Ditetradecyl peroxydicarbonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproduction and developmental toxicity effects of ditetradecyl peroxydicarbonate following oral administration were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:  
NOAEL for reproductive performance of the male and female rats: 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-04 until 2012-08-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 , before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females and males after mating 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 (°C): 22 ± 3
- 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 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.

Details on mating procedure:

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was analyzed using reverse phase HPLC method with UV detection.
The suitability of the chosen vehicle for the test item 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.

Details on study schedule:

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.

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

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

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

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

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

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

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

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.

Oestrous cyclicity (parental animals):

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. One pair was changed within the control group and within the high dose group after 14 day unsuccessful pairing.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

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

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

HISTOPATHOLOGY:
Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histological examination of kidneys was also performed in all animals because test item related changes were observed in the high dose treated male animals.

Postmortem examinations (offspring):

GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)

Statistics:

The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).

Reproductive indices:

The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Offspring viability indices:

The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

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.

Dermal irritation (if dermal study):

not examined

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:

not examined

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:

effects observed, non-treatment-related

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

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 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

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related differences between the control and dosed groups in the delivery data of dams.
Statistical significance was detected for the slightly less mean number of corpora lutea at 1000 and 300 mg/kg bw/day and for the slightly less percentage of viable pups at 100 mg/kg bw/day. However these differences were small and no statistically significant differences were observed for the percentage values of viable pubs in the higher 1000 and 300 mg/kg bw/day dose groups. Therefore, these findings were considered to be without toxicological significance and to be indicative of a normal reproductive function.

No test item effect on the reproductive performance was observed.
The percentage of fertile male and female animals was slightly less and the percentage of infertile male and female animals was slightly higher with respect to controls at 300 and 100 mg/kg bw/day, however it was not considered to be related to test item influence as there were no similar findings at 1000 mg/kg bw/day and these variations were within the historical control range.

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: No adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical signs were observed. The slightly higher percent of cold or not sucked pups at 1000 and 100 mg/kg bw/day was due to the insufficient nursing by some mothers (1/12 and 2/10, respectively).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item effect on the pup’s mortality.
The survival index was slightly less at 100 mg/kg bw/day with respect to control.
The mortality was slightly higher than in the control group at 1000 mg/kg bw/day between lactation days 0 and 4 and at 100 mg/kg bw/day on lactation day 0 and between lactation days 0 and 4. The higher mortality was originated from the inadequate nursing instinct of one dam at 1000 mg/kg bw/day (No. 429) and of two ones at 100 mg/kg bw/day (Nos. 221 and 230), which was considered to be individual variations and was not related to the test item effect.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A test item influence on the litter weight and litter weight gain and pup’s mean weight and weight gain were not detected at any dose level (1000, 300 and 100 mg/kg bw/day).
The mean litter weight gain was slightly but with statistical significance reduced when compared to the control at 1000 and 100 mg/kg bw/day between days 0 and 4, which however did not result in significant differences in the mean litter weight with respect the control. In addition the mean pup weight was comparable in all groups (1000, 300 and 100 mg/kg bw/day and control groups). Therefore, this variation was considered to be of no toxicological concern.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There was no test item related significant differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4.
Statistical significances noted for less mean and percentage of male offspring and for higher mean and percentage of female offspring (liveborns, viable pups on postnatal days 0 and 4) at 1000 mg/kg bw/day were due to the unusual sex distribution of control group. Collected historical control data indicates a sex distribution of 50 % male and 50 % female offspring. In this study, a higher percent of male pups (approximately 60 %) was found along with a less percentage of female pups (approximately 40 %) in the control group and vice versa at the 1000 mg/kg bw/day (approximately 40 % of male and 60 % of female pups.) The percentage of male live born pups was slightly below the control value but within the historical control ranges at 1000 mg/kg bw/day. These statistically significant differences were indicative of normal biological variations and were not considered to be related to the test item effect.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic alterations were found in offspring subjected to gross pathological examination. No signs of skeletal or visceral changes were observed in the offspring during the macroscopic examination.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The reproduction and developmental toxicity effects of ditetradecyl peroxydicarbonate following oral administration were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:  
NOAEL for reproductive performance of the male and female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 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.

Reproduction

There was no test item effect on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition).

Offspring

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

Conclusion

Under the conditions of the present study, 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 Levels (NO(A)EL) were determined as follows:

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

NO(A)EL for F1 Offspring:1000 mg/kg bw/day

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

In accordance with REACH Annex IX Section 8.7.3 Column 1 an extended one-generation reproductive toxicity study does not need to be conducted for the registered substance since none of the available data show any adverse effects on reproductive organs or tissues or reveal any other concerns in relation with reproductive toxicity.


In an OECD 408 compliant 90-day repeated dose toxicity study with the registered substance, there was no test item related mortality at any dose level (1000, 300 or 100 mg/kg bw/day). 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 this study, NOAEL was determined to be 1000 mg/kg bw/d. For more details please refer to IUCLID section 7.5.1

In an OECD 422 compliant study, the registered substance induced no mortalities at any of the dose levels tested (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. 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. There was no test item effect on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition). Negative effects of the test item on offspring development (mortality, clinical signs, body weight, and necropsy findings) were not detected between postnatal days 0 and 4.
In conclusion, under the conditions of this study, the registered substance 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 Levels (NO(A)EL) were determined as follows:
NO(A)EL for reproductive performance of the male and female rats: 1000 mg/kg bw/day
NO(A)EL for F1 Offspring:1000 mg/kg bw/day
For more details please refer to IUCLID section 7.8.1

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline and GLP compliant study. Reliable without restriction.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 422 study:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the 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.

Reproduction

There was no test item effect on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition).

Offspring

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

Conclusion

Under the conditions of the present study, 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 Levels (NO(A)EL) were determined as follows:

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

NO(A)EL for F1 Offspring:1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

Based on the observations in an OECD 414 and GLP compliant study in rats with the test item the following NOAELS were obtained:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-17 to 2019-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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: females: 8 weeks; males: 33-34 weeks
- Weight at study initiation: 169-230 g
- Fasting period before study: no
- Housing: Before mating: 1-3 females per cage 1-2 males per cage; During mating: 1 male and 1-3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (females); 21 weeks (males)

ENVIRONMENTAL CONDITIONS
- Temperature: 19.3 - 23.7 °C
- Humidity: 42-68 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on 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) was performed in the Analytical Laboratory of Test Facility twice during the study. Five samples from different places were taken from each concentration. Similarly, five samples were taken from the vehicle (Control group) and analyzed. 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 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).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 1-3 females/cage
- Length of cohabitation: The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieved at least twenty-five.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in the vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestational day 5 to 19.

Frequency of treatment:

daily, 7 days per week

Duration of test:

14 days

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:

25 sperm positive females per dose

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: The dose setting is based on findings obtained in Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the test item in the Rat. where the NOAEL for the adult animals (including reproductive performance) and offspring was determined as 1000 mg/kg bw/day which is the limit dose according to the OECD 414 test guideline.
- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing
- Cage side observations checked: behavior and general condition, duration and severity of the clinical signs, for signs of morbidity and mortality were made twice daily, at the beginning and end of the working period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
- gross pathology of dams´viscera
- organ weight: Thyroid glands together with the parathyroid glands of all sperm positive females

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females (no implantation, no corpora lutea), total exclusion
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.

Indices:

Pre-implantation loss:
Number of corpora lutea – Number of implantations / Number of corpora lutea * 100

Post-Implanation loss:
Number of Implantations – Number of live fetuses / Number of implantations * 100

Sex distribution:
Number of Male (Female) fetuses / Number of fetuses * 100

External abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses * 100

Visceral abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses examined * 100

Skeletal abnormalities/litter:
Number of fetuses with abnormality / Number of fetuses examined * 100

Clinical signs:

no effects observed

Description (incidence and severity):

None of the females died in the course of the study and there were no test item related clinical signs observed in the females of all dose groups. Soft faeces was observed in all females (including control animals) from gestation day 13 during the whole in-life phase. This was attributed as an effect of the vehicle and did not influence the general condition of the animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight, body weight gain in the different periods as well as corrected body weight of the females was similar in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Minimal (7.5%) but statistically significant decrease of the food consumption was observed in the 1000 mg/kg bw/day group if compared to the control between G.D. 11 and 14. This difference was considered as biologically not relevant considering the minimal manner and single incidence. Moreover the slightly reduced food consumption did not result in a reduced body weight development.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid weight: There were no significant differences in the thyroid weights among the experimental groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dilated renal pelvis were found in 3 females in the control group, two both in the 100 and 300 mg/kg bw/day groups and none in the high dose. This finding was not attributed to the treatment considering the lack of dose response. Diaphragmatic hernia as a developmental disorder (hence not related to the treatment) was observed in one female in the 300 mg/kg bw/day group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were not lesions observed upon histological examinations in the thyroid tissue.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The free thyroid hormone levels (FT3 and FT4) were not affected by the treatment. TSH levels were all below the level of detection, including the values of the control animals, and could thus not be further evaluated.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no increase indicated in the mean percent of pre- and post-implantation loss (early embryonic, late- and fetal death).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related differences in the body weight of the fetuses and placental weight.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable fetuses was similar in all dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses was similar in all dose groups.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Malformations
There were no malformations found in the test item treated groups.
Exencephaly was observed in one fetus in the control group.

Variations
Body weight retardation of the fetuses (below 2.39 g for males and 2.32 g for females) as well as paleness were evaluated as external variations. There was a statistically significant increased incidence of body weigh retardation indicated in the 100 mg/kg bw/day dose group. Considering that there was no dose response observed, this was not attributed to an effect of the test item. Further, there were two litters which were extraordinarily affected (dam#1238134 (6/7 pups retarded) and dam#1137192 (6/12 pups retarded)) which considerably affected the mean value evaluation, but should rather be regarded as biological variation.
Paleness was recorded for one single fetus in the 300 mg/kg bw/day group which was judged as incidental and not to be in relationship with the test item.

Placentas
There were no placental alterations recorded.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations:
The number of the affected fetuses was 1 each in the control and high dose group.
The parietal and frontal bones missed in the control fetus (the same as with exencephaly according to the external examination No.: 1559111/11). Also this fetus had a hemicentric ossification of a thoracic vertebra. The malformed fetus in the high dose group had the same vertebral abnormality. Considering that only a single malformation was found in the high dose and the same occurred in the control group, this finding was not attributed to the test item.

Variations:
Incompletely or not ossified skull bones, bipartite supra-occipital, not ossified hyoid, incompletely ossified sternum or bipartite and/or asymmetrically ossified sternebra, wavy ribs, bipartite or/and asymmetrically or incompletely ossified vertebral centra, incompletely or not ossified lumbar centra or arches, or dumb-bell shaped ossification of lumbar centra, asymmetric ossification or lack of ossification of sacral arches, unossified pubic bone or ischii, and asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination.
There were no significant differences in the incidence of the different type of skeletal variations or if summarized.

Visceral malformations:

no effects observed

Description (incidence and severity):

Malformations:
There were no malformations found at visceral examination of the fetuses.

Variations:
Slightly dilated lateral brain ventricle (unilateral) was recorded for one fetus in the control group. Hydroureter were found in all groups except the high dose. Hydroureter accompanied with dilated renal pelvis was observed in the groups with similar incidences. Considering the lack of dose response these variations were not attributed to the treatment.

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment related differences in the absolute or normalized ano-genital distance.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

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

Sex:

male/female

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Please refer to result tables attached.

Conclusions:

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and macroscopic changes could have been revealed at necropsy. The test item did not influence the food intake and body weight development of the maternal animals. Treatment with the test item had no impact on thyroid hormones (FT3 and FT4) levels or weight of thyroid glands. Treatment of the dams with the test item did not result in histological changes of the thyroids.
Treatment with the test item did not reveal any adverse effect on the pre- and post-implantation loss, number of implantations, sex distribution, body and placental weight, ano-genital distance, external, visceral and skeletal development of the fetuses.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Executive summary:

The test item was examined for its possible prenatal developmental toxicity in accordance with OECD TG 414 and under GLP.

Groups of 25 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle PEG 400. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in PEG 400 was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 200 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 93 and 98 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 19, 20, 21 and 21 evaluated litters in the control, 100, 300 and 1000 mg/kg bw/day group respectively. None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed. There were no adverse effects indicated in regards to food consumption or body weight development. Regarding FT3, FT4 and TSH level there was no test item effect indicated.

There was no significant differences in thyroid weights or histopathology among the groups. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. There were no test item related malformations found. The number of litters with malformations was one in the control and one in the high dose group. There was no increase of variations during fetal examinations indicated.

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

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline and GLP compliant study. Reliable without restriction.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414, rat

The test item was examined for its possible prenatal developmental toxicity in accordance with OECD TG 414 and under GLP.

Groups of 25 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle PEG 400. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in PEG 400 was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 200 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 93 and 98 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 19, 20, 21 and 21 evaluated litters in the control, 100, 300 and 1000 mg/kg bw/day group respectively. None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed. There were no adverse effects indicated in regards to food consumption or body weight development. Regarding FT3, FT4 and TSH level there was no test item effect indicated.

There was no significant differences in thyroid weights or histopathology among the groups. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. There were no test item related malformations found. The number of litters with malformations was one in the control and one in the high dose group. There was no increase of variations during fetal examinations indicated.

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

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

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 toxicity to reproduction the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).