Ditetradecyl peroxydicarbonate

Endpoint summary

Description of key information

The subchronic toxicity profile of the test item was assessed following oral administration in a study performed according to OECD Guideline 408 in rats. Based on the observations in this study the No Observed (Adverse) Effect Level (NO(A)EL) was determined as follows:  NOAEL for male and female rats: 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-07-04 until 2012-08-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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

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. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Hsd.Brl.Han:Wist

Sex:

male/female

Details on test animals or test system 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.

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.

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

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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in parental animals during the course of study (1000, 300 or 100 mg/kg bw/day or control groups).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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 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.

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.

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:

other: Apart from increased salivation shortly after administration no test item related effects were observed.

Key result

Critical effects observed:

no

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

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-06-01 until 2012-06-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

yes

Remarks:

14 day dose range finder

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

14 day dose range finder

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, July 2000

Deviations:

yes

Remarks:

14 day dose range finder

GLP compliance:

no

Remarks:

However, the principles of GLP were followed and all data were recorded and retained.

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: 54 - 60 days
- Weight at study initiation: Male animals: 220 – 239 g; Female animals: 127 – 151 g
- Fasting period before study: no
- Housing: 2 or 3 animals sex/cage in Type III polypropylene/polycarbonate cages.
- 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.
- Acclimatisation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 8-12 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in PEG 400, in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than three days before administration.

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, 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.
Analytical control of dosing solutions (control of concentration) was performed once during the study. Samples were taken on study day 4 and measurement was conducted the following day. The measured concentrations were between the 90 % and 110 % range of nominal concentrations.
The suitability of the chosen vehicle for the test item was analytically proven. 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:

14 days

Frequency of treatment:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time (+/- 2 hours). 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 21 days acclimatisation and was continued up to and including the day before necropsy.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose setting was based on findings obtained from a previously performed acute oral toxicity study with the test item in rats (LD50 > 2000 mg/kg).
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.

Positive control:

None

Observations and examinations performed and frequency:

Clinical Examination:
Detailed clinical observations were made on all animals once a day, after treatment, at approximately the same time. 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.

Weight Assessment:
All animals were weighed with an accuracy of 1 g on days 0, 7 and 13. Individual body weight changes were calculated between days 0-7, 7-13 and 0-13, latter for the total body weight gain. Fasted body weight was measured on day of necropsy (day 14).

Food Consumption:
The food consumption was determined with an accuracy of 1 g on days 7 and 13 by reweighing the non-consumed diet.

Clinical Pathology
Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment. Animals were food deprived for approximately 16 hours 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 Greiner Bio-One International AG), one for hematology (MiniCollect® K3EDTA, 0.25 mL, Greiner Bio-One International AG), and the third one (Vacuette® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.

Hematology:
- Time schedule for collection of blood: before sacrifice
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- 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:
- Time schedule for collection of blood: before sacrifice
- Animals fasted: Yes
- How many animals: all
- 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.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Pathology:
Gross necropsy was performed on each animal. Animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® one day after the last treatment. 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.

Organ Weight:
The following organ weights were determined and recorded:
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands; With precision of 0.001g: adrenals, ovaries

Other examinations:

None

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- hematology
- clinical chemistry
- organ weight data
The heterogeneity 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. Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The frequency of clinical symptoms and pathologic findings were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A test item related salivation was observed at 1000 mg/kg bw/day (5/5 male and 5/5 female) and at 300 mg/kg bw/day (4/5 male and 1/5 female) in slight or moderate degree in male animals and in a slight degree in female animals. Male animals proved to be more sensitive as salivation was observed with higher incidence than in females at 300 mg/kg bw/day. The behavior and physical condition were considered to be normal in all male and female animals in 100 mg/kg bw/day and control groups during the entire observation period.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in 1000, 300, 100 mg/kg bw/day or control groups during the course of 14-day treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was undisturbed in male and female animals administered with 1000, 300 and 100 mg/kg bw/day.
There were no differences between the test item treated and control animals in the mean body weight and body weight gain values during the entire observation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not affected by the treatment with the test item at any dose level (1000, 300 and 100 mg/kg bw/day).
The mean daily food consumption was comparable in all groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in the examined hematological parameters in male or female animals at any dose level.
All examined parameters were comparable with control value in male animals at 1000, 300 and 100 mg/kg bw/day. The only statistical significance was noted for the mean hematocrit value (HCT) in female animals dosed with 1000 mg/kg bw/day, however the value remained well within the historical control ranges, therefore it was judged to be without toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item or treatment related changes were detected in the investigated clinical chemistry parameters. The mean activity of aspartate aminotransferase (AST) was slightly less than in the control group in male animals at 1000 mg/kg bw/day and the phosphorous concentration (Pi) was slightly above the control value at 100 mg/kg bw/day. The mean creatinine (CREA) concentration was slightly higher with respect to control in female animals administered with 100 mg/kg bw/day. These statistical differences in AST activity, phosphorous and creatinine concentrations were not considered to be toxicologically meaningful because of the low magnitude and single occurrence, and all these changes were well within the historical control ranges.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item effect was observed relating to the weight of the investigated organs.
There were no toxicologically significant differences in the examined organ weights between the control and test item treated male or female animals at any dose level (1000, 300 and 100 mg/kg bw/day).
Statistical significances were only noted for female animals, at the mean brain weight relative to body weight and, vice versa, the mean body weight relative to the brain weight at 1000 and 100 mg/kg bw/day without any toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No specific macroscopic changes indicative of test item effect were observed in the organs and tissues at any dose levels (1000, 300 and 100 mg/kg bw/day). Necropsy observations revealed pale kidneys (1/5 male) and slight hydrometra (2/5 female) in 1000 mg/kg bw/day group, point-like hemorrhages (1/5 male) and pale kidneys (1/5 male) at 100 mg/kg bw/day and slight hydrometra (1/5 female) in the control group.
Point-like hemorrhages in the lungs, as consequence of euthanasia and exsanguination were only present in one animal of low dose group. Pale kidneys and hydrometra (latter indicative of sexual cycle of female animals) are frequent observations in experimental rats and were considered to be individual alterations irrespective of the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Basis for effect level:

other: 14 day dose range finding study oral

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

no

Conclusions:

Based on this 14 day dose range finder study, the doses for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study) were determined as follows: 100, 300 and 1000 mg/kg bw/day.

Executive summary:

The aim of this 14-Day toxicity study was to obtain first information on the toxic potential of ditetradecyl peroxydicarbonate in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study). The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5 animals/sex/dose) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses applied in concentrations of 20, 60 and 200 mg/mL, corresponding to a dose volume of 5 mL/kg bw for 14 days. Stability and concentration of test item in PEG 400 were analytically confirmed beforehand. Ditetradecyl peroxydicarbonate proved to be stable at room temperature for 24 hours and at 5 ± 3°C for 3 days. Content of formulations administered to animals were between a range of 90 and 110 % of nominal concentrations in the present study. Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (PEG 400) only.

Results

Mortality

No test item related mortality occurred at any dose level (1000, 300 and 100 mg/kg bw/day) during the observation period.

Clinical observations

Test item related salivation appeared in male and female animals dosed with 1000 or 300 mg/kg bw/day.

Body weight and body weight gain

There were no test item-related effects on the body weight and body weight gain.

Food consumption

The mean daily food consumption was similar in the control and test item treated groups (1000, 300 and 100 mg/kg bw/day).

Clinical pathology (Hematology and Clinical chemistry)

There were no test item related changes in the examined hematological an clinical chemistry parameters.

Organ pathology (Necropsy and Organ weight)

Specific macroscopic alterations related to the test item were not found during the necropsy and at the organ weight analysis.

Conclusion

Under the conditions of the present study, ditetradecyl peroxydicarbonate caused salivation in male and female Hsd.Brl.Han:Wistar rats during the course of a consecutive 14-day oral administration at 1000 and 300 mg/kg bw/day. At 100 mg/kg bw/day, no test item or treatment related changes were present.

Based on these observations the doses for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study) were determined as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-17 to 2020-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for toxicity 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 toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Male animals: 41 – 43 days old Female animals: 36 – 38 days old
- Weight at study initiation: Male animals: 181 – 207 g Female animals: 119 – 144 g
- Fasting period before study: no
- Housing: 5 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

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

Details on route of administration:

oral (gavage)

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the administration and stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: The test item was 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.
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL.
- Amount of vehicle: final volume applied: 5 mL/kg bw

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) were performed in the Analytical Laboratory of Test Facility three times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from the vehicle (Group 1) and analyzed.
Concentrations of the test item in the dosing formulations varied between ranges of 91 % and 93 % of nominal concentrations and formulations were homogeneous at each analytical occasion.
The suitability of the chosen vehicle for the test item at the intended concentrations was verified up front. Test item recovery was 105 and 99 % of nominal concentrations at 1 and 200 mg/mL in PEG 400, respectively. The test item proved to be stable in PEG 400 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:

90 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

vehicle control

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:

control and high dose groups:
10 animals/sex
+ 5 animals/sex as satellite groupe for recovery investigations

Mid and low dose:
10 animals/sex

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: The dose setting with 100, 300 and 1000 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study according to OECD 422 with the test item in the rat. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment: All animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups were as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups and cages according to the actual body weight.
- Fasting period before blood sampling for clinical biochemistry: 16 hours
- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first exposure, then once weekly
- parameters examined:
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 (treatment and recovery periods).

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, then twice weekly for four weeks (weeks 1-4) then once weekly (weeks 5-13) during the course of the treatment period and once weekly during the recovery period (weeks 13-17).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- reweighing of food in weekly intervals

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatisation period and at test termination (Day 89)
- Dose groups that were examined: control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatment or at the end of recovery period (at necropsy)
- 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: one day after last treatment or at the end of recovery period (at necropsy)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of exposure (Days 82)
- Dose groups that were examined: all animals of control and high dose; due to no effects observed mid and low dose animals were not examined
- Battery of functions tested: sensory activity / grip strength / motor activity


IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes; full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups.

Other examinations:

Determination of Serum Levels of Thyroid Hormones
Blood samples for measurements of thyroid hormones were collected as it is described in paragraph “Clinical Chemistry”. The quantitative determination of circulating FT3, FT4 and TSH in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immuno-chemistry analyzer.

Examination of Estrous Cycle
The estrous cycle of all female animals was examined during the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy). A vaginal smear was prepared from each female. After drying, smears were stained with 1 % aqueous methylene blue solution and were examined by a light microscope. Type of cycle (regular or irregular), cycle length, number of cycles during the two weeks, number of days in pro-estrous, estrous and diestrous, number of animals with prolonged diestrous, number of animals with prolonged estrous were evaluated.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- thyroid hormones
- sperm parameters
The heterogeneity 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.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse signs of systemic toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily clinical observations.

Treatment period
The behavior and physical condition of control male animals were considered to be normal at 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
Salivation was observed in male (15/15) and in female (13/15) animals administered with 1000 mg/kg bw/day with variable frequency and onset shortly after the daily treatment and with short duration from Day 28 up to the termination of the treatment period. Based on the short time frame of observation, this finding was judged to be test item related but not adverse. Animals in the control and lower dose groups did not salivate therefore salivation was considered to be related to the treatment with the high dose of the test item.
Softer than normal stool was seen in the bedding material in each cages of the control, 100, 300 and 1000 mg/kg bw/day groups during the observation period from Day 4 (male and female). The slight change in the consistency of the feces was also observed in control animals, therefore, it was considered to be related to the vehicle and not adverse as no related findings were detected at the examined parameters.

Recovery period
Clinical signs were not detected in animals of the control or 1000 mg/kg bw/day (male or female) dose group during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female animals – 1/10 at 100 mg/kg bw/day and 1/10 at 300 mg/kg bw/day – were found dead on Day 19. There were no preceding clinical signs referring to weak condition of these animals. Determining the cause of death was not feasible because the vital organs were cannibalized after the death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period.

Treatment period
The mean body weight was similar to their control in male and female animals treated with 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
Some statistically significant difference with respect to the control was detected at the mean body weight gain in male animals at 100 mg/kg bw/day between Days 84 and 89 (higher), at 300 mg/kg bw/day between Days 28 and 35 (higher) and at 1000 mg/kg bw/day between Days 70-77 (lower) and between Days 84 and 89 (higher). These minor changes in the body weight gain had no influence on the mean body weight or on the summarized body weight gain and therefore had no toxicological relevance.
The mean body weight and body weight gain were comparable – i.e. there were no statistically significant differences – in the control and test item treated female animals (100, 300 and 1000 mg/kg bw/day) during the entire treatment period.

Recovery period
The mean body weight and body weight gain was similar to the control in male animals at 1000 mg/kg bw/day during the recovery period.
The mean summarized body weight gain was slightly lower than in the control group in female animals at 1000 mg/kg bw/day during the post-treatment observation period (between Days 90-117). However, the mean body weight of these female animals was similar to the mean body weight of the control animals, therefore the slight changes in the mean body weight gain were toxicologically not relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item influence on the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day during the treatment period.

Treatment period
Statistically significant differences with respect to the control were observed for the slightly higher mean daily food consumption in male animals as follows:
- at 100 mg/kg bw/day on week 1;
- at 300 mg/kg bw/day on weeks 1, 6, 9 and 10;
- at 1000 mg/kg bw/day on weeks 1 and 6;
The mean daily food consumption was comparable with the control in female animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.
The minor differences were considered to be indicative of the biological variation and not related to the test item in the mean food consumption of male animals. The values met well the historical control values and there was no dose relevance.

Recovery period
During the course of the recovery period, the mean daily food consumption of male animals was similar in the control and high dose group.
In the female animals at 1000 mg/kg bw/day, the mean daily food consumption slightly exceeded the control value during the second and third weeks of the post-treatment period. This slight difference was considered to be toxicologically not relevant as similar findings were not detected during the course of the treatment period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 or 1000 mg/kg bw/day).

Treatment period
The examined hematological and blood coagulation parameters were similar to the control in male animals at 100 mg/kg bw/day.
Compared to the respective control group, slight reductions were detected in the mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet count (PLT) and in mean prothrombin time (PT) in male animals at 300 mg/kg bw/day and in the mean platelet count at 1000 mg/kg bw/day.
In the female animals, the mean prothrombin time was slightly but statistically significantly shorter than in the control group at 100 and 1000 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable to the respective value in the control group in female animals at 300 mg/kg bw/day.

Recovery period
The mean red blood cell count (RBC) and hematocrit value (HCT) were slightly lower than in the control group in male animals in 1000 mg/kg bw/day group at the end of the post-treatment period.
In the female animals in 1000 mg/kg bw/day recovery group, statistically significant changes were revealed with respect to the control group at the slightly lower mean percentage of neutrophil granulocytes (NEU), at the slightly higher mean percentage of lymphocytes (LYM) and at the lower mean hematocrit (HCT).
The slight changes in hematology or blood coagulation parameters were judged to have no toxicological relevance as these were with minor degree, not related to doses and values met well historical control (MCH, MCHC, PLT, PT in male animals and NEU, LYM, HCT and PT in female animals).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No adverse test item effect was detected at the evaluation of clinical chemistry parameters for all dose groups (100, 300 and 1000 mg/kg bw/day).

Treatment period
In the male animals treated with 100 mg/kg bw/day, elevated mean concentration of inorganic phosphorous (Pi) and lower mean concentration of low-density lipoproteins (LDL) were revealed in comparison with the control group.
The mean glucose concentration (GLUC) was slightly higher and the mean concentrations mean low-density lipoproteins and potassium (K+) were lower than in the control group in male animals at 300 mg/kg bw/day.
Statistically significant difference with respect to the control was detected at the lower mean concentrations of urea, blood urea nitrogen (BUN), cholesterol (CHOL), high-density lipoproteins (HDL), low-density lipoproteins and potassium and the slightly higher albumin: globulin ratio (A/G) in male animals at 1000 mg/kg bw/day.
The examined clinical chemistry parameters were comparable in the female animals in control and 100 and 300 mg/kg bw/day groups.
The mean activity of aspartate aminotransferase (AST) was higher and the mean concentrations of urea and BUN was lower with respect to the control in the female animals at 1000 mg/kg bw/day groups.

Recovery period
Statistical significance with respect to the control was observed at the slightly lower mean concentrations of urea and blood urea nitrogen in male animals in 1000 mg/kg bw/day group at the end of the recovery period.
In the female animals, the examined clinical chemistry parameters were comparable in the control and 1000 mg/kg bw/day groups at the termination of the recovery period.
The sporadic statistical differences detected at some parameters were considered to be of little or no biological relevance. All these changes were with minor degree and not related to doses (GLUC, Pi, LDL, K+). Slight changes in AST, UREA, BUN, CHOL, HDL and A/G in animals at 1000 mg/kg bw/day were judged to be toxicologically not significant as there were no supporting findings (hematology, histopathology, related biochemical parameters).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery
The functional observation battery did not demonstrate any test item related differences with respect to the controls in the behavior or in reactions to different type of stimuli at 100, 300 or 1000 mg/kg bw/day (male and female). The behavior and reactions to different type of stimuli or manipulations of animals were judged to be normal in the control and all test item treated groups at the end of the treatment period (on Day 82). Softer than normal stool was also detected at the cage-side examinations in each cage (control, 100, 300 and 1000 mg/kg bw/day).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day.

Treatment period
Statistically significant differences with respect to the control were noted for the slightly higher mean epididymides weights (absolute and relative to body weight) in male animals in 1000 mg/kg bw/day dose groups at termination of the treatment period.
In the female animals, the mean weights of the kidneys were slightly lower than in the control group at 1000 mg/kg bw/day.

Recovery period
The weights of the examined organs were similar in the male animals in control and 1000 mg/kg bw/day groups at the end of the recovery period.
In the female animals at 1000 mg/kg bw/day, statistical significances were revealed for the higher mean liver weights (absolute and relative to body and brain weights) and higher mean spleen weights (absolute and relative to body and brain weights) as well as for the slightly higher mean adrenal gland weight relative to brain weight. All these statistically significant differences during treatment and recovery period, with respect to the appropriate control, were of small degree and no related findings were detected at the histopathological examinations. Moreover, the weights of these organs met well – i.e. were well within or marginal to – the historical control in the female animals. The weights of epididymides also were within the historical control range except for four animals at 1000 mg/kg bw/day. However, in the lack of morphological lesions, these minor changes in the epididymides weights had no biological meanings. Therefore, these changes in the organ weights were not considered to be toxicologically relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.

Treatment period
In dead female animal at 100 mg/kg bw/day (1/10), the organs and tissues were cannibalized – except for head (brain, pituitary, eyes, Harderian gland) and spinal column with spinal cord. Therefore, macroscopic observations were not feasible. In dead female animal at 300 mg/kg bw/day (1/10), the heart, abdominal aorta, sternum, skin and mammary gland, kidneys, adrenal glands, urinary bladder, ovaries and uterus with vagina were cannibalized. Remaining organs and tissues were slightly autolyzed and did not show macroscopic changes. In the male animals, one or both sided pyelectasia was observed in each group at the terminal necropsy as follows: 1/10 control; 4/10 at 100 mg/kg bw/day; 2/10 at 300 mg/kg bw/day; 5/10 at 1000 mg/kg bw/day. Lack of seminal fluid was observed in one side of seminal vesicle in one control male animal (1/10). In the surviving female animals, one or both sided pyelectasia (2/10 control) and moderate or marked hydrometra – 1/10 control, 1/9 at 100 mg/kg bw/day, 2/9 at 300 mg/kg bw/day and 5/10 at 1000 mg/kg bw/day – were detected at the terminal necropsy.

Recovery period
In the male animals, both sided pyelectasia was observed in single animals in control (1/5) and in one high dose (1/5) group at the end of the recovery period.
One side pyelectasia (1/5) and moderate or marked hydrometra (2/5) were seen in the control female animals at the termination of the post-treatment observation period. The organs and tissues were judged to be normal in the female animals in 1000 mg/kg bw/day recovery group. These findings are common in untreated experimental rats and were not related to the test item. Lack of seminal fluid was only detected in one control male animal. Hydrometra is indicative of female sexual cycle. Histological findings were in accordance with macroscopic observation in the uterus. Pyelectasia is a common finding in experimental rats of this strain with similar age. Pyelectasia occurred in each group of male animals independently from doses. There were no pathological alterations in the uterus or kidneys at the histopathological examination, therefore these were considered toxicologically not relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histological examination did not reveal test item related adverse lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.
In control group, alveolar emphysema in the lungs (1/10 male and 2/10 female) occurred sporadically and was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in male animals terminally (1/10 control; 1/10 male at 1000 mg/kg bw/day) and in male and female animals at the end of the recovery period (1/5 control male, 2/5 male and 1/5 female at 1000 mg/kg bw/day). BALT is a physiological immunomorphological phenomenon, without toxicological significance. One or both sided pyelectasia was observed in the kidneys in several animals at termination of the treatment (1/10 male and 2/10 female control; 4/4 at 100 mg/kg bw/day; 2/2 at 300 mg/kg bw/day; 5/10 at 1000 mg/kg bw/day) and at the end of the recovery period (1/5 male and 1/5 female control, 1/5 male at 1000 mg/kg bw/day).
Pyelectasia without other histopathological lesions (degeneration, inflammation, fibrosis etc.) is a common slight individual disorder in laboratory rats, without toxicological significance. Histological examination revealed chronic progressive nephropathy (CPN) in two male animals (1/10 control and 1/10 at 1000 mg/kg bw/day) in minimal degree. CPN is an important spontaneous renal disease of the commonly used strains of laboratory rat. In one control male animal, decreased amount of secrete was observed in the seminal vesicle (one side, 1/10), which was considered as a slight functional disorder, without toxicological significance. The dilatation of the uterine horns in the female animals (1/10 control, 5/10 at 1000 mg/kg bw/day at termination and 2/5 control at the end of the recovery period) is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrus phase – of the inner genital organs.
Alveolar emphysema in connection with the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination procedure occurred with similar incidence in the control and treated animals.
Hyperplasia of bronchus associated lymphoid tissue – a physiological immunomorphological phenomenon – was without toxicological significance and was with similar incidence in control and high dose male animals and was only present in control female but not in the high dose females.
Dilatation of uterine horn related to hydrometra is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrous phase – of the inner genital organs. No morphological evidence of acute or subacute test item related injury (degeneration, inflammation, necrosis etc.) of the gastrointestinal tract, the liver, the pancreas, the respiratory system, the cardiovascular system, the urinary system, the lymphoid system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system, the eyes, the lachrymal glands, and the integumentary system, was observed. The structure and the cell morphology of the endocrine glands was the same in the control and treated animals.

Sperm Examinations
The sperm count, morphology and motility of sperm cells were not affected after three months administration of 1000 mg/kg bw/day of the test item.
The mean sperm count, percentage of motile cells and cells with normal morphology was similar in animals of the control and 1000 mg/kg bw/day groups at the termination of dosing period.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Clinical Biochemistry – Serum Levels of Thyroid Hormones
FT3, FT4 and TSH levels were not affected by the test item at 100, 300 or 1000 mg/kg bw/day (male and female) at the end of the treatment.
There were no significant differences with respect to the control in FT3, FT4 or TSH levels at 100 and 1000 mg/kg bw/day (male and female). The FT3 concentration slightly exceeded the control value in male animals at 300 mg/kg bw/day.
Statistical significance with respect to the control was observed in the male animals at 1000 mg/kg bw/day at the slightly higher FT3 level at the end of the recovery period. These slight differences with respect to the control were considered to be indicative of the biological variation and not related to the test item because there were no changes in the animals in the high dose at termination the treatment. The TSH concentrations were below the detection limit in all animals (0, 100, 300 and 1000 mg/kg bw/day), so it was considered to be in the physiological range of this strain, sex and age, as reflected by the historical control data.

Examination of Estrous Cycle
The estrous cycle was similar in the female animals in the control and test item administered groups during the last two weeks of the study. There were no significant differences between the control and test item treated groups in the percentage of female animals with regular or irregular cycles, mean number of cycles or mean cycle length, mean number of days in proestrous, estrous and diestrous or percentage of animals in prolonged diestrous.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested (limit dose)

Key result

Critical effects observed:

no

Please refer to result tables attached.

Conclusions:

Under the conditions of this OECD 408 compliant study the test item did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91-day oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL: 1000 mg/kg bw/day for male and female Han: WIST rats.

Executive summary:

The purpose of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by four weeks recovery period in order to assess persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). The suitability of the chosen vehicle – Polyethylene glycol 400 – for the test item was analytically proven up front. Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted at the end of the treatment. The body weight was determined twice weekly for four weeks (weeks 1-4) then once weekly during the course of the treatment and recovery periods. Food consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in male animals in the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, organs showing macroscopic changes were processed and evaluated histologically in the low and mid dose groups.

The results of the study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

There was no test item related mortality at any dose level (1000, 300 or 100 mg/kg bw/day). Two female animals (1/10 at 100 mg/kg bw/day and 1/10 at 300 mg/kg bw/day) were found dead for unknown reasons on Day 19.

No signs of toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily or detailed weekly clinical observations or during the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Salivation noted for male and female animals at 1000 mg/kg bw/day with different incidence during the treatment period was judged to be not adverse as animals salivated shortly after the daily administration of the test item for a short time period. Softer than normal stool in the bedding material was considered independent from the test item because it was seen in each cage in the control, 100, 300 and 1000 mg/kg bw/day groups from Day 4 up to the termination of the treatment. There were no clinical signs during the recovery period. The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period. The mean body weight was similar to the control in male and female animals at 1000 mg/kg bw/day during the recovery period. The mean daily food consumption was comparable in animals of the control and test item treated groups (100, 300 and 1000 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day).

Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day). No pathologic test item effect was detected at the evaluation of clinical chemistry parameters (100, 300 and 1000 mg/kg bw/day). FT3, FT4 and TSH levels were comparable in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) at the end of the treatment and recovery periods. The concentrations of TSH were below the detection limit in all animals (0, 100, 300 and 1000 mg/kg bw/day) in accordance with the historical control data, so it was considered to be in the physiological range of this strain, sex and age. Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 100, 300 or 1000 mg/kg bw/day. Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day. Histological examination did not reveal severe test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.

In conclusion, based on results of the study, NOAEL was determined to be 1000 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline and GLP compliant study. Reliable without restriction.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 422

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.

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

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. 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 examinations did not reveal any pathologic changes in the examined parameters. Specific macroscopic alterations related to the test item were not found during the necropsy. There were no test item related changes in the examined organ weights. Histological examination did not detect any toxic or test item related lesions in the genital and other organs of the experimental animals.

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

OECD 408

The purpose of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by four weeks recovery period in order to assess persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). The suitability of the chosen vehicle – Polyethylene glycol 400 – for the test item was analytically proven up front. Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted at the end of the treatment. The body weight was determined twice weekly for four weeks (weeks 1-4) then once weekly during the course of the treatment and recovery periods. Food consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in male animals in the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, organs showing macroscopic changes were processed and evaluated histologically in the low and mid dose groups.

The results of the study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

There was no test item related mortality at any dose level (1000, 300 or 100 mg/kg bw/day). Two female animals (1/10 at 100 mg/kg bw/day and 1/10 at 300 mg/kg bw/day) were found dead for unknown reasons on Day 19.

No signs of toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily or detailed weekly clinical observations or during the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Salivation noted for male and female animals at 1000 mg/kg bw/day with different incidence during the treatment period was judged to be not adverse as animals salivated shortly after the daily administration of the test item for a short time period. Softer than normal stool in the bedding material was considered independent from the test item because it was seen in each cage in the control, 100, 300 and 1000 mg/kg bw/day groups from Day 4 up to the termination of the treatment. There were no clinical signs during the recovery period. The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period. The mean body weight was similar to the control in male and female animals at 1000 mg/kg bw/day during the recovery period. The mean daily food consumption was comparable in animals of the control and test item treated groups (100, 300 and 1000 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day).

Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day). No pathologic test item effect was detected at the evaluation of clinical chemistry parameters (100, 300 and 1000 mg/kg bw/day). FT3, FT4 and TSH levels were comparable in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) at the end of the treatment and recovery periods. The concentrations of TSH were below the detection limit in all animals (0, 100, 300 and 1000 mg/kg bw/day) in accordance with the historical control data, so it was considered to be in the physiological range of this strain, sex and age. Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 100, 300 or 1000 mg/kg bw/day. Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day. Histological examination did not reveal severe test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.

In conclusion, based on results of the study, NOAEL was determined to be 1000 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).