Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
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

Test material

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

Test animals

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.

Administration / exposure

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.

Doses / concentrations
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

Examinations

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"

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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)

Effect levels (P0)

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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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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).

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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