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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-04 until 2012-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

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:
solid

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: 104 - 113 weeks old

- Weight at study initiation: Male animals: 351 - 450 g, Female animals: 203 – 248 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
Males after mating and pregnant females were housed 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: 48 days

ENVIRONMENTAL CONDITIONS
- Temperature: 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:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the administration.

VEHICLE
-The test item is not soluble in water therefore PEG-400 was used for preparing formulations appropriate for oral administration. PEG-400 was a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle :
A constant treatment volume of 5 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.
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 at the intended concentrations was verified up front. Ditetradecyl peroxydicarbonate recovery was 105 and 99 % of nominal concentrations at 1 and 200 mg/mL in PEG 400, respectively. Ditetradecyl peroxydicarbonate proved to be stable at room temperature for 24 hours (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL).
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 43 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 3 – 8 (for 44 – 48 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 44 days). Control animals were handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw.
Frequency of treatment:
Animals were treated once per day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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

Observations and examinations performed and frequency:
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.

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.

CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology and clinical chemistry were conducted 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).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 mL 9NC Microtube, 0.106 mol/L, Greiner Bio-One International AG, or equivalent), one for hematology (MiniCollect® EDTA tubes, spray-dried, 0.25 mL, Greiner Bio-One International AG, or equivalent), and the third one (VACUETTE® Serum Tube, 2.5 mL, Greiner Bio-One International AG, or equivalent) to obtain serum samples for clinical chemistry.
Tubes for hematology and coagulation should be filled up to the final volume (marked on the tubes) and at least 1.0 mL blood should be collected, if possible into clinical chemistry tubes.

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.
Sacrifice and pathology:
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.
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).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
increased salivation shortly after test item administration
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Mortality
There was no mortality in parental animals during the course of study (1000, 300 or 100 mg/kg bw/day or control groups).

Clinical Observations

Daily Observations
Test item related salivation was observed in male animals administered with 1000, 300 and 100 mg/kg bw/day with variable frequency within a group but in a dose related manner regarding the degree, incidence and onset (incidence: 9/12, 5/12, 2/12, respectively to doses of 1000, 300 and 100 mg/kg bw/day). Some female animals administered with 1000 mg/kg bw/day salivated shortly after the treatment for few days in the premating period (3/12) and during the gestation period (1/12).
Piloerection and dyspnea were observed in one dam (1/10) at 100 mg/kg bw/day on lactation days 0 and 1 probably due to an elaborated parturition. There were no similar signs in the animals administered with higher doses, therefore these signs were considered to be individual ones and test item effect was not supposed. Alopecia was detected on the abdomen of one control male animal from day 13 up to the end of the treatment, under the ear of one non-pregnant animal at 100 mg/kg bw/day (1/2) from Day 7 to Day 27 and on the forelimbs in one dam administered with 1000 mg/kg bw/day between lactation days 0 and 5. Scars were noted for one control female animal (on the thigh, 1/12) between Days 15 and 18 and for one non-pregnant animal (under the ear, 1/2) from Day 7 to Day 13. Alopecia and scars on the skin are common findings in this strain of experimental rats and were considered to be individual changes. These were present with low incidence in the control, low and high dose groups 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 and wounds on the skin as described above were observed and recorded at the weekly observations, too: alopecia on the abdomen in one male control animal (1/12) on Days 13, 20, 27, 34 and 41, as well as alopecia under the ear in one non-pregnant female animal at 100 mg/kg bw/day (1/12) on days 7, 13, 14 and 21 along with scars on Days 7 and 13.

Functional Observations
Functional observations did not demonstrate any test item related changes in the behavior, physical condition and reactions to different type of stimuli of animals selected for examination. (1000, 300 or 100 mg/kg bw/day, control).

Body Weight
The body weight development was undisturbed in the test item treated animals at 1000, 300 or 100 mg/kg bw/day with respect to controls.
There were no statistically or biologically significant differences between the control and test item treated groups in the mean body weight and body weight gain (male or female animals) during the entire observation period (premating, mating, gestation and lactation).

Food Consumption
The mean daily food consumption was not affected by the treatment with the test item in male or female animals at any dose level.
Statistical significances were not detected in male or female animals at any dose level (1000, 300 or 100 mg/kg bw/day) with respect to appropriate control during the entire observation period (premating, mating, gestation and lactation).

Hematology
Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (1000, 300 or 100 mg/kg bw/day).
Statistical significances were noted in male animals for a slightly higher mean percentage of basophil granulocytes (BASO) at 1000, 300 and 100 mg/kg bw/day and reticulocytes (RET) at 100 mg/kg bw/day. The mean percentage of monocytes (MONO) in female animals at 100 mg/kg bw/day showed some statistically significant variations when compared to the concurrent control. However, all these variations (BASO, RET and MONO) were with low magnitude and remained well within the historical control ranges. Therefore, these variations were not considered to be of biological or toxicological relevance.

Clinical Chemistry
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters.
In male animals administered with 1000 mg/kg bw/day, a statistically significant, higher mean activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) was observed. However, the histopathological findings and the respective organs weights did not indicate a test item effect on the liver. Moreover, when comparing the ALT values for the three dose levels, no clear dose-relationship is observable and, furthermore, the elevated ALT value remained within the historical control ranges. Therefore, this effect was considered to be of no toxicological relevance.
Furthermore, in the male animals, a less concentration of urea, cholesterol (CHOL), calcium (Ca2+) and sodium (Na+) were observed at 1000 mg/kg bw/day. Moreover, the cholesterol, calcium and chloride (Cl-) concentrations were below the control at 300 mg/kg bw/day and a lower calcium level was also noticed at 100 mg/kg bw/day.
In the female animals, statistical significance was noted for lower level of total bilirubin (TBIL) at 1000 and 300 mg/kg bw/day.
For CHOL, Ca2+, Na+ and Cl- the differences between the control and test item treated groups were statistically significant but were small and all values remained within the historical control ranges for these parameters. Therefore these findings were not considered to be of toxicological relevance.
The mean urea concentrations in male animals exceeded the historical control range in all test item treated groups and also in the control group. Therefore, the statistical significance noted for less mean urea concentration in male animals administered with 1000 mg/kg bw/day were considered to be of no toxicological importance.

Necropsy
Necropsy observation did not reveal any test item related macroscopic findings.
One side pyelectasia (1/12) was noted for one male animal (1/12) administered with 1000 mg/kg bw/day and alopecia on the abdomen was detected in one control male animal (1/12). Histopathological examination did not reveal any related degenerative, inflammatory or fibrotic lesion in the kidneys. Both of these observations were considered to be individual disorders commonly seen in untreated experimental rats of this strain therefore were without any toxicological relevance.
Marked hydrometra was observed in one of the non-pregnant female animals (1/2) administered with 100 mg/kg bw/day group. Hydrometra is also a species specific finding in relation with the sexual cycle of animals and there were no histopathological findings referring to test item related toxic effects. Therefore had no toxicological importance.

Organ Weight
The absolute and relative organ weights did not demonstrate any test item related alterations. There were no toxicologically significant differences between the control and test item treated groups (1000, 300 and 100 mg/kg bw/day) in the examined organ weights.
Statistical significance noted for the mean kidneys weight in female animals at 1000 mg/kg bw/day was considered to be without any toxicological relevance. This difference with respect to control was small (-12 %) and the value remained within the historical control ranges. Moreover, the mean kidney weights relative to body and brain weights of this group were comparable to control and there were no related findings in clinical chemistry parameters, necropsy or histopathology examinations.

Histopathology
Histological examination did not reveal any toxic or test item related lesions in the genital or other investigated organs.
The examined organs of reproductive system (testes and epididymides), were histologically normal and characteristic on the sexually mature organism in all animals at 1000, 300 and 100 mg/kg bw/day in the control group (n= 12, 1, 2 and 12). 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. The histological picture of epididymides and pituitary was normal in all cases as well.
In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups (n= 12, 1, 2 and 12 respectively to 1000, 300 and 100 mg/kg bw/day and control). 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 was 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. In one non-pregnant animal, dilatation of uterus was observed (1/2 at 100 mg/kg bw/day). This finding – without inflammation or other pathological lesion – was considered to be a physiological a physiological phenomenon in connection with the normal sexual cycle. The histological picture of pituitary was normal as well in the case of female treated and control animals.
In animals subjected to full histopathology examinations, some pulmonary alterations were observed in 1000 mg/kg bw/day and control groups. More specifically, focal alveolar emphysema was present in minimal degree in 2/5 males and 1/5 females at 1000 mg/kg bw/day and in 2/5 males and 1/5 females of the control group. Focal haemorrhage occurred in 1/5 male and 1/5 female animal in the control group. These pulmonary changes were considered to be consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguinations and not related to the test item.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in one control male animal (1/5) is a physiological phenomenon.
The pyelectasia of one male animal (1/5) treated with 1000 mg/kg bw/day was considered to be an individual disorder without toxicological significance in the absence of other pathological lesions (degeneration, inflammation and fibrosis).
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, kidney small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The subacute toxicity profile of ditetradecyl peroxydicarbonate was assessed following oral administration in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Level (NO(A)EL) was determined as follows: NOAEL for male and female rats: 1000 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 toxic potential of ditetradecyl peroxydicarbonate and on its possible effects 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=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 1000, 300 and 100 mg/kg bw/day at concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL, respectively, corresponding to 5 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Ditetradecyl peroxydicarbonate was stable at room temperature for one day and in a refrigerator (5 ± 3 °C) for 3 days. Concentration of the test item in the dosing formulations varied in the range of 90 % to 110 % in comparison to the nominal values, 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 with living pups, test item was administered through the gestation period and up to lactation days 3 – 8, 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. Five dams and males cohabited were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on days 4 postpartum and offspring were euthanized. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The reproductive organs and pituitary of non-pregnant and not mated female animals and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (PEG 400) only.

Results

Mortality

There was no test item related mortality at any dose level (1000, 300 or 100 mg/kg bw/day).

Clinical observation

Test item related salivation was observed in male animals at 1000, 300 and 100 mg/kg bw/day with variable occurrence within a group in a dose related onset and frequency after the daily treatment. Salivation appeared in less incidence in female animals administered with 1000 mg/kg bw/day. No toxic signs related to the test item were found at the detailed weekly and terminal 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). Individual dermal changes (alopecia and scars on the skin) were observed in single animals in control, 1000 and 100 mg/kg bw/day group, which are common findings in this strain of experimental rats and had no toxicological meaning. Piloerection and dyspnea was noted for one dam at 100 mg/kg bw/day during and one day after the parturition.

Body weight and body weight gain

The body weight development of parental male and female animals was undisturbed in the course of the entire study. No test item-related body weight, or body weight gain changes were observed with respect to controls at any dose level during the pre-mating, mating, post-mating, gestation and lactation periods.

Food consumption

The mean daily food consumption was not influenced by the test item.

Hematology

No test item-related changes were observed in investigated hematology parameters.

Clinical chemistry

Clinical chemistry examinations did not reveal any pathologic changes in the examined parameters.

Necropsy

Specific macroscopic alterations related to the test item were not found during the necropsy.

Organ weight

There were no test item related changes in the examined organ weights.

Histopathology

Histological examination did not detect any toxic or test item related lesions in the genital and other organs of the experimental animals.

Conclusion

Under the conditions of the present study, ditetradecyl peroxydicarbonate caused salivation (male and female animals) following an oral administration at 1000 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 300 and 100 mg/kg bw/day, salivation was observed in male animals. Ditetradecyl peroxydicarbonate did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day.

Based on these observations the No Observed (Adverse) Effect Level (NO(A)EL) were determined as follows:

NO(A)EL for male and female rats: 1000 mg/kg bw/day