Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-27 until 2012-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant 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:
, adopted 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 13 - 15 weeks old

- Weight at study initiation:
Male animals: 354 - 419 g
Female animals: 194 – 240 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet:
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water:
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
36 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20 mg/mL, 100 mg/mL and 300 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 3 days before the administration.
Analytical control of dosing solutions (control of concentration) was performed twice during the study.

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item
- Amount of vehicle :
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.

Details on mating procedure:
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was analyzed using reverse phase HPLC method with UV detection. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 97 and 104 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. Cyclohexylidenebis[tert-butyl] peroxide proved to be stable at room temperature for one day (112 and 105 % of the starting values at ca.1 and 500 mg/mL, respectively) and at 5 ± 3°C for 3 days (94 and 92 % of the starting values at ca. 1 and 500 mg/mL, respectively).
Duration of treatment / exposure:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks. Male animals were dosed for 46 days and satellite male animals for 43 days (14 days pre-mating, 14 days mating and 18 days post-mating; satellite animals: 14 days pre-mating, 4 days mating and 25 days post-mating), then they were subjected to necropsy one day after the last treatment.
Dams were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 – 10 (for 47 days; satellite animals for 43, 44 days. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (for 41 days). Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.
Frequency of treatment:
Animals were treated once per day.
Details on study schedule:
Male animals were dosed for 41 or 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 1-8 5 (for 42 – 60 days, depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 43 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Remarks:
Doses / Concentrations:
0, 40, 200, 600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex in the control and dose groups.

Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting was based on findings obtained in a previous oral repeated dose toxicity study. The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS:
General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs were recorded including onset, degree and duration of signs.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

BODY WEIGHT:
All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. Body weight of the female animals were additionally weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data were reported individually for adult animals. Individual body weight changes were calculated.
Body weight was measured on day of necropsy for each animal.

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period (pre-
mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4) except during mating phase.

EXAMINATION OF PLACENTAL SIGN:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

OBSERVATION OF THE DELIVERY PROCESS:
Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 post-partum with an accuracy of 0.1 g.
In addition to the observations on parent animals, any abnormal behavior of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all dead pups to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

HEMATOLOGY:
The hematology parameters were measured in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by SYSMEX XT-2000iV.
- Parameters checked:
White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential white blood cell count, Activated partial Thromboplastin Time, Prothrombin Time.

CLINICAL CHEMISTRY:
The clinical chemistry measurement were performed in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by Konelab 20i in all animals before the terminal necropsy.
- Parameters checked:
Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Total Protein concentration, , Albumin concentration, Albumin/globulin ratio.



Oestrous cyclicity (parental animals):
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. One pair was changed within the control group and within the high dose group after 14 day unsuccessful pairing.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.
Postmortem examinations (parental animals):
Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

PATHOLOGY:
Gross necropsy was performed on each animal one day after the last treatment). Animals were euthanized by exsanguination after verification of an Isofluran-narcosis.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality were recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions of all adult animals were preserved. Kidneys of all parental male and female animals were also preserved due to macroscopic findings in male animals dosed with 600 mg/kg bw/day. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected for blood collection from each group:
adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with vagina), gross lesions, heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve,
seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid, trachea, urinary bladder, Pups euthanized at day 4 post-partum, or shortly thereafter, were carefully examined for gross abnormalities externally.

ORGAN WEIGHT:
At the time of termination, body weight and weight of the testes, epididymides of all parental animals were determined with an accuracy of 0.01 g. Kidneys were also weighed in all male and female animals due to macroscopic findings in male animals dosed with 600 mg/kg bw/day.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
Paired organs were weighed individually; absolute organ weight was reported. Relative organ weight (to body and brain weight) was calculated and reported.

HISTOPATHOLOGY:
Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histological examination of kidneys was also performed in all animals because test item related changes were observed in the high dose treated male animals.
Postmortem examinations (offspring):
GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)


Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Offspring viability indices:
The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality
There was no test item related mortality during the course of study.
One dam dosed with 200 mg/kg bw/day died during the parturition. For this particular case there were no preceding toxic clinical signs and macroscopic changes were not found in the organs at the necropsy. Salivation was observed from Day 10 to Day 32 and on Days 36, 37 and 38. At the necropsy, signs of interrupted delivery were detected: the vaginal aperture was dilated and the fur around it was sanguineous. In the uterine horn, 7 dead fetuses were found. Histological examination revealed severe alveolar emphysema and moderate hemorrhage in the lungs and moderate hemorrhage in the uterus in connection with a probably shock, as cause of death, which cannot be linked to the test item exposure.

Clinical Observations

Daily Observations
Test item related salivation was observed in slight or moderate degree at 600 mg/kg bw/day (12/12 male and 12/12 female) and 200 mg/kg bw/day (16/17 male and 15/17 female) from Days 9 and 10 up to the end of the treatment period. The behavior and physical condition of animals were considered to be normal.
Alopecia was noted for two female animals (1/8 dam at 600 mg/kg bw/day from Day 3 up to gestation day 9 and 1/11 control dam from gestation day 6 up to lactation day 3) on the lefts side shoulder and on the back, respectively. Alopecia is a common findings in this strain of experimental rats and occurred in the control and high dose treated animals, therefore had no toxicological meaning in this study.

Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period (pre-mating, mating and post-mating).
Alopecia as described above was also observed and recorded for two female animals (1/8 dam at 600 mg/kg bw/day on Days 7 and 13 and on gestation day 6 and 1/11 control dam on gestation days 11 and 18 and on lactation day 1) at the weekly clinical observations.

Functional Observations
Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (600, 200 and 40 mg/kg bw/day, control).

Body Weight
A slight but statistically significant influence on the body weight gain was observed in male animals at 600 mg/kg bw/day but this minor variation was not considered to be of toxicological relevance. The mean body weight gain was depressed in the male animals administered with 600 mg/kg bw/day with respect to controls during the entire observation period but statistical significances were only noted for the mean values between days 7 and 13, 13 and 20, 20 and 27, 34 and 41, consequently the summarized mean body weight gain remained below the value of control group. The reduced mean body weight gain resulted in reduced mean body weight values on days 27, 34 and 41, however the differences with respect to control were small (≥ -6 %), therefore were not considered to be toxicologically relevant.
At 200 mg/kg bw/day, a transiently reduced body weight gain was observed in male animals between days 20 and 27, as well as between days 34 and 41. The difference with respect to control was only statistically but not biologically significant.
In the female animals, there were no test item related changes in the mean body weight or body weight gain. The mean body weight gain was slightly but statistically significantly higher in 200 mg/kg bw/day treated group between days 0 and 7 with respect to controls. This statistically significant difference with respect to control was not considered to be of toxicologically significant.

Food Consumption
The mean daily food consumption was slightly but statistically significantly reduced in male and female animals at 600 mg/kg bw/day during the second and first week, respectively, when comparing to the appropriate control value. This slight difference in the mean food consumption was not judged to be toxicologically significant, moreover there were no significant differences in the mean daily food consumption between the control and high dose treated groups in the course of following weeks of the treatment.
The mean daily food consumption was similar in the control and other test item treated groups (200 and 40 mg/kg bw/day) groups during the entire treatment period.

Hematology and Blood Coagulation
There were no test item related changes in the examined hematological or blood coagulation parameters in male or female animals at any dose level (600, 200 or 40 mg/kg bw/day).
Sporadic statistically significant differences were observed for some parameters with respect to controls: higher mean platelet count (PLT) and reduced mean prothrombin time (PT) in male animals administered with at 600 mg/kg bw/day and a reduced mean corpuscular hemoglobin concentration (MCHC) in female animals at 200 mg/kg bw/day.
However, these statistical significant differences were not considered toxicologically relevant. All these values were well within the historical control range.

Clinical Chemistry
A slight test item related elevation of cholesterol levels (CHOL) were detected in male and female animals at 600 mg/kg bw/day which could potentially be related to a test item influence on the hepatic system.
The other statistically significant variations between the control and treated groups were either judged not to be related to the treatment or to be of little or no biological relevance.
In male animals, slight changes were found in alkaline phosphatase activity (ALP, 600 and 200 mg/kg bw/day), in concentrations of total bilirubin (TBIL, 200 mg/kg bw/day), creatinine (CREA, 600 and 40 mg/kg bw/day), glucose (GLUC, 200 and 40 mg/kg bw/day), calcium (Ca2+, 600 and 200 mg/kg bw/day) and total protein (TPROT, 600 mg/kg bw/day).
In the female 600 mg/kg bw/day group, the mean creatinine concentration was lower while the mean sodium (Na+) concentration was higher than in the control group.
All these differences (in males and females) with respect to control were small and were considered to be independent from the treatment. All values were within the historical control ranges except the mean total bilirubin concentration in male rats administered with 200 mg/kg bw/day where no corresponding dose-response relationship was observed. More specifically, similar findings were not observed in the higher group and there were no related histopathological alterations therefore change in total bilirubin concentration was not considered to be related to the treatment.

Necropsy
Renal and hepatic alterations were observed at the gross necropsy. Pale kidneys (5/12 male animals), dark liver (11/12 males, 6/12 females), and enlarged liver (4/12 females) were observed at 600 mg/kg bw/day. Dark color of the liver was also noted for some male (2/17) and female (3/17) animals administered with 200 mg/kg bw/day.
In dead dam (200 mg/kg bw/day), dilated vaginal aperture, sanguineous fur around it and dead fetuses in the uterine horns were found.
Individual alterations were detected in all groups. Pyelectasia was present in one control (1/17) and one 600 mg/kg bw/day (1/12) treated male animals. Dark kidneys and spleen (1/12 male), yellow, compact sac adhered to the heart (1/8 dam) and swollen, pale upper part of the right side kidney (1/4 non-pregnant female) were observed in the 600 or 40 mg/kg bw/day group.

In one male animal dosed with 40 mg/kg bw/day (1/12), the liver was dark and hard if touching. In the control group, dark brown colored submandibular lymph nodes (1/17 male) and alopecia (1/11 dam) occurred. All these individual alterations are seen occasionally in experimental rats with similar age of this strain and were not related to the treatment in this study.
Hydrometra was present in several non-pregnant animals (4/4, 4/6, 4/4 and 2/5, respectively to 600, 200, 40 mg/kg bw/day and control groups), in one dam administered with 40 mg/kg bw/day (1/8) and in one not mated female (control, 1/1). Hydrometra due to the sexual cycle of animals is a common alteration in experimental rats and it was present in all female groups, so was considered to be irrespective of the treatment.

Organ Weight
Slightly higher mean weights of liver and kidneys (absolute and relative to body and brain weights) were indicative of test item influence on hepatic and renal function in male animals at 600 mg/kg bw/day. Also at 200 mg/kg bw/day the organ weights of liver and kidney were slightly elevated while reaching statistical significance but remaining within the range of the historical control values.
Similar changes were also noted for the liver weights (absolute and relative to body and brain weights) in female animals at 600 mg/kg bw/day. Again, in female animals a statistical significant increase in liver weight was observed at a dose 200 mg/kg bw/day but the values remained within the range of the historical control data.
Statistical significances were noted in male animals for higher spleen weights (absolute and relative to body and brain weights) at 600 and 200 mg/kg bw/day and for spleen weights relative to body and brain weights at 40 mg/kg bw/day. However there was no dose relevance and because of the low degree these spleen weight changes were judged to be of little or no biological significance. Moreover, the values were well within the historical control ranges.
At 600 mg/kg bw/day males, lower mean heart weight, higher mean brain and testes weight both relative to body weight and lower body weight absolute and relative to brain weight compared to the concurrent controls were not considered to be toxicologically significant. These statistically significant differences with respect to control were judged to be the consequence of the slightly lower fasted body weight of this group.
Also, statistical significance was observed at the mean brain weight and heart weight (absolute and/or relative to the body weight) in female animals administered with 600 mg/kg bw/day. However, the mean and individual heart weight remained within the historical control ranges, the degree of weight change for heart was only slight and no abnormal histopathological findings were observed.
Therefore these changes were not considered to be toxicologically relevant.

Histopathology
In dead animal (1/11 dam, 200 mg/kg bw/day), histological examination revealed alveolar emphysema (severe degree) and hemorrhage (moderate degree) in the lung, and moderate hemorrhage in the uterus, in connection with a probably shock, as cause of death which cannot be linked to the test item exposure. No degenerative or other lesions in connection with a possible toxic effect were detected in the investigated organs. It cannot be excluded however that the hemorrhage in the uterus caused the unscheduled death of this animal.
In the kidney of male animals treated with 600 mg/kg bw/day (8/8), hyaline-like droplets were observed in the epithel cells of some proximal convoluted tubules.
No hyaline like droplets were seen in the kidneys of animals belonging to the middle (5/5) or low (5/5) dose groups. The kidneys of female animals were negative regarding hyaline like droplets in all treated groups (5/5 dams per group at 600 and 40 mg/kg bw/day 6/6 dams in the 200 mg/kg bw/day group). Dilatation of tubuli in the distal area, at the border of cortical - medullary region, occurred in a proportion of male animals (5/8) belonging to the high dose group. These findings resembling on the so called “hyaline droplet nephropathy of male rats”.
Hyaline droplet nephropathy and “hydrocarbon nephropathy” are terms describe a spectrum of morphologic changes in the kidneys of male rats induced by a variety of structurally related and unrelated compounds. Hyaline droplet nephropathy is often associated with interference to α-2μ-globulin. If this is the case the observed nephropathy is specific to the male rat and has no relevance to humans. More specifically, there is abnormal accumulation of α-2μ-globulin phagolysosomes of tubular epithelium. One proposed mechanism suggests that the chemical or a metabolite binds with the α-2μ-globulin or alters the structure so that the tubular cell lysosomal enzymes cannot degrade the protein complex. Other proposed mechanisms include a direct cytotoxic effect. It is unlikely that the various chemicals associated with hyaline droplet nephropathy in the male rat act by the same mechanism. Some chemicals which produce hyaline droplet nepropathy in male rats produce renal toxicity (unassociated with α-2μ-globulin) in female rats, whereas other chemicals produce no effects in the kidney of female rats.
Histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides), and pituitary did not reveal any toxic or other test item related alterations at 600 mg/kg/bw/day dose.
In the male animals – including those which did not fertilize females – the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. Histologically, epididymides, and pituitary gland were normal in all cases as well.
In the female animals including not mated and non-pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. Dilatation of uterus was observed in one control dam (1/11), in non-pregnant animals (4/4 at 600 mg/kg bw/day, 2/6 at 200 mg/kg bw/day, 4/4 at 40 mg/kg bw/day and 2/5 in control group) and in the only not mated control female (1/1). The dilatation of uterine horns – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon in connection with the sexual function.
Focal alveolar emphysema in minimal or mild degree were present in animals of 600 mg/kg bw/day (2/5 male and 2/5 female) and in control animals (2/5 male and 3/5 female). Focal pulmonary hemorrhage was observed in one control male animal (1/5).
These pulmonary alterations occurred sporadically in the control and high dose group and were considered to be the consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The hyperplasia of bronchus associated lymphoid tissue (BALT) in control (1/5 male) and 600 mg/kg bw/day treated animals (1/5 male and 1/5 female) was a physiological phenomenon.
One side pyelectasia in the kidney (1/6 control male and 1/8 male animal at 600 mg/kg bw/day), hemorrhage in the submandibular lymph node (1/5 control male), focal congestion in the liver (1/5 male at 40 mg/kg bw/day), subacute epicarditis and angiofibroblast tissue formation in the thoracic cavity (1/5 female at 600 mg/kg bw/day) and one side lymphosarcoma in the kidney (1/1 non-pregnant female at 40 mg/kg bw/day) were considered to be individual disease.
Apart from the pathological changes observed in the kidney of male animals of the high dose group, all the above mentioned alterations, were not considered to be related to the test item exposure and therefore had no toxicological meaning.

Delivery Data of Dams
There were no test item related differences between the control and dosed groups in the delivery data of dams. Variations with regards to prolonged pregnancy parameters were without biological relevance. When compared to concurrent control data even lower values for prolonged pregnancy were observed for the 40 and 200 mg/kg bw/day dose groups.

Reproductive Performance
There were no test item related differences between the control and test item treated male animals in the examined parameters of reproductive performance. The percentage values of successfully mated males and females were slightly but statistically significantly higher in the test item treated groups (600, 200 and 40 mg/kg bw/day) with respect to control. However these differences had no toxicological meaning. The number and percent of fertile male and infertile male animals as well as the copulatory and fertility indices were similar in the control and test item treated groups.
There were no significant differences between the control and test item treated groups in the number and percentage of non-pregnant and pregnant animals, dams delivered and in the number of pregnant animals with live born pups or in the copulatory, fertility or gestation indices. The mean pre-coital interval and conceiving days were the shortest in the high dose group (600 mg/kg bw/day).
(Appendices XI/A and XI/B)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: salivation, reduced body weight gain and food consumption, changes in clinical pathology parameters and changes in organ pathology
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: salivation, reduced body weight gain and food consumption, changes in clinical pathology parameters and changes in organ pathology
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive performance: There were no test item related differences between the control and test item treated male animals in the examined parameters of reproductive performance.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Mortality
There was no test item effect on pup’s mortality.
Statistical significance was noted for the lower percent of viable pups at 200 mg/kg bw/day on lactation day 4 (i.e. for the survival index), which was due to the early euthanasia of pups (4/4) of dead dam.

Sex Distribution
There were no significant differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4.

Clinical Observations
Test item related clinical signs did not appear in the offspring.
The percent of pups without findings was the same in the control and 600 mg/kg bw/day group. Higher number and percent of pups with clinical observations (cold, no or little milk in the stomach) were found in the 200 mg/kg bw/day, but not at 600 mg/kg bw/day, therefore these were not considered toxicologically relevant.

Body Weight
There were no test item related significant differences in the mean litter weight and mean pup weight as well as in the weight gain of offspring.

Necropsy
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination (1/1 at 600 mg/kg bw/day, 1/1 at 200 mg/kg bw/day).
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Reproductive effects observed:
not specified
Conclusions:
The reproduction and developmental toxicity effects of cyclohexylidenebis[tert-butyl] peroxide following oral administration were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:  
NOAEL for male rats: 200 mg/kg bw/day;
NOAEL for female rats: 200 mg/kg bw/day.
NOAEL for reproductive performance of the male rats: 600 mg/kg bw/day
NOAEL for reproductive performance of the female rats: 600 mg/kg bw/day
NOAEL for F1 Offspring: 600 mg/kg bw/day
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was to provide initial information concerning the effect of the test item cyclohexylidenebis[tert-butyl] peroxide on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=17/sex in the control and middle dose group and n= 12 in the low and high dose groups) were administered orally (by gavage) once a day at 0 (vehicle only), 40, 200 and 600 mg/kg bw/day at concentrations of 20, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. Group of satellite animals (5 animals/sex/group; control and 200 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters in these groups. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations. Cyclohexylidenebis[tert-butyl] peroxide proved to be stable in sunflower oil formulations at room temperature for one day and at 5 ± 3°C for 3 days in a concentration range of 1 and 500 mg/mL. Concentration of the test item in the dosing solutions varied from 103 % to 109 % of nominal concentrations at all analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 10, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. The dams were allowed to litter, and rear their young up to termination on day 4 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups and in not mated and non-pregnant females and males cohabited with in the low and middle doses. Besides full histopathology examinations performed on preserved organs and tissue of randomly selected animals in the control and high dose group and on animals which were found to be dead, the kidneys and liver of animals at the low and medium doses were processed as well and evaluated histologically due to histopathology findings in kidneys of the high dose animals and macroscopic observations on the liver. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.

Results

Mortality

There was no test item related mortality at any dose level (600, 200 and 40 mg/kg bw/day). One dam administered with 200 mg/kg bw/day was found dead on the day of delivery. For this particular case, there were no preceding clinical signs and no macroscopic changes were found in the organs at the necropsy. Histological examination revealed severe alveolar emphysema and moderate hemorrhage in the lungs and moderate hemorrhage in the uterus, in connection with a probably shock, as cause of death, which cannot be linked to the test item exposure.

Clinical observation

Salivation related to the test item was observed in slight or moderate degree in male and female animals at 600 mg/kg bw/day and 200 mg/kg bw/day from Day 9 up to the end of the treatment period. No toxic signs related to the test item were found at general daily or detailed weekly clinical observations or at the functional observations. The behavior and physical condition of animals were considered to be normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).

Body weight and body weight gain

The mean body weight gain was reduced with respect to controls in male animals at 600 mg/kg bw/day resulting in a slightly reduced body weight towards the end of the treatment period (between Days 27 and 41) and a reduced total body weight gain. However the differences in body weight with respect to control were small (≥ -6 %). The body weight development of parental female animals was undisturbed in the course of the pre-mating, mating, post-mating, gestation and lactation periods.

Food consumption

The mean daily food consumption was transiently reduced in male and female animals at 600 mg/kg bw/day compared to their control group during the first two weeks of the study. There were no significant differences in the mean daily food consumption between the control and test item treated groups in the course of following weeks of the treatment. +

Hematology

There were no test item related changes in the examined hematological and blood coagulation parameters in male or female animals at any dose level (600, 200 or 40 mg/kg bw/day).

Clinical chemistry

A slightly higher mean concentration of cholesterol levels were detected in male and female animals at 600 mg/kg bw/day and a test item influence on the hepatic system cannot be excluded.

Necropsy

Test item related renal and hepatic changes were observed at 600 mg/kg bw/day: in male animals, the kidneys were found to be pale and the liver was dark; in female animals, dark and enlarged liver was detected with high incidence. Dark color of the liver was also noted for some male (2/17) and female (3/17) animals at 200 mg/kg bw/day.

Organ weight

The mean kidney weights (absolute and relative to body and brain weights) were statistically significantly elevated in male animals at 600 mg/kg bw/day with respect to controls. The mean liver weights (absolute and relative to body and brain weights) were statistically significantly higher than in the control group in male and female animals at 600 mg/kg bw/day. Also at 200 mg/kg bw/day, the weights of liver and kidney of male animals were slightly elevated while reaching statistical significance but remaining within the range of the historical control values. Similarly, in female animals a statistically significant increase in liver weight was observed at a dose 200 mg/kg bw/day but the values remained within the range of the historical control data.

Histopathology

Histopathology investigations revealed test item related renal lesions in male animals at 600 mg/kg bw/day. In the kidney of male animals treated with the high dose, hyaline-like droplets occurred in the epithel cells of some proximal convoluted tubules and dilatation of tubuli in the distal area, at the border of cortical - medullary region was detected (“hyaline droplet nephropathy of male rats”). These findings were not seen in the kidneys of high dose female animals as well as in male or female animals of the middle or low dose groups. Hyaline droplet nephropathy is often associated with interference to α-2μ-globulin. If this is the case the observed nephropathy is specific to the male rat and has no relevance to humans.

Reproduction

There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, body weight, and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study, cyclohexylidenebis[tert-butyl] peroxide caused salivation (male and female), slightly reduced body weight gain (male) and food consumption (male and female), higher level of serum cholesterol (male and female), and changes in organ pathology [pale kidneys in male animals, dark liver (male and female) enlargement of liver (female), higher kidney (male) and liver weights (male and female), hyaline-like droplets in the epithel cells of proximal convoluted tubules and dilatation of distal tubuli (male) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation and slight changes in organ pathology (dark color changes of the liver in male and female animals, higher kidney weights in male animals, and higher liver weights in male and female animals) were detected. However, no effect on liver and kidney function neither any histopathological effects were noted, and the observed weight changes remained within the range of the historical control data. At 40 mg/kg bw/day, there was no test item related effect. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male rats: 200 mg/kg bw/day

NOAEL for female rats: 200 mg/kg bw/day

NOAEL for reproductive performance of the male rats: 600 mg/kg bw/day

NOAEL for reproductive performance of the female rats: 600 mg/kg bw/day

NOAEL for F1 Offspring: 600 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline and GLP compliant study. Reliable without restriction.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422 study

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was to provide initial information concerning the effect of the test item cyclohexylidenebis[tert-butyl] peroxide on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=17/sex in the control and middle dose group and n= 12 in the low and high dose groups) were administered orally (by gavage) once a day at 0 (vehicle only), 40, 200 and 600 mg/kg bw/day at concentrations of 20, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. Group of satellite animals (5 animals/sex/group; control and 200 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters in these groups. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations. Cyclohexylidenebis[tert-butyl] peroxide proved to be stable in sunflower oil formulations at room temperature for one day and at 5 ± 3°C for 3 days in a concentration range of 1 and 500 mg/mL. Concentration of the test item in the dosing solutions varied from 103 % to 109 % of nominal concentrations at all analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 10, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. The dams were allowed to litter, and rear their young up to termination on day 4 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups and in not mated and non-pregnant females and males cohabited with in the low and middle doses. Besides full histopathology examinations performed on preserved organs and tissue of randomly selected animals in the control and high dose group and on animals which were found to be dead, the kidneys and liver of animals at the low and medium doses were processed as well and evaluated histologically due to histopathology findings in kidneys of the high dose animals and macroscopic observations on the liver. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.

 

Results

Mortality

There was no test item related mortality at any dose level (600, 200 and 40 mg/kg bw/day). One dam administered with 200 mg/kg bw/day was found dead on the day of delivery. For this particular case, there were no preceding clinical signs and no macroscopic changes were found in the organs at the necropsy. Histological examination revealed severe alveolar emphysema and moderate hemorrhage in the lungs and moderate hemorrhage in the uterus, in connection with a probably shock, as cause of death, which cannot be linked to the test item exposure.

 

Clinical observation

Salivation related to the test item was observed in slight or moderate degree in male and female animals at 600 mg/kg bw/day and 200 mg/kg bw/day from Day 9 up to the end of the treatment period. No toxic signs related to the test item were found at general daily or detailed weekly clinical observations or at the functional observations. The behavior and physical condition of animals were considered to be normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).

 

Body weight and body weight gain

The mean body weight gain was reduced with respect to controls in male animals at 600 mg/kg bw/day resulting in a slightly reduced body weight towards the end of the treatment period (between Days 27 and 41) and a reduced total body weight gain. However the differences in body weight with respect to control were small (≥ -6 %). The body weight development of parental female animals was undisturbed in the course of the pre-mating, mating, post-mating, gestation and lactation periods.

 

Food consumption

The mean daily food consumption was transiently reduced in male and female animals at 600 mg/kg bw/day compared to their control group during the first two weeks of the study. There were no significant differences in the mean daily food consumption between the control and test item treated groups in the course of following weeks of the treatment. +

 

Hematology

There were no test item related changes in the examined hematological and blood coagulation parameters in male or female animals at any dose level (600, 200 or 40 mg/kg bw/day).

 

Clinical chemistry

A slightly higher mean concentration of cholesterol levels were detected in male and female animals at 600 mg/kg bw/day and a test item influence on the hepatic system cannot be excluded.

 

Necropsy

Test item related renal and hepatic changes were observed at 600 mg/kg bw/day: in male animals, the kidneys were found to be pale and the liver was dark; in female animals, dark and enlarged liver was detected with high incidence. Dark color of the liver was also noted for some male (2/17) and female (3/17) animals at 200 mg/kg bw/day.

 

Organ weight

The mean kidney weights (absolute and relative to body and brain weights) were statistically significantly elevated in male animals at 600 mg/kg bw/day with respect to controls. The mean liver weights (absolute and relative to body and brain weights) were statistically significantly higher than in the control group in male and female animals at 600 mg/kg bw/day. Also at 200 mg/kg bw/day, the weights of liver and kidney of male animals were slightly elevated while reaching statistical significance but remaining within the range of the historical control values. Similarly, in female animals a statistically significant increase in liver weight was observed at a dose 200 mg/kg bw/day but the values remained within the range of the historical control data.

 

Histopathology

Histopathology investigations revealed test item related renal lesions in male animals at 600 mg/kg bw/day. In the kidney of male animals treated with the high dose, hyaline-like droplets occurred in the epithel cells of some proximal convoluted tubules and dilatation of tubuli in the distal area, at the border of cortical - medullary region was detected (“hyaline droplet nephropathy of male rats”). These findings were not seen in the kidneys of high dose female animals as well as in male or female animals of the middle or low dose groups. Hyaline droplet nephropathy is often associated with interference to α-2μ-globulin. If this is the case the observed nephropathy is specific to the male rat and has no relevance to humans.

 

Reproduction

There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

 

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, body weight, and necropsy findings) were not detected between postnatal days 0 and 4.

 

Conclusion

Under the conditions of the present study, cyclohexylidenebis[tert-butyl] peroxide caused salivation (male and female), slightly reduced body weight gain (male) and food consumption (male and female), higher level of serum cholesterol (male and female), and changes in organ pathology [pale kidneys in male animals, dark liver (male and female) enlargement of liver (female), higher kidney (male) and liver weights (male and female), hyaline-like droplets in the epithel cells of proximal convoluted tubules and dilatation of distal tubuli (male) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation and slight changes in organ pathology (dark color changes of the liver in male and female animals, higher kidney weights in male animals, and higher liver weights in male and female animals) were detected. However, no effect on liver and kidney function neither any histopathological effects were noted, and the observed weight changes remained within the range of the historical control data. At 40 mg/kg bw/day, there was no test item related effect. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male and female rats: 200 mg/kg bw/day

NOAEL for reproductive performance of male and female rats: 600 mg/kg bw/day

NOAEL for F1 Offspring: 600 mg/kg bw/day


Short description of key information:
The reproduction and developmental toxicity effects of cyclohexylidenebis[tert-butyl] peroxide following oral administration were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:  
NOAEL for reproductive performance of male and female rats: 600 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
Reproduction/developmental toxicity screening test recommended to simulate likely route of exposure.

Effects on developmental toxicity

Description of key information
The developmental toxicity  effects of  Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) following oral administration were assessed in a read across study according to OECD Guideline 414 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows: NOAEL: 1000 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20 December 2012 to 07 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, forestry abd Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147, (24 November 2000)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of one hundred and twenty time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 212 to 292g.


The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Appendix 22. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). There were no contaminants that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidity were included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2 ºC and 55 ± 15% respectively. Occasional deviations from the humidity target were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried
Details on exposure:
For the purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Dried Corn Oil. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. Results are given in Appendix 15 and show the formulations to be stable for four hours. Formulations were therefore prepared daily.


Samples were taken of each test item formulation and were analysed for concentration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 4% of the nominal concentration
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

Samples
The test item formulations were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 0.2 mg/ml.

Standards
Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.2 mg/ml.


Procedure

The standard and sample solutions were analysed by GC using the following conditions:

GC system :Agilent Technologies 5890, incorporating autosampler and workstation
Column :DB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program :initial 50 ºC for 0 mins
rate 10 ºC/min
temp 130 ºC for 0 mins
rate 50 ºC/min
final 260 ºC for 10 mins
Injection temperature :150 ºC
Flame ionisation detector temperature :250 ºC
Injection volume: 2 µl
Retention time : ~ 6.5 mins


Homogeneity Determinations
The test item formulations were assessed visually.

Stability Determinations
The test item formulations were sampled and analysed initially and then after storage at ambient conditions for four hours.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed on the day of preparation.
Details on mating procedure:
Not described in this study
Duration of treatment / exposure:
14 days (Between days 5 and 19 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.


Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.


Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).


Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.


Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.


3.4.5 Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation.



All implantations and viable foetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable foetus


Organ Weights
The liver and kidneys were removed from all animals that were killed at the end of the study and were dissected free from fat and weighed before fixation.


Histopathology
The liver and kidneys were removed from all parental animals and preserved in buffered 10% formalin. The tissues from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination.

Since there were indications of treatment-related liver changes, examination was subsequently extended to include similarly prepared sections of the liver from all animals from the low and intermediate groups.


Ovaries and uterine content:
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5 % ammonium poly sulphide to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/foetal and placental tissue visible

Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Fetal examinations:
The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were examined for skeletal development and anomalies. Following examination foetuses that were examined for skeletal development were placed in 100% glycerol.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and foetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Twenty-one females treated with 1000 mg/kg bw/day, seventeen females treated with 300 mg/kg bw/day and three females treated with 100 mg/kg bw/day had an enlarged liver at necropsy. One of the females treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver.

No such effects were detected in females treated with 30 mg/kg bw/day.

One female treated with 1000 mg/kg bw/day had a mass on the mammary gland. This was confirmed as a malignant tumour adenocarcinoma following histopathological examination. In isolation, this was considered not to be related to treatment.



Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight. Statistical significant was achieved in 100, 300 and 1000 mg/kg bw/day females (p<0.05 - 0.001).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects of any toxicological significance
Abnormalities:
not specified
Developmental effects observed:
not specified

              

Mortality

There were no unscheduled deaths.

 

Clinical Observations 

Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation from Day 6 and 14, respectively. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

  

               

Body Weight

No adverse effect on body weight development was detected. Statistical analysis did not reveal any significant differences.

Food Consumption

No adverse effect on dietary intake was detected.

 

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.001) in food consumption between Days 5 and 8. Recovery was evident thereafter, with values for these treated females actually being higher than control values therefore in isolation the intergroup difference was considered not to be of toxicological significance. 

 

               

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

 

No adverse effect on water consumption was detected.

Post Mortem Studies

Twenty-one females treated with 1000 mg/kg bw/day, seventeen females treated with 300 mg/kg bw/day and three females treated with 100 mg/kg bw/day had an enlarged liver at necropsy. One of the females treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver.  

 

No such effects were detected in females treated with 30 mg/kg bw/day.

 

One female treated with 1000 mg/kg bw/day had a mass on the mammary gland. This was confirmed as a malignant tumour adenocarcinoma following histopathological examination. In isolation, this was considered not to be related to treatment.

 

   

Organ Weights

Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight. Statistical significant was achieved in 100, 300 and 1000 mg/kg bw/day females (p<0.05 - 0.001).

 

         

Histopathology

Liver:Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups.

 

Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated inflammatory or degenerative changes is generally considered to be adaptive in nature and does not represent an adverse health effect.

 

Litter Responses

 

Litter Data and Litter Placental and Foetal Weights

There was no adverse effect onin-uterooffspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. There were also no adverse effects on pre-implantation losses or sex ratio.

 

Females treated with 300 mg/kg bw/day showed an increase in male and female foetal weights. Females treated with 30 mg/kg bw/day also showed an increase in male foetal weights. An increase in body weight is considered not to be of toxicological significance.

 

 Foetal Examination

For all dose groups, there were no significant treatment-related trends in the proportion of foetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study.

 

Females treated with 300 mg/kg bw/day showed a statistically significant increase in the number of foetuses showing greater than six ossified metacarpals. Subsequently these females also showed a statistically significant reduction in the number of foetuses showing six ossified metacarpals. The standard number of ossified metacarpals in Day 20 foetuses is greater than six. Therefore the intergroup differences indicate a higher number of foetuses showing a normal number of ossified metacarpals compared to controls and in the absence of a true dose related response as such, are considered of no toxicological importance.

 

Females treated with 1000 mg/kg/day showed a statistically significant increase in the percentage of foetuses showing one lumbar vertebral centre semi-bipartite. Females treated with 100 mg/kg bw/day showed a statistically significant increase in the percentage of foetuses showing one thoracic vertebral centre bipartite. The observation of one variant at a higher incidence compared with controls is not significant when evaluated in isolation. The number of parameters evaluated makes it highly likely that one finding will be seen at a higher level in dosed groups compared with controls. A true developmental effect is only seen when a number of variants or a syndrome of variance is observed. Therefore these intergroup differences are of no biological significance.

 

        
Conclusions:
The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

The read across study was performed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female during the period of organogenesis. 

 

The study was designed to comply with the following guidelines:

* US EPA Health Effects Test Guidelines OPPTS 870.3700, 'Prenatal Developmental Toxicity Stidy' (August 1998)

* Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147, (24 November 2000)

* OECD Guidelines for Testing of Chemicals, No 414, 'Prenatal Developmental Toxicity Study' (adopted 22 January 2001)

* Commission Regulation (ES) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

Methods

The test item was administered by gavage to four groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 30, 100, 300 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Dried Corn oil) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. Liver and kidney weights were recorded for all females at termination and histopathological evaluation of the liver and kidneys was performed.

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) in distilled water and then following examination for skeletal development transferred into 100% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and examined viscerally.

  

Results

  

Mortality

There were no unscheduled deaths.

 

Clinical Observations

Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

 

Body Weight

No treatment related effects in body weight development were detected.

 

Food Consumption

No toxicologically significant effects were detected in food consumption.

 

Water Consumption

No adverse effect on water consumption was detected.

  

Post Mortem Studies

Females treated with 1000, 300 and 100 mg/kg bw/day had an enlarged liver at necropsy. One female treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver. No such effects were detected in females treated with 30 mg/kg bw/day.

 

Organ Weights

Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight.

 

Histopathology

The following treatment related microscopic effects were detected:

 

Liver:Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups.

 

Litter Data and Litter Placental and Foetal Weights

No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development.

 

Foetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day. 

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline and GLP compliant 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
Additional information

The read across study was performed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female during the period of organogenesis. 

 

The study was designed to comply with the following guidelines:

* US EPA Health Effects Test Guidelines OPPTS 870.3700, 'Prenatal Developmental Toxicity Stidy' (August 1998)

* Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147, (24 November 2000)

* OECD Guidelines for Testing of Chemicals, No 414, 'Prenatal Developmental Toxicity Study' (adopted 22 January 2001)

* Commission Regulation (ES) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

Methods

The test item was administered by gavage to four groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 30, 100, 300 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Dried Corn oil) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. Liver and kidney weights were recorded for all females at termination and histopathological evaluation of the liver and kidneys was performed.

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) in distilled water and then following examination for skeletal development transferred into 100% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and examined viscerally.

  

Results

  

Mortality

There were no unscheduled deaths.

 

Clinical Observations

Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

 

Body Weight

No treatment related effects in body weight development were detected.

 

Food Consumption

No toxicologically significant effects were detected in food consumption.

 

Water Consumption

No adverse effect on water consumption was detected.

  

Post Mortem Studies

Females treated with 1000, 300 and 100 mg/kg bw/day had an enlarged liver at necropsy. One female treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver. No such effects were detected in females treated with 30 mg/kg bw/day.

 

Organ Weights

Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight.

 

Histopathology

The following treatment related microscopic effects were detected:

 

Liver:Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups.

 

Litter Data and Litter Placental and Foetal Weights

No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development.

 

Foetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day. 

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Guideline and GLP study

Justification for classification or non-classification

Based on the results of the reproduction and developmental toxicity screening test, the test item was not classified and labelled according to Regulation (EC) No 1272/2008 (CLP).