Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-29 until 2012-04-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant 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), adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 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:
Male and female animals: 85 -90 days old

- Weight at study initiation:
Male animals: 322 - 383 g
Female animals: 182 – 225 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 25 mg/mL, 75 mg/mL and 155 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:
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. TBPPI was stable at room temperature for 24 hours and in a refrigerator (5 ± 3 °C) for 3 days.
Concentration of the test item in the dosing formulations varied in the range of 98 % to 104 % in comparison to the nominal values, thereby confirming proper dosing.
A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPPI was stable at room temperature for 24 hours (recovery: 98 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, both) and in a refrigerator (5 ± 3 °C) for 3 days (recovery: 105 and 103 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, respectively).
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 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 3-6 (for 42 – 47) 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 42 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 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 3-6(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 42 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 310 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex in the control and dose groups. 7 animals were used as randomization reserve – these served for base level measurements of clinical pathology.
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).
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: survival 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
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
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:
no effects observed

Details on results (P0)

Mortality
There was no mortality in parental animals during the course of study (310, 150 or 50 mg/kg bw/day, or control groups).

Clinical Observations
Daily Observations
Test item related salivation appeared in male and female animals administered with 310 and 150 mg/kg bw/day groups with a dose related onset and incidence. 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 150 mg/kg bw/day.
Salivation was observed in male animals at 310 mg/kg bw/day (12/12) and 150 mg/kg bw/day (11/12) with variable occurrence after the daily treatment from days 4 and 7, respectively.
In female animals, salivation was detected at 310 mg/kg bw/day (12/12 and 6/8) and 150 mg/kg bw/day (4/12 and 3/10) during the premating and gestation period but not in the lactation period.
One dam, no 325 (150 mg/kg bw/day), showed transiently clinical signs on gestation days 20, 21, and on lactation day 0 (piloerection, decreased muscle tone, decreased activity, hunched back, squatting position and yellowish soiled fur around the anus). These were considered to be individual finding as no similar signs were observed in the higher dose group.
Alopecia was noted for some female animals at 310 mg/kg bw/day (3/12) on the fore limbs and hind limbs, thigh, head and neck from premating or gestation up to termination. One non-pregnant female administered with 150 mg/kg bw/day (1/12) also showed alopecia for some days. Alopecia is a common finding in this strain of experimental rats and was considered to be an individual change/alteration with no toxicological meaning in this study. For detail please refer to the table 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 and recorded at the weekly observations, too.

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. (310, 150 and 50 mg/kg bw/day, control).

Body Weight
A test item related depression of the body weight gain was detected with respect to controls at 310 mg/kg bw/day in male animals during the entire study and in female animals during the lactation period.
In the male animals dosed with 310 mg/kg bw/day, the body weight gain was less than in the control group during the entire observation period with statistical significances on weeks 1, 2, 3 and 6, thus the total body weight gain also remained below the control value. The reduced body weight gain resulted in a slightly but statistically significantly lower body weight values with respect to controls from day 20 (days 20, 27, 34 and 41).
A transiently higher mean body weight gain was found in male animal at 150 mg/kg bw/day between days 20 and 27, without toxicological relevance.
The mean body weight gain was less in the female animals dosed with 310 mg/kg bw/day than in the control group during the lactation period (no statistical significance) resulting in a less body weight on lactation day 4 (p < 0.01).
In the females, the mean body weight and body weight gain was similar to that of the control group in all test item treated groups during the premating and gestation periods. For detail please refer to the table in the attachment.

Food Consumption
A test item influence on the mean daily food consumption was observed in male and female animals at 310 and 150 mg/kg bw/day.
The mean daily food consumption was less than in the control group at 310 and 150 mg/kg bw/day doses during the first week of treatment (male), during the premating (female) and at 310 mg/kg bw/day between lactation days 0 and 4. For detail please refer to the table in the attachment.

Hematology
There were no test item related changes in the examined hematological parameters in male or female animals at any dose level (310, 150 and 50 mg/kg bw/day).
Statistically significant differences between the control and dosed groups in some hematological parameters were not considered toxicologically relevant as these were with low magnitude and values remained well within the historical control ranges or the expected dose-response relationship was not observed (less white blood cell count (WBC) in male animals administered with 310 or 50 mg/kg bw/day; lower percentage of neutrophil granulocytes (NEU) at 310 mg/kg bw/day and monocytes (MONO) at 50 mg/kg bw/day and a higher percentage of lymphocytes (LYM) at 310 mg/kg bw/day in the female animals, with respect to controls). For detail please refer to the table in the attachment.

Clinical Chemistry
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters for the male animals.
In the female animals treated with 310 mg/kg bw/day, a slightly reduced glucose concentration may be indicative of a test item related effect however the finding was considered to be of no toxicological concern.
The mean concentration of total bilirubin (TBIL) was slightly less and the mean calcium (Ca2+) levels were higher than in the control group in the male animals dosed with 310 mg/kg bw/day. However, these changes were within the historical control ranges and all other examined clinical chemistry parameters were comparable with the appropriate control value.
In the female animals treated with 310 mg/kg bw/day, the mean creatinine concentration (CREA) was slightly reduced but remained within the historical control ranges. Also the glucose (GLUC) concentrations were slightly but significantly below the value of control and historical control ranges in this treatment group.
One dam (no. 430) administered with 310 mg/kg bw/day showed an extreme high activity of liver enzymes: AST activity was approximately 9 fold of the mean control and ALT reached value of approximately two fold of the mean control. Histopathological examination of liver and kidneys did not reveal any morphological changes related to the elevated enzyme levels. The enzyme activities of other 4 animals in this group were in the normal ranges, therefore this kind of alteration was considered to be an individual one. For detail please refer to the table in the attachment.

Necropsy
Necropsy observation did not reveal any test item related macroscopic findings in male or female animals at any dose level (310, 150 and 50 mg/kg bw/day).
No macroscopic changes were detected in male animals at 310, 150 mg/kg bw/day and in the control group.
Congenital absence of left side kidney and seminal vesicle and compensatory enlargement of the right side kidney, smaller than normal epididymis were observed in one male animal at 50 mg/kg bw/day, which was considered to be an individual alteration commonly occurring also in untreated experimental rats of this strain.
One side pyelectasia was noted for single dam (1/10) at 150 mg/kg bw/day. Pale liver (1/8), smaller than normal thymus (1/8) and alopecia (forelimbs, hind limbs, neck, head and thigh) were observed in dams (3/8) administered with 310 mg/kg bw/day.
In non-pregnant females one side pyelectasia (1/4 at 310 mg/kg bw/day) and hydrometra (1/3 control; 1/2 at 150 mg/kg bw/day) were observed.
Pyelectasia, pale liver and smaller than normal thymus were only seen in single female animals in the high dose group. These alterations are also commonly seen in untreated experimental rats. In this study, there were no histopathological findings referring to toxic effects, therefore these findings had no toxicological importance.
Alopecia was an individual change occurring also commonly in untreated experimental rats of this strain, therefore was considered to be individual changes without any toxicological relevance.

Organ Weight
A test item influence on renal function was observed as mean weights of kidneys (absolute, and relative to body and brain weights) were slightly higher with respect to the controls and above the historical control ranges in male animals at 310 mg/kg bw/day dose. The increases in absolute, relative to body, and brain weights were determined to be 26, 31 and 23%, respectively.
Slightly higher mean weights of kidneys (absolute and relative to body and brain weights) were observed in male animals at 310 mg/kg bw/day dose. Minor changes were also noted for the kidney weights (absolute and relative to body and brain weights) at 150 and 50 mg/kg bw/day, however in the lack of any dose relevance and because of the low degree, these were judged to be of little or no biological significance even if values were out of the historical control ranges in some cases.
Statistical significance noted for the mean liver weight relative to body weight at 310 mg/kg bw/day probably was due to the significantly less fasted mean body weight of this group since no similar changes occurred in the liver weight relative to brain weight.
Slightly but statistically significantly higher spleen weights in male animals at 310 mg/kg bw/day (relative to body weight) and at 150 mg/kg bw/day (absolute and relative to body and brain weight) were not considered to be toxicologically significant in the lack of dose-response and any histopathological findings, moreover all values were well within the historical control range.
The fasted body weight was slightly less than in the control group at 310 mg/kg bw/day, consequently, the brain weight relative to body weight was higher and the body weight relative to brain weight was less with respect to control.
In female animals, higher mean weights of kidneys (absolute and relative to body and brain weights) were detected at 150 mg/kg bw/day, but not in the 310 mg/kg bw/day. The kidney weight relative to body weight exceeded the control vale in females at 310 mg/kg bw/day however this was probably due to the significantly less mean fasted body weight of this group.
Statistical significances were noted in female animals for the higher mean absolute liver weight at 150 mg/kg bw/day and for liver weights relative to body and brain weight at 310, 150 and 50 mg/kg bw/day doses. The liver weight changes were not considered to be toxicologically or biologically significant because the values all remained within the historical control ranges, there was no dose dependency and there were no related pathological findings at the clinical chemistry and histopathology investigations.
The fasted body weight was slightly less than in the control group also in female animals at 310 mg/kg bw/day consequently, the brain weight relative to body weight was higher and the body weight relative to brain weight was less with respect to control. For detail please refer to the table in the attachment.

Histopathology
Histological examination did not reveal any toxic or test item 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 and pituitary was normal in all cases as well.
In animals subjected to full histopathology examinations, in the lungs focal alveolar emphysema in minimal degree (2/5 male control) and hyperplasia of bronchus associated lymphoid tissue (BALT; 310 mg/kg bw/day: 1/5; control: 2/5) were observed.
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 four control animals dilatation of uterus (2/9 dams and 2/3 non pregnant females) was observed. The histological picture of pituitary was normal as well in the female treated and control animals.
In the female animals subjected to full histopathology examinations, in the lungs focal alveolar emphysema (1/5 at 310 mg/kg bw/day and 1/5 control) and focal hemorrhage (1/5 at 310 mg/kg bw/day and 1/5 control) were observed in minimal degree. Unilateral pyelectasia was noted for two females (1/5 at 310 mg/kg bw/day, 1/5 at 150 mg/kg bw/day). The hyperplasia of bronchus associated lymphoid tissue was present in the control (1/5) and high dose 5 (1/5) treated animals.
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 were the same at the control and treated animals.
The pulmonary changes (emphysema and hemorrhages) were considered to be a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The hyperplasia of bronchus associated lymphoid tissue is a physiological phenomenon and was detected in some control and treated animals, too.
Unilateral pyelectasia occurring in two test item treated female animals (at 310 and 150 mg/kg bw/day) without other pathological lesions (inflammation or fibrosis) is a slight individual disorder without toxicological significance.
The uterus dilatation - without inflammation or other pathological lesion - is a physiological phenomenon in connection with the normal sexual cycle. For detail please refer to the table in the attachment.

Effect levels (P0)

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Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic effects: salivation, reduced body weight development, changes in clinical pathology parameters and changes in organ pathology
Dose descriptor:
NOAEL
Remarks:
Reproductive perfomance
Effect level:
310 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Mortality
A higher extra uterine mortality was ascribed to the treatment at 310 mg/kg bw/day but it was probably related to maternal toxicity (changes in body weight and food consumption during the lactation period).

The extra uterine mortality of pups was significantly higher in 310 mg/kg bw/day group than in the control group between postnatal days 0 and 4. 40 % of live born pups died between postnatal days 0 and 4 (ie., from this 40 %, 62.5 % were cannibalized and 37.5 % were found dead). The litter mean of viable pups was also less at 310 mg/kg bw/day with respect to control on postnatal day 4. For detail please refer to the table in the attachment.

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
Cold, not suckled (no milk in the stomach) pups and cachexia were observed with high incidence in litters at 310 mg/kg bw/day. The number and litter means of these signs were significantly higher than in the control group. In two pups of this group cyanotic skin were also observed.
The higher incidence of clinical signs in offsprings was related to the test item effect at 310 mg/kg bw/day and was considered to be as a consequence of maternal toxicity. For detail please refer to the table 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 310 mg/kg bw/day.
The mean litter weight was significantly less than in the control group at 310 mg/kg bw/day on postnatal day 4, and the litter weight gain was also less than in the control between days 0 and 4. The mean pup weight on day 4 and the mean weight gain of pups between days 0 and 4 was significantly reduced for some litters (3/6) at 310 mg/kg bw/day. The latter effects may be linked to the significantly reduced body weight and food consumption observed in females during the lactation period. For detail please refer to the table in the attachment.

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 dead offspring during the macroscopic examination. Cachexia was detected in offspring as mentioned above.




Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mean litter weight and mean pub weight; increased extra uterine mortality.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The subacute toxicity oral of TBPPI were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for male and female rats: 150 mg/kg bw/day, based on Systemic effects: salivation, reduced body weight development, changes in clinical pathology parameters and changes in organ pathology
NOAEL for reproductive performance of the male and female rats: 310 mg/kg bw/day, No effects up to the highest dose.
NOAEL for F1 Offspring: 150 mg/kg bw/day, based on Mean litter weight and mean pub weight; increased extra uterine mortality.
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 TBPPI 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), 50, 150 and 310 mg/kg bw/day at concentrations of 25 mg/mL, 75 mg/mL and 155 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. TBPPI was stable at room temperature for 24 hours and in a refrigerator (5 ± 3 °C) for 3 days. Concentration of the test item in the dosing formulations varied in the range of 98 % to 104 % 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 – 6, 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 – 7 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. 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 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 (310, 150 and 50 mg/kg bw/day).

Clinical observation

Test item related salivation appeared in male and female animals administered with 310 and 150 mg/kg bw/day groups with a dose related 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 at 310 mg/kg bw/day in male animals during the entire observation period (with statistical significances on weeks 1, 2, 3 and 6 and if summarized) and in dams during lactation period. These changes resulted in a slightly less body weight in the range of -5 to -7 % in male animals with respect to control from day 20 up to the termination and -13 % in dams on lactation day 4.

Food consumption

The mean daily food consumption was slightly less comparing to the control group at 310 and 150 mg/kg bw/day doses during first week of premating period (male), during the premating (female) and at 310 mg/kg bw/day between lactation days 0 and 4.

Hematology

Hematology examinations did not reveal any test item related changes in the evaluated hematological parameters at any dose level (310, 150 and 50 mg/kg bw/day).

Clinical chemistry

No test item-related changes were observed in investigated clinical chemistry parameters for the male animals. In the female animals treated with 310 mg/kg bw/day, the glucose (GLUC) concentration was slightly but significantly below the value of control and historical control ranges in this treatment group and a test item related effect cannot be ruled out. However, as the reduction in GLUC was very slight, the finding was considered to be of no toxicological concern.

Necropsy

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

Organ weight

A test item influence on renal function was observed as mean weights of kidneys (absolute, and relative to body and brain weights) were slightly higher with respect to the controls and above the historical control ranges in male animals at 310 mg/kg bw/day dose. The increases in absolute, relative to body, and brain weights were determined to be 26, 31 and 23%, respectively.

Histopathology

Histopathology investigation did not detect any microscopic changes related to the test item effect.

Reproduction

There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

Offspring

A test item effect on the offspring development was observed in the significantly higher number and percentage of extra uterine mortality in 310 mg/kg bw/day group between postnatal days 0 and 4, and in the significantly less litter weight and litter weight gain and mean pup’s weight and weight gain at 310 mg/kg bw/day. These effects were probably a consequence of the observed maternal systemic effect (reduced body weight and food consumption during lactation period).

Conclusion

Under the conditions of the present study, TBPPI caused salivation (male and female), changes in body weight (male and female), food consumption (male and female), slightly reduced glucose concentration (female) and elevated kidney weights (male) following an oral administration at 310 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 150 mg/kg bw/day, salivation and slightly reduced food consumption were observed in male and female animals. At 50 mg/kg bw/day, no test item related adverse effects were observed. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male and female rats: 150 mg/kg bw/day

NOAEL for reproductive performance of the male and female rats: 310 mg/kg bw/day

NOAEL for F1 Offspring: 150 mg/kg bw/day