Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-23 until 2012-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 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.

Administration / exposure

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.

Doses / concentrationsopen allclose all
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

Examinations

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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily Observations
Test item 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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

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

Applicant's summary and conclusion

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