Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-02 to 2016-08-11
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:
Batch No.: 1198394-01
Physical state: liquid
Colour: colourless
Density: 0.87 g/cm³
Purity: 75%
Solvent: 20-25% Isododecan, CAS No. 93685-81-5
Storage Conditions: -8 to 0 °C, protected from light
Expiry date: 30-January-2016

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: 11-12 weeks
- Body weight range: males before initiation of pairing: 330 - 351 g; females before initiation of pairing: (batch 1): 200-230 g, (batch 2): 244-270g; females (batch 3), at time of mating: 214-236 g
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102150411) (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. 290716/0631)
- 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: at least 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
Vehicle:
paraffin oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared with paraffinum perliquidum. The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. The test item formulations were prepared freshly on each administration day before the administration procedure. 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 vehicle and vortexing it. Formulations or control item were stored at 2 to 8 °C on crushed ice and were administered within 6 hours after preparation (see stability data in study no. 114850). Prior to administration test item formulations were brought to room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): paraffinum perliquidum
- Amount of vehicle (if gavage): 3 mL/kg bw
- Lot/batch no. (if required): 000055558748

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, 15 mL of MD and 65 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:
Females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the mating a detailed clinical observation outside the home cage was made. One female without vagina opening was excluded. 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’. Due to an increased number of non-pregnant females in the LD and the HD group, additional females from newly delivered batches of animals were mated and assigned to these treated groups in order to obtain a sufficient number of at least 17 pregnant females per group.
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)
Remarks:
Control group (C)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group (LD)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Middle dose (MD)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose (HD)
No. of animals per sex per dose:
The 4 groups comprised 25 female Wistar rats in the control group, 30 in the LD group, 25 in the MD group and 30 in the HD group, respectively.
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 3 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 was processed by Alizarin red staining and examined for skeletal alterations. The remaining litter was examined for soft tissue anomalies by a microdissection technique. Craniofacial examination of the heads of the foetuses used for the soft tissue examination was performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.
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. 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:
effects observed, treatment-related
Description (incidence and severity):
see box "Details on results"
Mortality:
mortality observed, non-treatment-related
Description (incidence):
see box "Details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see box "Details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see box "Details on results"
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:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Mortality:
Female no. 86 of the HD group was found dead on the respective study day 8. Its death was a single case and happened after only 3 days of treatment without any preceding signs of morbidity. Thus, the cause of death was unclear but might be considered incidental and not related to an effect of the test item. One female of the control group and one of the LD group were euthanized on study day 17 due to littering before the scheduled sacrifice (no. 12 with 13 pups, no. 38 with 2 pups). Apparently these animals had already been pregnant before the first detection of sperm in the vaginal smear. All remaining animals survived until the end of the study.

Clinical Observations:
At the end of the treatment period, slight to severe piloerection was noted in 5/30 females of the HD group. Furthermore ataxia and hypothermia were observed on gestation day 20 in one single female (no. 98) of the HD group. As these signs were not observed in the lower dose groups or controls, they were considered to be related to the treatment with the test item at a dose of 1000 mg/kg bw/day. Low incidences of the slight clinical signs of alopecia or crust on various body parts were noted in single females without dose dependency and were considered incidental in nature. None of the females showed signs of abortion prior to the scheduled sacrifice. However, female no. 12 of the control group and female no. 38 of the LD group littered normally in the course of the study before terminal sacrifice.

Body Weight 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 not statistically significantly lower in the HD group when compared to controls during gestation days 5-8 (24 % of controls) and 8-11 (78 % of controls). This was based on a loss of body weight of some of the HD females. Significantly (p< 0.01) lower mean body weight gain was observed at the end of the treatment period (58 % of controls on gestation days 14-17; 62 % of controls on gestation days 17-20) and when the entire study period was evaluated (p< 0.001, 74 % of controls). Lower body weight gain resulted in statistically significantly lower mean body weight of the HD group at the end of the treatment period. 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 (91 % of controls, p< 0.01). Effects on mean body weight and body weight gain in the HD group were considered to be test item related. Throughout the whole study period, slightly lower mean body weight was noted for the MD group (6 % to 7 % below controls) with statistical significance except on day 14. As this slight difference was already apparent on gestation day 0 before initiation of treatment, it was considered to be incidental and not related to the treatment with the test item. Mean body weight of the LD group was also unaffected by the test item administration. Mean body weight gain of the LD and the MD group was comparable to the control group throughout the whole study period and was within the normal range of variation for this strain.

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 at the end of the treatment period on gestation days 14-17 (86 % of controls) and 17-20 (74 % of controls); differences from the control group were significant (p< 0.01 and p< 0.001). This resulted in a moderately, statistically significantly lower mean food consumption of the HD group when the entire study period was evaluated (90 % of controls, p< 0.01). 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 unaffected by the treatment with the test item. Differences to the control group were slight and not statistically significant.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
see box "Details on maternal toxic effects"
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see box "Details on maternal toxic effects"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see box "Details on maternal toxic effects"
Early or late resorptions:
no effects observed
Description (incidence and severity):
see box "Details on maternal toxic effects"
Dead fetuses:
no effects observed
Description (incidence and severity):
see box "Details on maternal toxic effects"
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
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): see box "Details on maternal toxic effects"
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 18/30 pregnancies in the LD group, 20/25 in the MD group and 20/30 in the HD group compared to 23/25 pregnancies in the control group. The low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 60 % in the LD group and of 67 % in the HD group were considered to be a biological variation.
Other effects:
no effects observed
Details on maternal toxic effects:
Test item related, moderately, statistically significantly lower mean terminal body weight was observed in the HD group when compared to the control group (91 % of controls, p< 0.01). Furthermore, moderately lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was noted in the HD group (91 % of controls, p< 0.01). This was considered as an effect of the test item. Slightly but not statistically significantly lower mean adjusted maternal weight of the MD group was attributed to a slightly lower mean body weight from the start (before initiation of treatment) until the end of the study. Thus, no toxicological relevance was assumed. Marginally lower values for uterus weight in the LD, the MD and the HD group when compared to the control group were seen without a dose dependent pattern and without statistical significance and were considered incidental.
No test item related effects of toxicological relevance were noted for number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, male and female foetuses, sex ratio and percent pre- and post-implantation loss. Slightly but statistically significantly lower number of implantation sites was observed in the LD group when compared to the control group. However, without dose dependency and as treatment in this study was not initiated before actual nidation, this was considered as incidental in nature. Equally, the slightly but statistically significantly lower number of foetuses in the right uterine horn of the LD group when compared to the control group was considered incidental. 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:
clinical signs
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):
see box "Details on embryotoxic/teratogenic effects"
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
Description (incidence and severity):
see box "Details on embryotoxic/teratogenic effects"
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see box "Details on embryotoxic/teratogenic effects"
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
see box "Details on embryotoxic/teratogenic effects"
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Low incidences of haematoma on various body parts were noted in isolated females of the control group and/or the dose groups without dose dependency. A malrotated hindleg was noted in one single foetus of the control group and a bent tail in one single foetus of the MD group which was considered 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 2nd sternebra (31.58 % compared to 4.76 % in controls, p< 0.05), unossified 5th sternebra (47.37 % compared to 9.52 % in controls, p < 0.05) and unossified 6th sternebra (21.05 % compared to 0.00 % in controls, p< 0.05) were observed in the HD group when compared to the concurrent control group. Incomplete ossification of 3rd and 4th sternebra was seen without statistical significance in the HD group (each 10.53 %) but was not observed in the other groups. Besides, marginally, statistically insignificantly higher litter incidence of incomplete ossification of 1st sternebra was noted in the HD group in 2 litters (10.53 %) and in one litter (one foetus) of the MD group (5.00 %) when compared to 0.00 % in the control group. Furthermore, a slightly but statistically significantly higher litter incidence of incomplete ossification of sacral arch(es) was noted in the HD group (26.32 % (5 litters) compared to 0.00 % in controls, p< 0.05) and without statistical significance in the MD group (15.00 %). The finding of unossified sacral centrum(s) was noted in 2 litters of the HD group (10.53 %) which was not seen in the other groups. Values for incomplete ossification of sacral arch(es) and unossification of sacral centrum(s) were noted to be above historical control data. A moderately higher litter incidence of unossified metacarpal(s) was observed with statistical significance in the HD group (63.16 %, p< 0.05) when compared to the control group (23.81 %). This was shown to be marginally higher compared to maximum litter incidence of historical control data (62.50 %). 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 generalized 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. Incomplete ossification of bones of the pelvic girdle was noted without statistical significance in single foetuses of the dose groups. Incomplete ossification of the pubis was seen in 5 foetuses (2 litters) of the HD group and one foetus of the MD group. Besides, incomplete ossification of the ischium was seen in each one foetus of the same 2 litters of the HD group. The delayed ossification was associated with fetal growth retardation as only foetuses with markedly reduced foetal weight (≤ 2.5 g) were affected.
A slightly but statistically significantly higher litter incidence of incomplete ossification of squamosal (left side) in the MD group (20.00 %) when compared to 0.00 % in the control group showed no dose dependency (LD group 5.88%, HD group 10.53 %). Value was within maximum litter incidence of historical control data and was considered incidental.
Statistically significantly lower litter incidence of 14th rudimentary rib (right side) was seen without dose dependency in the MD group when compared to the control group and was not considered as an effect of the test item.
Rudimentary cervical ribs were noted throughout all groups including control without statistical significant difference. Cervical ribs are skeletal alterations that can occasionally be seen in animals of this strain. Rudimentary/short ribs are considered as transient abnormalities. In contrast, full/long cervical ribs are considered permanent and may cause health effects in humans; however, postnatal consequences in rats are unknown but assumed to be minimal. As overall rudimentary cervical ribs were seen throughout all groups including control without a dose dependent pattern, this finding was not considered to be treatment related but spontaneous in nature. A full unilateral cervical rib was seen in one single foetus of the MD group and one single foetus of the HD group. Considering the low fetal incidence (0.88 % and 0.86 %, respectively) without clear dose dependency, this was also considered as incidental.
The finding of bent scapula (right side) was seen in single foetuses of the dose groups without dose dependency (litter incidence 5.88 % in the LD, 10.00 % in the MD and 5.26 % in the HD group). Thus, this finding was considered incidental without relation to the treatment and was considered as post-natally reversible.
Fusion of centrum(s) and arch(es) of sacral and caudal vertebrae were seen without dose dependency in single foetuses of all groups including control and was considered incidental. Furthermore, the malformations of misshapen scapula, 6th sternebra and thoracic centrum were observed in each one foetus of the control group without relation to the treatment with the test item. Fused zygomatic arch was seen in two single foetuses of the HD group and two of the control group and likewise was considered incidental. There was no statistical significance and no indication of a test item related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.
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. All litter incidences were statistically insignificant
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed few findings (retinal fold, dilated third ventricle, dilated lateral ventricle, subcutaneous edema, small pituitary gland) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency in the dose groups compared to the controls. Statistical analysis of the data revealed no significant effect. These findings were considered to be spontaneous in nature and not related to the treatment with the test item.
Details on embryotoxic / teratogenic effects:
Mean foetus weight of the HD group (3.16 g) was moderately, statistically significantly lower when compared to the control group (3.67 g) (86 % of controls, p< 0.001). This resulted in a moderately lower total litter weight when compared to controls (82 % of controls, p< 0.05). The lower mean foetus weight of the HD group was considered to be related to the treatment with the test item.
Evaluation of individual dams and litters indicated that predominantly dams with the lowest total food consumption of the HD group were associated with markedly reduced total body weight gain as seen e.g. in dam no. 79, 87, 89, 100 and 107. No correlation was found between the comparably low body weight gain of dam no. 108 and 109 and their total food consumption which was in the normal range when compared to control. Dams of the HD group which were seen with the lowest total body weight gain were linked to the individual litters with the lowest mean foetal body weights (below 3.00 g) within the HD group regardless of litter size (dam no. 79, 87, 89, 100, 107, 108 and 109).
Moderately, statistically significantly lower total litter weight and female litter weight of the LD group (80 % of controls, p< 0.01; 75 % of controls, p< 0.05) were attributed to a slightly lower mean number of foetuses in the LD group (10.00) when compared to the control group (11.86). Without dose dependency this was considered incidental. Mean foetus weight of the LD and the MD group was comparable to the control group.
There were no test item related effects of toxicological relevance for the total number of foetuses and number of male and female foetuses in any of the groups.

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:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: Observed foetal effects at 1000 mg/kg body weight/day were considered to be secondary to maternal toxicity.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: at 1000 mg/kg bw/day statistically significantly reduced ossification of some bones (2nd, 5th and 6th sternebra, sacral arch(es) and metacarpal(s).

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 in study week 1 and the last study week for all dose groups. The mean recoveries observed in the LD, the MD and the HD group were 105.0 %, 102.6 % and 96.1 % of the nominal concentration, respectively. Homogeneity of formulation samples was determined in study week 1 and the last study week for the LD and the HD group. The mean recovery observed was between 107.7 % and 107.9 % of nominal value for the LD group and between 91.4 % and 97.6 % for the HD group. The coefficients of variation of the different sampling locations (top, middle, bottom) were between 1.4 % and 1.9 % in the LD group and between 0.8 % and 0.2 % in the HD group.

Applicant's summary and conclusion

Conclusions:
In a developmental toxicity study (OECD 414) Bis(3,5,5-trimethylhexanoyl) peroxide (75% purity) was administered to 25 female Wistar rats in the control group and mid dose group, 30 female animals in the low and high dose group, in paraffinum perliquidum 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 the test item in this study is considered to be 300 mg/kg body weight/day.
Executive summary:

In a developmental toxicity study (OECD 414) Bis(3,5,5-trimethylhexanoyl) peroxide (75% purity) was administered to 25 female Wistar rats in the control group and mid dose group, 30 female animals in the low and high dose group, in paraffinum perliquidum 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.

At a dose level of 1000 mg/kg bw/day test item related clinical signs like slight to severe piloerection and moderately, statistically significantly reduced body weight, body weight gain and food consumption were observed towards the end of the treatment period. Females treated with 1000 mg/kg bw/day also showed moderately, statistically significantly reduced adjusted maternal weight. At a dose level of 1000 mg/kg bw/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 (2nd, 5th and 6th sternebra, sacral arch(es) and metacarpal(s)) was observed which was indicative of a generalized delayed ossification associated with fetal growth retardation. Observed foetal effects at 1000 mg/kg bw/day were considered to be secondary to maternal toxicity. No effects on prenatal data, foetal external, visceral and craniofacial parameters were observed at 1000 mg/kg bw/day.

No effects of Bis(3,5,5-trimethylhexanoyl) peroxide on females and foetuses were found at dose levels up to 300 mg/kg bw/day. The NOAEL for both maternal toxicity and foetal toxicity of Bis(3,5,5-trimethylhexanoyl) peroxide in this study is considered to be 300 mg/kg bw/day.