Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reprotoxic properties of TBPND 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:   NOEL for male rats: 60 mg/kg bw/day; NOAEL for male rats: 60 mg/kg bw/day; NOAEL for female rats: 60 mg/kg bw/day; NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day; NOAEL for F1 Offspring: 60 mg/kg bw/day.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-23 until 2012-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 86 -91 days old

- Weight at study initiation:
Male animals: 331 - 392 g
Female animals: 190 – 216 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 30 mg/mL, 100 mg/mL and 300 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 3 days before the administration.
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item
- Amount of vehicle (if gavage):
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.

Details on mating procedure:
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL.
Concentration of the test item in the dosing formulations varied in the range of 97 % to 106 % in comparison to the nominal values.
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 41 or 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 1-8 5 (for 42 – 60 days,
depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 43 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.
Frequency of treatment:
Animals were treated once per day.
Details on study schedule:
Male animals were dosed for 41 or 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 1-8 5 (for 42 – 60 days, depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 43 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
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). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Offspring viability indices:
The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily Observations
Test item related salivation was observed in animals of all dosed groups (12/12 male and 12/12 female at 600 mg/kg bw/day; 12/12 male and 12/12 female at 200 mg/kg bw/day and 11/12 male, 5/12 female at 60 mg/kg bw/day) in a dose related manner regarding the degree, onset and frequency. Marked salivation only occurred in the high dose treated animals. The frequency of observation was the highest in 600 mg/kg bw/day group. Male animals proved to be more sensitive than females as number of animals showing salivation and frequency of observation were higher in males than in females at 60 mg/kg bw/day.
Alopecia was noted on the left forelimb for one male animal (1/12) dosed with 60 mg/kg bw/day from day 13 up to the termination; on the left side of the back for one female (1/12) at 60 mg/kg bw/day and on the limbs and abdomen for single control female animal (1/12) during the gestation and lactation period. Alopecia is a common finding in this strain of experimental rats and was present in the control and low dose only therefore had no toxicological meaning in this study.

Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period (pre-mating, mating and post-mating). Alopecia as described above was observed at the weekly observations, too.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item related depression of the body weight development was detected in male and female animals at 600 and 200 mg/kg bw/day with respect to controls.
In the male animals dosed with 600 mg/kg bw/day, the body weight gain was less than in the control group with statistical significances on weeks 1, 2 and 6, thus the total body weight gain remained below control value, too. The reduced body weight gain resulted in a slightly but statistically significantly lower body weight values during the study from day 13 (days 13, 20, 27, 34 and 40) with respect to controls.
In the females, a less body weight gain was observed during the premating and gestation period with respect to control with statistical significances at the summarized body weight gain during the premating, on gestation weeks 2 and 3, and the total body weight gain during the gestation period. The body weight remained below the control value on gestation day 21 and during lactation period (lactation days 0 and 4).
At 200 mg/kg bw/day, in the male animals the mean body weight gain was significantly less than in the control between day 27 and 40 and if summarized (between day 0 and 40).
In the females, significantly less body weight gain was found during the premating period (no statistical significance on week 1) and the mean body weight was also lower than in the control on lactation days 0 and 4.
The body weight development was slightly less than in the control in male animals at 60 mg/kg bw/day between days 27 and 34 however there was no significant difference in the body weight and in the mean summarized body weight gain comparing to the control.
In the females, the mean body weight and body weight gain was similar to that of the control group during the entire observation period (premating, gestation and lactation)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A test item influence on the mean daily food consumption was observed in male and female animals at 600 and 200 mg/kg bw/day. The statistical significances indicated only slight differences between the control and 200 mg/kg bw/day groups and were not considered to be biologically significant.
The mean daily food consumption was lower than in the control group at 600 and 200 mg/kg bw/day doses, during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.
In male animals, the higher mean food consumption at 600 mg/kg bw/day between days 27 and 34 and the less mean food consumption at 60 mg/kg bw/day between days 7 and 13 were not judged to be toxicologically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the examined hematological parameters in male animals at any dose level. Statistically significant differences between the control and dosed groups in some hematological parameters (neutrophil granulocytes (NEU), lymphocytes (LYM), monocytes (MONO), eosinophil granulocytes (EOS) and activated partial thromboplastin time (APTT) were not considered toxicologically relevant as these were with low magnitude and values remained well within the historical control ranges.
In the female animals, the hemoglobin concentration (HGB) and hematocrit vale (HCT) were slightly below the control value at 600 mg/kg bw/day and the percentage of reticulocytes (RET) was higher than in the control group at 600, 200 and 60 mg/kg bw/day (latter without statistical significance).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and concentration of creatinine and urea were also indicative of the test item effect.
The statistically significant differences in levels of some parameters were judged to be of little or no biological significance as the mean values were within or marginal to the historical control ranges (inorganic phosphorous, potassium, alkaline phosphatase) or these were without any dose-response relationship (total bilirubin, calcium, cholesterol and aspartate aminotransferase).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group as compared with the middle or high dose groups.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns, increased extra uterine mortality and higher numbers of dams with prolonged duration of pregnancy.
Although there were no statistically significant differences with respect to control, the mean of post-implantation loss and total intrauterine mortality, mean number of stillborns per litter, number of dams with prolonged pregnancy were higher, and the mean of total births per litter, mean number of live-borns per litter and viable pups per litter, were less than in the control at 600 mg/kg bw/day.
Statistical significances noted for the higher number and percent of implantations and less number and percent of pre-implantation loss were not relevant toxicologically in the 200 and 60 mg/kg bw/day groups and for total intrauterine mortality in the 200 mg/kg bw/day group.
There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance. The copulatory and fertility indices were similar at each dose level.
There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval were similar to or were the same as in the control group in all test item treated groups.
Mortality
There was no test item related mortality during the course of study.

Clinical Observations
Daily Observations
Test item related salivation was observed in animals of all dosed groups (12/12 male and 12/12 female at 600 mg/kg bw/day; 12/12 male and 12/12 female at 200 mg/kg bw/day and 11/12 male, 5/12 female at 60 mg/kg bw/day) in a dose related manner regarding the degree, onset and frequency. Marked salivation only occurred in the high dose treated animals and only slight degree of salivation was noted for the low dose treated animals. The frequency of observation was the highest in 600 mg/kg bw/day group. Male animals proved to be more sensitive than females as number of animals showing salivation and frequency of observation were higher in males than in females at 60 mg/kg bw/day.
Alopecia was noted on the left forelimb for one male animal (1/12) dosed with 60 mg/kg bw/day from day 13 up to the termination; on the left side of the back for one female (1/12) at 60 mg/kg bw/day and on the limbs and abdomen for single control female animal (1/12) during the gestation and lactation period. Alopecia is a common finding in this strain of experimental rats and was present in the control and low dose only therefore had no toxicological meaning in this study. For detail please refer to tables in the attachment.

Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period (pre-mating, mating and post-mating).
Alopecia as described above was observed at detected at the weekly observations, too.

Functional Observations
Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (600, 200 and 60 mg/kg bw/day, control).

Body Weight
A test item related depression of the body weight development was detected in male and female animals at 600 and 200 mg/kg bw/day with respect to controls.
In the male animals dosed with 600 mg/kg bw/day, the body weight gain was less than in the control group with statistical significances on weeks 1, 2 and 6, thus the total body weight gain remained below control value, too. The reduced body weight gain resulted in a slightly but statistically significantly lower body weight values during the study from day 13 (days 13, 20, 27, 34 and 40) with respect to controls.
In the females, a less body weight gain was observed during the premating and gestation period with respect to control with statistical significances at the summarized body weight gain during the premating, on gestation weeks 2 and 3, and the total body weight gain during the gestation period. The body weight remained below the control value on gestation day 21 and during lactation period (lactation days 0 and 4).
At 200 mg/kg bw/day, in the male animals the mean body weight gain was significantly less than in the control between day 27 and 40 and if summarized (between day 0 and 40).
In the females, significantly less body weight gain was found during the premating period (no statistical significance on week 1) and the mean body weight was also lower than in the control on lactation days 0 and 4.
The body weight development was slightly less than in the control in male animals at 60 mg/kg bw/day between days 27 and 34 however there was no significant difference in the body weight and in the mean summarized body weight gain comparing to the control.
In the females, the mean body weight and body weight gain was similar to that of the control group during the entire observation period (premating, gestation and lactation). For detail please refer to tables in the attachment.

Food Consumption
A test item influence on the mean daily food consumption was observed in male and female animals at 600 and 200 mg/kg bw/day. The statistical significances indicated only slight differences between the control and 200 mg/kg bw/day groups and were not considered to be biologically significant. The mean daily food consumption was lower than in the control group at 600 and 200 mg/kg bw/day doses, during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.
In male animals, the higher mean food consumption at 600 mg/kg bw/day between days 27 and 34 and the less mean food consumption at 60 mg/kg bw/day between days 7 and 14 were not judged to be toxicologically significant.

Hematology
There were no test item related changes in the examined hematological parameters in male animals at any dose level. Statistically significant differences between the control and dosed groups in some hematological parameters (neutrophil granulocytes (NEU), lymphocytes (LYM), monocytes (MONO), eosinophil granulocytes (EOS) and activated partial thromboplastin time (APTT) were not considered toxicologically relevant as these were with low magnitude and values remained well within the historical control ranges.
In the female animals, the hemoglobin concentration (HGB) and hematocrit vale (HCT) were slightly below the control value at 600 mg/kg bw/day and the percentage of reticulocytes (RET) was higher than in the control group at 600, 200 and 60 mg/kg bw/day (latter without statistical significance).
For detail please refer to tables in the attachment.

Clinical chemistry
In the male animals dosed with 600 mg/kg bw/day, a slightly higher mean activity of alanine-aminotransferase (ALT) and higher mean concentration of creatinine (CRE), inorganic phosphorous (Pi) and potassium (K+), less activity of aspartate-aminotransferase (AST) and concentration of total bilirubin (TBIL), calcium (Ca2+) were observed. No statistical significance was noted for the mean urea concentrations, although it was higher than the control value and also exceeded the historical control range.
In the female animals of this group, significantly higher activity of alanine-aminotransferase and alkaline phosphatase (ALP), higher concentration of urea and potassium were detected.
At 200 mg/kg bw/day, in male animals a less mean concentration of total bilirubin and potassium, in female animals a higher activity of alanine-aminotransferase and higher concentration of urea and cholesterol (CHOL) was observed.
In group of 60 mg/kg bw/day, a slightly less activity of aspartate-aminotransferase and less concentration of total bilirubin was noted for male animals, while in female there were no statistically significant changes with respect to controls, however the alanine-aminotransferase activity slightly exceeded the control value (no statistical significance).
In summary, the higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and concentration of creatinine and urea were also indicative of the test item effect.
The statistically significant differences in levels of some parameters were judged to be of little or no biological significance as the mean values were within or marginal to the historical control ranges (inorganic phosphorous, potassium, alkaline phosphatase) or these were without any dose-response relationship (total bilirubin, calcium, cholesterol and aspartate aminotransferase). For detail please refer to tables in the attachment.

Necropsy
Enlarged (5/12) and pale (3/12) kidneys reflected a test item influence in male animals dosed with 600 mg/kg bw/day. In one male animal (1/12) pyelectasia occurred.
Hydrometra (indicative of sexual cycle of female animals) is a frequent observation in experimental rats, was present in one high dose treated animal (1/12) and has no toxicological meaning without pathological changes.
Alopecia was an individual change occurring in single animals of 60 mg/kg bw/day (1/12, male, 1/11 dams) and control (1/12) groups. For detail please refer to tables in the attachment.

Organ weight
In the male animals, the liver weights were higher than in the control at 600 mg/kg bw/day (absolute and relative to body and brain weights) and at 200 mg/kg bw/day (relative to body weight). The kidney weights (absolute and relative to body and brain weights) also exceeded the control value at 600 and 200 mg/kg bw/day. The fasted body weight, the thymus weights (absolute and relative to body and brain weights) and adrenal weights (absolute and relative to brain weight) were less than in the control at 600 and 60 mg/kg bw/day, respectively. Spleen (200 mg/kg bw/day) and testes (600 and 200 mg/kg bw/day) weights relative to body weigh were above the control value.
In the female animals, liver weights were higher than the control at 600 and 200 mg/kg bw/day (absolute and relative to body and brain weights) and at 60 mg/kg bw/day (relative to body weight). Statistical significances were also noted for less fasted body weight, body weight relative to brain weight, and brain weight relative to body weight, kidney weights relative to body weight at 600 and 200 mg/kg bw/day and heart weight relative to body weight at 600 mg/kg bw/day.
In summary, a test item related higher liver and kidney weights were found at 600 and 200 mg/kg bw/day with a difference between the genders and in compliance with changes in clinical chemistry parameters. Changes noted for the liver weight were present both in males and females at 600 mg/kg bw/day and in females at 200 mg/kg bw/day. Alterations in kidney weights at 600 and 200 mg/kg bw/day were pronounced in male animals, however it was only present in the kidney weight relative to body weight in females in which the fasted body weight was slightly (but statistically significantly) less with respect to controls, therefore a test item influence in females may be questionable.
The liver weight relative to body weight of female animals administered with 60 mg/kg bw/day was slightly above the control value in compliance with the slight changes of alanine aminotransferase activities.
The lower thymus weights (absolute and relative to body and brain weights) at 600 mg/kg bw/day might be also related to test item.
The significantly higher organ weights of heart, spleen and testes relative to body weight with respect to controls were considered to be independent from the test item, these were probably due to the slightly lower body weight values moreover no supporting findings were found in clinical pathology, necropsy or histopathology observations. For detail please refer to tables in the attachment.

Histopathology
Hyaline-like droplets in the epithelial cells of some proximal convoluted tubules (12/12, 12/12, 11/12), segmental tubular basophilia (10/12, 10/12, 3/12) accompanied with slight inter-tubular lymphocytic infiltration and dilatation of tubuli in the distal area at the border of cortical - medullary region (12/12, 8/12, 1/12) were observed in male animals in all dose groups (600, 200 and 60 mg/kg bw/day, respectively). Minimal alveolar emphysema (2/5, 2/5) and mild hyperplasia of bronchus associated lymphoid tissue (BALT; 0/5, 2/5) were detected in the lungs of examined male animals in 600 mg/kg bw/day and control group, respectively.
The evaluated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all investigated male animals of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. The histological picture of epididymides and pituitary was normal in all cases as well.
Minimal alveolar emphysema (2/5) and mild hyperplasia of bronchus associated lymphoid tissue (1/5) in the lungs and dilatation of uterus (1/12) were detected in female animals of the control group.
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. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation, the epithelial capsule and ovarian stroma were normal in all cases as well.
The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. The histological picture of pituitary was normal as well in the case of treated and control animals.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, 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 (male and female).
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals (male and female). For detail please refer to tables in the attachment.

In summary, test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group as compared with the middle or high dose groups.
Hyaline droplet nephropathy and “hydrocarbon nephropathy” are terms that describe a spectrum of morphologic changes in the kidneys of male rats induced by a variety of structurally related and unrelated compounds. There is abnormal accumulation of α-2µ-globulin phagolysosomes of tubular epithelium. One proposed mechanism suggests that the chemical or a metabolite binds with the α-2µ-globulin - which is only present in the male species - or alters the structure of the protein so that the tubular cell lysosomal enzymes cannot degrade the protein complex. In this case the observed nephropathy is specific to the male rat and has no relevance to humans. Other proposed mechanisms include a direct cytotoxic effect. It is unlikely that the various chemicals associated with hyaline droplet nephropathy in the male rat act by the same mechanism. Some chemicals which produce hyaline droplet nephropathy in male rats produce renal toxicity (unassociated with α-2µ-globulin) in female rats, whereas other chemicals produce no effects in the kidney of female rats.

The focal alveolar emphysema in the lungs occurred sporadically and was considered as consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. The hyperplasia of bronchus associated lymphoid tissue is a physiological phenomenon occurred in some control and treated animals. Uterus dilatation – without inflammation or other pathological lesion – is a physiological phenomenon in connection with the normal sexual cycle.

Delivery Data of Dams
The number and percentage of post-implantation loss and stillborns, duration of pregnancy was higher and the number of live-born pups was less than the appropriate values of control group at 600 mg/kg bw/day. Although there were no statistically significant differences with respect to control, the mean of post-implantation loss and total intrauterine mortality, mean number of stillborns per litter, number of dams with prolonged pregnancy were higher, and the mean of total births per litter, mean number of live-borns per litter and viable pups per litter, were less than in the control at 600 mg/kg bw/day.
Statistical significances noted for the higher number and percent of implantations and less number and percent of pre-implantation loss were not relevant toxicologically in the 200 and 60 mg/kg bw/day groups and for total intrauterine mortality in the 200 mg/kg bw/day group.

Reproductive Performance
There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance. The copulatory and fertility indices were similar at each dose level. Spermatogenesis in the rate male appeared unaffected by the test item.

There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval were similar to or were the same as in the control group in all test item treated groups.
However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns, increased extra uterine mortality and higher numbers of dams with prolonged duration of pregnancy.

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEL
Effect level:
< 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systhemic effects: Rat specific hyaline nephropathy
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related clinical signs did not appear in the pups. The number and percent of cold and not suckled pups were slightly higher than in the control group at 600 and 200 mg/kg bw/day but these were considered to be independent from the treatment as these signs occur in not treated animals with similar percentage.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number and percentage of extra uterine mortality was slightly higher than in the control group in 600 mg/kg bw/day group between postnatal days 0 and 4. The mean number of dead pups was also higher than in the control group. The survival indices were similar between all groups (600, 200 and 60 mg/kg bw/day and control groups).
The higher mean of female viable pups at 60 mg/kg bw/day and litter mean has no toxicological significance.
Body weight and weight changes:
effects observed, treatment-related
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 detected at 600 and 200 mg/kg bw/day.
The mean litter weight was significantly less than in the control group at 600 mg/kg bw/day on postnatal day 4, and the litter weight gain between days 0 and 4 was also less than in the control at 600 and 200 mg/kg bw/day. The mean pup weight on days 0 and 4 as well as the mean weight gain of pups between days 0 and 4 were significantly less than in the control group at 600 and 200 mg/kg bw/day.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Mortality
The number and percentage of extra uterine mortality was slightly higher than in the control group in 600 mg/kg bw/day group between postnatal days 0 and 4. The mean number of dead pups was also higher than in the control group. The survival indices were similar between all groups (600, 200 and 60 mg/kg bw/day and control groups).
The higher mean of female viable pups at 60 mg/kg bw/day and litter mean has no toxicological significance.

Sex Distribution
There were no significant differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4

Clinical Observations
Test item related clinical signs did not appear in the pups. The number and percent of cold and not suckled pups were slightly higher than in the control group at 600 and 200 mg/kg bw/day but these were considered to be independent from the treatment as these signs occur in not treated animals with similar percentage. For detail please refer to tables in the attachment.

Body Weight
A test item influence on the litter weight and litter weight gain and pup’s mean weight and weight gain were detected at 600 and 200 mg/kg bw/day.
The mean litter weight was significantly less than in the control group at 600 mg/kg bw/day on postnatal day 4, and the litter weight gain between days 0 and 4 was also less than in the control at 600 and 200 mg/kg bw/day. The mean pup weight on days 0 and 4 as well as the mean weight gain of pups between days 0 and 4 were significantly less than in the control group at 600 and 200 mg/kg bw/day.
The mean litter weight and mean pup weight were similar in the control and test item treated groups, no test item related changes were found. For detail please refer to tables in the attachment.

Necropsy
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination. More specifically, no signs of a teratogenic effect of the test item were noted and no structural or visceral malformations were observed in the offspring during the macroscopic examination. For detail please refer to tables in the attachment.

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
no
Conclusions:
The reprotoxic properties of TBPND 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:  
NOEL for male rats: < 60 mg/kg bw/day; NOAEL for male rats: 60 mg/kg bw/day; NOAEL for female rats: 60 mg/kg bw/day; NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day; NOAEL for F1 Offspring: 60 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 TBPND 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), 60, 200 and 600 mg/kg bw/day at concentrations of 30, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 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.

The first five dams and males cohabited with 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 – 9 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. The kidneys of all animals were also processed and evaluated histologically due to macroscopic observations at the necropsy. 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 female animal and male cohabited with in the low dose group were also processed and evaluated histologically.  

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

Results

Mortality

There was no test item related mortality at any dose level (600, 200 and 60 mg/kg bw/day).  

Clinical observation

Test item related salivation appeared in male and female animals administered with 600, 200 and 60 mg/kg bw/day groups in all with a dose related degree, onset and incidence. 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 normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).  

Body weight and body weight gain

The body weight gain was reduced with respect to controls in male and female animals at 600 and 200 mg/kg bw/day resulting in a slightly less body weight at 600 mg/kg bw/day in male animals from day 13 up to the termination and in female animals on gestation day 21 and lactation days 0 and 4. The summarized body weight gain also remained below the control value in male animals administered with 600 and 200 mg/kg bw/day and in females at 600 and 200 mg/kg bw/day during the premating and at 600 mg/kg bw/day during the gestation period.  

Food consumption

The mean daily food consumption was less comparing to the control group at 600 and 200 mg/kg bw/day doses during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.  

Hematology

Hematology examinationsrevealed test item related higher percentage of reticulocytes in female animals administered with 600, 200 and 60 mg/kg bw/day and less hemoglobin concentration and hematocrit value at 600 mg/kg bw/day with respect to controls.  

Clinical chemistry

A higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and higher concentration of creatinine and urea were also indicative of the test item effect.  

Necropsy

Test item related renal changes (enlarged and pale kidneys) were observed in male animals dosed with 600 mg/kg bw/day.Specific macroscopic alterations related to the test item were not found in female animals during the necropsy.

Organ weight

Higher kidneys weights of male animals administered with at 600 and 200 mg/kg bw/day and higher liver weights in males at 600 mg/kg bw/day, in females at 600, 200 and 60 mg/kg bw/day reflected a test item influence on renal and hepatic functions in accordance with clinical chemistry and necropsy findings.  

Histopathology

Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated male groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group with respect to the middle or high dose groups. Hyaline droplet nephropathy was associated with interference to α-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans.  

Reproduction  

There were no differences between the control and test item treated groups in the reproductive ability of male and female animals. Spermatogenesis in the rate male appeared unaffected by the test item. However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns and higher numbers of dams with prolonged duration of pregnancy  

Offspring

A test item effect on the offspring development was observed in the higher number and percentage of extra uterine mortality in 600 mg/kg bw/day group between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight and weight gain at 600 and 200 mg/kg bw/day.  

Conclusion

Under the conditions of the present study, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected.  Dam’s delivery was affected by the test item at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed.  At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day.

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

NOEL for male rats: < 60 mg/kg bw/day

NOAEL for male rats: 60 mg/kg bw/day

NOAEL for female rats: 60 mg/kg bw/day  

NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day  

NOAEL for F1 Offspring: 60 mg/kg bw/day

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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:
other:
Justification for type of information:
According to Annex IX, 8.7.3 column 1 of REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT (REACH), an extended one-generation reproductive toxicity study having regard to the likely route of human exposure, shall be proposed for substances within the tonnage band 100 - 1000 tpa, if the 28-day or 90-day study or other available studies on reproduction indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

As discussed in more detail below, there is no evidence of substance-related effects with regard to reproductive toxicity as demonstrated in the available Combined Repeated Dose Toxicity Study with Reproduction/Developmental Screening Test (OECD 422), the Prenatal Developmental Toxicity study (OECD 414), as well as the Repeated Dose 90-Day Toxicity study (OECD 408, read across), and the Reproduction/Developmental Toxicity Screening study (OECD 421, read across) in rats.

The OECD 422 study was conducted to provide initial information concerning the toxic potential of TBPND and on its possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as development of the F1 offspring. In this study, four groups of Wistar rats (12 animals/sex) were administered orally (by gavage) once a day with test item concentrations of 60, 200 and 600 mg/kg bw. The control groups received the vehicle only. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 8, i.e. up to the day before the necropsy.
A test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns, increased extra uterine mortality and higher numbers of dams with prolonged duration of pregnancy. Although there were no statistically significant differences with respect to control, the mean of post-implantation loss and total intrauterine mortality, mean number of stillborns per litter, number of dams with prolonged pregnancy were higher, and the mean of total births per litter, mean number of live-borns per litter and viable pups per litter, were less than in the control at 600 mg/kg bw/day. No toxicological relevant findings with regard to offspring were noted at 200 and 600 mg/kg bw/day.
This observed test item related influence on dam’s delivery at the highest dose group (600 mg/kg bw/day) was noted at clear and pronounced maternal toxicity only. Females exhibited marked salivation only after treatment with 600 mg/kg bw/day. Furthermore, the mean daily food consumption was significantly less in comparison to the control group during the first week of premating as well as on gestation weeks 2 and 3. Concomitantly, significantly less body weight gain was observed during the premating and gestation period with respect to control. Statistical significances were noted at the summarized body weight gain during the premating, on gestation weeks 2 and 3, and the total body weight gain during the gestation period. The body weight remained below the control value on gestation day 21 and during lactation period (lactation days 0 and 4). Females also showed higher percentage of reticulocytes and less hemoglobin concentration and hematocrit value with respect to controls. Test item related renal and hepatic changes are reflected by higher liver and kidney weight in accordance with clinical parameters, i.e. higher mean activity of alanine aminotransferase and mean concentrations of urea.
The described prolonged pregnancy, the higher percentage and litter mean of post-implantation loss and stillborns at 600 mg/kg bw/day is a result of an insufficient supply of the fetus caused by the maternal systemic toxicity.
The systemic toxicity observed is partially due to the problems of the dose setting for pregnant and non-pregnant animals in one study. It is well known, that pregnant animals are more sensitive and doses showing only slight effects in non-pregnant animals can have more pronounced effects in dams causing secondary effects in the offspring due to malnutrition.
This phenomenom is generally known for human health: A deficient supply for the mother can lead to placental insufficiency resulting in an inadequate development of the fetus. Effects on dam’s delivery are in line with the observations concerning the offspring development showing higher extra uterine mortality between postnatal days 0 and 4, and a less litter weight/weight gain and pup’s weight/weight gain.
In contrast, no effects were noted on reproductive organs. Spermatogenesis in the male rat was unaffected by the test item. In more detail, the evaluated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all investigated male animals of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. The histological picture of epididymides and pituitary was normal in all cases as well. Regarding the reproductive organs of female rats, 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. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation, the epithelial capsule and ovarian stroma were normal in all cases as well. The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. The histological picture of pituitary was normal as well in the case of treated and control animals. Taken into account that sperm analysis and detailed histopathology of organs of the reproductive system are the most sensitive parameters to detect substances toxic to reproduction, the results clearly indicate that effects on dam’s and progeny are linked to the systemic effects noted in dams.

Further studies are available supporting that the effect on reproduction and development of the progeny is not induced directly by TBPND itself but rather a consequence of pronounced maternal toxicity, if noted at all.

The test item was examined for its possible prenatal developmental toxicity according to the requirements of an OECD 414 Guideline. Therefore, groups of 22, 22 and 25 sperm-positive female Wistar rats were treated with TBPND by oral (gavage) administration daily at three dose levels 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum.
The administration of 200 mg/kg bw/day TBPND to dams resulted in a slightly to moderately reduced body weight gain and food consumption from gestation day 17 onwards. In the same dose group, fetal weight was slightly lower and the incidence of body weight retardation increased moderately, both were considered to be related to the treatment of the dams.
The mean number of implantation, the post-implantation loss as well as sex distribution of fetuses was not influenced by the treatment up to the highest concentration tested (200 mg/kg bw/day) at which maternal toxicity was observed. These results fit well with the results of the OECD 422 study at a test item concentration of 200 mg/kg bw/day and support the statement that there is no concern in relation to reproductive toxicity, if doses without pronounced toxicity are investigated.

Furthermore, the analogue substance TBPIN (CAS 13122-18-4) was administered to Wistar rats at 50, 160 and 400/500 mg/kg bw/day in a Reproductive/developmental Toxicity Screening study (please refer to read-across justification attached to IUCLID section 13). Just as observed in the OECD 422 study conducted with TBPND, the number of pregnant animals with liveborns was less than the control value after administration of 500/400 mg/kg bw/day TBPIN, which is most likely due to the maternal toxicity of TBPIN, i.e. two rats died before the delivery. As a consequence of maternal toxicity, observations of offspring revealed slightly higher mortality, clinical signs and depression of body weight development at 160 and 500/400 mg/kg bw/day. The following findings further support that the effect of TBPIN on reproductive performance is related to maternal toxicity and thus is considered to be an indirect test item effect. The number and percentage of mated and fertile male animals, as well as the copulatory and fertility indices were not affected by the treatment. The number and percentage of fertile males, copulatory and fertility indices were similar in the control and all test item treated groups. Furthermore, histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles and pituitary) did not reveal any toxic or other test item related lesions at 50, 160 or 500/400 mg/kg/bw/day doses.

A Repeated Dose 90-Day Toxicity study (OECD 408) conducted with the structural analogue substance TBPIN is available, taking into account possible effects on spermatogenesis and estrous cycle (please refer to read-across justification attached to IUCLID section 13). TBPIN was administered orally (by gavage) to Wistar rats once a day at doses of 0 (vehicle control), 10, 40 and 160 mg/kg bw/day for 90 days.
As a result, there was no test item related influence on the estrous cycle up to and including the highest dose tested (160 mg/kg bw/day). Sperm analysis did not reveal changes in sperm count, sperm motility as well as sperm morphology in control animals and the high dose group (160 mg/kg bw/day). Thus, there were no reproductive effects reported in this OECD 408 study with the structural analogue substance TBPIN. As already discussed above, no adverse effects on reproductive performance are expected by TBPND and further testing would not be improved the hazard assessment.

A comprehensive set of data is available to cover the endpoint reproductive toxicity using a weight of evidence approach and allows the conclusion that there is no indication or need for an Extended One-Generation Study (OECD 443).
Although a test item related influence on the dam’s delivery and consequently on pups appeared with respect to the controls at the highest dose group (600 mg/kg bw/day) in the OECD 422 study, this effect is associated with clear maternal toxicity and thus considered to be a secondary effect. No effects on pups and dam’s delivery were observed at lower concentrations (60 and 200 mg/kg bw/day) also confirmed by the results of the OECD 414 study. All available studies confirm the lack of fertility effects or effects on reproductive organs even at systemic toxic doses. A link between maternal toxicity and fetal development is also demonstrated in the OECD 414 study as a poor general state (reduced body weight and food consumption) caused lower fetal weights.
Thus, for TBPND there is no concern regarding reproductive toxicity.
This is further supported by the findings with the analogue substance TBPIN which shares besides structural similarities also a comparable toxicity profile. Neither adverse effects on sperm parameters (counts, motility, morphology) and on estrous cycle nor histopathological changes of reproductive organs were observed after repeated treatment with systemic doses of TBPIN in an OECD 408 study or OECD 421 study, respectively. Similar to TBPND, effects on dam’s delivery and consequently to pups were only noted for clear systemic doses.

Under consideration of the data described above, no indication for potential reproductive toxicity are obtained in the extended data package available above and consequently an Extended One-generation Study is not required, also taking animal welfare reasons into account.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 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 TBPND 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), 60, 200 and 600 mg/kg bw/day at concentrations of 30, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 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.

The first five dams and males cohabited with 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 – 9 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. The kidneys of all animals were also processed and evaluated histologically due to macroscopic observations at the necropsy. 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 female animal and male cohabited with in the low dose group were also processed and evaluated histologically.  

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

Results

Mortality

There was no test item related mortality at any dose level (600, 200 and 60 mg/kg bw/day).  

Clinical observation

Test item related salivation appeared in male and female animals administered with 600, 200 and 60 mg/kg bw/day groups in all with a dose related degree, onset and incidence. 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 normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).  

Body weight and body weight gain

The body weight gain was reduced with respect to controls in male and female animals at 600 and 200 mg/kg bw/day resulting in a slightly less body weight at 600 mg/kg bw/day in male animals from day 13 up to the termination and in female animals on gestation day 21 and lactation days 0 and 4. The summarized body weight gain also remained below the control value in male animals administered with 600 and 200 mg/kg bw/day and in females at 600 and 200 mg/kg bw/day during the premating and at 600 mg/kg bw/day during the gestation period.  

Food consumption

The mean daily food consumption was less comparing to the control group at 600 and 200 mg/kg bw/day doses during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.  

Hematology

Hematology examinationsrevealed test item related higher percentage of reticulocytes in female animals administered with 600, 200 and 60 mg/kg bw/day and less hemoglobin concentration and hematocrit value at 600 mg/kg bw/day with respect to controls.  

Clinical chemistry

A higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and higher concentration of creatinine and urea were also indicative of the test item effect.  

Necropsy

Test item related renal changes (enlarged and pale kidneys) were observed in male animals dosed with 600 mg/kg bw/day.Specific macroscopic alterations related to the test item were not found in female animals during the necropsy.

Organ weight

Higher kidneys weights of male animals administered with at 600 and 200 mg/kg bw/day and higher liver weights in males at 600 mg/kg bw/day, in females at 600, 200 and 60 mg/kg bw/day reflected a test item influence on renal and hepatic functions in accordance with clinical chemistry and necropsy findings.  

Histopathology

Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated male groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group with respect to the middle or high dose groups. Hyaline droplet nephropathy was associated with interference to α-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans.  

Reproduction  

There were no differences between the control and test item treated groups in the reproductive ability of male and female animals. Spermatogenesis in the rate male appeared unaffected by the test item. However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns and higher numbers of dams with prolonged duration of pregnancy  

Offspring

A test item effect on the offspring development was observed in the higher number and percentage of extra uterine mortality in 600 mg/kg bw/day group between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight and weight gain at 600 and 200 mg/kg bw/day.  

Conclusion

Under the conditions of the present study, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected.  Dam’s deliverywas affected by the test itemat 600 mg/kg bw/dayas the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed.  At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day.

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

NOEL for male rats: < 60 mg/kg bw/day

NOAEL for male rats: 60 mg/kg bw/day

NOAEL for female rats: 60 mg/kg bw/day  

NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day  

NOAEL for F1 Offspring: 60 mg/kg bw/day

OECD 421 with structural analogue substance CAS 13122 -18 -4

A Reproduction/Developmental toxicity screening test with the structural analogue substance tert-Butylperoxy-3,5,5-trimethylhexanoat (CAS 13122 -18 -4) was performed according to OECD 421 (for more details on the read-across approach please refer to the read-across justification attached to IUCLID section 13). The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to rats at repeated doses of 50, 160 and 500/400 mg/kg bw/day compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonad function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses. In this study, 12 animals/sex/dose were involved with exception of the 500/400 mg/kg bw group (13 males) and the 160 mg/kg bw/day group (13 females). All animals of the P generation were treated prior to mating (14 days) and throughout mating (16 days). Male animals were treated 17 days post mating. For females, treatment was continued through the gestation period and up to lactation day 3 or shortly thereafter, i.e. up to the day before the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, oestrous cycle, mating, and pregnancy and delivery process.

Tert-Butylperoxy-3,5,5-trimethylhexanoat caused death of pregnant Hsd.Brl.HAn: Wistar rats at 160 and 400 mg/kg bw/day at the end of pregnancy and at early lactation due to acute-subacute tubular damage in the kidneys and related pulmonary alterations.

Reproductive performance of males was unaffected by treatment. Reproductive performance of female animals was affected by the treatment at 160 and 400 mg/kg bw/day through the parturition and nourishing and limited to the systemic toxicity at those doses. In the F1 generation, the higher mortality, clinical signs and depressed body weight development was considered to be the consequence of maternal toxicity at 160 and 400 mg/kg bw/day. Effects on reproduction and/or developmental of pups were thus only at doses pronounced in concentration with maternal toxicity and are in conclusion not regarded as relevant or indication for a specific hazard.

The no observed adverse effect level (NOAEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for parental effects was 50 mg/kg bw /day. For reproduction parameters, the NOAELs were 400 mg/kg bw/day for male rats and 50 mg/kg bw/day for female rats. For F1 offspring, the NOAEL was 50 mg/kg bw/day.


Effects on developmental toxicity

Description of key information

The test substance was examined for its possible prenatal developmental toxicity. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day NOAEL (developmental toxicity): 60mg/kg bw/day NOAEL (teratogenicity): 200 mg/kg bw/day (high dose)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-11 to 2016-09-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: Young adult and nulliparous females, 11-12 weeks of age at start of the mating period
Males: experienced males, 38-39 weeks of age at start of the mating period
- Fasting period before study: None
- Housing: Before mating: 1-3 females per cage 1-2 males per cage
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: Animals will receive 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 500 mL bottle ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 10 mg/mL, 30 mg/mL and 100 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before administration and were stored in the refrigerator (5 +/- 3°C) until application.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil (Helianthii annui oleum raffinatum), The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of the nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively.
- Lot/batch no.: 1506-4604
- Purity: 98 and 102 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility at least twice during the study.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL). A separate analytical report provided this information.
Details on mating procedure:
- Impregnation procedure: The females will be 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 achieves at least twenty two.
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male to three females
- Length of cohabitation: two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19. 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.
Frequency of treatment:
Daily
Duration of test:
The sperm positive females were treated from gestational day 5 to 19. Gross pathology afterwards.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
10 mg/mL
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
30 mg/mL
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
100 mg/mL
No. of animals per sex per dose:
80 males, 130 females to achieve at least 22 sperm positive females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in a GLP Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422). 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.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured at least once in the pre-mating period, but will not be statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All sperm positive females were sacrificed by decapitation after anesthetizing with Isofluranum on day 20 of gestation.
- Organs examined: The abdomen was opened, the uterus with cervix and the left ovary was removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.

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:
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) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Indices:
Pre-implanation 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
Historical control data:
Findings were compared to historical control data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related clinical observations. Alopecia and small wound on the skin were observed with a low incidence without a relationship to the test item. Piloerection was recorded in one dam in the high dose group on the day before Caesarean section. Considering that this occurred only in one female, this sign was not attributed to the treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no significant differences in the body weight values among the dose groups. Statistically significantly, slight to moderate lower body weight gain was indicated in the 60 (-11%) and 200 (-24%) mg/kg bw/day groups for the days between 17 and 20 (p<0.05) and in the 200 mg/kg bw/day group for between days 0 to 20 (-12%). The differences in the body weight parameters in the 200 mg/kg bw/day dose group were considered as treatment-related as there was also a statistically significant (p<0.05), moderate reduction of the corrected body weight gain as most relevant weight parameter in this group. The slightly lower body weight gain in the 60 mg/kg bw/day group was considered as non-adverse as no other weight parameter was affected accordingly.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight but statistically significant reduction of the food consumption was observed (p<0.05, -8%) between gestation days 17 and 20 in the 200 mg/kg bw/day dose group. Considering that this reduction correlated with a moderately lower body weight- and corrected body weight gain, a relationship to the treatment was considered as likely. There were no significant differences indicated in the food consumption of the animals in the 20 and 60 mg/kg bw/day groups relative to the control.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related macroscopic alterations recorded. At necropsy the stomach was found to be empty in case of three dams in the 200 mg/kg bw/day group. Also an empty stomach except some bedding was recorded for two dams in the 60 mg/kg bw/day group. These females were considered to have no macroscopic alterations. There was one female in the 200 mg/kg bw/day group where pin-prick sized dark coloured points were to see in the glandular stomach. Clotted bloody stomach content with intact stomach surface was observed in one dam in the 20 mg/kg bw/day dose group. These findings were judged not to be in relationship with the treatment considering the isolated occurrence .
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect related to the administration of the test item in the mean percent of pre-implantation loss, early embryonic, late- and fetal death, the mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses in the test item treated groups. Moreover the preimplantation loss as non-relevant finding was statistically significantly lower in the 200 mg/kg bw/day group. Statistically significantly (p<0.05) higher mean number of early embryonic death in all test item treated groups as well as postimplantation loss in the 60 mg/kg bw/day group and in the 200 mg/kg bw/day group both without a dose response-relationship considering the mean percentage values was indicated only by the Chi square test and not in case the mean percentage was calculated. Therefore, the observed differences were finally judged to be not related to the treatment.
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of sperm positive females was 25 in the control and high dose as well as 22 both in the low and mid dose groups. There were two non- pregnant females each in the control and 200 mg/kg bw/day and five in the 20 mg/kg bw/day group. In the 60 mg/kg bw/day dose group all females were pregnant. Two females which had less than 3 implantations (one each in the control and 200 mg/kg bw/day group) were excluded from the data evaluation. Data of one litter (with one fetus) were excluded from the data evaluation in the 200 mg/kg bw/day dose group. In total, on gestation day 20 there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 20, 60 and 200 mg/kg bw/day groups respectively.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the fetuses and absolute placental weight (but not the most relevant relative placental weight) was slightly but statistically significantly (p<0.05 to (p<0.01) lower in the 200 mg/kg bw/day group compared to the control. The reduction of the mean fetal weights was judged to be in relationship with the treatment. In contrast, the observation regarding the reduced mean absolute placental weight was considered as not relevant as the most relevant relative placental weight due to its relation to body weight was not affected and moreover, the mean relative placental weights were slightly higher, although without statistically significance, than the respective, control values.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
The number of evaluated fetuses was 253, 198, 225 and 253 in the control, 20, 60 and 200 mg/kg bw/day groups, respectively.

Malformations
One fetus was found with a malformation (umbilical hernia) at external examination in the 200 mg/kg bw/day group. According to the experience with this species in this laboratory, umbilical hernia occurs sporadically without a relationship to the treatment. This is in line with historical control data of other Wistar rats (Crl:WI(Han), Ginkis and Clifford, 2009) and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993). Both are known to have low incidences of umbilical hernia as spontaneous finding. Consequently, this single malformation in the high dose group in this study was judged to be incidental.

Variations
Body weight retardation (below 3.03 g for males and 2.79 g for females) was evaluated as an external variation. The incidence of body weight retarded fetuses increased statistically significantly (p<0.01) in the 200 mg/kg bw/day dose group which was considered to be in a relationship with the treatment of the dams.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 126, 99, 113 and 128 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of fetuses with abnormalities among the experimental groups.

Malformations:
Malformations were found only in single cases and distributed among allthe experimental groups. Slightly split or split xiphoid process was recorded for one fetus both in the control and 200 mg/kg bw/day group. Split sternum, bent scapula in two separate fetuses in the 20, dumb-bell shaped cartilage of thoracic vertebral centrum in the 60 mg/kg bw/day dose group and a hemicentric thoracic centrum in one control fetus were considered to be without any relationship to the treatment of the dams with the test item.

Variations:
Incompletely ossified skull bones, bipartite supra occipital, unossified hyoid, incompletely or not ossified, misaligned or bipartite sternebra, wavy, interrupted ribs, shorter 13th rib, neck rib anlage, bipartite (with or without asymmetric ossification), incomplete ossification or unossified vertebral arches, incompletely ossified pubic bones, asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination. Slightly but statistically significantly (p<0.05) increased incidence if only 3 or less sternebra were ossified in the high dose group.
Opposed to this the incidence of fetuses with incomplete ossification of the skull bones which was statistically significantly lower in the 200 mg/kg bw/day group as well as the occurrence of wavy ribs was statistically significantly lower in the 60 and 200 mg/kg bw/day groups. In summary the slight increases and decreases in the incidence of skeletal variations were judged to be not related to the treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 127, 99, 112 and 125 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of variations and malformations among the experimental groups.

Malformations:
Besides the umbilical hernia discussed above at the section external examination, an isolated case of unilateral microphthalmia was found in one fetus in the high dose group. According to the background database of Toxi-Coop Zrt. (Appendix XXIV/A and B) this finding may occur sporadically in control fetuses. Considering the background data and that it was a single fetus with this abnormality in this study, this isolated observation was judged to be not related to the test item. Hydronephrosis was recorded for one fetus in the 20 mg/kg bw/day dose group and none in the higher dose groups hence was judged to be incidental.

Variations:
Hydroureter as variation was found in all groups including control. In addition dilated renal pelvis was found in one single fetus in the high dose group. Considering the low incidence and lack of dose response both variations were found to be without a test item response
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
NOAEL
Remarks:
Teratogenicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Only slightly lower fetal weight was observed at 200 mg/kg bw/day
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows:
NOAEL (maternal toxicity): 60 mg/kg bw/day
NOAEL (developmental toxicity): 60 mg/kg bw/day
NOAEL (teratogenicity): 200 mg/kg bw/day (high dose)
Executive summary:

The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 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 sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % 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. Caesarean section and gross pathology were performed on gestational day 20. 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, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 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

The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 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 sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % 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. Caesarean section and gross pathology were performed on gestational day 20. 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, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).

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 reproduction and developmental toxicity screening test the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.