Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxic effects on Sprague-Dawley rats of both sexes after repeated dosing with Luperox 801 (97% tert-butyl cumyl peroxide), as well as any effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and development of offspring up to Day 4 post partum were evaluated in an OECD 422 study (Liberati, 2013. The test item, suspended in corn oil, was administered by oral gavage to 3 groups of 10 males and 10 females each at doses of 0, 75, 150 or 600 mg/kg. Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy. Females were treated for 2 weeks before pairing, thereafter during pairing, post coitum and lactation periods until Day 3 post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis), litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs and macroscopic observations of pups were also performed.

The parental toxicity is detailed in the section 7.5 of the IUCLID, repeated dose toxicity. 

One control female judged to be in extremis was killed on post coitum Day 18. A total of 5 females were found not pregnant at necropsy: 2 in the mid-dose group and 3 in the high dose group. One mid-dose female had total litter loss on Day 0 post partum and was sacrificed on Day 3 post partum. The number of females with live pups on Day 4 post partumwas: 9 in the control group, 10 in the low dose group, 7 each in the mid-dose and high dose groups.

All females mated both in the control and treated groups. The decrease in the number of oestrus cycles and in the pre-coital interval observed in high dose females with respect to the control females were not considered of toxicological relevance. Two females in the mid-dose group and 3 females in the high dose group were found not pregnant at necropsy.The copulatory index of males and females did not show differences between treated and control groups. On the contrary, fertility index of males and females receiving the dose levels = 150 mg/kg/day was decreased compared to the control group. Corpora lutea, implantations and total litter size were statistically decreased in the high dose females respect to the controls. In addition, an increase in pre-birth loss percentage was also detected in the same group. Gestation periods were similar in treated groups and controls. There was no difference sex ratio. In the high dose group, decreased litter weight was noted on Day 1post partum with respect to the controls. On Day 4 post partum, live litter size, number of females and litter weight were still significantly reduced compared to controls and no difference in sex ratio were detected. 

On the basis of the results obtained in the study, the NOAEL for both systemic toxicity and reproduction/developmental toxicity is considered 150 mg/kg/day for males and females. For systemic toxicity, this NOAEL is based on clinical signs, reduced body weight and bodweight gain at 600 mg/kg/day and on kidney macroscopic and microscopic changes on male rats. For reproduction/developmental toxicity this NOAEL is based on the decrease of fertility index, decrease of corpora lutea and implantation sites, litter weight and litter size at 600 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2012 to 25 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and with a weight range of approximately 188 to 214 g for males and 160 to 176 g for females, were received from Charles River Italy S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 13 days was allowed before the start of treatment, during which time the health status of the animals was assessed by
thorough observations.

Animal room controls were set to maintain temperature and relative humidity at 22°C +- 2°C and 55% +- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

In-life data from 26 April 2012 to 25 June 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.

The required amount of Ter-Butyl Cumyl Peroxide was suspended in the vehicle (corn oil).
The formulations were prepared daily (concentrations of 15, 30 and 120 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied.
The formulations were gently mixed using mechanic stirrer.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 15 to 120 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).
In RTC Study no. 88760, stability after 24 hours at room temperature was verified in the range from 1 to 200 mg/mL. According to RTC’s SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (90%-110% for concentration and CV<10% for homogeneity).
Samples of the formulations prepared on Weeks 1 and 6 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC’s SOPs for suspensions (90-110% for concentration and CV <10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 88760), in the range from 1 to 200 mg/mL.
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum.


Frequency of treatment:
once a day
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 groups of 10 males and 10 females each . A similar constituted control group received the vehicle alone during the treatment period.

Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected as it is a possible route of exposure of the test item in man.
The dosages, selected in consultation with the Sponsor, were 75, 150 and 600 mg/kg/day.
Dose levels were selected in agreement with the Sponsor based on information from a preliminary non GLP compliant study (RTC Study No.: 88780EXT).
Parental animals: Observations and 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.
One control animal (no. 91770009) judged to be in extremis was killed.

Clinical signs

All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions

Clinical observations (Functional Observation Battery Tests)

Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males the tests were performed the day before necropsy and for females on Day 3 post partum.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the tests were performed the day before necropsy and for females on Day 3 post partum

Body weight

Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to pairing and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

Food consumption

The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Clinical pathology investigations

As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation.

The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count

Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests

Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (Only males)

At the same time interval as the clinical pathology investigations, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle]
Litter observations:
Parturition and gestation length

A parturition check was performed from Day 20 to Day 25 post coitum.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Pups identification, weight and observation

As soon as possible after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.

Postmortem examinations (parental animals):
Parental animals were killed by exsanguination under isofluorane anaesthesia. All animals were subject to necropsy

Parental males:
The males were killed after the mating of all females, after 33 days of treatment.

Parental females:
The females with live pups were killed on Day 4 post partum.
The females which did not give birth 25 days after positive identification of mating were sacrificed on Days 26 or 27 post coitum.

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding one animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.

All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Organ weights

From all animals (with the exception of animal humane killed) the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissue fixed and preserved

Samples of all the tissues listed in Annex 1 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

The examination was restricted as detailed below:

a) Tissues specified in Annex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
b) Tissues specified in Annex 1 from all animals killed during the treatment period.
c) All females sacrificed because no delivery, to investigate possible causes.
d) All abnormalities in all groups

Postmortem examinations (offspring):
Pups were euthanised by intraperitoneal injection of Thiopenthal on Day 4 post partum
All pups found dead in the cage were examined for external and internal abnormalities (including gonadal inspection).
All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. 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.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
The criterion for statistical significance was p<0.05 and p<0.01.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
Reproductive indices:
The following reproductive indices were calculated:

Males
Copulatory Index (%) = no. of animals mated / no. of animals paired x 100
Fertility Index (%) = no. of males which induced pregnancy / no. of males paired x 100


Females
Copulatory Index (%) = no. of animals mated / no. of animals paired x 100
Fertility Index (%) = no. of pregnant females /no. of females paired x 100

Males and females
Pre-coital Interval = Mean number of days between pairing and mating
Offspring viability indices:
Females

Pre-birth loss was calculated as a percentage from the formula:

(No. of visible implantations - total litter size at birth ) / No. of visible implantations x 100


Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size) / Total litter size x 100


Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth - live litter size at Day 4) / Total litter size at birth x 100

Pre-implantation loss was calculated as a percentage from the formula:

(no. corpora lutea- no. implantations) / no. corpora lutea x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
Description (incidence and severity):
See section 7.5.1.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mid-dose female receiving 150 mg/kg bw/day (animal no. 91770043) had total litter loss on Day 0 post partum and was sacrificed on Day 3 post partum.
The number of females with live pups on Day 4 post partum was: 9 in the control group, 10 in the low dose group (75 mg/kg bw/day), 7 each in the mid-dose (150 mg/kg bw/day) and high dose groups (600 mg/kg bw/day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In pregnant females, the decrease in body weight in Group 4 was statistically significant during the post coitum period starting from Day 7 (approximately 6%) to Day 20 (approximately 18%) and on Day 1 post partum (approximately 15%) and Day 4 post partum (approximately 18%). Before pairing there was no effect on body weight in females although a very slight decrease, not statistically significant, was observed in Group 4 ( up to 3.6%).

In females, statistically significant decrease in body weight gain in Group 4 was recorded on days 14 and 20 post coitum (up to approximately 51%). Although not statistically significant, a trend to decrease in body weight gain was also observed in Group 2 and 3 ( up to 12.4%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly reduced food consumption was recorded in high dose pregnant females starting from Day 14 post coitum (approximately 18%) up to Day 4 post partum (approximately 35%). Although not statistically significant, a trend to decrease in food consumption was also observed in Group 3 (up to 5.6%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See section 7.5.1.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See section 7.5.1.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
See section 7.5.1.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See section 7.5.1.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See section 7.5.1.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
The number of oestrus cycles was decreased in high dose females with respect to the control females. A trend to decrease was also observed in Groups 2 and 3. However, these values were comparable with the control historical data, therefore they were not considered to be of toxicological relevance.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All females mated both in the control and treated groups.
A total of 5 females were found not pregnant at necropsy: 2 in the mid-dose group (animal nos. 91770047, 91770059) and 3 in the high dose group (animal nos. 91770065, 91770073, 91770077).
Decrease in pre-coital interval observed in high dose females was not considered to be of toxicological relevance.
The copulatory index did not show differences between treated and control groups.
Fertility index of males and females receiving the dose levels = 150 mg/kg/day was decreased compared to the control group. Although the index of 80% detected in the 150 mg/kg/day level was comparable with the historical control data
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
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
histopathology: non-neoplastic
reproductive performance
other: Decrease of corpora lutea
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Apparently no food intake (milk) and small appearance were the clinical signs generally noted in control and treated groups. In addition, cold to touch was observed in treated groups (2 litters in Group 2, 1 pup in Group 3 and 4 litters in Group 4).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased litter weight was noted on Days 1 and 4post partum in the high dose group.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No milk in stomach and autolysed organs in the abdomen and thoracic cavity were observed at necropsy in the decedent pups of control and treated groups. Most of the organs cannibalized were observed in all pups of one mid-dose female (animal no. 91770043)
Malrotated forelimb and polysyndactyly were confirmed respectively in one pup of low dose group and one pup of mid-dose group sacrificed on Day 4. These malformations were considered incidental and not treatment-related.
No abnormalities were found in all other pups of control and treated groups sacrificed on Day 4.
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
At birth, significant reduced litter size (total and live, respectively 35% and 37%), together with a significantly reduced number of females (approximately 50%), were detected at statistical analysis in the high dose group compared to controls. However, no significant differences in sex ratio (calculated as percentage of males) were detected in the same group.
On Day 1 post partum a significantly reduced mean litter weight (46%) was found in the high dose group compared to controls.
On Day 4 post partum, live litter size, number of females and litter weight were still significantly reduced in the high dose group (respectively 50%, 63% and 54%) compared to controls and no difference in sex ratio were detected.
Cumulative % pup loss on Day 4 post partum was considerably increased in the high dose group, although without a statistical significance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
other: Decrease of implantation sites
Critical effects observed:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

BODY WEIGHT

 

FEMALES

 

Day of post coitum phase

Body weight(g)

7

14

20

Dose levels

(mg/kg)

Control

294.69

333.91

424.19

75

294.84 (+0.05%)

332.21 (-0.5%)

417.67 (-1.5%)

150

285.93 (-3.0%)

323.24 (-3.2%)

400.86 (-5.5%)

600

275.69 (-6.4%)*

303.34 (-9.2%)**

346.43 (-18.3%)**

 

Day of post partum phase

1

4

Dose levels

(mg/kg)

Control

319.12

315.56

75

317.85 (-0.4%)

318.44 (+0.9%)

150

306.85 (-3.8%)

293.81 (-6.9%)

600

272.97 (-14.5%)**

258.13 (-18.2%)**

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

BODY WEIGHT GAIN

FEMALES

 

Day of post coitum phase

Body weightgain(g)per day

14

20

Dose levels

(mg/kg)

Control

5.602

14.761

75

5.339 (-4.7%)

14.244 (-3.5%)

150

5.329 (-4.9%)

12.938 (-12.4%)

600

3.951 (-29.5%)**

    7.181 (-51.4%)**

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

FOOD CONSUMPTION

FEMALES

 

Day of post coitum phase

Day of post partum phase

Food consumption

(g/animal/day)

14

20

4

Dose levels

(mg/kg)

Control

24.87

25.69

30.54

75

25.35 (+1.9%)

25.83 (+0.5%)

33.40 (+9.4%)

150

23.75 (-4.5%)

25.31 (-1.5%)

28.82 (-5.6%)

600

20.37 (-18.1%)**

21.10 (-17.9%)*

19.79 (-35.2%)*

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

OESTRUS CYCLE

 

 

Number of

oestrus cycles

 

Fertility index

(%)

Precoital interval

(days)

Dose levels

(mg/kg)

Control

3.6

100

2.7+1.3

75

3.4 (-5.6%)

100

2.6+1.4

150

3.3 (-8.3%)

80

2.9+2.1

600

2.7 (-25.0%)

70

1.6+0.5

REPRODUCTIVE PARAMETERS

 

 

Corpora Lutea

(Mean number)

Implantations

(Mean number)

Total litter

size

(Mean number)

Pre-birth

 loss

(%)

Cumulative

 loss

(%)

Dose levels

(mg/kg)

Control

16.22

15.78

15.00

4.82

2.96

75

16.20 (-0.1%)

16.10 (+2.0%)

15.40 (+2.7%)

5.13 (+6.4%)

1.89 (-36.1%)

150

16.38 (+1.0%)

16.00 (+1.4%)

14.88 (-0.8%)

6.58 (+36.5%)

3.64 (+23.0%)

600

10.43 (-35.7%)*

10.43 (-33.9%)*

9.71 (-35.3%)*

8.37 (+73.7%)

20.81 (+603.0%)

*  group mean is significantly different from control at level p < 0.05

LITTER DATA

 

 

At birth

 

Day 1 post partum

Day 4 post partum

Live litter size

(Mean number)

Litter weight

(g)

Live litter size

 (Mean number)

Litter weight

 (g)

Dose levels

(mg/kg)

Control

14.89

99.59

14.56

134.71

75

15.40 (+3.4%)

102.26 (+2.7%)

15.10 (+3.7%)

142.32 (+5.6%)

150

14.57 (-2.1%)

97.39 (-2.2%)

14.14 (-2.9%)

135.43 (+0.5%)

600

9.43 (-36.7%)*

53.30 (-46.5%)*

7.29 (-49.9%)*

61.46 (-54.4%)*

*  group mean is significantly different from control at level p < 0.05

Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproductive/developmental toxicity could be considered 150 mg/kg/day.
Executive summary:

The toxic effects on Sprague-Dawley rats of both sexes after repeated dosing with Luperox 801 (97% tert-butyl cumyl peroxide), as well as any effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and development of offspring up to Day 4 post partum were evaluated in an OECD 422 study. The test item, suspended in corn oil, was administered by oral gavage to 3 groups of 10 males and 10 females each at doses of 0, 75, 150 or 600 mg/kg. Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy. Females were treated for 2 weeks before pairing, thereafter during pairing, post coitum and lactation periods until Day 3 post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis), litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs and macroscopic observations of pups were also performed. The parental toxicity is detailed in the section 7.5 of the IUCLID, repeated dose toxicity. 

One control female judged to be in extremis was killed on post coitum Day 18. A total of 5 females were found not pregnant at necropsy: 2 in the mid-dose group and 3 in the high dose group. One mid-dose female had total litter loss on Day 0 post partum and was sacrificed on Day 3 post partum. The number of females with live pups on Day 4 post partumwas: 9 in the control group, 10 in the low dose group, 7 each in the mid-dose and high dose groups.

All females mated both in the control and treated groups. The decrease in the number of oestrus cycles and in the pre-coital interval observed in high dose females with respect to the control females were not considered of toxicological relevance. Two females in the mid-dose group and 3 females in the high dose group were found not pregnant at necropsy.The copulatory index of males and females did not show differences between treated and control groups. On the contrary, fertility index of males and females receiving the dose levels = 150 mg/kg/day was decreased compared to the control group.

Corpora lutea, implantations and total litter size were statistically decreased in the high dose females respect to the controls. In addition, an increase in pre-birth loss percentage was also detected in the same group. Gestation periods were similar in treated groups and controls.

There was no difference sex ratio. In the high dose group, decreased litter weight was noted on Day 1post partum with respect to the controls. On Day 4 post partum, live litter size, number of females and litter weight were still significantly reduced compared to controls and no difference in sex ratio were detected.

On the basis of the results obtained in the study, the NOAEL for both systemic toxicity and reproduction/developmental toxicity is considered 150 mg/kg/day for males and females. For systemic toxicity, this NOAEL is based on clinical signs, reduced body weight and bodyweight gain at 600 mg/kg/day and on kidney macroscopic and microscopic changes on male rats. For reproduction/developmental toxicity this NOAEL is based on the decrease of fertility index, decrease of corpora lutea and implantation sites, litter weight and litter size at 600 mg/kg/day.

 

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
other:
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

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.

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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)
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.
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.
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
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
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.
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
Abnormalities:
effects observed, treatment-related
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.
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
Abnormalities:
no effects observed
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
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Key study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), Tert-butyl-a,a-dimethylbenzyl peroxide (TBCP) is not classified for fertility/developmental toxicity.