fetal Examination

External Findings

A statistically significant increase in the incidence of small fetuses (102 fetuses out of 16 litters) was evident from females treated with 1000 mg/kg bw/day.  No such findings were evident in litters from females treated with 300 or 100 mg/kg bw/day.

Detailed Visceral Examinations

At 1000 mg/kg bw/day, there was an increased incidence of fetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and left sided umbilical artery

compared with control.  No such findings were evident in litters from females treated with 300 or 100 mg/kg bw/day.

Detailed Skeletal Examination

At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control.  No treatment-related adverse effects were detected in the type and incidence of skeletal findings in fetuses from females treated with 300 or 100 mg/kg bw/day.

Conclusion

The oral (gavage) administration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil to pregnant Sprague-Dawley rats from gestation Days 5 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation (-23% cumulative gain at Day 20 of gestation) and an initial reduction (-19%) on food consumption at 1000 mg/kg bw/day.  No similar effects were apparent at 300 or 100 mg/kg bw/day.  Consequently, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day, although reduced fetal and placental weights and an increased incidence of visceral (kinked/dilated ureters, increased renal pelvic cavitation and left sided umbilical artery) and skeletal findings (reduced ossification) indicated an adverse effect on fetal growth.  No adverse treatment-related changes in the measured fetal parameters or embryofoetal development were detected at 300 or 100 mg/kg bw/day.  The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1
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

Justification for selection of Effect on developmental toxicity: via oral route:
Data from an OECD 414 study in rat via oral administration is available and have provided the NOAEL for the substance..

Justification for classification or non-classification

The NOAEL for developmental toxicity was 300 mg/kg bw/day due to adverse effects on fetal growth at 1000 mg/kg bw/day. The toxicologically significant findings from the 90 -day repeated dose study (with extended reproductive parameters) have shown adverse effects on male fertility. The latter would require the substance to be classified as reproductive toxicant Category 1B. These data are conclusive, and no further animal testing is deemed to be necessary for this endpoint.