Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-13 to 2018-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-11-13 to 2018-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 13 weeks at premating, 15 weeks at mating
- Weight at study initiation: The weight variation in animals involved at the starting point of the study will not exceed ± 20 % of the mean group weight of each sex.
- Housing: 2 animals of the same sex / cage (before mating); 1 male and 1 female / cage (mating); females individually and males 2 animals / cage (postmating)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 -70 %
- Air changes: > 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 12 mg/mL, 40 mg/mL and 120 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the use.

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 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 12, 40 and 120 mg/mL
- Amount of vehicle: a constant treatment volume of 5 mL dose preparation/kg was administered in all groups
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility once during the study. Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and measured once during the study. Similarly, five samples were taken from the control solution (Group 1) and analyzed.

The measured concentrations varied between the range of 84, 91 and 88 % of the nominal concentrations of 12, 40 and 120 mg/mL.
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 at the appropriate frequency of preparation. (Recovery from sunflower oil: 92 % and 96 % of nominal concentrations at ca. 1 mg/mL and ca. 200 mg/mL). The test item was stable at the intended concentrations for at least 4 hours at room temperature and for three days in a refrigerator.
Duration of treatment / exposure:
males: 29 days
females: 63 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on a preliminary dose range finding study.
The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Rationale for animal assignment: random by body weight and estrus cycle (females)
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily after treatment;
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 behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: study day 0 (before treatment), weekly thereafter. Fasted body weight was measured on day of necropsy (day 14).

FOOD CONSUMPTION: Yes
- time schedule: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatement
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
The following organ weights were determined and recorded (paired organs were weighed together):
With precision of 0.01g: liver, kidneys, testes, epididymides, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands, as a whole;
With precision of 0.001g: adrenal glands

HISTOPATHOLOGY: No
Other examinations:
Estrous Cycle:
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles were included in the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy.
Statistics:
The statistical evaluation of appropriate data was 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 is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily observations
Test item or treatment related clinical signs were observed at 600 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were observed in several male and female animals administered with 600 mg/kg bw/day after the daily administration. These signs appeared shortly after the treatment and ceased after a short period of time. Therefore, they were considered to be toxicologically not relevant. Clinical signs of moribund and dead animals (1/12 control; 2/12 at 600 mg/kg bw/day; male, each) are listed in section “Mortality”. There were no clinical signs in the control (11/12 male and 12/12 female), in the 60 mg/kg bw/day group (12/12 male and 12/12 female), and in the 200 mg/kg bw/day group (12/12 male and 11/12 female) during the course of the study, i.e. these parental animals exhibited normal behavior and physical condition with no abnormalities during the treatment period. Alopecia on the neck was noted for one female animal at 200 mg/kg bw/day (1/12) from Day 7 until the end of the study.
At 600 mg/kg bw/day, nuzzling up the bedding material (10/10 males, 12/12 females) and salivation (6/10 surviving males, 3/12 females) were detected in surviving male animals and in female animals after the daily treatment at the clinical observations.
In one male animal at 600 mg/kg bw/day (1/10 of survivors), piloerection, decreased activity and noisy breathing was also observed transiently from Day 41 until Day 43 or 47. These clinical signs were probably individual findings, which were only observed for a few days and in single animal. However, test item effect cannot be excluded entirely.

Detailed weekly observations
There were no clinical signs in animals of control (11/12 male and 12/12 female), 60 mg/kg bw/day (12/12 male and 12/12 female) or 200 mg/kg bw/day group (12/12 male and 11/12 female) at the detailed weekly observations. One male animal in the control group (1/12, no. 111) showed severe activity decrease, piloerection, sanguineous hairs around the nose and eye on Day 27 and it was found to be moribund state and euthanized on Day 29. These clinical signs were indicative of individual disease of this control animal.
Alopecia on the neck was noted for one female animal at 200 mg/kg bw/day (1/12) from Day 7 until the end of the study. Alopecia is a common observation in experimental rats with similar age of this strain. Piloerection, decreased activity and noisy breathing was observed in one male animal at 600 mg/kg bw/day (1/10 surviving) on Day 41. Dyspnea was noted in one male animal at 600 mg/kg bw/day (1/2 of dead animals) on Days 48 and 54 at the detailed weekly observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three male animals (one control and two ones at 600 mg/kg bw/day) were subjected to early necropsy due to moribund condition or death.
One control male animal (no. 111) was judged to be in moribund state (severe activity decrease, piloerection, prone position, decreased body tone, and sanguineous hair around the nose and eye were observed) and it was euthanized and subjected to necropsy on Day 29. Histopathological examination revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach as individual lesions causing death of this animal.
In animal no. 402 at 600 mg/kg bw/day, slight activity decrease (Day 7) salivation and nuzzling up the bedding material, (from Days 3 or 6 to day 49, respectively) were observed and this animal was found dead on Day 50. Sign of diarrhea (hairs were contaminated with faeces) were observed at the necropsy. Histological examinations revealed fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs as the probable cause of death. These findings refer to para-gastric treatment. No toxic or any other lesions were observed in the other investigated organs of this animal. Therefore, dead of this animal was regarded not test item related.
In animal, no. 408, nuzzling up the bedding material (between Days 6 and 54), noisy breathing or dyspnea (between Days 45 and 54) and significant weight loss (-152 g between Days 41 and 54) were observed and this animal was found dead on Day 55. Cachexia, autolyzed organs, gas filled intestines and smaller than normal prostate and seminal vesicles were detected at the necropsy. Histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. With the results of this study, the cause of the death of this animal cannot be determined. A test item effect cannot be verified but cannot be excluded either.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in male animals at 600 mg/kg bw/day during the entire observation period. The mean body weight gain was significantly higher than in the control in male animals at 60 mg/kg bw/day between Days 27 and 34. Statistical significance with respect to the control was detected in male animals at 200 mg/kg bw/day at the lower mean body weight gain between Days 0 and 7 and at the higher mean body weight gain between Days 27 and 34. The mean body weight was significantly lower than in the control in male animals at 600 mg/kg bw/day from Day 7 up to the end of the study (Day 54). The mean body weight gain was also significantly reduced in this group on Weeks 1, 2, 4, 6 and 8 and for the entire treatment period (between Day 0 and 54). There were no statistically significant differences between the control and test item treated groups in the mean body weight and mean body weight gain in females at 60, 200 and 600 mg/kg bw/day in the pre-mating, gestation or lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of male animals was transiently reduced in male animals at 600 mg/kg bw/day on week 1 in full compliance with the body weight alterations.
The food consumption was comparable in the control and in the 60 and 200 mg/kg bw/day groups, i.e. statistically significant differences were not seen in the mean daily food consumption of male or female animals with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals). Therefore, intermittently found significant higher body gain is considered to be incidental and not of biological relevance or test item treatment related.
In male animals at 600 mg/kg bw/day, the daily mean food consumption was reduced during the first week; however it was similar to their control during subsequent weeks.
Statistical significance noted for the daily mean food consumption of female animals at 600 mg/kg bw/day was of with minor degree and considered to be toxicologically relevant on week 1. The daily mean food consumption of female animals was comparable in the control and test item treated groups at 600 mg/kg bw/day during the gestation and lactation periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant elevation of percentage of reticulocytes was observed in male animals at 600 mg/kg bw/day along with a decrease in the red blood cell count and hemoglobin concentration. The increased percentage of reticulocytes might be indicative of loss or damage of erythrocytes in circulation, which was accompanied by an accelerated erythropoiesis. This finding was in accordance with increase of the spleen weights in male animals at 600 mg/kg bw/day. Although there were no supporting clinical chemistry or histopathological findings, alterations in these parameters might refer to slight anemic changes and were considered to be treatment related. All examined hematological and blood coagulation parameters were comparable with the control in male animals at 60 and 200 mg/kg bw/day.
In the male animals at 600 mg/kg bw/day, the mean percentage of monocytes (MONO) and eosinophil granulocytes (EOS), the mean red blood cell (erythrocyte) count (RBC), the mean hemoglobin concentration (HGB) and mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) was statistically significantly lower compared to their control. Statistical significance was also noted for the higher mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) content and for the higher mean percentages of reticulocytes (RET). Changes in RET, RBC an HGB referred to damage or loss of red blood cells accompanied by restoration mechanisms, although values of RBC and HGB remained within historical control ranges. Slight differences with respect to the control in MONO, EOS, MCV, MCH and MCHC were judged to have no or little biological significance. All examined hematological and blood coagulation parameters were comparable with their controls in female animals at 60, 200 and 600 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related adverse alterations were detected in the examined clinical chemistry parameters in male or female animals at 60, 200 or 600 mg/kg bw/day. Statistical significance was detected in male animals at 200 mg/kg bw/day at the lower mean total bilirubin (TBIL) concentration and at the higher potassium concentration (K+) in female animals at 60 and 200 g/kg bw/day. These differences were only seen at the lower doses therefore were considered to be indicative of biological variation and toxicologically not relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any treatment-related alterations at the end of the treatment period (selected male and female, 60, 200 or 600 mg/kg bw/day groups, on Day 50). The physical condition, behavior or reactions to different types of stimuli were similar in the male or female animals of control and test item treated groups in the examined parameters.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item effect was supposed on the weights of liver, kidneys and spleen in male animals administered with 600 mg/kg bw/day.

Elevation of the spleen weights in male animals at 600 mg/kg bw/day along with changes in red blood cell parameters (RBC, HGB, RET) might refer to increased loss or damage of erythrocytes in the circulation. Slight changes in the liver and kidney weights might be related to a test item influence on hepatic or renal functions. However, the degree of all of these changes was small and there were no supporting histopathological findings. Therefore, it was considered to be indication of the adaptation process and the toxicological significance is equivocal. In the male animals, statistical differences compared to their control were detected at the organ weight evaluation as follows:

- liver weight (absolute) was higher at 200 mg/kg bw/day;
- the fasted body weight (absolute and relative to brain weight) was lower and the brain weight relative to body weight was higher at 600 mg/kg bw/day;
- weights of liver, spleen and adrenal glands (absolute, relative to body and brain weight) were higher at 600 mg/kg bw/day;
- kidney weights (relative to body and brain weight) were higher at 600 mg/kg bw/day;
- heart weights (absolute and relative to brain weight) were lower at 600 mg/kg bw/day;
- weights of thymus, epididymides and seminal vesicles (absolute) were lower at 600 mg/kg bw/day;
- testes weight relative to body weight was higher at 600 mg/kg bw/day;

In female animals, the following statistical differences were observed in the organ weights when compared to their control:
- weight of adrenal glands relative to the body weight at 200 mg/kg bw/day was higher;
- liver weight relative to body weight was higher at 200 and 600 mg/kg bw/day was;
- kidney weights relative to body and brain weight were higher at 600 mg/kg bw/day;
- weights of adrenal glands (absolute, relative to body and brain weight) were higher at 600 mg/kg bw/day;

Alterations in the weights of adrenal glands at 600 mg/kg bw/day (male and female) were with small degree and there were no histological changes to substantiate their relevance. Statistical significances noted for weights (absolute or relative to body weight) of heart, thymus, testes, epididymides and prostate were mainly due to body weight changes in male animals at 600 mg/kg bw/day and were not associated with any clinical pathological or histopathological findings. Elevation in weights of the liver and kidneys in female animals were with small degree and were not associated with any clinical pathological or histopathological findings therefore were not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings related to the effect of the test item were not detected in organs or tissues of male or female animals at 60, 200 or 600 mg/kg bw/day at the necropsy.

Dead and moribund animals:
One male animal (no. 111), which was judged to be in moribund condition on Day 29, red discharge on the hairs around the eyes and nose, hematoma in the brain (starting from bulbus olfactorius), approximately 0.5x1cm greyish white erosion at the cardia of the stomach, black content in the intestines were observed. In dead animal no. 402 at 600 mg/kg bw/day, sign of diarrhea (i.e. faeces on the hairs around the anus) was observed at the necropsy. Dead animal no. 408 was cachectic and the prostate and seminal vesicle were smaller than normal, the intestines were filled up with gas and the organs were autolyzed.

Surviving animals:
There were no macroscopic changes in the organs or tissues of most of surviving male animals terminated as scheduled (11/11 in the control, 12/12 at 60 mg/kg bw/day, 11/12 at 200 mg/kg bw/day, 10/10 at 600 mg/kg bw/day). In one male animal at 200 mg/kg bw/day, pale kidneys were observed. In dams, there were no macroscopic changes in the organs or tissues in the control (10/10), at 60 mg/kg bw/day (10/11), at 200 mg/kg bw/day (10/11) and at 600 mg/kg bw/day (9/10). Hydrometra (1/11 at 60 mg/kg bw/day) and pale kidneys (1/11 at 200 mg/kg bw/day and 1/10 at 600 mg/kg bw/day) were detected in single dams at the necropsy. There were no macroscopic changes in not delivered (1/1 at 200 mg/kg bw/day) or in any other non-pregnant (1/2 control, 1/1 at 60 mg/kg bw/day, 2/2 at 600 mg/kg bw/day) or female animals. Hydrometra was noted for one non-pregnant control female animal (1/2).
Pale kidneys were judged to be toxicologically not relevant in the lack of related histopathological alterations. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these were judged not to be toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no pathologic changes detectable by histological examination in the examined reproductive organs or tissues of male or female animals (control and 600 mg/kg bw/day). Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at 600 mg/kg/bw/day.

Moribund and dead animals:
In the control male animal (no. 111) which was found in moribund condition, histopathological examination revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach. Mononuclear cell leukemia is a common disease causing early death among laboratory rats, therefore this was considered to be incidental. In dead male animal no. 402, fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs was observed as the probable cause of death due to para-gastric treatment. No toxic or any other lesions were observed in the other investigated organs of this animal. In dead animal no. 408, the histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. Based on these findings, the cause of death of this animal cannot be determined and test item influence cannot be excluded or verified.

Surviving animals
In all male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. 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 control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
In two female animals (1/2 control; non-pregnant; 1/10 dam at 60 mg/kg bw/day), dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In selected animals, alveolar edema in minimal degree was observed in the lungs of one control female (1/5) animal. This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted in one male animal of the control group (1/5). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance.

There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals. The cyto-morphology of the endocrine glands was the same in the control and the treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle:
The estrous cycle was not affected by the test item at any dose level (60, 200 or 600 mg/kg bw/day). There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-experimental or pre-mating period.

Serum T4 Level:
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4) level in parental male animals or in PN13 offspring at any dose levels.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other: no adverse effects observed at highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this OECD 422 compliant study, Methyl-isobutyl-ketone-peroxide (MIKP) administered at 60, 200 or 600 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats. The development of the F1 offspring was not impaired to post-natal day 13 at any dose level after repeated oral administration of dams. 600 mg/kg bw/day caused clinical signs (male and female animals), reduced body weight and body weight gain (during the entire observation period) and food consumption (on week 1), changes in some red blood cell parameters (red blood cell count, hemoglobin concentration and percentage of reticulocytes) and in spleen weight in male animals. At 60 or 200 mg/kg bw/day, there were no test item related alterations.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 200 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 600 mg/kg bw/day
NOAEL for reproductive performance of male/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 obtain initial information on the toxic potential of Methyl-isobutyl-ketone-peroxide (MIKP) and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 60 mg/kg bw/day, 200 mg/kg bw/day and 600 mg/kg bw/day compared to control animals. Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 60, 200 and 600 mg/kg bw/day doses corresponding to concentrations of 0, 12, 40 and 120 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Methyl-isobutyl-ketone-peroxide (MIKP) concentrations in the dosing formulations varied in the acceptable range between 95 % and 102 % of the nominal values confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 55 days). Females were additionally exposed through the gestation period and up to lactation days 13-17 (altogether for 51-55 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups and in non-pregnant or not delivered females, but not the males these females cohabited with in the low and middle dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. Full histopathology was also performed in one moribund male animal in the control and in two found dead males at 600 mg/kg bw/day.

In addition, kidneys were also processed and examined for one male and one female at 200 mg/kg bw/day due to the necropsy observation (pale, both side). Uterus of one female in the low dose group were also processed and examined histopathologically on the basis of necropsy findings (hydrometra).

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

Mortality

One control male animal was in moribund state and subjected to early necropsy on Day 29 of the study. Severe activity decrease, piloerection, sanguineous hairs around the nose and eye were noted for this animal before the euthanasia. Histological examinations revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach as individual lesions causing death of this animal.

One male animal administered with 600 mg/kg bw/day was found dead due to para-gastric treatment on Day 50. Histological examinations revealed fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs as the probable cause of death.

Another male animal administered with 600 mg/kg bw/day was found dead on Day 55. Clinical signs (noisy breathing, dyspnea) and significant weight loss were observed during the preceding days. Histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. With these results of this study, the cause of the death of this animal cannot be determined. A test item effect may be assumed but cannot be substantiated with these findings.

Clinical observation

Test item related clinical signs were observed in male and female animals at 600 mg/kg bw/day (nuzzling up the bedding material and salivation) with short duration after the daily administration. Clinical signs of systemic toxicity related to the test item were not detected at any dose level (60, 200 or 600 mg/kg bw/day) at the weekly detailed clinical observations or at the terminal functional observations.

Body weight and body weight gain

The body weight gain and body weight were reduced in male animals at 600 mg/kg bw/day from week 1 up to the termination of the study.

Food consumption

The daily mean food consumption was transiently reduced in male animals at 600 mg/kg bw/day on week 1 in full compliance with the body weight alterations.

The mean daily food consumption was not affected at 60, 200 or 600 mg/kg bw/day in female animals (during the pre-mating, gestation and lactation periods).

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (60, 200 and 600 mg/kg bw/day).

Reproductive performance

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

Delivery data of dams

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (60, 200 and 600 mg/kg bw/day).

Hematology

Hematological investigations revealed elevated percentage of reticulocytes and lower red blood cell count and hemoglobin concentration in male animals at 600 mg/kg bw/day with respect to their control. These findings in accordance with an increased spleen weights might be indicative of loss or damage of erythrocytes in circulation, which was accompanied by an accelerated erythropoiesis.

Clinical chemistry

Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 60, 200 or 600 mg/kg bw/day).

Serum thyroid hormone

There were no test item related changes in the serum thyroid hormone levels at any dose (parental male animals or PN13 offspring).

Necropsy

Macroscopic alterations related to the effect of the test item were not found in male or female animals at 60, 200 or 600 mg/kg bw/day at the necropsy.

Organ weight

Test item effect was assumed on the weights of liver, kidneys and spleen in male animals administered with 600 mg/kg bw/day.

Elevation of the spleen weights in male animals at 600 mg/kg bw/day along with increased percentage of reticulocytes might refer to increased loss or damage of erythrocytes in the circulation.

Slight changes in the liver and kidney weights might be related to a test item influence on hepatic or renal functions. However the degree of all of these changes was small and there were no supporting histopathological findings.

Histopathology

Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 600 mg/kg bw/day.

There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or in the 600 mg/kg bw/day group.

Offspring

The offspring’s development was not adversely influenced at any dose level.

Conclusion

Under the conditions of the present study, Methyl-isobutyl-ketone-peroxide (MIKP) administered at 60, 200 or 600 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats. The development of the F1 offspring was not impaired to post-natal day 13 at any dose level after repeated oral administration of dams. 600 mg/kg bw/day caused clinical signs (male and female animals), reduced body weight and body weight gain (during the entire observation period) and food consumption (on week 1), changes in some red blood cell parameters (red blood cell count, hemoglobin concentration and percentage of reticulocytes) and in spleen weight in male animals. At 60 or 200 mg/kg bw/day, there were no test item related alterations.

 

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male rats: 200 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 600 mg/kg bw/day

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

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

 

Data source

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

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:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 13 weeks at premating, 15 weeks at mating
- Weight at study initiation: The weight variation in animals involved at the starting point of the study will not exceed ± 20 % of the mean group weight of each sex.
- Housing: 2 animals of the same sex / cage (before mating); 1 male and 1 female / cage (mating); females individually and males 2 animals / cage (postmating)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 -70 %
- Air changes: > 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 12 mg/mL, 40 mg/mL and 120 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the use.

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 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 12, 40 and 120 mg/mL
- Amount of vehicle: a constant treatment volume of 5 mL dose preparation/kg was administered in all groups
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred or two weeks elapsed. Three female animals (no. 425, 428, 431) at the high dose failed to mate during the 14-days period, therefore mating period was prolonged and their partners were exchanged with each other.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility once during the study. Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and measured once during the study. Similarly, five samples were taken from the control solution (Group 1) and analyzed.

The measured concentrations varied between the range of 84, 91 and 88 % of the nominal concentrations of 12, 40 and 120 mg/mL.
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 at the appropriate frequency of preparation. (Recovery from sunflower oil: 92 % and 96 % of nominal concentrations at ca. 1 mg/mL and ca. 200 mg/mL). The test item was stable at the intended concentrations for at least 4 hours at room temperature and for three days in a refrigerator.
Duration of treatment / exposure:
males: 29 days
females: 63 days
Frequency of treatment:
daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on a preliminary dose range finding study.
The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Rationale for animal assignment: random by body weight and estrus cycle (females)
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily after treatment;
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 behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: study day 0 (before treatment), weekly thereafter. Fasted body weight was measured on day of necropsy (day 14).

FOOD CONSUMPTION: Yes
- time schedule: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatement
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles were included in the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testes weight, epididymis weight, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (see table 4)
The following organ weights were determined and recorded (paired organs were weighed together):
With precision of 0.01g: liver, kidneys, testes, epididymides, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands, as a whole;
With precision of 0.001g: adrenal glands

HISTOPATHOLOGY: No
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
The statistical evaluation of appropriate data was 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 is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Parental Males:
- Percentage of pairings
- Percentage of fertile pairings
- Percentage of infertile males
- Male copulatory index: Number of males with confirmed mating / total number of males cohabited x 100
- Male fertility index: Number of males impregnating for a female / total number of males with confirmed mating x 100

Parental Females:
- Percentage of pairings / sperm positive females
- Percentage of pregnant females
- Percentage of sperm positive, but non-pregnant females
- Percentage of non-mated females
- Female copulatory index: Number of females with confirmed mating / total number of females cohabited x 100
- Female fertility index: Number of pregnant female / number of spermpositive females x 100
- Gestation index: Number of females with liveborn pubs / number of pregnant females x 100
- Duration of pregnancy (days)
- Number of implantations / dams
- Number of dams with live pups on lactation days 0, 4 and 13
- Post-implantation mortality: Number of implantations - Number of liveborns / Number of implantations x 100
Offspring viability indices:
Offspring:
Litter weight on postnatal days 0, 4 and 13
- Mean body weight gain per litter between postnatal days 0-4, 4-13 and for overall between post-natal days 0 and 13
- Number of live births per litter, and number of viable pups per litter on postnatal days 0, 4 and 13
- Survival Index of pups on postnatal day 13: Number of live pups on postnatal days 13 / Number of live borns x 100
- Sex ratio % (on postnatal days 0 and 13): Number of pups examined - Number of males (females) / Number of pups examined x 100
- Normalized anogenital distance
- Number of nipples/areolae in male pups
- T4 on post-partum day 13
- postnatal mortality: Number of liveborns - Number of live pups on PND 13 / Number of liveborns x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily observations
Test item or treatment related clinical signs were observed at 600 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were observed in several male and female animals administered with 600 mg/kg bw/day after the daily administration. These signs appeared shortly after the treatment and ceased after a short period of time. Therefore, they were considered to be toxicologically not relevant. Clinical signs of moribund and dead animals (1/12 control; 2/12 at 600 mg/kg bw/day; male, each) are listed in section “Mortality”. There were no clinical signs in the control (11/12 male and 12/12 female), in the 60 mg/kg bw/day group (12/12 male and 12/12 female), and in the 200 mg/kg bw/day group (12/12 male and 11/12 female) during the course of the study, i.e. these parental animals exhibited normal behavior and physical condition with no abnormalities during the treatment period. Alopecia on the neck was noted for one female animal at 200 mg/kg bw/day (1/12) from Day 7 until the end of the study.
At 600 mg/kg bw/day, nuzzling up the bedding material (10/10 males, 12/12 females) and salivation (6/10 surviving males, 3/12 females) were detected in surviving male animals and in female animals after the daily treatment at the clinical observations.
In one male animal at 600 mg/kg bw/day (1/10 of survivors), piloerection, decreased activity and noisy breathing was also observed transiently from Day 41 until Day 43 or 47. These clinical signs were probably individual findings, which were only observed for a few days and in single animal. However, test item effect cannot be excluded entirely.

Detailed weekly observations
There were no clinical signs in animals of control (11/12 male and 12/12 female), 60 mg/kg bw/day (12/12 male and 12/12 female) or 200 mg/kg bw/day group (12/12 male and 11/12 female) at the detailed weekly observations. One male animal in the control group (1/12, no. 111) showed severe activity decrease, piloerection, sanguineous hairs around the nose and eye on Day 27 and it was found to be moribund state and euthanized on Day 29. These clinical signs were indicative of individual disease of this control animal.
Alopecia on the neck was noted for one female animal at 200 mg/kg bw/day (1/12) from Day 7 until the end of the study. Alopecia is a common observation in experimental rats with similar age of this strain. Piloerection, decreased activity and noisy breathing was observed in one male animal at 600 mg/kg bw/day (1/10 surviving) on Day 41. Dyspnea was noted in one male animal at 600 mg/kg bw/day (1/2 of dead animals) on Days 48 and 54 at the detailed weekly observations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three male animals (one control and two ones at 600 mg/kg bw/day) were subjected to early necropsy due to moribund condition or death.
One control male animal (no. 111) was judged to be in moribund state (severe activity decrease, piloerection, prone position, decreased body tone, and sanguineous hair around the nose and eye were observed) and it was euthanized and subjected to necropsy on Day 29. Histopathological examination revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach as individual lesions causing death of this animal.
In animal no. 402 at 600 mg/kg bw/day, slight activity decrease (Day 7) salivation and nuzzling up the bedding material, (from Days 3 or 6 to day 49, respectively) were observed and this animal was found dead on Day 50. Sign of diarrhea (hairs were contaminated with faeces) were observed at the necropsy. Histological examinations revealed fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs as the probable cause of death. These findings refer to para-gastric treatment. No toxic or any other lesions were observed in the other investigated organs of this animal. Therefore, dead of this animal was regarded not test item related.
In animal, no. 408, nuzzling up the bedding material (between Days 6 and 54), noisy breathing or dyspnea (between Days 45 and 54) and significant weight loss (-152 g between Days 41 and 54) were observed and this animal was found dead on Day 55. Cachexia, autolyzed organs, gas filled intestines and smaller than normal prostate and seminal vesicles were detected at the necropsy. Histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. With the results of this study, the cause of the death of this animal cannot be determined. A test item effect cannot be verified but cannot be excluded either.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in male animals at 600 mg/kg bw/day during the entire observation period. The mean body weight gain was significantly higher than in the control in male animals at 60 mg/kg bw/day between Days 27 and 34. Statistical significance with respect to the control was detected in male animals at 200 mg/kg bw/day at the lower mean body weight gain between Days 0 and 7 and at the higher mean body weight gain between Days 27 and 34. The mean body weight was significantly lower than in the control in male animals at 600 mg/kg bw/day from Day 7 up to the end of the study (Day 54). The mean body weight gain was also significantly reduced in this group on Weeks 1, 2, 4, 6 and 8 and for the entire treatment period (between Day 0 and 54). There were no statistically significant differences between the control and test item treated groups in the mean body weight and mean body weight gain in females at 60, 200 and 600 mg/kg bw/day in the pre-mating, gestation or lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of male animals was transiently reduced in male animals at 600 mg/kg bw/day on week 1 in full compliance with the body weight alterations.
The food consumption was comparable in the control and in the 60 and 200 mg/kg bw/day groups, i.e. statistically significant differences were not seen in the mean daily food consumption of male or female animals with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals). Therefore, intermittently found significant higher body gain is considered to be incidental and not of biological relevance or test item treatment related.
In male animals at 600 mg/kg bw/day, the daily mean food consumption was reduced during the first week; however it was similar to their control during subsequent weeks.
Statistical significance noted for the daily mean food consumption of female animals at 600 mg/kg bw/day was of with minor degree and considered to be toxicologically relevant on week 1. The daily mean food consumption of female animals was comparable in the control and test item treated groups at 600 mg/kg bw/day during the gestation and lactation periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant elevation of percentage of reticulocytes was observed in male animals at 600 mg/kg bw/day along with a decrease in the red blood cell count and hemoglobin concentration. The increased percentage of reticulocytes might be indicative of loss or damage of erythrocytes in circulation, which was accompanied by an accelerated erythropoiesis. This finding was in accordance with increase of the spleen weights in male animals at 600 mg/kg bw/day. Although there were no supporting clinical chemistry or histopathological findings, alterations in these parameters might refer to slight anemic changes and were considered to be treatment related. All examined hematological and blood coagulation parameters were comparable with the control in male animals at 60 and 200 mg/kg bw/day.
In the male animals at 600 mg/kg bw/day, the mean percentage of monocytes (MONO) and eosinophil granulocytes (EOS), the mean red blood cell (erythrocyte) count (RBC), the mean hemoglobin concentration (HGB) and mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) was statistically significantly lower compared to their control. Statistical significance was also noted for the higher mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) content and for the higher mean percentages of reticulocytes (RET). Changes in RET, RBC an HGB referred to damage or loss of red blood cells accompanied by restoration mechanisms, although values of RBC and HGB remained within historical control ranges. Slight differences with respect to the control in MONO, EOS, MCV, MCH and MCHC were judged to have no or little biological significance. All examined hematological and blood coagulation parameters were comparable with their controls in female animals at 60, 200 and 600 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related adverse alterations were detected in the examined clinical chemistry parameters in male or female animals at 60, 200 or 600 mg/kg bw/day. Statistical significance was detected in male animals at 200 mg/kg bw/day at the lower mean total bilirubin (TBIL) concentration and at the higher potassium concentration (K+) in female animals at 60 and 200 g/kg bw/day. These differences were only seen at the lower doses therefore were considered to be indicative of biological variation and toxicologically not relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any treatment-related alterations at the end of the treatment period (selected male and female, 60, 200 or 600 mg/kg bw/day groups, on Day 50). The physical condition, behavior or reactions to different types of stimuli were similar in the male or female animals of control and test item treated groups in the examined parameters.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no pathologic changes detectable by histological examination in the examined reproductive organs or tissues of male or female animals (control and 600 mg/kg bw/day). Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at 600 mg/kg/bw/day.

Moribund and dead animals:
In the control male animal (no. 111) which was found in moribund condition, histopathological examination revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach. Mononuclear cell leukemia is a common disease causing early death among laboratory rats, therefore this was considered to be incidental. In dead male animal no. 402, fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs was observed as the probable cause of death due to para-gastric treatment. No toxic or any other lesions were observed in the other investigated organs of this animal. In dead animal no. 408, the histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. Based on these findings, the cause of death of this animal cannot be determined and test item influence cannot be excluded or verified.

Surviving animals
In all male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. 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 control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
In two female animals (1/2 control; non-pregnant; 1/10 dam at 60 mg/kg bw/day), dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In selected animals, alveolar edema in minimal degree was observed in the lungs of one control female (1/5) animal. This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted in one male animal of the control group (1/5). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance.

There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals. The cyto-morphology of the endocrine glands was the same in the control and the treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 Level:
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4) level in parental male animals or in PN13 offspring at any dose levels.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle was not affected by the test item at any dose level (60, 200 or 600 mg/kg bw/day). There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-experimental or pre-mating period.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item at 60, 200 or 600 mg/kg bw/day in male or female animals. Statistical significance was observed at the higher fertility index of male and female animals at 200 mg/kg bw/day because two male animals (2/12) in the control did not fertilize their females. There was a statistically significant difference between the control and the 200 mg/kg bw/day group of female animals in the gestation index as one pregnant female animal in mid dose group did not deliver. This minor change was considered to be an isolated finding and not related to the test item. There were no significant differences in the copulatory indices in males and females between the control and test item treated groups. The percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) were comparable in female animals of control and test item treated animals. The mean pre-coital interval and the mean conceiving days of the low and high doses were slightly higher than in the control group however no statistical significance was obtained. These values exceeded the mean historical control value as well. As no dose dependence was observed, these changes were considered to have no toxicological relevance.

Delivery Data of Dams:
There were no relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (60, 200 or 600 mg/kg bw/day). The number of pregnant females and dams delivered, the mean number of implantations, duration of pregnancy, and pups births (total, live, viable) and live birth index were comparable in all groups. Statistical significance was noted with respect to the control for the higher mean post-implantation loss and for the higher mean number of stillborn in dams at 60 and 600 mg/kg bw/day. There was no dose dependence in these alterations therefore these findings were considered to have no toxicological meaning and to be indicative of biological variation. Moreover, all of the stillborn pups/dose belonged to one dam in both of these doses and this kind of intrauterine mortality also occurs in untreated experimental rats.

Effect levels (P0)

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Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other: no adverse effects observed at highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse clinical signs in the offspring between postnatal days 0 and 13. The percentage of offspring with signs (cold, not suckled) at 600 mg/kg bw/day was higher than in the control group. However, these findings were not considered to be toxicologically relevant as these were transient i.e. the signs were detected mainly after the delivery. Occasionally, other clinical signs were also noted for some pups: smaller than normal (1 % in control, 2 % in low dose, 4 % in mid dose, 1 % in high dose), thin (1 % in mid dose), cyanotic skin (1 % in mid dose), pale (3 % at mid dose) and thickened, red tail (1 % in mid dose), which were not considered to have toxicological meaning because of the sporadic occurrence and lack of dose dependency.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality. The mean number of dead offspring per litter was slightly higher with respect to the control at 600 mg/kg bw/day on post-natal day 0 and at 200 and 600 mg/kg bw/day between postnatal day 0 and 13. However, no statistical significance was attained and there was no dose-response relationship in the mean number of dead pups per litter for the observation period (between post-natal days 0 and 13. Therefore, these slight differences were considered to be toxicologically not relevant.

There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
The mean number of liveborns, the mean number of viable pups per litter on postnatal days 0, 4 and 13 was comparable in the control and in all test item treated groups (60, 200 and 600 mg/kg bw/day). There were no statistically significant differences between the control and test item treated (60, 200 or 600 mg/kg bw/day) groups in the survival indices.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not detected. The mean litter weight and litter weight gain were comparable in the control and in all test item treated groups (60, 200 and 600 mg/kg bw/day) on postnatal days 0, 4 and 13. The mean body weight of pups at 60 mg/kg bw/day slightly exceeded that of the control group on postnatal day 0.
The mean body weight gain of pups at 600 mg/kg bw/day was slightly but statistically significantly lower between postnatal days 0-4. The mean body weight was also statistically significantly lower than in the control group in offspring at 600 mg/kg bw/day on postnatal days 4 and 13. Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the higher male and female pup weight at 60 mg/kg bw/day on postnatal day 4 and for the lower male and female pup weights at 200 and 600 mg/kg bw/day on postnatal day 4. These differences with respect to the control in the body weight of offspring were with minor degree (<10 %) and were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
There were no macroscopic changes in the organs or tissues of stillborn offspring (7/7 at 60 mg/kg bw/day and 7/7 at 600 mg/kg bw/day) subjected to necropsy on postnatal day 0.
The following findings were observed in dead pups at the necropsy:
- missing mandible – 1/1 control on PN0; This malformation occurs with low incidence in untreated experimental rats and has no toxicological relevance in this study but was indicative of biological variations.
- autolyzed visceral organs – 2/7 at 200 mg/kg bw/day on PN0;
- gas filled stomach – 2/7 at 200 mg/kg bw/day on postnatal day 0;
- empty stomach and yellowish, foamy content in the intestines – 1/7 at 200 mg/kg bw/day on PN9;
- partially cannibalized (visceral organs, left limb) – 1/7 at 200 mg/kg bw/day on PN 9;
- gas filled intestines and stomach - 1/7 at 200 mg/kg bw/day on PN12;

There were no macroscopic changes in the organs or tissues of dead pups (5/5) at 600 mg/kg bw/day on postnatal day 0.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance:
The anogenital distances in male or female offspring or nipple retention in male offspring were not affected by the test item at 60, 200 and 600 mg/kg bw/day).
In male pups, statistical significances were observed at the slightly longer mean normalized anogenital distances at 600 mg/kg bw/day, and at the slightly longer mean absolute anogenital distances at 60 mg/kg bw/day. The absolute anogenital distances were slightly but statistically significantly shorter than in the control group in female pups at 200 and 600 mg/kg bw/day.
However, these statistical differences in the absolute or normalized anogenital distance of pups were not considered to be toxicologically relevant because of the slight degree. These minor alterations were in compliance with the minor body weight alterations and were considered to be indicative of biological variations and not related to the test item. Nipples/areoles were not visible in any of the examined male offspring in the control or 60, 200 or 600 mg/kg bw/day groups on postnatal day 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed at the highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this OECD 422 compliant study, Methyl-isobutyl-ketone-peroxide (MIKP) administered at 60, 200 or 600 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats. The development of the F1 offspring was not impaired to post-natal day 13 at any dose level after repeated oral administration of dams. 600 mg/kg bw/day caused clinical signs (male and female animals), reduced body weight and body weight gain (during the entire observation period) and food consumption (on week 1), changes in some red blood cell parameters (red blood cell count, hemoglobin concentration and percentage of reticulocytes) and in spleen weight in male animals. At 60 or 200 mg/kg bw/day, there were no test item related alterations.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 200 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 600 mg/kg bw/day
NOAEL for reproductive performance of male/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 obtain initial information on the toxic potential of Methyl-isobutyl-ketone-peroxide (MIKP) and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 60 mg/kg bw/day, 200 mg/kg bw/day and 600 mg/kg bw/day compared to control animals. Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 60, 200 and 600 mg/kg bw/day doses corresponding to concentrations of 0, 12, 40 and 120 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Methyl-isobutyl-ketone-peroxide (MIKP) concentrations in the dosing formulations varied in the acceptable range between 95 % and 102 % of the nominal values confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 55 days). Females were additionally exposed through the gestation period and up to lactation days 13-17 (altogether for 51-55 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups and in non-pregnant or not delivered females, but not the males these females cohabited with in the low and middle dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. Full histopathology was also performed in one moribund male animal in the control and in two found dead males at 600 mg/kg bw/day.

In addition, kidneys were also processed and examined for one male and one female at 200 mg/kg bw/day due to the necropsy observation (pale, both side). Uterus of one female in the low dose group were also processed and examined histopathologically on the basis of necropsy findings (hydrometra).

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

Mortality

One control male animal was in moribund state and subjected to early necropsy on Day 29 of the study. Severe activity decrease, piloerection, sanguineous hairs around the nose and eye were noted for this animal before the euthanasia. Histological examinations revealed mononuclear cell leukemia (tumor cells in the liver and brain) and ulceration in the stomach as individual lesions causing death of this animal.

One male animal administered with 600 mg/kg bw/day was found dead due to para-gastric treatment on Day 50. Histological examinations revealed fibrinous exudate in the trachea, congestion and diffuse alveolar edema in the lungs as the probable cause of death.

Another male animal administered with 600 mg/kg bw/day was found dead on Day 55. Clinical signs (noisy breathing, dyspnea) and significant weight loss were observed during the preceding days. Histological examinations revealed mild alveolar emphysema and congestion in the lungs, decreased amount of secretum in the prostate and cervical vesicles. With these results of this study, the cause of the death of this animal cannot be determined. A test item effect may be assumed but cannot be substantiated with these findings.

Clinical observation

Test item related clinical signs were observed in male and female animals at 600 mg/kg bw/day (nuzzling up the bedding material and salivation) with short duration after the daily administration. Clinical signs of systemic toxicity related to the test item were not detected at any dose level (60, 200 or 600 mg/kg bw/day) at the weekly detailed clinical observations or at the terminal functional observations.

Body weight and body weight gain

The body weight gain and body weight were reduced in male animals at 600 mg/kg bw/day from week 1 up to the termination of the study.

Food consumption

The daily mean food consumption was transiently reduced in male animals at 600 mg/kg bw/day on week 1 in full compliance with the body weight alterations.

The mean daily food consumption was not affected at 60, 200 or 600 mg/kg bw/day in female animals (during the pre-mating, gestation and lactation periods).

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (60, 200 and 600 mg/kg bw/day).

Reproductive performance

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

Delivery data of dams

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (60, 200 and 600 mg/kg bw/day).

Hematology

Hematological investigations revealed elevated percentage of reticulocytes and lower red blood cell count and hemoglobin concentration in male animals at 600 mg/kg bw/day with respect to their control. These findings in accordance with an increased spleen weights might be indicative of loss or damage of erythrocytes in circulation, which was accompanied by an accelerated erythropoiesis.

Clinical chemistry

Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 60, 200 or 600 mg/kg bw/day).

Serum thyroid hormone

There were no test item related changes in the serum thyroid hormone levels at any dose (parental male animals or PN13 offspring).

Necropsy

Macroscopic alterations related to the effect of the test item were not found in male or female animals at 60, 200 or 600 mg/kg bw/day at the necropsy.

Organ weight

Test item effect was assumed on the weights of liver, kidneys and spleen in male animals administered with 600 mg/kg bw/day.

Elevation of the spleen weights in male animals at 600 mg/kg bw/day along with increased percentage of reticulocytes might refer to increased loss or damage of erythrocytes in the circulation.

Slight changes in the liver and kidney weights might be related to a test item influence on hepatic or renal functions. However the degree of all of these changes was small and there were no supporting histopathological findings.

Histopathology

Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 600 mg/kg bw/day.

There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or in the 600 mg/kg bw/day group.

Offspring

The offspring’s development was not adversely influenced at any dose level.

Conclusion

Under the conditions of the present study, Methyl-isobutyl-ketone-peroxide (MIKP) administered at 60, 200 or 600 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats. The development of the F1 offspring was not impaired to post-natal day 13 at any dose level after repeated oral administration of dams. 600 mg/kg bw/day caused clinical signs (male and female animals), reduced body weight and body weight gain (during the entire observation period) and food consumption (on week 1), changes in some red blood cell parameters (red blood cell count, hemoglobin concentration and percentage of reticulocytes) and in spleen weight in male animals. At 60 or 200 mg/kg bw/day, there were no test item related alterations.

 

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male rats: 200 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 600 mg/kg bw/day

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

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