Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase (first day of mating):14 February 2017 End of experimental phase (last day of necropsy): 15March 2017 Study completation (date of study director's signature on the report): 24 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 weeks old (females)
- Weight at study initiation: 180-203 g (females)
- Fasting period before study: no
- Housing: Before and after mating, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate,
Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0Mucedola) was provided as necessary and changed at least 2 times a week.
During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: An acclimatisation period of 26 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): the rooms were lit by artificial light for 12 hours each day.

IN-LIFE DATES:
From: 14 February 2017 (first day of mating) To: 15 March 2017 (Last day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment, analysis was performed in RTC Study no. 88760 to confirm that the proposed formulation procedure was acceptable (range of 1-200mg/mL) and that the stability of the formulation was satisfactory (24 hours and 8 days at room temperature).
Samples of the formulations prepared during the first, second and last week of treatment, were analysed to check the homogeneity and concentration. Results of the analyses were within the limits of acceptance stated in RTC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the males and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were
maintained.
Duration of treatment / exposure:
from Day 6 through Day 19 post coitum.
Frequency of treatment:
daily
Duration of test:
until day 20 p.c.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected on the basis of a preliminary, non GLP compliant study, oral prenatal developmental toxicity study in rats at the dose levels of 50, 150, 450 and 750mg/kg/day (RTC study no. Y0050). In this study, the dose level of 450mg/kg/day induced a slight maternal toxicity and 750 mg/kg/day was in excess of the maximal tolerated dose.

- Rationale for animal assignment:
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the
groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Maternal examinations:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CLINICAL SIGNS
All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.
Presence of subcutaneous mass was reported as individual data, only.

BODY WEIGHT
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

NECROPSY
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were notedand the abnormalities preserved in 10% neutral buffered formalin.
The ovaries and uteri were examined to determine:
– gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible;
– late resorptions: placental and foetal remnants visible.


ORGAN WEIGHTS
From all animals the kidneys and liver were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

TISSUES FIXED AND PRESERVED
Stomach, kidneys, liver and any abnormalities were fixed and preserved in 10% neutral buffered formalin. No histopathological examination was performed.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
– number, sex and weight of all live foetuses
– number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing)
– number of intra-uterine deaths
– Gross evaluation of placentae
Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e.,routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination. Skeletal and fixed-visceral examinations were performed in all groups.
Structural deviations were classified as follows:
Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies
Minor abnormalities that are detected relatively frequently.
Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
A comparison with the RTC historical control data was carried out, even if these data are not QA audited.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:
(no.o f corpora lutea -no. of impl antations/no. of corpora lutea) ×100
Post-implantation loss was calculated as a percentage from the formula:
(no. of implantations -no. of live foetuses/no. of implantations)×100
Total implantation loss was calculated as a percentage from the formula:
(no.of corpora lutea -no.of live foetuses/no. of corpora lutea)×100
Sex ratios of the foetuses were calculated as the percentage of males per litter.
Historical control data:
See attached file

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ataxia and piloerection were detected in all females receiving 450mg/kg/day. In few of these animals, these signs were associated with decreased activity, decreased muscle tone, tremor, hunched posture (cyphosis), pale appearance and staining of perigenital region.
Piloerection, associated with ataxia in one animal, was also noted in 4 out of 24 females receiving 150mg/kg/day and in 1 out of 24 females receiving 50mg/kg/day.
The subcutaneous mass (thoracic dorsal region) observed in one control female was considered spontaneous in origin.
Mortality:
no mortality observed
Description (incidence):
A total of five females were found not pregnant at necropsy: two in the control group and one in each of the low, mid- and high dose groups. In addition, one female in the low dose group had unilateral implantation and one in the high dose group had total resorption. The number of
females with live foetuses on gestation Day 20 was 22 in the control and high dose groups and 23 in the low and mid-dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in bodyweight was noted in females receiving 450mg/kg/day, starting from Day 12 post coitum throughout the study (up to -13%), when compared to controls. A statistically significant reduction in body weight (up to -6%) was also seen in females receiving 150mg/kg/day on Days 12, 18 and 20 post coitum. No significant change in body weight was seen in females receiving 50mg/kg/day, when compared to controls.
Body weight gain was significantly decreased at statistical analysis in females receiving 450mg/kg/day, starting fromDay 9 toDay 20 post coitum and in females receiving 150mg/kg/day on Days 9, 18 and 20 post coitum. No toxicological relevance was attributed to the slightly significant reduction in body weight gain seen in females receiving 50mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in food consumption was seen in females receiving 450mg/kg/day, starting from Day 9 (-40%) up to Day 20 post coitum (-29%), compared to controls. The statistically significant decrease, detected on Day 9 post coitum in females receiving 50 and 150mg/kg/day (-13% and -28%, respectively), compared to controls, was not considered of toxicological significance, since a recovery was noted throughout the study.
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:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in terminal body weight (-12%), gravid uterus weight (-28%) and absolute weight gain (-38%) was seen in females receiving 450mg/kg/day, when compared to controls. A slight but statistically significant decrease was also seen in terminal body weight, gravid uterus weight and absolute weight gain of females receiving 150mg/kg/day (-5%, -13% and -14%, respectively). No significant differences were seen in females receiving 50mg/kg/day, when compared to controls.

A statistically significant increase in absolute and relative kidneys and liver weights was seen in females receiving 450mg/kg/day, when compared to controls.
A statistically significant increase in absolute and relative liver weights was also detected in females receiving 150mg/kg/day, when compared to controls.
No statistically significant differences were recorded in females receiving 50mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of swollen liver was observed in mid- and high dose treated females, when compared to the control and low dose groups. In addition, enlarged liver was observed in few instances in low and high dose treated females. The remaining changes observed are suggested to be incidental or characteristically seen in untreated Sprague Dawley rats of the same age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
LITTER DATA
A statistically significant increase of total implantation loss (post-implantation loss) associated to statistically significant increase of total intra-uterine deaths (early resorption) and decrease in total viable foetuses, were noted in females receiving 450mg/kg/day, when compared to controls. In addition, in the same group, statistically significant decreases of mean litter and foetal weight were also detected.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
PREGNANCY OF FEMALES
A total of five females were found not pregnant at necropsy: two in the control group and one in each of the low, mid- and high dose groups. In addition, one female in the low dose group had unilateral implantation and one in the high dose group had total resorption. The number of females with live foetuses on gestation Day 20 was 22 in the control and high dose groups and 23 in the low and mid-dose groups.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Abnormalities:
effects observed, treatment-related

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 450mg/kg/day statistically significant decreases of mean litter and foetal weight were detected.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
A decrease in total viable foetuses, were noted in females receiving 450mg/kg/day, when compared to controls.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
External malformations:
no effects observed
Description (incidence and severity):
A total of 15 small foetuses (<2.7 g) were detected, only in treated groups, without a dose relationship: 9 in low dose (6 in litter from dam no. X0400053, 1 in litter from dam no.X0400067 and 2 in litter from dam no. X0400095) 2 in mid-dose (litter from dam no. X0400107) and 4 in high dose females (1 each in litter from dam nos. X0400159, X0400167, X0400169 and X0400181).
No abnormalities were detected in the other foetuses examined.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Major findings (arches of cervical vertebrae fused, absence of ribs, radius/ulna/scapula misshaped) were seen in one foetus in each of low (litter from dam no. X0400095), mid- (litter
from dam no. X0400107) and high (litter from dam no. X0400169) dose groups, respectively.
These findings were observed in single foetuses from different litters and without dose relationship, therefore a treatment-related effect is unlikely.
Anomalies and variants (14th rib short, incomplete ossification of skull bones, no/incomplete ossification of 5th/6th sternebrae) were seen in treated and control foetuses but with a slightly higher incidence in high dose group (450mg/kg/day), compared to control.
Other findings were seen in treated and control foetuses with similar or higher incidence in the controls, and therefore were considered incidental.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Major findings (persistent truncus arteriousus, cardiomegaly, agenesia/hypoplasia of kidney, ureter, lungs and thymus) were seen only in one high dose foetus (litter from dam no. X0400181). This foetus was found small (body weight < 2.7 g) at the external examination. Absence of innominate artery and presence of a supernumerary lobe in the lung were noted in two (one each in litter from dam no. X0400113 and dam no.X0400121) and in one (litter from dam no. X0400125) mid-dose foetuses, respectively. The above mentioned findings were seen in single foetuses from different litters without a dose relationship, therefore treatment-related effect is unlikely. The ocular malformation (eye hypoplastic/misshapen) seen in one control foetus (litter from dam no. X0400001), was considered incidental in origin.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

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

Applicant's summary and conclusion

Conclusions:
On the basis of the above results, it can be concluded that the dosage of 450mg/kg/day induced a significant maternal toxicity, dosage of 150mg/kg/day induced slight maternal toxicity and dosage of 50mg/kg/day was well tolerated. The NOAEL (No Observed Adverse Effect Level) for maternal toxicity could be considered at 50mg/kg/day.
Retarded foetal growth and post implantation losses secondary to maternal toxicity were observed at dosage of 450mg/kg/day. Foetal development was not affected at dosages of 50 and 150mg/kg/day. The NOAEL for foetal development could be considered at 150mg/kg/day.
Executive summary:

The effects of Tert-butyl a,a-dimethylbenzyl peroxide were investigated after oral administration in female rats during pregnancy and on embryo-foetal development (Liberati, 2018). Groups of 24 Sprague-Dawley SD female rats were administered during the gestation period, starting from Day 6 through Day 19 post coitum at the dosing volume of 5 mL/kg, with dose levels of 0, 50, 150 and 450 mg/kg/day. Body weight, clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination and organ weights. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animals died during the study. The number of females with live foetuses on gestation Day 20 was 22 in the control and high dose groups and 23 in the low and mid-dose groups. During the dosing period, ataxia and piloerection were detected in all females receiving 450 mg/kg/day. In few of these animals, these signs were associated with decreased activity, decreased muscle tone, tremor, hunched posture (cyphosis), pale appearance and staining of perigenital region. Piloerection and ataxia were occasionally recorded in few females receiving 150 and 50 mg/kg/day. Statistically significant decreases in body weight (starting from Day 12 post coitum) and body weight gain (starting from Day 9 post coitum) were noted in females receiving 150 and 450 mg/kg/day, when compared to controls. No toxicologically relevant change in body weight and in body weight gain were seen in females receiving 50 mg/kg/day, when compared to controls. A statistically significant reduction in food consumption was seen in females receiving 450 mg/kg/day, compared to controls.

A statistically significant decrease in terminal body weight, gravid uterus weight and absolute weight gain was seen in females receiving 450 (-12%, -28% and -38%) and 150 mg/kg/day (-5%, -13% and -14%, respectively), when compared to controls. No significant differences were seen in females receiving 50 mg/kg/day, when compared to controls.

A statistically significant increase of total implantation loss (post-implantation loss (23.89% vs. 0.91 %) associated to statistically significant increase of total intra-uterine deaths (early resorptions, 2.52% vs. 0.14%) and decrease of total viable fetuses (10.26 vs. 15), were noted in females receiving 450 mg/kg/day, when compared to controls. In addition, in the same group statistically significant decreases of mean litter (38.46 g vs. 56.67 g) and foetal weight (3.53 g vs. 3.80 g) were also detected.

A statistically significant increase in kidneys and liver weights was seen in females receiving 450 mg/kg/day when compared to controls. A statistically significant increase in liver weights was also detected in females receiving 150 mg/kg/day, when compared to controls. No statistically significant differences were recorded in females receiving 50 mg/kg/day.

An increased incidence of swollen liver was observed in mid- and high dose treated females, when compared to the control and low dose groups. In addition, enlarged liver was observed in few instances in low and high dose treated females.

No findings that could be considered treatment-related were noted at the external examination of the foetuses in any group. Anomalies and variants (14th rib short, incomplete ossification of skull bones, no/incomplete ossification of 5th/6th sternebrae) were seen in treated and control foetuses but with a slightly higher incidence in high dose group (450 mg/kg/day), compared to control. Other findings were seen in treated and control foetuses with similar or higher incidence in the controls or seen in treated groups only but with low incidence and therefore were considered incidental. Other major findings were seen in one foetus each in the low, mid- and high dose groups, respectively.

These findings were observed in single foetuses from different litters and without dose relationship, therefore the correlation with treatment remains unclear. Anomalies and variants (moderate/slight pelvic dilatation and moderate/slight enlarged and kinked ureters, slight enlarged heart ventricle) were seen in treated and control foetuses but with a slightly higher incidence in mid- and/or high dose groups, respect to control. The other changes were seen in treated and control foetuses, with similar or higher incidence in the controls, and therefore were considered incidental. Other major findings (persistent truncus arteriousus, cardiomegaly, agenesia/hypoplasia of kidney, ureter, lungs and thymus) were seen only in one high dose foetus. This foetus was found small (body weight < 2.7 g) at the external examination. Absence of innominate artery and presence of a supernumerary lobe in the lung were noted in two and in one mid-dose foetuses, respectively. The above mentioned findings were seen in single foetuses from different litters without a dose relationship, therefore the correlation with treatment remains unclear.

On the basis of the above results, it can be concluded that the dosage of 450 mg/kg/day induced a significant maternal toxicity, dosage of 150 mg/kg/day induced slight maternal toxicity and dosage of 50 mg/kg/day was well tolerated. The NOAEL (No Observed Adverse Effect Level) for maternal toxicity could be considered at 50 mg/kg/day.

Retarded foetal growth and post implantation losses secondary to maternal toxicity were observed at dosage of 450 mg/kg/day. Foetal development was not affected at dosages of 50 and 150 mg/kg/day. The NOAEL for foetal development could be considered at 150 mg/kg/day.