Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-08 to 2016-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Btach No.: 1198395-01
Physical state: liquid
Colour: colourless
Density: 0.995 g/cm³
Purity: 98.00 %
Storage conditions: -15 to -35 °C, protected from light
Expiry date: 2015-11-30

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age of the females at arriving at test facility: 11-12 weeks
- Age of the males at the start of pairing: 12-13 weeks
- Body weight range: males before initiation of pairing: 320 - 347 g; females before initiation of pairing: 196-227 g
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102150820) (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0631)
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations. For preparation, the test item was weighed into a tarred plastic vial on a precision balance. The test item formulations were prepared at room temperature by adding the required volume of the control item and vortexing it. Formulations or control items were stored at 2 to 8 °C or on crushed ice and were administered within 6 hours after preparation. Prior to administration test item formulations were brought to room temperature. The application volume for all groups was 5 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of approx. 10 mL of HD, 20 mL of MD and 50 mL of LD were retained from all groups once in the first and last week of the study (in total 8 samples). Sample analysis was performed on the same day. Samples for testing of homogeneity were taken from the top, middle and bottom of the HD and LD preparation and analysed on the same day (in total 12 samples). Samples were taken once in the first and last week of the study.
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day 0.
Duration of treatment / exposure:
between gestation day 5 and gestation day 19
Frequency of treatment:
daily between gestation day 5 and 19
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
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:
25 female animals each in control, low and medium dose group. 35 female animals in the high dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: according to OECD 414
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Animals from each batch were weighed once before initiation of pairing to ensure that the body weights were within + 20% variation, except for animals from batch 2 which were weighed at the time of mating. The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males during the entire study or for both male and females during the mating period

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The position and number of foetuses in each uterine horn was also recorded.
Fetal examinations:
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies. One half of each litter will be examined for soft tissue anomalies by a microdissection technique. Craniofacial examination of the heads of the foetuses used for the soft tissue examination will be performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.
The remaining half of each litter will be processed by Alizarin red staining and examined for skeletal alterations. For interpretation of significant external, visceral and skeletal findings, they were described as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life)
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Indices:
number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post-implantation loss.
Historical control data:
yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicological relevance in the dose groups when compared to the control group.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the control or any of the dose groups during the treatment period of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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 gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of any group.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
BODYWEIGHT DEVELOPMENT:
The mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group. However, after initiation of treatment the mean body weight gain was noted to be moderately but statistically significantly lower (p < 0.05 and p < 0.01) in the HD group when compared to controls during gestation days 5-8 (16 % of controls),14-17 (81 % of controls), 17-20 (79 % of control) and 0-20 (83 % of controls). Mean body weight of the HD group was slightly lower on gestation day 17 (94 % of controls, p< 0.05) and moderately lower on gestation day 20 (92 % of controls, p < 0.01). Throughout the whole study period, slightly lower mean body weight was noted for the MD group (1 % to 4 % below controls). As this slight difference was already apparent on gestation day 0 before initiation of treatment, it was considered to be incidental and not related effects of toxicological relevance to the treatment with the test item. Mean body weight of the LD group was also unaffected by the test item administration.

FOOD CONSUMPTION:
In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be moderately lower compared to the control group on various occasions throughout the treatment period but statistical significance was achieved only during the treatment period on gestation days 11-14 (85 % of controls) and gestation days 0-20 (91% of Controls). This effect on the food consumption of the HD group was considered to be test item related.
Mean food consumption of the LD and the MD group was slightly lower without achieving statistical significance compared to the controls. As differences with the control group were marginal and did not have an impact on the mean body weights in either group, the lower food consumption was not considered to be adverse.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion prior to the scheduled sacrifice.
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
Description (incidence and severity):
Successful mating resulted in 19/25 pregnancies in the LD group, 18/25 in the MD group and 22/35 in the HD group compared to 21/25 pregnancies in the control group. The low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 76 % in the LD group and of 72 % in the MD group were considered to be a biological variation. However, the low pregnancy rate of 62 % in the HD could be attributed to treatment with test item.
Other effects:
not examined
Details on maternal toxic effects:
PRENATAL DATA:
No test item related effects of toxicological relevance were noted for adjusted maternal weight (terminal body weight - gravid uterus weight), number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post-implantation loss. No dead foetuses were noted in any of the groups.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean foetus weight of the HD group (3.27 g) was moderately, statistically significantly lower when compared to the control group (3.70 g) (88 % of controls, p< 0.001).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
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):
Based on the lower mean foetus weight of the HD group, a moderately, statistically significantly lower total litter weight was observed when compared to controls (78 % of controls, p< 0.001). Moderately, statistically significantly lower female litter weight of the HD group (73 % of controls, p< 0.01) was attributed to a slightly lower mean number of foetuses in the HD group (10.27) when compared to the control group (11.67).
There were no test item related effects of toxicological relevance for the mean litter weight, total number of foetuses and number of male and female foetuses in any of the groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Low incidences of haematoma on right hind leg (1 each in Control and MD group), discoloured red focus on right hind leg (1 in MD), small lower jaw (1 in control) which was confirmed by skeletal evaluation and discoloured red snout (1 in MD and 2 in HD) were noted in isolated females of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to the treatment.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase for litter incidences of incomplete ossification of parietal -bilateral (59.09 % compared to 23.81 % in controls, p< 0.05), incomplete ossification of 4th sternebra (72.73 % compared to 38.10 % in controls, p< 0.05), incomplete ossification of xiphoid (100 % compared to 71.43 % in controls, p< 0.01), rudimentary left 7th cervical rib (31.82 % compared to 0.0 % in controls, p< 0.01), unossified metacarpals (81.82% compared to 14.29% in control, p< 0.001), and ilium (bilateral) caudal offset (31.82 % compared to 4.76 % in controls, p< 0.05) were observed in the HD group when compared to the concurrent control group.
The observed statistically significantly reduced ossification of several bones in the HD group that normally exhibit rapid ossification in the last days of gestation indicates a generalised skeletal delay in the HD group. This delayed ossification was considered to be associated with the observed maternal toxicity (lower body weight and food consumption) and reduced foetal body weight of the HD group. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore not considered to be adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control. Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.
Other effects:
not specified

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced ossification of several bones (sternebra, xiphoid, cervical rib, metacarpals and ilium)

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in the first and last study week. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 87.8%, 102.5% and 115.9% of the nominal concentration, respectively. Homogeneity of formulation samples was determined at two concentrations, 20 mg/mL and 200 mg/mL, in the first and last study week. The mean recoveries observed for the LD dose group was 83.5% and 90.6% of the nominal value and 129.1% and 102.6% of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 2.7% and 1.2% in LD dose group, 0.3% and 0.2% in HD dose group.

Applicant's summary and conclusion

Conclusions:
In developmental toxicity study (OECD 414) Bis(2-ethylhexyl)peroxydicarbonate (98% purity) was administered to 25 female Wistar rats/dose, 35 female animals in the high dose group, in corn oil at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 5 through 19 of gestation. On day 20 the animals were sacrificed. Based on the results, the NOAEL for both maternal toxicity and foetal toxicity of Bis(2-ethylhexyl)peroxydicarbonate in this study is considered to be 300 mg/kg body weight/day.
Executive summary:

In a developmental toxicity study (OECD 414) Bis(2-ethylhexyl)peroxydicarbonate (98% purity) was administered to 25 female Wistar rats/dose, 35 female animals in the high dose group, in corn oil at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 5 through 19 of gestation. On day 20 the animals were sacrificed.

No treatment-related effects in mortality, clinical signs or gross pathological changes were seen in any maternal animals. Moreover, no test item related effects of toxicological relevance were noted for adjusted maternal weight (terminal body weight - gravid uterus weight), number of corpora lutea, implantation sites, early and late resorptions and pre- and post-implantation loss. But, in the high dose group (1000 mg/kg bw/day) toxicologically relevant adverse effects on body weight development and food consumption were noted.

At a dose level of 1000 mg/kg body weight/day mean foetus weight of the HD group was moderately, statistically significantly lower when compared to the control group. This resulted in a statistically significantly lower total litter weight when compared to controls. Furthermore, at 1000 mg/kg bw/day statistically significantly reduced ossification of some bones was observed which was indicative of a generalized delayed ossification associated with foetal growth retardation. The observed foetal effects at 1000 mg/kg body weight/day might be secondary to maternal toxicity. No effects on prenatal data, foetal external, visceral and craniofacial parameters were observed at 1000 mg/kg body weight/day.

No effects of Bis(2-ethylhexyl)peroxydicarbonate on females and foetuses were found at dose levels up to 300 mg/kg body weight/day. Based on the results, the NOAEL for both maternal toxicity and foetal toxicity of Bis(2-ethylhexyl)peroxydicarbonate in this study is considered to be 300 mg/kg body weight/day.