Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-18 to 2017-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22th January 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/Lot no.:1224545-01
- Physical state: solid
- Colour: white
- Molecular weight: 398.54 g/mol
- Purity: 95.6%
- Storage conditions: 2-8 °C, protected from light
- expiry date: not applicable

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: 309 - 364 g; females before initiation of pairing: 179 - 240 g
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (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): ad libitum, free access to Altromin 1324 maintenance diet
- Water (e.g. ad libitum): 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
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance. The test item formulations were prepared at room temperature by adding the vehicle to give the appropriate final concentrations and vortexing it. The test item formulations were prepared freshly on each administration day before the administration procedure. After preparation, the test item formulations and control item were stored at 2 to 8 °C or on crushed water ice and was administered within 6 hours after preparation. Shortly before administration, formulations were brought to room temperature. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle (corn oil) was selected as suggested by the sponsor based on the test item´s characteristics, testing guideline and a previously performed 28 day study.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): MKBQ9948V, MKBV2080V, MKBW9504V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study samples were collected for the investigation of homogeneity and substance concentration. Samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 and in the week before the last week of the treatment period (in total 20 samples). After transfer to the analytical department samples were stored at 2-8 °C and were measured the same day within 6 hours after arrival.
Details on mating procedure:
Prior to the start of the mating a detailed clinical observation outside the home cage was made. No animal showed pathological signs before the mating. After the acclimatisation period of 5 days, females were paired with males as per the 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. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. After getting 100 sperm-positive females, the remaining females and males were discarded without any observations.
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:
20 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:
Medium dose group (MD)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group (HD)
No. of animals per sex per dose:
25 female Wistar rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The 3 dose groups were selected based on the results of a previous 28-day study of the test item and in consultation with the sponsor.
- 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 if present.

BODY WEIGHT: Yes
- All animals were weighed once before initiation of pairing to ensure that the body weights were within +/- 20% variation. 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:
- External examinations: Yes: [all per litter] -- Each foetus was examined for external anomalies. Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

- Soft tissue examinations: Yes: [half per litter] -- One half of each litter was examined for soft tissue anomalies by a microdissection technique. Foetuses scheduled for the visceral examination were pinned to a paraffin covered petri dish with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid. The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect. The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula. The position, size, colour and shape of the heart was recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.

- Skeletal examinations: Yes: [half per litter; 20 litters per group] -- Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95% ethanol. These foetuses were processed using the Alizarin red staining technique. After fixation in 95 % ethanol, the foetuses were macerated with a 1% aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025% in 1% aqueous potassium hydroxide) for 1 day. After that the foetuses were again transferred to 1 % KOH. Alizarin stained foetuses were then cleared and dehydrated in a solution containing 2 parts of 70 % ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

- Head examinations: Yes: [half per litter; 20 litters (same as those used for soft tissue examinations) per group] -- After the completion of the visceral examination by the microdissection technique, foetuses were transferred to plastic bottles containing formalin-aceto-alcohol for later craniofacial examination by the razor-blade-serial-section 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. Before initiating the serial sectioning with a razor blade, foetuses were transferred to the beaker containing tap water for deformalisation. After deformalisation, a single foetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.
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, non-treatment-related
Description (incidence and severity):
Moving the bedding was noted occasionally and dose dependently in 4/25 females of the MD group and in 8/25 females of the HD group. Furthermore, slight salvation was seen on single days of treatment in 2/25 females of the MD group and 1/25 females of the HD group. Moving the bedding and salivation were seen transiently in few animals in timely relation to dose administration and were considered as slight clinical signs elicited by local effects of the test item formulation and/or discomfort of the animals due to oral administration, but not systemic toxicity.

Local alopecia on various body parts was noted in few females of the control group and the dose groups without dose dependency and was considered incidental in nature. Transient nasal discharge in a single female of the control group was considered as incidental.
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):
The mean body weight increased in all groups before initiation of treatment and further increased with the progress of the study comparably in the control and the LD group. After initiation of treatment a statistically significant mean loss of body weight was noted dose dependently in the MD and the HD group during gestation days 5-8 when compared to controls (-1.17 g and -7.88 g, respectively, compared to a gain of 4.50 g in controls). Throughout the remaining treatment period, mean body weight gain of the MD and the HD group was comparable to controls without statistical significant difference. When related to the entire study period (gestation day 0-20), slightly but statistically significantly lower body weight gain was observed in the MD group (11% below controls) and the HD group (12% below controls). Marginally lower mean body weight was noted in the HD group when compared to controls from gestation day 8 onwards. This achieved statistical significance on gestation day 8 (5% below controls), day 11 (4% below controls), day 17 (4% below controls) and day 20 (4% below controls). Marginally but statistically significantly lower mean body weight was also noted in the MD group on gestation day 20 when compared to controls (4% below controls).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to the body weight loss after initiation of treatment, mean food consumption in the MD and HD group was dose dependently, moderately and statistically significantly lower on gestation day 5-8 when compared to the control group (24 and 40% below controls). A slight but still statistically significant effect was noted in the MD and the HD group on gestation day 8-11 (16 and 22% below controls) and day 11-14 (12 and 13% below controls). Food consumption of the MD and HD group was comparably to the controls throughout the remaining study period. When related to the entire study period (gestation period day 0-20) mean food consumption was 11% below controls in the MD and the HD group with achieving statistical significance. Mean food consumption of the LD was unaffected by the treatment with the test item.
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):
At necropsy, extra growth in fat tissue (additional amount of fat tissue with different consistency) was noted close to the left ovary in one female of the HD group. This single finding was considered as incidental.

No further gross pathological changes were observed during the macroscopic examination of females of any of the groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of implantation sites and percent pre- and post-implantation loss. There were no statistically significant differences between the dose groups and controls.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of total resorptions. There were no statistically significant differences between the dose groups and controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of early and late resorptions. There were no statistically significant differences between the dose groups and controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any of the groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 25/25 pregnancies in the LD group, 24/25 in the MD group and 25/25 in the HD group when compared to 24/25 pregnancies in the control group.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other:
Remarks:
Effects on body weight development and food consumption were only transiently noted in the MD and HD groups. Furthermore, there was only a marginal impact on mean body weight (less than 5% difference to controls). Therefore, these effects were considered as slight effects of the treatment but not as adverse.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of live foetuses. There were no statistically significant differences between the dose groups and controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for sex ratio. There were no statistically significant differences between the dose groups and controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of foetuses and total litter weight (as well as mean fetal body weight). Statistical analysis showed no significant differences to the control group.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of litter incidences showed no significant differences to the control group.

By external examination, an extra toe on the right hindlimb was observed in a single foetus of one litter (female no. 56) of the MD group. As only one foetus was affected, this was considered incidental. No extra ossification site was seen at skeletal examination. Furthermore, the same foetus and a littermate were observed with curled or bent tail at external examination.

As only one litter of an intermediate group was affected, this was not considered to be related to the treatment with the test item.

By external examination, in a single foetus of one litter (female no. 3) of the control group a depression on the left side of the head was noted. No skeletal correspondence for this finding was observed at skeletal examination.

A single foetus of one litter (female no. 83) of the HD group was observed with the malformation of an omphalocele which was also confirmed at visceral examination. However, as only one foetus was affected, this finding was considered as incidental and not related to the treatment with the test item.

Low incidences of haematoma on various body parts were noted in single foetuses of of the control group and the dose groups without dose dependency and were considered to be spontaneous in nature without relation to the treatment with the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings in all groups including control. However, none of these findings was considered as toxicologically relevant.

Statistical analysis showed no significant differences to the control group except for a higher litter incidence of incomplete ossification of 5th sternebra in the MD group (95.00%) when compared to the control group (55.00%). This finding was only marginally below maximum historical data (95.83%) and did not show any dose dependency (HD group 70.00%). Thus, it was considered as incidental. Incidences for remaining findings for sternebra such as incomplete ossification of other sternebra, increased and misaligned ossification and unossification in the dose groups were comparable to the control group and also showed no dose dependency. There was no further statistical significance and no indication of a test item related trend in the type and/or incidences of foetal findings. They were considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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. Litter incidences were statistically insignificant.

The malformation of an omphalocele in a single foetus of one litter (female no. 83) of the HD group was considered as incidental and not related to the treatment with the test item.

As the remaining 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. Incidences of observed findings were well within historical control data.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed few findings (dilated third ventricle, dilated lateral ventricle, retinal fold and small pituitary gland) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls without any dose dependent trend. 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.

The finding of a subcutaneous haematoma was seen in a single foetus of the control group and of a subcutaneous oedema in a single foetus of the MD group. These findings were considered as incidental based on the low incidence without dose dependency.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
Observed effects were either incidental or spontaneous in nature.

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Dose Formulation Analysis:

Homogeneity and concentration analysis of formulation samples was performed for all groups in week 1 and the week before the last week of the treatment period. Nominal concentrations for the prepared suspensions were confirmed for all dose groups as measured weekly mean concentration did not differ from nominal concentration by more than 15% in the LD, MD and the HD group. The mean recoveries observed for the LD group were 115.0% and 113.0% of the nominal value, 105.0% and 97.8% for the MD group, and two times 106.7% of the nominal value for the HD group. Overall homogeneity was confirmed as weekly mean coefficient of variation for all dose groups of the different sampling locations (top, middle, bottom) (2.1% and 3.0% in the LD group, 0.8% and 1.5% in the MD group and 0.9% and 1.6% in the HD group).

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study (performed in accordance with OECD 414) the test item bis(4-tert-butylcyclohexyl) peroxydicarbonate was prepared using corn oil as vehicle and orally administered to 25 female Wistar rats/dose group at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 5 through 19 of gestation. On gestation day 20 the animals were sacrificed and examined. No test item-specific adverse effects were found in pregnant females and foetuses up to 1000 mg/kg body weight/day. Based on the results, the NOAEL for both maternal toxicity and foetal toxicity of the test item in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

In a prenatal developmental toxicity study (performed in accordance with OECD 414) the test item bis(4-tert-butylcyclohexyl) peroxydicarbonate (95.4% purity) was prepared using corn oil as vehicle and administered to 25 female Wistar rats/dose group by at dose levels of 0, 100, 300 and 1000 mg/kg bw/day by oral gavage from days 5 through 19 of gestation. On gestation day 20 the animals were sacrificed and examined. No clinical signs of maternal systemic toxicity or mortality were observed in this study. At the doses of 300 and 1000 mg/kg bw/day a tendency towards dose-dependent attenuation in body weight gain and food intake was observed. However, effects on body weight development and food consumption was only transiently noted. Furthermore, there was just a marginal impact on the mean body weight (no more than 5% difference from the controls). Therefore, these maternal effects were considered as slight but not adverse effects of the treatment. No effects of the test item on foetuses were found up to 1000 mg/kg bw/day. Thus, the NOAEL for both maternal and foetal toxicity is considered to be 1000 mg/kg bw/day.

The prenatal developmental toxicity study in the rat is classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rat.