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:
2012-02-24 until 2012-04-18
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:
adopted 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid

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: 82 - 87 days
- Weight at study initiation: Male animals: 356– 411 g; Female animals: 196 – 234 g
- Fasting period before study: no
- Housing: 2 or 3 animals sex/cage in Type III polypropylene/polycarbonate cages.
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimatisation period: 22 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
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 4 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. Concentration of the test item in the dosing formulations varied in the range of 97 % to 102 % in comparison to the nominal values at both analytical occasions.

VEHICLE
- Justification for use and choice of vehicle:
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 :
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. 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 99 and 101 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPIB proved to be stable room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 98 % at 500 mg/mL) and at 5 +/- 3°C for 4 days (recovery was 105 % of starting concentration at 1 mg/mL and 98 % at 500 mg/mL).
Duration of treatment / exposure:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 40 or 41 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 1-9 (for 41 – 54 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 41 or 46 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 40 or 41 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 1-9 (for 41 – 54 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 41 or 46 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 (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals/sex in the control and dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting was based on findings obtained in a previous oral repeated dose toxicity study. The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
Positive control:
Not applicable

Examinations

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):
Test item related salivation was observed in male and female animals at 600, 200 or 60 mg/kg bw/day with variable occurrence within a group in a dose related onset and frequency after the daily treatment. Salivation appeared immediately before the daily treatment in some cases at 600 mg/kg bw/day (male and female). 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).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no mortality in parental animals during the course of study (600, 200 or 60 mg/kg bw/day, or control groups).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight and body weight gain was reduced with respect to controls in male animals at 600 mg/kg bw/day. The less body weight gain resulted in a slightly less body weight with respect to controls from day 7 up to the termination of the study.
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 animals at 600 mg/kg bw/day during the premating.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology examinations revealed slightly less number of red blood cell count and higher reticulocyte count in male animals administered with 600 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters.
In male animals, a statistically significant higher mean activity of alanine aminotransferase (ALT), was observed. However, the histopathological findings and the respective organs weights did not indicate a test item effect on the liver. Therefore, this effect was considered to be of no toxicological relevance.
Furthermore, in the male animals, a higher total bilirubin level (TBIL) and less total protein concentration (TPROT) were observed at 600 mg/kg bw/day. Moreover, the chloride (Cl-) concentration was below the control at 60 mg/kg bw/day. Although statistically significant, for these parameters the differences between the control and test item treated groups were small and all values remained within the historical control ranges. Therefore these findings were not considered to be of toxicological relevance.
In female animals, statistical significances were noted for less concentrations of creatinine (CREA) at 600 mg/kg bw/day, and of glucose (GLUC) and bile acids (BAC) at 60 mg/kg bw/day. Also here. these statistical significances indicated small differences between the control and test item treated groups and values were within the historical control ranges. Therefore, these effects were considered to be of no toxicological importance. All other examined clinical chemistry parameters were comparable with the appropriate control value.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related changes in the behavior, physical condition and reactions to different type of stimuli of animals selected for examination. (600, 200 or 60 mg/kg bw/day, control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination did not reveal any toxic or test substance related lesions in the genital and other organs of the experimental animals.
In all investigated male animals the examined organs of reproductive system (testes, epididymides), were histologically normal and characteristic on the sexually mature organism in all cases 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 was normal in all cases as well.
In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma was normal in all cases, as well. The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. In two non-pregnant animals dilatation of uterus was observed (1/1 at 200 mg/kg bw/day and 1/2 at 60 mg/kg bw/day). This finding - without inflammation or other pathological lesion – was considered a physiological phenomenon in connection with the normal sexual cycle.
The histological picture of pituitary was normal as well in the case of male and female treated and control animals.
In animals subjected to full histopathology examinations, some pulmonary alterations were observed in 600 mg/kg bw/day and control groups. More specifically, focal alveolar emphysema was present in minimal or mild degree in 2/5 males at 600 mg/kg bw/day and in 1/5 males and 1/5 females of the control group. Focal haemorrhage occurred sporadically in 2/5 males of the 600 mg/kg bw/day treatment group and in the control group in 2/5 males and 1/5 females. However, these pulmonary changes were considered to be consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguinations and not related to the test item.
The slight dilatation of some tubuli in the kidney of two male animals (2/5) treated with at 600 mg/kg bw/day was considered to be an individual disorder without toxicological significance in the absence of other pathological lesions (degeneration, inflammation and fibrosis).
The hyperplasia of bronchus associated lymphoid tissue (BALT) and the brown-red material in the sinusoidal macrophages in the mesenteric lymph nodes in some control and treated animals was considered a physiological phenomenon.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, kidney small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed.
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test item effect on the reproductive performance was observed.
Variations were noted for less number and percentage of fertile males and pregnant females, dams delivered and pregnant with live borns and higher number and percent of infertile males and non-pregnant females at 600 mg/kg bw/day which reached statistical significance when compared to the control groups. However these variations were within the historical control range. The copulatory index was similar in all groups and no relevant effect was noted on fertility indices.
(Appendices XI/A and XI/B)

Details on results (P0)

Mortality
There was no mortality in parental animals during the course of study (600, 200 or 60 mg/kg bw/day, or control groups).

Clinical Observations
Daily Observations
Test item related salivation was observed in male and female animals at 600, 200 or 60 mg/kg bw/day with variable occurrence within a group and in a dose related onset and frequency after the daily treatment. Some high dose treated male (6/12) and female (8/12) animals already showed salivation sporadically immediately before the treatment. Male animals proved to be slightly 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 for some female animals (1/12 on the abdomen at 600 mg/kg bw/day; 3/12 on the neck, back or on abdomen in the control group) from premating to gestation (1/3 control) or from gestation up to and including lactation days (1/1 at 600 mg/kg bw/day and 2/3 control). Alopecia is a common finding in this strain of experimental rats and was considered to be an individual change. It was present with higher incidence in the control group, 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 and recorded at the weekly observations, too. “Salivation before treatment” appeared also at the handling of animal at the detailed weekly observations.

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

Body Weight
A test item related depression of the body weight and body weight gain was observed in male animals at 600 mg/kg bw/day with respect to controls.
In the male animals dosed with 600 mg/kg bw/day, the mean body weight decreased with statistical significance in comparison to the preceding value during the first two weeks of treatment and the mean body weight gain was significantly less than in the control group during weeks 3, 4 and 6, thus the total body weight gain also remained significantly less with respect to control. The reduced body weight gain resulted in a slightly but statistically significantly less body weight values with respect to controls from day 7 up to termination (days 7, 13, 20, 27, 34 and 39) .
A transiently, slight but statistically ignificant reduction in mean body weight gain was found in male animals at 200 mg/kg bw/day between days 34 and 39. However, as the total body weight gain of this dose group remained comparable to the control group, this effect was not considered to be of toxicological relevance. The body weight gain was significantly higher than the control at 60 mg/kg bw/day between days 27 and 34 and this was considered to be without any toxicological relevance.
There were no biologically significant differences in the mean body weight and body weight gain in female animals between the control and test item treated groups during the entire observation period (premating, gestation and lactation). Statistical significance was noted for a slightly higher body weight gain at 60 mg/kg bw/day between gestation days 7 and 14.

Food Consumption
A test item influence on the mean daily food consumption was observed in male animals at 600 mg/kg bw/day. More specifically, the mean daily food consumption was significantly less than in the control group at 600 mg/kg bw/day during the premating and post mating week 1.
A slight but statistically significant reduction in food consumption in female animals at 600 mg/kg bw/day during week 1 of premating period was not considered toxicologically relevant in female animals as the value did not deviate more than 10% to the value obtained from the respective concurrent control animals.

Hematology
A slightly but statistically significant reduced mean number of red blood cell count (RBC) and related higher reticulocyte count (RET) was noted for male animals administered with 600 mg/kg bw/day and a test item effect cannot be excluded.
In female animals these changes were present in a lesser degree but statistical significance was not reached when compared to the concurrent control values. Therefore the female related changes were not considered to be toxicologically relevant.
The mean white blood cell count (WBC) exceeded significantly the control value due to an extreme high value of one male animal (no.405) only with significantly elevated percentage of neutrophil granulocytes, therefore this was considered to be an individual change and not test item related.
Statistical significances were noted for a reduced mean percentage of monocytes (MONO), eosinophil granulocytes (EOS), higher mean platelet count (PLT) and corpuscular (erythrocyte) volume (MCV) in male animals dosed with 600 mg/kg bw/day. The mean percentage of monocytes in male animals at 200 mg/kg bw/day and mean percentage of eosinophil granulocytes in female animals at 60 mg/kg bw/day showed some statistically significant variations when compared to the concurrent control. However, all these variations (MONO, EOS, PLT MCV) were within low magnitude and remained well within the historical control ranges. Therefore, these variations were not considered to be of biological or toxicological relevance.

Clinical Chemistry
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters.
In male animals, a statistically significant higher mean activity of alanine aminotransferase (ALT), was observed. However, the histopathological findings and the respective organs weights did not indicate a test item effect on the liver. Therefore, this effect was considered to be of no toxicological relevance.
Furthermore, in the male animals, a higher total bilirubin level (TBIL) and less total protein concentration (TPROT) were observed at 600 mg/kg bw/day. Moreover, the chloride (Cl-) concentration was below the control at 60 mg/kg bw/day. Although statistically significant, for these parameters the differences between the control and test item treated groups were small and all values remained within the historical control ranges. Therefore these findings were not considered to be of toxicological relevance.
In female animals, statistical significances were noted for less concentrations of creatinine (CREA) at 600 mg/kg bw/day, and of glucose (GLUC) and bile acids (BAC) at 60 mg/kg bw/day. Also here. these statistical significances indicated small differences between the control and test item treated groups and values were within the historical control ranges. Therefore, these effects were considered to be of no toxicological importance. All other examined clinical chemistry parameters were comparable with the appropriate control value.

Necropsy
Necropsy observation did not reveal any test item related macroscopic findings.
Brownish-red mesenteric lymph nodes were observed in 600 mg/kg bw/day group (11/12 male, 5/8 dam and 2/4 non-pregnant females). Histopathological investigation revealed brown-red material in the sinusoidal macrophages in the mesenteric lymph nodes which also occurred in some control animals and was considered to be a physiological phenomenon.
No macroscopic changes were detected in male animals at 200 and 60 mg/kg bw/day and in the control group.
Alopecia was an individual change in single dam (1/8) treated with 600 mg/kg bw/day and in two control dams (2/11). Alopecia is commonly seen in untreated experimental rats of this strain, therefore was considered to be without any toxicological relevance.
In non-pregnant females, hydrometra was detected at 600 (1/4), 200 (1/2) and at 60 mg/kg bw/day (1/1). Hydrometra is also a species specific finding in relation with the sexual cycle of animals and there were no histopathological findings referring to test item related toxic effects. Therefore these occurrences had no toxicological importance.

Organ Weight
Slightly but statistically significant higher mean weights of spleen and adrenal glands (absolute and relative to body and brain weights) were observed and although no histopathological effect was noted, in cannot be excluded that these findings are related to a test item effect on function of these organs in male animals dosed with 600 mg/kg bw/day.
It has to be highlighted that the fasted body weight was slightly less than in the control group at 600 mg/kg bw/day male animals, consequently, the brain weight relative to body weight was higher and, vice versa, the body weight relative to brain weight was less with respect to control. Statistical significances noted for decreased weights of thymus (absolute), and increased weights of kidney, liver and testes (all relative to body weight) at 600 mg/kg bw/day, were due to the slightly but significantly less fasted body weight with respect to control. These values remained close (kidney) or within (thymus, liver and testes) the ranges of the historical control values and furthermore no significant differences appeared in the respective organ weights relative to brain weight ratios. Thus these findings were not considered to be toxicologically significant.
In female animals treated with 60 mg/kg bw/day, the liver weights (absolute and relative to body and brain weights) were slightly but with statistical significance reduced than in the control. This finding is considered to be without any toxicological relevance as no dose-response relationship could be established.

Histopathology
Histological examination did not reveal any toxic or test substance related lesions in the genital and other organs of the experimental animals.
In all investigated male animals the examined organs of reproductive system (testes, epididymides), were histologically normal and characteristic on the sexually mature organism in all cases 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 was normal in all cases as well.
In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma was normal in all cases, as well. The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. In two non-pregnant animals dilatation of uterus was observed (1/1 at 200 mg/kg bw/day and 1/2 at 60 mg/kg bw/day). This finding - without inflammation or other pathological lesion – was considered a physiological phenomenon in connection with the normal sexual cycle.
The histological picture of pituitary was normal as well in the case of male and female treated and control animals.
In animals subjected to full histopathology examinations, some pulmonary alterations were observed in 600 mg/kg bw/day and control groups. More specifically, focal alveolar emphysema was present in minimal or mild degree in 2/5 males at 600 mg/kg bw/day and in 1/5 males and 1/5 females of the control group. Focal haemorrhage occurred sporadically in 2/5 males of the 600 mg/kg bw/day treatment group and in the control group in 2/5 males and 1/5 females. However, these pulmonary changes were considered to be consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguinations and not related to the test item.
The slight dilatation of some tubuli in the kidney of two male animals (2/5) treated with at 600 mg/kg bw/day was considered to be an individual disorder without toxicological significance in the absence of other pathological lesions (degeneration, inflammation and fibrosis).
The hyperplasia of bronchus associated lymphoid tissue (BALT) and the brown-red material in the sinusoidal macrophages in the mesenteric lymph nodes in some control and treated animals was considered a physiological phenomenon.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, kidney small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed.
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
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:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item effect on the pup’s mortality.
The number and percentage of extra uterine mortality (missing or dead pups) was similar in the control and test item treated groups (600, 200 and 60 mg/kg bw/day) between postnatal days 0 and 4. The survival indices were the same in all groups (600, 200 and 60 mg/kg bw/day and control groups).
Body weight and weight changes:
effects observed, non-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 not detected at any dose level (600, 200 and 60 mg/kg bw/day).
The mean litter weight gain was slightly but with statistical significance reduced when compared to the control at 600 mg/kg bw/day between days 0 and 4, which however did not result in significant differences in the mean litter weight with respect the control. In addition the mean pup weight was comparable in all groups (600, 200 and 60 mg/kg bw/day and control groups). Therefore, this variation was considered to be of no toxicological concern.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination. No signs of skeletal or visceral changes were observed in the offspring during the macroscopic examination.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Mortality
There was no test item effect on the pup’s mortality.
The number and percentage of extra uterine mortality (missing or dead pups) was similar in the control and test item treated groups (600, 200 and 60 mg/kg bw/day) between postnatal days 0 and 4. The survival indices were the same in all groups (600, 200 and 60 mg/kg bw/day and control groups).

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
No test item related clinical signs were observed.

Body Weight
A test item influence on the litter weight and litter weight gain and pup’s mean weight and weight gain were not detected at any dose level (600, 200 and 60 mg/kg bw/day).
The mean litter weight gain was slightly but with statistical significance reduced when compared to the control at 600 mg/kg bw/day between days 0 and 4, which however did not result in significant differences in the mean litter weight with respect the control. In addition the mean pup weight was comparable in all groups (600, 200 and 60 mg/kg bw/day and control groups). Therefore, this variation was considered to be of no toxicological concern.

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

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The reproductive- and developmental-toxic properties of the test item were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:
NOAEL for male rats: 200 mg/kg bw/day; NOAEL for female rats: 600 mg/kg bw/day.
NOE(A)L for reproductive performance of the male and female rats: 600 mg/kg bw/day
NO(A)EL for F1 Offspring:600 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 TBPIB 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 mg/mL, 100 mg/mL and 300 mg/mL 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. TBPIB was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 4 days. Concentration of the test item in the dosing formulations varied in the range of 97 % to 102 % in comparison to the nominal values, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females with living pups, test item was administered through the gestation period and up to lactation days 3 – 8, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. Five dams and males cohabited 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 postpartum and offspring were euthanized. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The reproductive organs and pituitary of non-pregnant and not mated female animals and males cohabited with in the low and mid dose groups were also processed and evaluated histologically.

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

Results

Mortality

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

Clinical observation

Test item related salivation was observed in male and female animals at 600, 200 or 60 mg/kg bw/day with variable occurrence within a group in a dose related onset and frequency after the daily treatment. Salivation appeared immediately before the daily treatment in some cases at 600 mg/kg bw/day (male and female). 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 and body weight gain was reduced with respect to controls in male animals at 600 mg/kg bw/day. The less body weight gain resulted in a slightly less body weight with respect to controls from day 7 up to the termination of the study.

Food consumption

A test item influence on the mean daily food consumption was observed in male animals at 600 mg/kg bw/day during the premating and post mating week 1.

Hematology

Hematology examinations revealed slightly less number of red blood cell count and higher reticulocyte count in male animals administered with 600 mg/kg bw/day.

Clinical chemistry

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

Necropsy

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

Organ weight

Slightly higher mean weights of spleen and adrenal gland (absolute and relative to body and brain weights) were indicative of test item influence on function of these organs in male animals at 600 mg/kg bw/day dose.

Histopathology

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

Conclusion

Under the conditions of the present study, TBPIB caused salivation (male and female), changes in body weight (male), food consumption (male), red blood cell parameters (RBC, RET; male) and in weight of spleen and adrenal glands (male) 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 and 60 mg/kg bw/day, salivation was observed in male and female animals. No indication of a toxic effect on the reproduction performance was noted in this study up to the highest concentration tested. No test item related adverse effect on the offspring development was observed. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:

NO(A)EL for male rats: 200 mg/kg bw/day

NO(A)EL for female rats:600 mg/kg bw/day

NOE(A)L for reproductive performance of the male and female rats: 600 mg/kg bw/day

NO(A)EL for F1 Offspring:600 mg/kg bw/day