Registration Dossier
Registration Dossier
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EC number: 247-955-1 | CAS number: 26748-41-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
A reproduction/ developmental toxicity screening test according OECD TG 422 was performed in 2012 with TBPND using dosages of 60, 200 and 600 mg/kg bw/day. The following NOAELs were obtained: NOAEL for male rats: 60 mg/kg bw/day; NOAEL for female rats: 60 mg/kg bw/day; NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day; NOAEL for F1 Offspring: 60 mg/kg bw/day.
A modified study according OECD TG 421 in rats was performed in 2021 with TBPND, in which the parent (P) generation was dosed 10 weeks prior to mating. The dosages tested were 50, 150 and 300 mg/kg bw/day. This study was performed to conclude on the dose setting for the subsequent OECD TG 443 study. In this pre-study, the NOAEL for systemic toxicity of male/ female rats was 300 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats was 150 mg/kg bw/day and the NOAEL for F1 Offspring is 50 mg/kg bw/day.
The main study according to OECD TG 443 was performed in Han:Wist rats including Cohort 3 (ECHA request, CCH-D-2114493102-56-01/F). Based on the results of the pre-study the parent (P) generation was dosed 10 weeks prior to mating with dosages of 50, 150 or 450 mg/kg bw/day. The reproductive performance was not impaired in male or female rats after administration of TBPND via oral gavage. No treatment-related adverse changes were noted in any of the reproductive parameters investigated (i.e. mating and fertility indices, sperm parameters, precoital time, number of implantations, estrous cycle). Test-item related systemic effects in the body weight, food consumption and in the kidneys- related parameters (urine and clinical chemistry, organ weights, macroscopic and microscopic findings) were observed in high dose parental male animals. Signs of systemic effect manifested in changes and of clinical chemistry parameters and increased liver weight in parental female animals in the high dose group.
In F1 offspring pre-weaning, depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. However, this is considered secondary to maternal toxicity and as a nonspecific effect. No treatment-related effect on anogenital distance, areola/nipple retention, surface righting reflex and thyroid hormone levels (FT4 and TSH in PND 22 pups) were detected in F1 offspring.
In F1 adult (F1 Cohort 1A, Cohort 1B, Cohort 3, post-weaning) at 450 mg/kg bw/day reduction in body weight and food consumption (Cohorts 1A, 1B and 3) in male or female animals and kidneys related parameters (urine and clinical chemistry parameters, organ weight, macroscopic and microscopic findings) in male animals of F1 generation (Cohorts 1A) was detected. No treatment-related effects were recorded for developmental parameters in F1-animals, including balano-preputial separation, vaginal opening, occurrence of first estrus, sperm parameters, ovarian follicle counts and histopathology of reproductive organs.
At 150 mg/kg bw/day of male animals in F1 generation (Cohorts 1A and B), body weight and kidney-related findings were observed in a lesser degree. No test item-related adverse effects were detected at 50 mg/kg bw/day in F1 adults (Cohorts 1A, 1B and 3).
No treatment-related immunotoxic effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2012-02-23 until 2012-04-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), adopted 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Hsd.Brl.Han:Wist
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 86 -91 days old
- Weight at study initiation:
Male animals: 331 - 392 g
Female animals: 190 – 216 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
21 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m. - Route of administration:
- oral: gavage
- Vehicle:
- other: Sunflower oil (Helianthii annui oleum raffinatum)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 30 mg/mL, 100 mg/mL and 300 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 3 days before the administration.
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study
VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item
- Amount of vehicle (if gavage):
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0. - Details on mating procedure:
- Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL.
Concentration of the test item in the dosing formulations varied in the range of 97 % to 106 % in comparison to the nominal values. - Duration of treatment / exposure:
- The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 41 or 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 1-8 5 (for 42 – 60 days,
depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 43 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw. - Frequency of treatment:
- Animals were treated once per day.
- Details on study schedule:
- Male animals were dosed for 41 or 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 1-8 5 (for 42 – 60 days, depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 43 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw. - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 600 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 12 animals/sex in the control and dose groups.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The dose setting was based on findings obtained in a previous oral repeated dose toxicity study. The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.
DETAILED CLINICAL OBSERVATIONS:
General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs were recorded including onset, degree and duration of signs.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
BODY WEIGHT:
All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. Body weight of the female animals were additionally weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data were reported individually for adult animals. Individual body weight changes were calculated.
Body weight was measured on day of necropsy for each animal.
FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period (pre-
mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4) except during mating phase.
EXAMINATION OF PLACENTAL SIGN:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.
OBSERVATION OF THE DELIVERY PROCESS:
Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 post-partum with an accuracy of 0.1 g.
In addition to the observations on parent animals, any abnormal behavior of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all dead pups to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.
CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology and clinical chemistry were conducted in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 mL 9NC Microtube, 0.106 mol/L, Greiner Bio-One International AG, or equivalent), one for hematology (MiniCollect® EDTA tubes, spray-dried, 0.25 mL, Greiner Bio-One International AG, or equivalent), and the third one (VACUETTE® Serum Tube, 2.5 mL, Greiner Bio-One International AG, or equivalent) to obtain serum samples for clinical chemistry.
Tubes for hematology and coagulation should be filled up to the final volume (marked on the tubes) and at least 1.0 mL blood should be collected, if possible into clinical chemistry tubes.
HEMATOLOGY:
The hematology parameters were measured in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by SYSMEX XT-2000iV.
- Parameters checked:
White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential white blood cell count, Activated partial Thromboplastin Time, Prothrombin Time.
CLINICAL CHEMISTRY:
The clinical chemistry measurement were performed in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by Konelab 20i in all animals before the terminal necropsy.
- Parameters checked:
Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Total Protein concentration, , Albumin concentration, Albumin/globulin ratio. - Oestrous cyclicity (parental animals):
- Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. One pair was changed within the control group and within the high dose group after 14 day unsuccessful pairing.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. - Sperm parameters (parental animals):
- Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. - Litter observations:
- Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery. - Postmortem examinations (parental animals):
- Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
PATHOLOGY:
Gross necropsy was performed on each animal one day after the last treatment). Animals were euthanized by exsanguination after verification of an Isofluran-narcosis.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality were recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions of all adult animals were preserved. Kidneys of all parental male and female animals were also preserved due to macroscopic findings in male animals dosed with 600 mg/kg bw/day. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected for blood collection from each group:
adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with vagina), gross lesions, heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve,
seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid, trachea, urinary bladder, Pups euthanized at day 4 post-partum, or shortly thereafter, were carefully examined for gross abnormalities externally.
ORGAN WEIGHT:
At the time of termination, body weight and weight of the testes, epididymides of all parental animals were determined with an accuracy of 0.01 g. Kidneys were also weighed in all male and female animals due to macroscopic findings in male animals dosed with 600 mg/kg bw/day.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
Paired organs were weighed individually; absolute organ weight was reported. Relative organ weight (to body and brain weight) was calculated and reported.
HISTOPATHOLOGY:
Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histological examination of kidneys was also performed in all animals because test item related changes were observed in the high dose treated male animals. - Postmortem examinations (offspring):
- GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4) - Statistics:
- The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. - Reproductive indices:
- The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
- Offspring viability indices:
- The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Daily Observations
Test item related salivation was observed in animals of all dosed groups (12/12 male and 12/12 female at 600 mg/kg bw/day; 12/12 male and 12/12 female at 200 mg/kg bw/day and 11/12 male, 5/12 female at 60 mg/kg bw/day) in a dose related manner regarding the degree, onset and frequency. Marked salivation only occurred in the high dose treated animals. The frequency of observation was the highest in 600 mg/kg bw/day group. Male animals proved to be more sensitive than females as number of animals showing salivation and frequency of observation were higher in males than in females at 60 mg/kg bw/day.
Alopecia was noted on the left forelimb for one male animal (1/12) dosed with 60 mg/kg bw/day from day 13 up to the termination; on the left side of the back for one female (1/12) at 60 mg/kg bw/day and on the limbs and abdomen for single control female animal (1/12) during the gestation and lactation period. Alopecia is a common finding in this strain of experimental rats and was present in the control and low dose only therefore had no toxicological meaning in this study.
Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period (pre-mating, mating and post-mating). Alopecia as described above was observed at the weekly observations, too. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test item related depression of the body weight development was detected in male and female animals at 600 and 200 mg/kg bw/day with respect to controls.
In the male animals dosed with 600 mg/kg bw/day, the body weight gain was less than in the control group with statistical significances on weeks 1, 2 and 6, thus the total body weight gain remained below control value, too. The reduced body weight gain resulted in a slightly but statistically significantly lower body weight values during the study from day 13 (days 13, 20, 27, 34 and 40) with respect to controls.
In the females, a less body weight gain was observed during the premating and gestation period with respect to control with statistical significances at the summarized body weight gain during the premating, on gestation weeks 2 and 3, and the total body weight gain during the gestation period. The body weight remained below the control value on gestation day 21 and during lactation period (lactation days 0 and 4).
At 200 mg/kg bw/day, in the male animals the mean body weight gain was significantly less than in the control between day 27 and 40 and if summarized (between day 0 and 40).
In the females, significantly less body weight gain was found during the premating period (no statistical significance on week 1) and the mean body weight was also lower than in the control on lactation days 0 and 4.
The body weight development was slightly less than in the control in male animals at 60 mg/kg bw/day between days 27 and 34 however there was no significant difference in the body weight and in the mean summarized body weight gain comparing to the control.
In the females, the mean body weight and body weight gain was similar to that of the control group during the entire observation period (premating, gestation and lactation) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- A test item influence on the mean daily food consumption was observed in male and female animals at 600 and 200 mg/kg bw/day. The statistical significances indicated only slight differences between the control and 200 mg/kg bw/day groups and were not considered to be biologically significant.
The mean daily food consumption was lower than in the control group at 600 and 200 mg/kg bw/day doses, during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.
In male animals, the higher mean food consumption at 600 mg/kg bw/day between days 27 and 34 and the less mean food consumption at 60 mg/kg bw/day between days 7 and 13 were not judged to be toxicologically significant. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related changes in the examined hematological parameters in male animals at any dose level. Statistically significant differences between the control and dosed groups in some hematological parameters (neutrophil granulocytes (NEU), lymphocytes (LYM), monocytes (MONO), eosinophil granulocytes (EOS) and activated partial thromboplastin time (APTT) were not considered toxicologically relevant as these were with low magnitude and values remained well within the historical control ranges.
In the female animals, the hemoglobin concentration (HGB) and hematocrit vale (HCT) were slightly below the control value at 600 mg/kg bw/day and the percentage of reticulocytes (RET) was higher than in the control group at 600, 200 and 60 mg/kg bw/day (latter without statistical significance). - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and concentration of creatinine and urea were also indicative of the test item effect.
The statistically significant differences in levels of some parameters were judged to be of little or no biological significance as the mean values were within or marginal to the historical control ranges (inorganic phosphorous, potassium, alkaline phosphatase) or these were without any dose-response relationship (total bilirubin, calcium, cholesterol and aspartate aminotransferase). - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group as compared with the middle or high dose groups.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns, increased extra uterine mortality and higher numbers of dams with prolonged duration of pregnancy.
Although there were no statistically significant differences with respect to control, the mean of post-implantation loss and total intrauterine mortality, mean number of stillborns per litter, number of dams with prolonged pregnancy were higher, and the mean of total births per litter, mean number of live-borns per litter and viable pups per litter, were less than in the control at 600 mg/kg bw/day.
Statistical significances noted for the higher number and percent of implantations and less number and percent of pre-implantation loss were not relevant toxicologically in the 200 and 60 mg/kg bw/day groups and for total intrauterine mortality in the 200 mg/kg bw/day group.
There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance. The copulatory and fertility indices were similar at each dose level.
There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval were similar to or were the same as in the control group in all test item treated groups. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- clinical signs
- body weight and weight gain
- gross pathology
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- clinical signs
- body weight and weight gain
- organ weights and organ / body weight ratios
- Dose descriptor:
- NOEL
- Effect level:
- < 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Systhemic effects: Rat specific hyaline nephropathy
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 200 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- reproductive performance
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Test item related clinical signs did not appear in the pups. The number and percent of cold and not suckled pups were slightly higher than in the control group at 600 and 200 mg/kg bw/day but these were considered to be independent from the treatment as these signs occur in not treated animals with similar percentage.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- The number and percentage of extra uterine mortality was slightly higher than in the control group in 600 mg/kg bw/day group between postnatal days 0 and 4. The mean number of dead pups was also higher than in the control group. The survival indices were similar between all groups (600, 200 and 60 mg/kg bw/day and control groups).
The higher mean of female viable pups at 60 mg/kg bw/day and litter mean has no toxicological significance. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test item influence on the litter weight and litter weight gain and pup’s mean weight and weight gain were detected at 600 and 200 mg/kg bw/day.
The mean litter weight was significantly less than in the control group at 600 mg/kg bw/day on postnatal day 4, and the litter weight gain between days 0 and 4 was also less than in the control at 600 and 200 mg/kg bw/day. The mean pup weight on days 0 and 4 as well as the mean weight gain of pups between days 0 and 4 were significantly less than in the control group at 600 and 200 mg/kg bw/day. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 200 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- no
- Conclusions:
- The reprotoxic properties of TBPND were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:
NOEL for male rats: < 60 mg/kg bw/day; NOAEL for male rats: 60 mg/kg bw/day; NOAEL for female rats: 60 mg/kg bw/day; NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day; NOAEL for F1 Offspring: 60 mg/kg bw/day. - Executive summary:
The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the toxic potential of TBPND and on its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 60, 200 and 600 mg/kg bw/day at concentrations of 30, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 8, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups.
The first five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on days 4 – 9 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. The kidneys of all animals were also processed and evaluated histologically due to macroscopic observations at the necropsy. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The reproductive organs and pituitary of non-pregnant female animal and male cohabited with in the low dose group were also processed and evaluated histologically.
The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.
Results
Mortality
There was no test item related mortality at any dose level (600, 200 and 60 mg/kg bw/day).
Clinical observation
Test item related salivation appeared in male and female animals administered with 600, 200 and 60 mg/kg bw/day groups in all with a dose related degree, onset and incidence. No toxic signs related to the test item were found at the detailed weekly and terminal functional observations. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).
Body weight and body weight gain
The body weight gain was reduced with respect to controls in male and female animals at 600 and 200 mg/kg bw/day resulting in a slightly less body weight at 600 mg/kg bw/day in male animals from day 13 up to the termination and in female animals on gestation day 21 and lactation days 0 and 4. The summarized body weight gain also remained below the control value in male animals administered with 600 and 200 mg/kg bw/day and in females at 600 and 200 mg/kg bw/day during the premating and at 600 mg/kg bw/day during the gestation period.
Food consumption
The mean daily food consumption was less comparing to the control group at 600 and 200 mg/kg bw/day doses during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.
Hematology
Hematology examinationsrevealed test item related higher percentage of reticulocytes in female animals administered with 600, 200 and 60 mg/kg bw/day and less hemoglobin concentration and hematocrit value at 600 mg/kg bw/day with respect to controls.
Clinical chemistry
A higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and higher concentration of creatinine and urea were also indicative of the test item effect.
Necropsy
Test item related renal changes (enlarged and pale kidneys) were observed in male animals dosed with 600 mg/kg bw/day.Specific macroscopic alterations related to the test item were not found in female animals during the necropsy.
Organ weight
Higher kidneys weights of male animals administered with at 600 and 200 mg/kg bw/day and higher liver weights in males at 600 mg/kg bw/day, in females at 600, 200 and 60 mg/kg bw/day reflected a test item influence on renal and hepatic functions in accordance with clinical chemistry and necropsy findings.
Histopathology
Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated male groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group with respect to the middle or high dose groups. Hyaline droplet nephropathy was associated with interference to α-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans.
Reproduction
There were no differences between the control and test item treated groups in the reproductive ability of male and female animals. Spermatogenesis in the rate male appeared unaffected by the test item. However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns and higher numbers of dams with prolonged duration of pregnancy
Offspring
A test item effect on the offspring development was observed in the higher number and percentage of extra uterine mortality in 600 mg/kg bw/day group between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight and weight gain at 600 and 200 mg/kg bw/day.
Conclusion
Under the conditions of the present study, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected. Dam’s delivery was affected by the test item at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed. At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day.
Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:
NOEL for male rats: < 60 mg/kg bw/day
NOAEL for male rats: 60 mg/kg bw/day
NOAEL for female rats: 60 mg/kg bw/day
NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day
NOAEL for F1 Offspring: 60 mg/kg bw/day
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2021-06-15 to 2021-06-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29th July 2016
- Deviations:
- yes
- Remarks:
- 10 weeks pre-treatment, no hematology and clinical biochemistry measurements, histopathology only performed for ovaries
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han:WIST
- Details on species / strain selection:
- The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and well known fertility parameters.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt.Cserkesz u. 90. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 46- 50 days old
Female animals: 41 - 46 days old
- Weight at study initiation:
Male animals: 200 – 240 g
Female animals: 126 – 156 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Mated females were housed individually
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, Spezialdiäten GmbH, D-59494 Soest, Germany
- Water, ad libitum, tap water
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- vegetable oil
- Remarks:
- Sunflower oil (Helianthi annui oleum raffinatum)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in vehicle in concentrations of 25 mg/mL, 75 mg/mL and 150 mg/mL in the formulation laboratory of the Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility three times during the treatment period.
VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 25, 75, 150 mg/mL
- Amount of vehicle: 2 mL - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Any other deviations from standard protocol: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of dosing formulations was between the range of 95 % to 109 % of the nominal concentrations. A sufficient stability and homogeneity in the chosen vehicle have been verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 ± 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL).
- Duration of treatment / exposure:
- All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35.
- Frequency of treatment:
- daily
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose setting was based on results obtained in previous studies performed with TBPND in the rat: 14-Day Oral Gavage Dose Range Finding Study with TBPND in the Rat andCombined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with TBPND in the Rat.
- Positive control:
- none
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the treatment at approximately the same time
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 behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma
BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter and on the day of the necropsy. Parental females were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter, then on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7, 14 and 21 post-partum. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption of parent animals was determined weekly by weighing the given and non-consumed diet by weekly interval with an accuracy of 1 g during the treatment period except mating phase: during the premating period and during post-mating period for male animals; premating period, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.
WATER CONSUMPTION AND COMPOUND INTAKE: No
DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as follows:
- from all dams on lactation/ post-natal day 22;
- from all parent male animals at termination on Day 99;
- non-pregnant female animals on Day 96; - Oestrous cyclicity (parental animals):
- Estrous cycle was monitored by examining vaginal smears daily before the mating started from each animal for two weeks and from the beginning of the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.
- Sperm parameters (parental animals):
- Parameters examined in male parental generation:
testis weight, epididymis weight, weight of prostate and seminal vesicles with coagulating glands, spermatogenesis, histopathology - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as
follows:
- from 2-7 pups per litter on post-natal day 4 (except for litters with 9 or less pups)
- from 3-8 pups per litter on lactation/ post-natal day 22;
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum FT4 and TSH levels on post-natal days 4 and 22.
OTHER:
One or two male and one or two female pups per litter were selected on post-natal day 21 and were identified by individual numbers written with a marker pen on the tail, were caged (2-3 pups/cage) and were administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight were determined twice weekly and were recorded. The food consumption was determined on post-natal day 22, 29 and 35. All pups were subjected to macroscopic necropsy observation on post-natal day 36. - Postmortem examinations (parental animals):
- SACRIFICE
Gross necropsy was performed on each parental animal one day after the last treatment. Animals were euthanized by exsanguination after verification of Isofluran-narcosis and were subjected to gross necropsy as follows:
- Parental male animals: on Day 99;
- Dams: on post-partum days 22, 23 or 24 (on Days 114-127);
- Non-pregnant (but mated) female animals on Day 96
GROSS NECROPSY
- After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.
HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Paired organs were weighed together. The thyroid weight was not determined because there were no test item related changes in the hypothalamic-pituitary-thyroid function at the thyroid hormone examinations. Histopathological processing and quantitative examinations of the ovaries were performed in female animals in the control and high dose groups. Follicular enumeration (primordial and small growing follicles – primary, secondary, tertiary follicles, corpora lutea, atresia) was performed in the largest surface section of the ovaries. The fixed ovaries were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy. - Postmortem examinations (offspring):
- GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS: Not determined - Statistics:
- 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.
- Reproductive indices:
- See table in "Any other information on materials and methods".
- Offspring viability indices:
- See table in "Any other information on materials and methods".
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related adverse clinical signs in any group (50, 150 or 300 mg/kg bw/day). Some male animal at 300 mg/kg bw/day showed salivation shortly after the administration for some minutes. Although the behavior and physical condition of these animals were also normal and therefore, salivation was considered to be not adverse. Salivation was noted for some male animal (7/12) at 300 mg/kg bw/day from Day 32 up to and including 65 or 98. Dermal changes were detected in one male animal at 300 mg/kg bw/day (1/12; scar at the neck region, left side, 0.5-1 cm in diameter, in slight, moderate or marked degree between Days 56 and 97) and in one female animal at 50 mg/kg bw/day (1/11 dam; alopecia on the fore limbs between lactation days 8 and 22). Similarly, reddish discoloration of the hair around eye is frequently observed in non-treated animals of this strain of experimental rats with similar age. This finding was detected in one dam from Day 33 up to and including lactation day 21. Scar, alopecia and reddish discoloration around the eye are common findings occurring also in non-treated animals of this strain of experimental rats with similar age. Therefore, these have no toxicological relevance.
- Mortality:
- no mortality observed
- Description (incidence):
- There was no mortality in control, 50, 150 or 300 mg/kg bw/day groups during the course of study (male and female).
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of gestation and lactation periods. The mean body weight gain was also reduced in dams at 300 mg/kg bw/day during the gestation period and during the first few days of lactation period.The mean body weight was comparable in the male animals in the control and all test item administered groups on each measuring day from Day 0 up to and including Day 97. Statistical significance with respect to the control was observed at the higher mean body weight gain of male animals at 150 and 300 mg/kg bw/day between Days 0-3 and at the lower mean body
weight gain of male animals at 300 mg/kg bw/day between Days 90-97. These changes in the body weight gain did not influence the mean body weight values of male animals. In the female animals, the mean body weight was comparable in the control and all dosed groups during the entire pre-mating period in-spite of the lower mean body weight gain at 300 mg/kg bw/day between Days 28-35,
56-63 and 0-69. At 150 mg/kg bw/day, the mean body weight gain exceeded the control between lactation day 7-14 in female animals.During the course of the gestation and lactation periods, statistical significance with respect to the control was observed at the lowered mean body weight and body weight gain of dams at 300 mg/kg bw/day as follows:
- lower mean body weight on gestation days 14 and 21 and lower mean body weight gain between gestation days 7-14, 14-21 and 0-21;
- lower mean body weight on lactation days 0, 4, 7, 14 and lower mean body weight gain between lactation days 0-4 and 7-14;
This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related adverse changes in the mean daily food consumption of male or female animals at 50, 150 or 300 mg/kg bw/day. The mean daily food consumption was comparable in male animals in the control and test item treated groups at 50, 150 and 300 mg/kg bw/day during the pre-mating and post-mating periods except for week 13 (between Days 83-90), where statistical significance with respect to the control was observed at the slightly higher mean daily food consumption of male animals at 300 mg/kg bw/day. In the female animals, statistical significances were detected at the slightly lower mean daily food consumption at 50 and 300 mg/kg bw/day on week 9 of pre-mating period and at 300 mg/kg bw/day between gestation days 14 and 21.
These changes in the mean daily food consumption were considered to be toxicologically not relevant because of the minor degree and transient occurrence. - 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
- Endocrine findings:
- not specified
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 300 mg/kg bw/day group. The mean number of primordial, primary, secondary and tertiary follicle, corpora lutea were similar in the control and 300 mg/kg bw/day. Statistical significance with respect to the control was observed at the higher mean number of follicular atresia. Follicular atresia is a normal - physiological - process in the ovary to regulate the number of follicles in the developing pool. Increase in follicular atresia can be observed secondary due to different causes. Since the number of primary, secondary and tertiary follicles was not changed compared to the control the increased follicular atresia can be judged as a biological variation.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related adverse changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in the parental male animals. The FT4 levels were slightly lower than in the control group in parental male animals at 150 and 300 mg/kg bw/day groups. The TSH concentration was higher than in the control group in parent male animals at 150 and 300 mg/kg bw/day. These minor changes were considered to be of no toxicological relevance as values met well the historical control in the most animals. The changes in FT4 were considered to be toxicologically not relevant considering the lack of significant relevant changes in TSH values.
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The examined parameters of the estrous cycle were not affected by the test item at 50, 150 or 300 mg/kg bw/day. There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular/ irregular cycles, in the mean number or length of cycles, mean number of days in proestrus or estrous or diestrous during pre-mating period. The number or percentage of animals in prolonged diestrous was slightly higher in the control group than in the test item treated groups as an indication of biological variation.
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The examined parameters of reproductive performance were not adversely affected by the treatment with the test item in male or female animals at 50, 150 or 300 mg/kg bw/day. Statistical significances with respect to the control were detected at the lower mean percentage of fertile male and female animals (fertility indices) at 50 and 300 mg/kg bw/day as a consequence of infertile mating of one pair (1/12) at 50 mg/kg bw/day and two pairs (2/12) at 300 mg/kg bw/day. The values were within the historical control range. The copulatory index and gestation indices were 100 % in all groups.
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 300 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to highest dose tested
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- reproductive
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: reduction of mean number of births (total, live and alive)
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Offspring: There were no adverse test item related clinical signs in the offspring from post-natal day 0 to 22. Slightly higher percentage of cold pups were detected at 150 and 300 mg/kg bw/day (16 and 24 %, respectively) with respect to the control. Not suckled pups were observed with the highest incidence at 150 and 300 mg/kg bw/day (35 and 29 %, respectively). Alopecia was observed on the whole body of pups in one litter (6 %) at 50 mg/kg bw/day. Additionally, smaller than normal, pale pups, hemorrhage (on the nose, abdomen), bite mark on the abdomen were noted for some pup. These signs were considered to be toxicologically not relevant as the signs were transient.
F1: There were no clinical signs in male or female animals of F1 generation in the control and 50, 150 or 300 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including postnatal day 35). - Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- There was no test item related effect on offspring’s extra uterine mortality. The extrauterine mortality was comparable in the control, 50, 150 and 300 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21.
The survival of offspring were not affected by the test item at 50, 150 or 300 mg/kg bw/day on post-natal days 0, 4 and 21. Statistical significances with respect to the control was detected at 300 mg/kg bw/day at the lower mean number of pups (live and alive) on post-natal day 0 and mean number of pups per litter on post-natal day 4. Although, the extrauterine mortality was low and similar in each group. There was no significant difference between the control and test item treated (50, 150 and 300 mg/kg bw/day) groups in the survival indices on post-natal day 4. The survival index was higher at 300 mg/kg bw/day than in the control group on post-natal day 21 due to the lower number of offspring euthanized on postnatal day 4. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Offspring: The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 up to postnatal day 21. The mean litter weight, litter weigh gain, body weight and body weight gain were comparable in the control and 50 mg/kg bw/day after birth up to and including post-natal day 21. At 150 mg/kg bw/day, statistical significance with respect to the control was detected at the lower mean litter weight gain between post-natal days 0 and 4 however the mean litter weight was similar to the control. The mean pup weight was lower than in the control at 150 mg/kg bw/day on post-natal
days 4, 7 and 14 as a consequence of the lower mean body weight gain between post-natal days 0-4 and 4-7. The mean litter weight and mean litter weight gain were significantly lower than in the control in offspring at 300 mg/kg bw/day from post-natal day 0 up to and including post-natal day 21 reaching statistical significances in the most cases. Compared to the control, statistically significant difference was observed at the lower mean body weight of pups at 300 mg/kg bw/day on post-natal days 7, 14 and 21 as well as at lower mean body weight gain of pups between postnatal days 4-7 and 7-14, as well as between post-natal days 0-21. Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly lower mean weight of male pups at 50, 150 and 300 mg/kg bw/day and of female pups at 150 mg/kg bw/day on post-natal day 4.
F1: The body weight development of the F1 animals was reduced at 150 and
300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. The body weight and body weight gain were similar in the control and 50 mg/kg bw/day administered male animals during the observation period. In the male animals at 150 mg/kg bw/day, the mean body weight was lower than in the control group during the two weeks observation period with statistical significance on post-natal days 22, 25, 29. The mean body weight gain for these animals was comparable to their control. At 300 mg/kg bw/day, the mean body weight and body weight gain of male animals were significantly lower (up to 17% lower body weight) than in the control reaching statistical significances in the most cases.
The body weight and body weight gain were comparable to the control in
female animals at 50 mg/kg bw/day during the observation period. At 150 mg/kg bw/day, the mean body weight of female animals was lower than in the control group during the two weeks observation period with statistical significance on each measuring days. Statistical significance with respect to the control was detected at the lower mean body weight gain for these animals between post-natal days 25 and 29. In the female animals at 300 mg/kg bw/day, the mean body weight and body weight gain were significantly lower than in the control reaching statistical significances for body weight on each measuring days and for body weight gain between postnatal days 22-25 and 25-29. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- F1: The mean daily food consumption was lowered at 150 and 300 mg/kg bw/day in F1 male and female animals in accordance with the body weight
changes. The mean daily food consumption was similar in the control and 50 mg/kg bw/day groups (male and female) during the 14-day observation period.
Statistical significance with respect to the control was detected at the lower mean daily food consumption of male animals at 150 mg/kg bw/day between post-natal days 22-29 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35. In the female animals, lower mean daily food consumption was observed at 150 mg/kg bw/day between post-natal days 29-35 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Description (incidence and severity):
- The sex distribution of male and female pups (mean percentage of pups per litter) was comparable in all groups on post-natal days 0, 4 and 21.
- Anogenital distance (AGD):
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related changes in the anogenital distances at 50, 150 or 300 mg/kg bw/day in male or female offspring. Statistical significance was observed at the slightly shorter mean absolute anogenital distance in female pups at 150 and 300 mg/kg bw/day. The mean normalized anogenital distance exceeded the control in male pups at 300 mg/kg bw/day and was below the control in female pups at 300 mg/kg bw/day. These differences were with minor degree and within the range of the historical control data. Therefore, were considered to have no toxicological relevance.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- Nipples/areoles were not seen in any of the examined male offspring in the control or 50, 150 or 300 mg/kg bw/day groups on post-natal day 13.
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Offspring: Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination (stillborn or dead pups on postnatal day 4). In dead pup in the control group (1/1 male), empty stomach and slightly
autolyzed visceral organs were observed. At 50 mg/kg bw/day, there were no macroscopic findings in one dead pup (1/1). In one stillborn pup at 150 mg/kg bw/day (1/7), malformations – missing orifices of eyes, nose and mouth – were observed. There were no macroscopic findings in organs or tissues in stillborn pups: 1/1 at 50 mg/kg bw/day, 6/7 at 150 mg/kg bw/day and 3/3 at 300 mg/kg
bw/day.
F1: Macroscopic alterations in connection with the test item were not detected in male or female animals of F1 generation at the necropsy. Species specific macroscopic findings – renal changes (male and female) and hydrometra (female) – were observed. In male animals, pale kidneys (1/12 control, 1/11 at 300 mg/kg bw/day) and right side pyelectasia (3/12 at 150 mg/kg bw/day; 1/11 at 300 mg/kg bw/day) were observed. In female animals, slight, moderate or marked hydrometra was detected as follows: 1/11 in control group; 1/11 at 50 mg/kg bw/day; 3/14 at 150 mg/kg bw/day). Additionally, pale kidneys (2/11 at 50 mg/kg bw/day; 1/14 at 150 mg/kg bw/day; 1/10 at 300 mg/kg bw/day) and one or both sided pyelectasia (1/11 at 50 mg/kg bw/day; 2/10 at 300 mg/kg bw/day) were seen. Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age. - Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- The FT4 and TSH levels were not adversely affected in PN22 offspring at any dose levels.
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- TBPND caused reduction of mean number of births (total, live and alive) after administration of 300 mg/kg bw/day by oral gavage to Han:WIST rats. 50 or 150 mg/kg bw/day did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female animals. At 300 mg/kg bw/day, the body weight development of dams was reduced during the gestation and lactation period. There were no signs of systemic toxicity at 50, 150 mg/kg bw/day (male and female) or 300 mg/kg bw/day (male). The body weight development of the offspring was depressed between post-natal day 0 and 21 after repeated oral administration of dams at 150 or 300 mg/kg bw/day. The body weight development and food consumption of the F1 animals were reduced at 150 and 300 mg/kg bw/day during the 14-day post-weaning treatment. Based on these observations the NOAEL for systemic toxicity of male/ female rats is 300 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats is 150 mg/kg bw/day and the NOAEL for F1 Offspring is 50 mg/kg bw/day.
- Executive summary:
A modified OECD 421 study in rats was performed. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 150 and 300 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.
All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.
There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.
The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.
The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.
The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day
NOAEL for overall reproductive performance of male/ female rats: 150 mg/kg bw/day
NOAEL for F1 Offspring: 50 mg/kg bw/day
- Endpoint:
- extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-03-26 to 2022-05-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- 25 June 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- 15 July 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 10 weeks, as requested by ECHA.
- Basis for dose level selection: Based on a pre-test conduced with TBPND (Modified OECD 421 with 10 weeks pre-mating treatment); furthermore, results from a OECD 422 toxicity study in rats was taken into consideration for dose level selection (see supporting information, RSS).
- Exclusion of extension of Cohort 1B: not requested by ECHA based on available data and not necessary based on study results
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: not request by ECHA based on available data
- Inclusion of developmental immunotoxicity Cohort 3, as requested by ECHA
- Route of administration: oral (gavage) - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males and females:
Male animals: 41 - 43 days (6 weeks) old
Female animals: 39 - 42 days (6 weeks) old
- Weight at study initiation:
(P) Male animals: 158 – 222 g
(P) Female animals:120 – 147 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 animals of the same sex/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-70 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light - Route of administration:
- oral: gavage
- Vehicle:
- vegetable oil
- Remarks:
- Sunflower oil (Helianthi annui oleum raffinatum)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 25, 75 and 225 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on 5 occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 25, 75 and 225 mg/mL
- Amount of vehicle: 2 mL/kg bw - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- A sufficient stability and homogeneity in the chosen vehicle have been verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 ± 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL).
- Duration of treatment / exposure:
- Parental males were dosed for 70 days pre-mating and up to 14 days mating and until to the weaning of offspring. Overall treatment period is therefore up to 18 weeks for male parental animals.
Parental females were dosed for 70 days pre-mating, through up to 14 days mating period and throughout pregnancy and at least up to and including post-partum day 21 or up to the day before sacrifice. The day of birth (viz. when parturition is complete) is defined as day 0 post-partum. Overall treatment period is therefore up to 18 weeks for female parental animals.
Dosing of F1 offspring selected for follow-up examinations began on PND 22 up to post-natal day 90 (Cohort 1A) or at least up to post-natal day 97 (Cohort 1B).
F1 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen in Cohort 3, were administered and observed individually up to and including the day before euthanasia on PND 61 ± 3. - Frequency of treatment:
- daily, 7 days/week
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 450 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- P0: 26
F1, Cohort 1A: 20
F1, Cohort 1B: 20
F1, Cohort 3: 20 - Control animals:
- yes, concurrent vehicle
- yes, historical
- Details on study design:
- - Dose selection rationale:
The dose setting for the OECD TG 443 study is based on findings obtained in previous studies with TBPND in rats. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.
A reproduction/ developmental toxicity screening test according OECD TG 422 was performed in 2012 with TBPND using dosages of 60, 200 and 600 mg/kg bw/day. Here, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected. Dam’s delivery was affected at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed. At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day. Based on these observations the following NOAELs were determined:
NOAEL systemic for male rats: 60 mg/kg bw/day
NOAEL systemic for female rats: 60 mg/kg bw/day
NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day
NOAEL for F1 Offspring: 60 mg/kg bw/day
Based on these results a modified study according OECD TG 421 was performed in Hsd.Brl.Han:Wistar rats with TBPND in 2021, in which the parent (P) generation was dosed 10 weeks prior to mating. The highest dose was reduced to 300 mg/kg bw/day based on significant reductions in body weight/body weight gain during gestation and lactation in females observed in the OECD TG 422 study from 2012. In males, effects in kidney especially in the high dose of 600 mg/kg bw/day were the basis for high dose reduction to 300 mg/kg bw/day. Therefore, the dosage levels tested were 50, 150 and 300 mg/kg bw/day. This study was performed to conclude on the dose setting for the subsequent OECD TG 443 study and to investigate also the effect of longer pre-treatment period on systemic toxicity and fertility. At 300 mg/kg bw/day, the body weight development of dams was reduced during the gestation and lactation period. There were no signs of systemic toxicity at 50, 150 mg/kg bw/day (male and female) or 300 mg/kg bw/day (male). TBPND caused reduction of mean number of births (total, live and alive) after administration of 300 mg/kg bw/day by oral gavage to Han:WIST rats as far as investigated in this study. 50 or 150 mg/kg bw/day did not adversely influence the reproductive performance (gonad function, mating behaviour, conception, parturition) in parental male and female animals. The body weight development of the offspring was depressed between post-natal day 0 and 21 after repeated oral administration of dams at 150 or 300 mg/kg bw/day. The body weight development and food consumption of the F1 animals were reduced at 150 and 300 mg/kg bw/day during the 14-day post-weaning treatment. Based on these observations the NOAELs were determined as following:
NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats:150 mg/kg bw/day
NOAEL for F1 Offspring: 50 mg/kg bw/day
Since no significant systemic toxicity was observed at 300 mg/kg bw/day in the modified OECD 421 study, the highest dose for the OECD TG 443 study was elevated to 450 mg/kg bw/day. The doses which were chosen for the main study have been the following:
Group 1: 0 mg/kg bw/day
Group 2: 50 mg/kg bw/day
Group 3: 150 mg/kg bw/day
Group 4: 450 mg/kg bw/day
- Fasting period before blood sampling for clinical biochemistry: yes - Positive control:
- not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the female animals were additionally weighed on gestation day 10 in order to give accurate treatment volumes, but these data were not be evaluated statistically. F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning (on PND 25, 29, 32, 36, 39 and 42), and once weekly thereafter (Cohorts 1A, 1B and Cohort 3). For selected F1offspring, the body weight was recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency). Fasted body weight was measured on the day of necropsy for all animals (P and F1)
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was determined weekly by reweighing the non consumed diet with a precision of 1 g during the treatment period except mating phase as follows:
- by weekly interval during premating and post-mating periods for P male animals and for F1 animals (Cohorts 1A, 1B and Cohort 3) after weaning;
- by weekly interval during premating period, on gestation days 0, 7, 14 and 21, on lactation days 0, 7, 14 and 21, then weekly if needed, for P female animals
WATER CONSUMPTION AND COMPOUND INTAKE: No
URINALYSIS: Yes
The following parameters were evaluated in selected test animals of P and F1 Cohort 1A generation:
Nitrite (NIT), pH, Glucose (GLUC), Urobilinogen (UBG), Bilirubin (BIL), Ketone (KET), Blood, Leucocytes (LEU), Specific Gravity (SG), Protein (PROT), Volume (VOL), Sediment (SED), Colour, Clarity
HEMATOLOGY/BLOOD COAGULATION
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
White blood cell (leukocyte) count (WBC), Red blood cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) hemoglobin (MCH), Mean Corpuscular (erythrocyte) hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).
CLINICAL CHEMISTRY
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total protein concentration (TPROT)
DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- from 10 parent animals/sex/ group - Oestrous cyclicity (parental animals):
- Estrous cycle was monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating starts.Vaginal smears were also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation. Vaginal smear was prepared on the day of the necropsy of parental animals.Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear is recorded thus determining the time interval between these events. Estrous cycle of F1 adult female animals was examined for a period of two weeks commencing on PND77 and PND84 in Cohort 1A and Cohort 1B, respectively, including necropsy days. Vaginal smears were stained with 1 % aqueous methylene blue solution. After drying, the smears were examined with a light microscope.
- Sperm parameters (parental animals):
- Sperm parameters were measured in all control and high dose male animals in P generation and in F1 generation in Cohort 1A. The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded. Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms were determined. A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails). The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperm in homogenization was enumerated. The testis and epididymidis were frozen and enumeration were performed later.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on PND13, FT3, FT4 and TSH in surplus pups at PND 4 (pooled by litters) and in pups not selected for Cohorts on post-natal day 22.
Sexual maturity of selected F1 animals was examined by observing balano-preputial separation (between post-natal days 25 and 35) or vaginal patency (between post-natal days 28 and 40; or until PND45). The body weight was determined on the day when balanopreputial separation or vaginal patency is completed
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes, Cohort 3
Animals of Cohort 3 were administered daily from weaning up to the day before the termination. The following observations will be conducted:
- Checking of mortality/ morbidity – twice daily;
- General clinical observations – daily;
- Detailed clinical observations were made outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
- Body weight measurement – on PND 22, then twice a week during the two weeks following weaning and once weekly thereafter;
- Food consumption – on PND 22 and weekly thereafter
- Blood sampling for T-cell dependent antibody response assay (TDAR) on PND 56±3 and PND 61±3;
On PND 61±3 the primary IgM antibody response to a T-cell dependent antigen – keyhole limpet hemocyanin (KLH) – was assessed after subcutaneous immunization of all Cohort 3 animals with KLH (0.2 mL at a concentration of 1.5 mg/mL on PND56±3; prior dosing with test-item). Anti-KLH IgM antigen concentrations in serum was measured before immunization and 5 days after immunization using a validated ELISA method (Rat Anti-KLH IgM ELISA kit, Life Diagnostics, Inc.). Responses typically peak five days after immunization.
DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- in surplus pups at PND 4 (pooled by litters)
- from 10 adult F1 male and female animals/group (Cohort 1A ) at termination
- from 10 F1 pups/group not selected for cohorts on PND22 - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: after mating period
- Maternal animals: on PND 22.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively, for high dose and control animals:
Organ weights (all parental (P) animal and all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands
The organs were fixed in 4% buffered formaldehyde solution. Paired organs were weighed together except for organs with macroscopically visible difference in size between the two organs. Absolute organ weight was recorded and reported.
Full histological examinations was performed on the above listed organs and tissues of control and high dose treated parental animals. Reproductive organs were examined in all animals suspected of reduced fertility (not mated, non pregnant or not delivered) in the low and mid dose group.
SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis: CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) T lymphocytes, B lymphocytes and natural killer (NK) cells were identified by a validated flow cytometry method. Immuno-staining and immunophenotyping of spleen lymphocytes were carried out after preparation of single cell suspensions from one half of each provided spleen. For further information please refer to the attached background material. - Postmortem examinations (offspring):
- SACRIFICE
- Offspring selected for Cohort 1A and Cohort 1B: on post-natal days 91 and 98, respectively
- Offspring selected for Cohort 3 on PND 61±3
- Offspring not selected: on post-natal day 22
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights ( all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands
In animals of Cohort 1B, the weight of following organs was determined:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain,
- pituitary
For 10 male and 10 female pups per group, not selected for Cohorts, – from as many litters as possible – brain, spleen and thymus was weighed.
In animals of Cohort 3, the final body weight was determined.
Full histological examinations was performed on the above listed organs and tissues of control and high dose treated adult F1 animals in Cohort 1A. In adult F1 animals in Cohort 1B, the uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands as one unit (with their fluids), brain, pituitary will be processed to the block stage. In the ovaries of F1 adult females of control and high dose groups, a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was performed.
Detailed histological examination of testis was conducted with special emphasis on stages of spermatogenesis in the F1 male gonads and histopathology of interstitial testicular cell structure. Rete testis – where feasible – caput, corpus, and cauda of the epididymides were examined. All gross lesions were examined histologically. The fixed tissues were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy. - Statistics:
- The homogeneity of variance between groups was checked by Bartlett’s
homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of intergroup differences. Getting significant results 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. - Reproductive indices:
- Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100
Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100
Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100 - Offspring viability indices:
- Formulas for Calculation of Pup Mortality and Sex Ratio Indices:
Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100
Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100
Survival Index: Number of live pups on PND 13 / Number of liveborns x 100
Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100 - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day).
Salivation was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration immediately after the administration. Therefore, salivation was judged to be toxicologically not relevant in this study. There were no preceding clinical signs in dead animal at the daily or at the detailed weekly clinical observations. The death occurred after working hours as animal was found dead at the morning checking.
Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day).
Salivation was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration immediately after the administration. Therefore, salivation was judged to be toxicologically not relevant in this study.
There were no preceding clinical signs in dead animal at the daily or at the detailed weekly clinical observations. The death occurred after working hours as animal was found dead at the morning checking.
The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 450 mg/kg bw/day) during the entire treatment period.
In the male animals, slight salivation was observed at 450 mg/kg bw/day with variable onset and duration from Day 13 (9/26).
In the female animals at 450 mg/kg bw/day, salivation was detected during the pre-mating (5/26), gestation and lactation (4/24, both) periods.
Additionally, the following clinical signs were seen in some animals:
-Reddish colored hairs:
-around the right eye – 1/26 control male from Day 14 up to termination
-around the neck: from Day 87 to 98, 1/26 female at 150 mg/kg bw/day
-Scar:
-behind left ear and on right side scapula from Day 7 up to Day 27 or 34, 1/26 male at 50 mg/kg bw/day
-on right side scapula between Day 21 and 27, 1/26 female at 50 mg/kg bw/day
-Alopecia in female animals:
-1/24 control, 1/26 at 50 mg/kg bw/day, 2/23 at 150 mg/kg bw/day, 1/24 at 450 mg/kg bw/day during the gestation period and 2/24 control, 4/23 at 150 mg/kg bw/day and 1/24 at 450 mg/kg bw/day during the lactation period
-Soft swelling on pudenda 1/26 at 150 mg/kg bw/day from Day 76 to 82
These dermal changes and discoloration of hair are common findings in experimental rats of this strain with similar age. These were detected independently from doses in this study. Swelling at pudenda was an individual alteration occurring also in untreated rats and was due to the congenital absence of vaginal orifice (introitus vaginae). - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There was no test item related fatal death at any dose level (50, 150 or 450 mg/kg bw/day) during the course of the observation period. One male animal at 450 mg/kg bw/day was found dead on Day 64. Based on necropsy and histological findings, this animal died as a consequence of suffocation and shock.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weight development was depressed in a dose related manner in parental male animals at 150 and 450 mg/kg bw/day during the entire treatment period.
Slightly reduced mean body weight gain resulted in only minor changes in the mean body weight in male animals at 150 mg/kg bw/day (less than 5 % relative to control). Therefore, these findings were considered to have little or no toxicological relevance. There was no significant difference between the control and 50 mg/kg bw/day groups in the mean body weight of male animals during the entire observation period.
The mean body weight gain was lower than in the control at 50 mg/kg bw/day during the first week (Day 0-7). However, the summarized body weight gain was comparable to the control in male animals at 50 mg/kg bw/day.
The mean body weight and body weight gain were slightly lowered in male animals at 150 mg/kg bw/day comparing to the control during the observation period. Statistical significances with respect to the control were detected at the lower mean body weight (-4, -5 %) on Days 63, 69, 83, 97, 104 and 111, as well as at the lower mean body weight gain at 150 mg/kg bw/day between Days 0-7, 14-21, 28-35, 49-56, 97-104 and for the study overall (0-111) in male animals.
At 450 mg/kg bw/day, the mean body weight of male animals was significantly lower than in the control from Day 21 onwards (-5, -13 %) in consequence of the lower mean body weight gain during the entire observation period with statistical significance on week 1, 3, 4, 5, 8, 9, 10, 15, 16 and for the study overall. The mean body weight gain slightly exceeded the control in male animals at 450 mg/kg bw/day on week 11.
In the female animals at 50 and 150 mg/kg bw/day, the mean body weight was predominantly comparable with the control during the entire observation period. Statistically significant difference with respect to the control was observed occasionally at the lower mean body weight of female animals at 50 mg/kg bw/day on lactation day 14 and at 150 mg/ kg bw/day on gestation day 21 and on lactation day 0 when compared to the control.
At 450 mg/kg bw/day, the mean body weight of female animals exceeded the control (4-6 %) from Day 14 up to the end of pre-mating period and was lower than in the control on gestation day 21, lactation days 0, 4, 14 and 21 (up to -6 %),
Statistical significances were detected at the lower or higher mean body weight gain of female animals when compared to the control as follows:
-at 50 mg/kg bw/day: lowered mean body weight gain on week 6 and between lactation days 7-14
-at 150 mg/kg bw/day: higher mean body weight gain on weeks 1, 2 and 7, as well as lower mean body weight gain on week 6 and during 3rd week of gestation and for the entire gestation period (0-21, ca. 10%)
-at 450 mg/kg bw/day: higher mean body weight gain on weeks 1, 2 and for pre-mating period overall (Days 0-69) and lower mean body weight gain between gestation days 14 and 21 and during the entire gestation period (0-21, ca. 22%) - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- The mean daily food consumption was not adversely affected in parental male or female animals at 50, 150 or 450 mg/kg bw/day.
Slight, toxicologically not relevant changes in the mean daily food intake were in accordance with the body weight variations in male and female animals at each dose level and were not strictly dose related or consistent.
Statistical significance with respect to the control was detected at the lower mean daily food consumption of parental male animals at 50 mg/kg bw/day on weeks 1 and 3 (-4, -5%) and at 150 mg/kg bw/day on weeks 1, 3, 4 and 10 (-5, -19 %).
At 450 mg/kg bw/day, the mean daily food consumption was lower than in the control group in parental male animals on weeks 1, 2, 3, 7 and 10 (-6, -10 %).
In the female animals the mean daily food intake was comparable to the control at 50 mg/kg bw/day during pre-mating, gestation and lactation periods.
Higher mean daily food consumption was detected in female animals at 150 mg/kg bw/day on week 2 (6 %) and at 450 mg/kg bw/day on weeks 2, 5 and 8 (8, 13 %) when compared to the control.
Statistical significance with respect to the control was observed at the lower mean daily food consumption in the female animals at 150 mg/kg bw/day and at 450 mg/kg bw/day on gestation week 3 (-9 %, both) and between lactation day 4-7, on lactation weeks 2 and 3 (-8, -16 %).
These slight differences with respect to the control were of low degree and not consistent during the treatment period. Therefore, these were considered to be of low or no toxicologically relevance. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item-related adverse changes in the examined hematological or blood coagulation parameters in parental male or female animals at 50, 150 or 450 mg/kg bw/day. The examined hematological and blood coagulation parameters were comparable in the control, 50 mg/kg bw/day (female) and 150 mg/kg bw/day (male). Statistical significance with respect to the control was observed at lower mean percentage of eosinophil granulocytes (EOS) at 50 and 450 mg/kg bw/day and at the shorter mean prothrombin time (PT) at 450 mg/kg bw/day in male animals.
In female animals, statistical significance was detected at the higher mean percentage of neutrophil granulocytes (NEU) along with lowered mean percentage of lymphocytes (LYM) at 150 mg/kg bw/day and at the elevated mean percentage of reticulocytes (RET) at 450 mg/kg bw/day when compared to the control. The differences with respect to the control group reached statistical significance at the mentioned parameters. However, there were no dose relevance and the individual values met the historical control range (i.e., were within or close to the range) in male and female animals.
Therefore, the differences in these hematological parameters were considered to have little or no toxicological relevance. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical chemistry investigations revealed elevated activity of alanine aminotransferase (male and female), creatinine (male), urea (male and female) and cholesterol (female) at 450 mg/kg bw/day. Slight alterations in creatinine (male), cholesterol (female) indicated test item influence at 150 mg/kg bw/day in a lesser degree.
The examined clinical chemistry parameters were comparable with their control in male and female animals at 50 mg/kg bw/day.
Statistically significant differences with respect to the control were detected at the higher mean creatinine (CREA) and sodium (Na+) levels at 150 mg/kg bw/day and at the elevated mean activity of alanine aminotransferase (ALT), creatinine, urea and mean lowered mean concentrations of cholesterol, sodium and potassium (K+) at 450 mg/kg bw/day in male animals.
In the female animals at 150 mg/kg bw/day, the mean concentration of CREA and cholesterol (CHOL) was higher and the concentration of glucose (GLUC) was lower than in the control group.
At 450 mg/kg bw/day, higher mean activity of alanine aminotransferase, higher mean level of urea, cholesterol and potassium, lower mean concentration of total bilirubin (TBIL), creatinine and glucose were observed when compared to their control in female animals.
Although findings were of low degree, test item influence in the high dose animals is presumed. Elevated levels of creatinine and urea in the males may be based on renal lesions, as there was an indication for renal alterations in the same sex noted at the histopathological examinations. The increase in alanine aminotransferase in both sexes and in cholesterol of the females may indicate liver function alterations.
The differences to the control of the above-mentioned parameters reached statistical significance, the values of most of these parameters corresponded well to the historical control values – Na +, K+ in male animals, TBIL, CREA, GLUC, K+ in female animals. - Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals at any dose levels.
Neither a dose-dependent effect nor statistically significant differences were detected between the control and test item administered animals in the serum levels of FT3 and FT4. The FT3 and FT4 levels were comparable in male and female parental animals in the control, 50, 150 and 450 mg/kg bw/day groups.
Statistically significant difference was detected at the higher mean TSH level at 450 mg/kg bw/ in parental male animals. The degree of changes was minor and individual and mean values corresponds to the historical control ranges. In the absence of related changes in reproductive performance, organ weight or histopathology observations, these findings in female animals were considered to be toxicologically not relevant.
The cytomorphology of endocrine glands were the same in the control and treated animals. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The pH of urine was slightly lowered, protein and ketone were present in urine in male animals at 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations.
Statistical significances were detected at the lower mean pH of the urine in male animals at 450 mg/kg bw/day and in female animals at 150 mg/kg bw/day.
Ketone test was positive in 3/10 male animals at 150 mg/kg bw/day and 8/10 male animals at 450 mg/kg bw/day groups. Normally, ketones are totally resorbed by renal tubules. Elevated level in the urine may refer to tubular damage or exceedance of the tubular resorption threshold.
Proteinuria is well known in experimental rats; however, protein was detected with high incidence (100 %) in male animals at 450 mg/kg bw/day. - Behaviour (functional findings):
- not examined
- Immunological findings:
- effects observed, non-treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathology investigation did not reveal toxic or other test item-related lesions of investigated genital organs of the experimental male and female parental animals at 50, 150 or 450 mg/kg bw/day doses.
Renal changes – distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat – were detected only in male animals at 150 and 450 mg/kg bw/day (50 and 100% of examined animals, respectively). CPN is a spontaneous renal disease of laboratory rat with similar age, therefore 150 and 450 mg/kg bw/day doses of test item were assumed to be a predisposing factor in the development of pathogenesis of CPN in male animals.
In dead animal at 450 mg/kg bw/day (1/26 male), histological examination revealed acute alveolar emphysema and edema in the lungs (probably in connection with suffocation and shock), distended tubules with hyaline cats in the kidneys. No degenerative or other toxic lesions were observed in the investigated organs of this animal.
The investigated organs of the male reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all parental male animals in the control and 450 mg/kg bw/day groups (26/26 for both groups, including dead animal) as well as in not mated male (1/1) and in males not impregnated females (2/2) at 150 mg/kg bw/day. Sperm granuloma in one side epididymis (1/26 at 450 mg/kg bw/day, including dead animal) is a spontaneous individual disorder occurring in laboratory rats.
In the female animals at 450 mg/kg bw/day and control groups and in not mated (1/1), non-pregnant (2/2) female animals at 150 mg/kg bw/day of parental generation, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was detected in some female animals in each group: 5/26, 6/6, 2/4 and 2/26, respectively to groups of control, 50, 150 and 450 mg/kg bw/day. Dilatation of uterine horns is a slight neuro-hormonal phenomenon in connection with sexual function – pro-estrous phase – of the inner genital organs.
Some incidental individual findings – occurring commonly in experimental rats – were also detected histologically in female reproductive organs: cyst at the oviducts (1/26 at 450 mg/kg bw/day) or in the vagina (1/4 at 150 mg/kg bw/day), hemangioma in the uterus (1/26 at 450 mg/kg bw/day) and endometritis (1/26 at 450 mg/kg bw/day).
Tubular basophilia and lymphocytic infiltration were detected in one female animal at 450 mg/kg bw/day (1/26).
One or both sided renal pyelectasia occurred in each group of male and female animals independently from doses as follows:
-male 6/26 control, 3/26 at 50 mg/kg bw/day, 3/26 at 150 mg/kg bw/day, 9/26 at 450 mg/kg bw/day
-female: 2/26 control, 4/4 at 50 mg/kg bw/day, 2/2 at 150 mg/kg bw/day, 1/26 at 450 mg/kg bw/day
Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is common in laboratory rats of this strain and is considered to be an individual disorder without pathological significance.
Histopathology investigation also revealed spontaneous individual disorders, which are well-known lesions and commonly observed in experimental rats of this strain and not related to the test item:
-hepatic granuloma 1/3 male at 150 mg/kg bw/day
-vacuolation of hepatocytes in the liver 1/3 male at 150 mg/kg bw/day
-focal hyperplasia in the spleen 1/26 female at 450 mg/kg bw/day
-focal fibrosis in the spleen 1/1 female at 150 mg/kg bw/day
Focal fibrosis in the Glisson’s capsule of the liver develops due to the diaphragmatic hernia because of mechanical irritation (2/26 male control, 1/1 female at 50 mg/kg bw/day, 1/3 male and 1/1 female at 150 mg/kg bw/day,
The focal hemorrhage (1/26, 2/5, 3/6) and erosion (2/26, 2/5, 3/6, respectively to groups control, 50 and 150 mg/kg bw/day) in the stomach of female animals could be in connection with mechanical origin (gastric tube) and may be considered as individual disorder.
The atrophy of hair follicles (1/26 female control, 1/1 female at 150 mg/kg bw/day) is common finding in laboratory rats in accordance with dermal alopecia.
There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals. - Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The estrous cycle was not influenced by TBPND at 50, 150 or 450 mg/kg bw/day.
There were no statistically or biologically significant differences between the control and test item treated groups (50, 150 or 450 mg/kg bw/day) in the examined parameters of estrous cycle: the percentage of animals with regular cycles, the mean number of cycles, the mean length of cycles, mean number of days pro-estrous, estrous or diestrus were similar in all groups and there were no animals with prolonged estrous during the pre-mating period. - Reproductive function: sperm measures:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Sperm examinations did not reveal any test item-related influence on the sperm cells at 450 mg/kg bw/day.
Statistical significances were detected at the lowered mean percentage of immotile sperm cells at 450 mg/kg bw/day.
The mean percentage of sperms with normal morphology was similar in the control and 450 mg/kg bw/day groups.
Differences in the percentage of immotile sperm cells was considered to be indicative of biological variation and normal function of male genital organs. Since no test-item related influence on sperm examinations was detected at the highest dose the lower dose groups have not been examined. - Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The reproductive performance of male and female animals was not affected by the treatment at 50, 150 or 450 mg/kg bw/day with respect to their control.
Statistical significances detected at some parameters (copulatory, fertility and gestation indices, percentage of pregnant/ non-pregnant female animals, mean number of conceiving days) were indicative of biological variation and not related to the test item.
In the male animals, statistical significance with respect to the control was detected at the higher copulatory index (higher percentage of mated male animals) at 50 and 150 mg/kg bw/day and at the higher fertility index (higher percentage of fertile male and lower percentage of infertile animals) at 50 mg/kg bw/day.
In the female animals, the copulatory index (higher percentage of mated animals) exceeded the control in each group, while the fertility index (percentage of fertile animals and lower percentage of infertile animals) was above the control only at 50 mg/kg bw/day and the gestation index (percentage of dams delivered) was below the control at 450 mg/kg bw/day.
Statistical significance was also detected at the higher percentage of pregnant female animals at 50 mg/kg bw/day and lower mean number of conceiving days at 450 mg/kg bw/day when compared to their control.
The differences between the control and test item treated groups are without any biological relevance and lay well within the biological range of variation. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- reproductive performance
- Effect level:
- 450 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: highest dose tested
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- clinical biochemistry
- organ weights and organ / body weight ratios
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- urinalysis
- gross pathology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. The percentage of offspring showing signs (no milk in the stomach, cold, missing) was higher in litters of dams treated with 450 mg/kg bw/day than in the control. Most of these observations were detected on the day of delivery and corresponded with the findings of inadequate nursing behavior of the respective dams. Some other sporadic clinical signs were detected with similar incidence (smaller than normal, pale, hemorrhage, found dead) in the control and 50, 150 or 450 mg/kg bw/day dose groups.
There were no clinical signs in male or female animals in F1 Cohort 1A generation in 50, 150 or 450 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including post-natal day 91-97). The behavior and physical condition of all F1 Cohort 1A animals was normal at each dose level (50, 150 or 450 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
There were no test item related clinical signs in male or female animals in F1 Cohort 1B generation in 50, 150 or 450 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including post-natal day 96-105). Most of the male and female animals were normal in the control, 50, 150 and 450 mg/kg bw/day during the observation period. Alopecia was noted on the fore-limbs for only one female animal at 150 mg/kg bw/day from post-natal day 98 up to post-natal day 105. Alopecia on the skin is a species-specific finding detectable in non-treated experimental rats of this strain. Animals in the F1 Cohort 1B showed normal behavior and physical condition at each dose level (control, 50, 150 or 450 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period. Alopecia was detected on single female animal at 150 mg/kg bw/day on post-natal days 98 and 105 at the detailed weekly clinical observation, too.
Clinical signs were not detected in male or female animals in F1 Cohort 3 in control, 50, 150 or 450 mg/kg bw/day groups.
All animals in the F1 Cohort 3 showed normal behavior and physical condition at each dose level (control, 50, 150 or 450 mg/kg bw/day) based on the daily general and weekly detailed clinical observations during the entire treatment period. - Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- There was no test item-related effect on offspring’s extra uterine mortality. The extrauterine mortality was low and comparable in the control, 50, 150 and 450 mg/kg bw/day group on post-natal day 0 and from birth to post-natal day 21.
There was no mortality in F1 Cohort 1A animals in control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).
There was no test item related mortality in F1 Cohort 1B animals in 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).
One female animal at 150 mg/kg bw/day (1/20) died immediately after the daily administration on post-natal day 76 presumably due to a probably mis-gavage during the administration with gastric tube. No preceding clinical observations or body weight loss were observed.
No animal died in F1 Cohort 3 in the control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female). - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weight development of the F1 offspring was depressed at 50, 150 and 450 mg/kg bw/day. The reduction in body weight gain and body weight was with low degree but was consistent at 50 and 150 mg/kg bw/day and was therefore considered to be related to the test item. However, the values for 50 mg/kg bw/day were within the historical control ranges and therefore it was considered to have low or no toxicological relevance. The dose of 450 mg/kg bw/day significantly and consistently lowered the body weight and body weight gain of offspring from birth to post-natal day 21.Statistical significances with respect to the control were observed at the lower mean body weight and body weight gain of offspring predominantly at each measuring day at 50, 150 and 450 mg/kg bw from birth up to post-natal day 21. An exception is 50 mg/kg bw/day group, where the mean body weight exceeded the control on post-natal days 0 and 4 and the mean body weight gain was similar to the control between PND0 and 4. Similarly, the mean body weight was comparable with the control at 150 mg/kg bw/day at birth (PND0).When evaluating the body weight separately by gender, lowered mean body weight was detected at 150 mg/kg bw/day (male and female pups) and at 450 mg/kg bw/day (male and female pups) on PND4, when compared to their control. The mean litter weight and litter weight gain were comparable with control and 50 mg/kg bw/day groups during the 21-day observation period.
Statistical significance with respect to the control was detected at the lower mean litter weight at 150 mg/kg bw/day from post-natal day 7 up to PND21 (-8, -10 %) and lower mean litter weight gain from post-natal day 0 up to PND14 (-10, -16 %) and if summarized between post-natal day 0 and 21 (-9%). At 450 mg/kg bw/day, the mean litter weight and litter weight gain were significantly lower than in the control at each measuring occasion.
The body weight development was depressed in F1 Cohort 1A (male and female animals) at 450 mg/kg bw/day during the entire treatment period. Similar change was observed in the body weight at 150 mg/kg bw/day, however in a lesser degree however., Here, the difference from the control was lower than 10 % the and therefore, body weight changes in the mid dose group was considered to have little or no toxicological relevance.
Statistically significant difference with respect to the control were detected in the body weight and body weight gain per dose as follows:
Male animals
- 50 mg/kg bw/day: lower mean body weight on post-natal day 22 (-7 %), higher mean body weight gain between post-natal days 25-29
- 150 mg/kg bw/day: lower mean body weight on post-natal days 22, 25, 29, 36, 39, 63, 70, 77, 84, 91 (4-9 %), lower mean summarized body weight gain (between postnatal days 22 and 91)
- 450 mg/kg bw/day: lower mean body weight from post-natal day 22 up to and including post-natal day 91 (14-24 %) and lower mean body weight gain between post-natal days 22-36, 42-70, 77-91 and if summarized
-The changes in the body weight gain at 450 mg/kg bw/day resulted in significant changes in the body weight of male animals, and therefore, were considered to be toxicologically relevant.
Female animals:
-50 mg/kg bw/day: lower mean body weight on post-natal day 22 (-6 %);
-150 mg/kg bw/day: lower mean body weight on post-natal days 22, 25, (8-10 %),
-450 mg/kg bw/day: lower mean body weight from post-natal day 22 up to and including post-natal day 91 (9-27 %) lower mean body weight gain between post-natal days 22-25, 25-29 and higher mean body weight gain between post-natal days 36-39
These minor changes in the body weight gain observed for females resulted in significant changes in the body weight, however, were not consistent and therefore, were considered to be toxicologically not relevant.
The mean body weight was reduced in F1 Cohort 1B at 150 mg/kg bw/day (male) and at 450 mg/kg bw/day (male and female) during the entire treatment period (from post-natal day 22 up to post-natal day 98). The difference from the control remained consistent in male animals and lowered in female animals by the end of treatment period. The difference from the control was lower than 10 % at 150 mg/kg bw/day and therefore, body weight changes in the mid dose group were considered to have little or no toxicological relevance. The mean body weight was comparable in male and female animals in the control and 50 mg/kg bw/day body during the entire observation period. Statistical significances with respect to the control was detected at the lower mean body weight of male animals at 150 and 450 mg/kg bw/day in a dose related manner from weaning up to the termination of the treatment period. The body weight gain was also reduced in male animals at 150 and 450 mg/kg bw/day (-8% and -19% between PND22 and 98, respectively) and was comparable with control in female animals at 450 mg/kg bw/day, if summarized (between PND22 and 98).
Changes in the mean body weight gain comparing to the control showed sporadic occurrence in male animals as follows:
-50 mg/kg bw/day: lowered between PND49-56, elevated between PND56-63
-150 mg/kg bw/day. lowered between PND49-56 and if summarized (between PND22-98)
-450 mg/kg bw/day: lowered during the entire observation period reaching statistical significances between PND22-36, each occasion, between 42-56, 63-70 and if summarized
In the female animals, the mean body weight was statistically significantly lower than in the control group at 150 mg/kg bw/day on PND22 and at 450 mg/kg bw/day at each occasion between PND22 and 98.
The following statistically significant differences with respect to the control were detected in the mean body weight gain in the female animals:
-50 mg/kg bw/day: higher mean body weight gain between PND56-63
-150 mg/kg bw/day: lower mean body weight gain between PND49-56
-450 mg/kg bw/day: lowered between PND22-29, elevated between PND39-42 and between PND 91-98
The mean body weight was reduced in F1 Cohort 3 at 450 mg/kg bw/day (male and female) during the entire treatment period (from post-natal day 22 up to post-natal day 56). The mean body weight was mostly comparable in male animals in the control, 50 and 150 mg/kg bw/day body during the entire observation period. The mean body weight was lower than in the control in male animals at 50 mg/kg bw/day on starting day (PND22) and the mean body weight gain exceeded the control at 150 mg/kg bw/day between post-natal day 25-29.
Statistical significances with respect to the control was detected at the lower mean body weight of male animals at 450 mg/kg bw/day from weaning up to the termination on post-natal day 56. The mean body weight gain of these animals was also lowered when compared to control between post-natal days 22-25, 25-29, 29-32, 32-36, 42-49, 49-56 and if summarized between post-natal days 22-56. In the female animals, the mean body weight was similar in the control and at 50 mg/kg bw/day during the observation period and the mean body weight gain was higher than in the control between post-natal days 36-39. At 150 mg/kg bw/day, the mean body weight was lowered comparing to the control in female animals on post-natal days 22, 25 and the mean body weight gain was comparable with that of the control group during the entire observation period. At 450 mg/kg bw/day, the mean body weight remained below the control between post-natal day 22-56 and the body weight gain was lower than in the control between post-natal day 22-25, 25-29 and was higher than in the control between post-natal days 32-36, 36-39 and 39-42. Thus, the summarized body weight gain was comparable with the control in female animals administered with the high dose of the test item. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- The mean daily food consumption of F1 Cohort A male animals was reduced at 450 mg/kg bw/day in accordance of body weight changes during the observation period. The mean daily food consumption was comparable with the control in all treated female animals and in male animals at 50 and 150 mg/kg bw/day during the entire observation period.At 450 mg/kg bw/day, statistically significant difference with respect to the control was detected at the lower mean daily food consumption in male animals by weekly interval between post-natal days 22-91 (-7, -29 %, no statistical significance between PND70-77) and in female animals on weeks 1 and 2 (-30, -15 %, respectively). Transient difference with respect to the control in female animals at 450 mg/kg bw/day was judged to be toxicologically not relevant.
The mean daily food consumption was reduced in male animals at 150 and 450 mg/kg bw/day during the entire treatment period and in female animals at 450 mg/kg bw/day during the course of the first two weeks of the treatment in F1 Cohort 1B. The difference from the control was -9 up to -33% in male animals at 450 mg/kg bw/day. The difference from the control was minor (≤11 %) in male animals at 150 mg/kg bw/day and was transient in female animals therefore these were considered to have little or no toxicological significance. The mean daily food consumption was similar to their control in male animals of F1 Cohort 1B at 50 mg/kg bw/day during the entire observation period (between PND22 and PND98). A lowered mean daily food consumption was observed compared to control in male animals at 150 and 450 mg/kg bw/day during the entire study with statistical significance between PND22-29, at each occasion between PND42-77 and PND84-91 in mid dose group and at each occasion between PND22-77 and PND91-98 in high dose treated animals. In the female animals, the mean daily food consumption was higher than in the control group at 50 mg/kg bw/day between PND56-63 and PND 91-98 and at 150 mg/kg bw/day between PND91 and 98. At 450 mg/kg bw/day, the mean daily food consumption of female animals was lower than in the control group between PND22-29 and PND29-36 and was higher than in the control between PND91-98.
The mean daily food consumption was lowered at 450 mg/kg bw/day during the first two weeks of the treatment in female animals and during the entire treatment period in female animals. The mean daily food consumption was mostly similar to their control in male and female animals of F1 Cohort 3 at 50 and 150 mg/kg bw/day during the entire observation period (between PND22 and PN56). Statistical significance with respect to the control was only detected at the higher mean daily food intake of female animals at 150 mg/kg bw/day between post-natal 36-42. At 450 mg/kg bw/day, the mean daily food consumption remained below the control between post-natal days 22 and 56 in male animal and between post-natal days 22 and 36 in female animals. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item-related adverse changes in the examined hematological and blood coagulation parameters in male or female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1A. Statistical significance with respect to the control was detected at the elevated percentage of neutrophil granulocytes (NEU) along with lowered percentage of lymphocytes (LYM) and lower mean hemoglobin concentration (HGB) in male animals at 50 mg/kg bw/day. At 150 mg/kg bw/day, the mean hemoglobin concentration and hematocrit (HCT) were lower than in the control in male animals. In the female animals, the mean white blood cell count was higher at 50, 150 or 450 mg/kg bw/day and the mean prothrombin time (PT) and activated partial thromboplastin time (APTT) were slightly shorter when compared to the control. The minor changes noted in the hematological and blood coagulation parameters were considered to have little or no toxicological relevance. Statistical significances at NEU, LYM, HGB and HCT were detected at the lower dose group but not at high dose treated animals. White blood cell count was relatively low in the female control group resulting statistical significances in all treated groups. Decreased in blood coagulation times has no toxicologic meaning. All individual values were either close to or within the historical control range.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Pathologic alterations were not detected at the evaluation of clinical chemistry parameters in F1 Cohort 1A male or female animals at 50, 150 or 450 mg/kg bw/day.
An elevated mean concentration of creatinine may be associated with the renal changes in male animals at 450 mg/kg bw/day.
In the male animals, higher mean activity of alanine aminotransferase (ALT) was detected in all treatment groups being statistically significant at 150 and 450 mg/kg bw/day compared to the control. The concentrations of creatinine (CREA) and UREA exceeded the control in male animals at 450 mg/kg bw/day.
The examined clinical chemistry parameters were comparable in the female animals in the control, 50 and 150 mg/kg bw/day groups.
Statistical significance with respect to the control was detected at the higher mean activity of alanine aminotransferase and higher mean concentration of total bilirubin (TBIL) and urea in female animals at 450 mg/kg bw/day.
The individual values of most of these parameters corresponded well to the historical control values – ALT, UREA, TBIL – in male or female animals. Elevation in alanine aminotransferase activity may indicate liver function alterations in both sexes and has no toxicological relevance in the absence of histological changes.
Higher concentration of creatinine and urea in the males may be based on renal lesions, as there was an indication for renal alterations detected at the histopathological examinations.
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in male or female animals in the F1 Cohort 1A at 50, 150 or 450 mg/kg bw/day.
In the male animals, the thyroid hormone concentrations were mostly comparable with their control at each dose level. Statistical significance was only detected at the slightly lower mean FT4 level at 450 mg/kg bw/day. Individual values were close or within the historical control of FT4 .
In the female animals, statistical significances with respect to the control were detected at the higher mean concentrations of FT3 at 50 and 150 mg/kg bw/day and of FT4 at 50, 150 and 450 mg/kg bw/day. In the lack of dose relevancy and related findings (macroscopic or microscopic changes in pituitary or thyroid gland, changes on estrous cycle) a relation to treatment is unlikely. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The examined urine parameters were predominantly comparable in the F1 Cohort 1A male and female animals in the control and 50, 150 and 450 mg/kg bw/day groups. However, the pH of urine was slightly but statistical significant lowered and ketone was present in urine in male animals at 150 and 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations.
Ketone test was positive in 7/10 male animals at 150 mg/kg bw/day and 9/10 male animals and 1/10 female at 450 mg/kg bw/day groups. Normally, ketones are totally resorbed by renal tubules. Elevated level in the urine may refer to tubular damage or exceedance of the tubular resorption threshold.
These minor changes were considered to have little or no toxicological significance as vales of pH in male animals remained within the control ranges. - Sexual maturation:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The sexual maturity was not adversely affected in F1 male or female animals at 50, 150 or 450 mg/kg bw/day (Cohort 1A, Cohort 1B, Cohort 3, N=60).
- Anogenital distance (AGD):
- effects observed, non-treatment-related
- Nipple retention in male pups:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item related changes were observed in the weights of kidneys in male animals and in the liver in female animals at 450 mg/kg bw/day in F1 Cohort 1A. The changes in kidneys weights were supported by histological findings (chronic progressive nephropathy) in male animals at 150 and 450 mg/kg bw/day. However, there were no accompanying histological lesions in the kidneys at the low dose administered male animals or in female animals at any dose level, therefore, elevation in the kidneys or liver weights may be interpreted as an adaptational phenomenon.
In the male animals at 50 mg/kg bw/day, the mean weights of kidneys exceeded the control and the mean weights of submandibular lymph nodes were lower than in the control (absolute and relative to body and brain weights, for both organs).
At 150 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean fasted body weight in males (absolute and relative to brain weight), at the higher mean weights of kidneys (relative to body and brain weights) and at the lower mean pituitary weights (absolute and relative to brain weight). The weight of thymus (absolute) was lower, while the weights of several organs relative to body weight was higher than in the control (brain, liver, spleen, testes, epididymides, thyroid glands) in consequence of the lowered mean body fasted weight of animals in this group.
At 450 mg/kg bw/day, lower mean fasted body weight (absolute and relative to brain weight), higher mean weights of kidneys (absolute and relative to body and brain weights) and liver (relative to body and brain weights), lower mean thymus weights (absolute and relative to brain weight) were observed in males when compared to their control.
In relation to the lower mean fasted body weight in male animals at 450 mg/kg bw/day, statistical significances with respect to the control was detected at the lower mean absolute organ weights (seminal vesicles, prostate, pituitary), at lower mean absolute weights along with higher body weight related mean organ weights (brain, heart, testes, epididymides) at higher mean organ weight relative to brain and body weights (heart, spleen, thyroid gland, popliteal lymph nodes and adrenal glands).
In the female animals at 50 mg/kg bw/day, the mean weights of liver (absolute and relative to body and brain weights) were elevated with respect to their control.
At 150 mg/kg bw/day, statistical significance with respect to the control was observed at lower mean fasted body weight, higher mean liver and heart weights (relative to body and brain weights, both organs), higher mean kidney weights relative to body weight and at the lower mean weight of ovaries (absolute).
At 450 mg/kg bw/day, statistical significance with respect to the control was observed at lower mean fasted body weight and brain weight (absolute for both organs), at higher mean liver and heart weights (absolute and relative to body and brain weights, both organs), at the lower mean weight of thymus and pituitary (absolute, both) in the female animals. The mean thyroid weight (relative to body weight), the mean weights of kidneys, ovaries, spleen and adrenal glands (relative to body and brain weight, each) also exceeded the control value in female animals at 450 mg/kg bw/day.
The most of statistically significant differences with respect to the control were due to the lowered body weight of male animals at 150 mg/kg bw/day and in male and female animals at 450 mg/kg bw/day as well as to the lowered brain weight in male and female animals at 450 mg/kg bw/day. Accompanying morphological changes were not detected at the histopathological examination and hematology investigations. Clinical chemistry parameters did not reveal test item-related abnormalities. Therefore, changes in these organ weights were judged to have little or no toxicological relevance due to the minor degree and in the lack of dose dependency (where relevant) or associated histopathological alterations.
The weights of the examined organs were not adversely affected in male and female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1B.
In the male animals, the weights of all examined organs were comparable with the control at 50 mg/kg bw/day. At 150 mg/kg bw/day, the mean fasted body weight and the mean thymus weight (absolute and relative to brain weight, both) were lower and the mean brain weight relative to body weight was higher than in the control. In the male animals at 450 mg/kg bw/day, lower mean fasted body weight (absolute and relative to brain weight), lower mean weights (absolute and relative to brain weight, each) of thymus, prostate and pituitary were observed when compared to the control. The mean weights (absolute) of brain, testes, epididymides, seminal vesicles were below the control and was higher than in the control if referred to body weight in case of brain, spleen and testes in male animals administered with high dose. In the female animals at 50 mg/kg bw/day, statistical significances with respect to the control was detected at the higher mean weights of uterus (absolute and relative to body and brain weights) and ovaries (absolute and relative to brain weight). At 150 mg/kg bw/day, elevated mean weights of spleen and ovaries both relative to body and brain weights were observed when compared to the control. Statistical significances with respect to the control was detected at the lower mean fasted body weight, at lower mean weights of brain and pituitary (absolute), at higher mean spleen weights (relative to body and brain weights) and higher mean weights of ovaries (absolute and relative to body and brain weights) in female animals at 450 mg/kg bw/day.
Minor changes in thymus weights in male animals at 150 and 450 mg/kg bw/day may be due to a slightly enhanced involution of the organ or to body and brain weight changes. The lowered fasted body weight and brain weight may result in statistical significances in the absolute and relative organ weights both in male and female animals (brain, spleen, testes, epididymides, seminal vesicles, prostate, pituitary, ovary, uterus). The values were close or within the historical control ranges. Therefore, these minor changes were considered to be toxicologically not relevant. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no test item related specific macroscopic findings in offspring subjected to early necropsy (before weaning) or terminally (at the weaning).
Before the weaning, pups were subjected to necropsy because of death (stillborn and pups found dead) or after euthanasia on PND4 (surplus pups). Gas content (1/35) or milk (2/35) in the stomach at 50 mg/kg bw/day, autolyzed organs (2/43) at 150 mg/kg bw/day, pale body (4/47) and pale lungs (1/47), not cleaned pup with umbilical cord (1/47) at 450 mg/kg bw/day were detected. In offspring subjected to necropsy at weaning, both sided pyelectasia was observed in single pup at 150 mg/kg bw/day (1/69). Pyelectasia is a common finding in this strain of experimental rat with similar age.
Macroscopic alterations related to the effect of the test item were detected in the kidneys in male animals at 450 mg/kg bw/day in Cohort 1A (paleness, white area between the cortex and medulla) at the necropsy.
A test item effect may be supposed but cannot be proved with the results of this study in development of yellowish deposition on serous membrane of several organs in the thoracic cavity in male or female animals at 50, 150 or 450 mg/kg bw/day.
These necropsy observations were in full accordance with histopathological findings.
Male
-control: right or both sided pyelectasia (9/20)
-50 mg/kg bw/day: right or both sided pyelectasia (10/20), pale kidneys (1/20) yellowish deposition at bifurcation of the lungs (1/20), on lower part or right side of thymus (2/20) or on the muscle on larynx/ trachea (1/20)
-150 mg/kg bw/day: right or both sided pyelectasia (7/20); thickened bifurcation in lungs (1/20), brownish-red colored thymus (1/20), hard knot in the thymic tissue (1/20), hernia diaphragmatica with small part of liver (1/20 two peppers-sized) and yellow lentil sized subcutaneous formula at sternum (1/20);
-450 mg/kg bw/day: right or left sided pyelectasia (10/20), pale kidneys (5/20), white area between renal cortex and medulla (4/20), yellowish deposition on the lungs (1/20), thymus (1/20) and on ribs under lungs (1/20), adhesion of lungs to the diaphragm (1/20), hard knot in the thymic tissue (1/20), larger than normal kidneys (1/20)
Female
-control: right or both sided pyelectasia (5/20), slight, moderate or marked hydrometra (6/20);
-50 mg/kg bw/day: one or both sided pyelectasia (6/20), slight, moderate or marked hydrometra (7/20), adhesion of lungs to ribs and white granules on the surface (1/20), yellow deposit on the right upper side of thymus (1/20), white granules on the surface of diaphragm (1/20)
-150 mg/kg bw/day: right or both sided pyelectasia (10/20), slight, moderate or marked hydrometra (8/20), yellowish thick deposit on the lungs (1/20), brownish red colored thymus (1/20), greyish-yellow hard formation in the thymus (1/20), yellowish thick deposit on the wall of heart (1/20)
-450 mg/kg bw/day: right or both sided pyelectasia (4/20), slight, moderate or marked hydrometra (6/20), yellowish deposition in one (1/20, each) or two animals on the following organs: esophagus, trachea, thoracic aorta, thorax, thymus (2/20), adhesion of thoracic aorta to spine (1/20), adhesion of lungs to ribs (1/20) and diaphragm (1/20), brownish-red colored thymus (1/20), thickened spleen with rounded margins (1/20) and thickened thymus (left side, 1/20)
Renal alterations (paleness, white area between the cortex and medulla) in male animals and yellowish deposition on several organs in the thoracic cavity in male and female animals were in full accordance with histopathological findings. Some findings (adhesions, thickening, knots or different formations) on thoracic organs were consequences of accumulation of yellow or grayish yellow tissue. Regarding the incidence of these findings in groups, a test item influence cannot be verified or excluded with the results of this study. Yellowish deposition was only detected in test item administered animals independently from doses.
Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, these findings were considered as individual lesion without toxicological significance. Hernia diaphragmatica were individual alterations and are considered to be also species-specific change and not treatment-related.
Splenic signs, brownish-red thymus and cyst near to sternum in individual animals were considered to be incidental individual findings.
Necropsy observations revealed test item-related alterations in the kidneys in male animals at 150 and 450 mg/kg bw/day in Cohort 1B. Yellow deposition and adhesion of organs mainly in the thoracic cavity were observed only in single animals at 50 and 450 mg/kg bw/day.
Dead animal
Brownish-red colored thymus and pea-sized erosion on the fundic part of the stomach were observed in dead female animal (1/1) at 150 mg/kg bw/day.
Surviving animals
In the male animals, necropsy observations revealed the following findings:
-control group: brownish-red thymus (1/20), right or both sided pyelectasia in the kidneys (8/20) and
-50 mg/kg bw/day: one or both sided pyelectasia in the kidneys (9/20)
-150 mg/kg bw/day: one or both sided pyelectasia in the kidneys (10/20), pale color of kidneys and white area between the renal cortex and medulla (1/20);
-450 mg/kg bw/day: right or both sided pyelectasia in the kidneys (10/20); pale color of kidneys (7/20), white area between the renal cortex and medulla (2/20) and swelling of kidneys (1/20), adhesion of right lung-lobe to heart and thymus by a yellowish deposition and thickened pericardium and adhesion to heart muscle (1/20), Hernia diaphragmatica with a part of liver (1/20), smaller than normal testes and epididymides (1/20)
In the female animals, necropsy observations revealed the following findings:
-control group: brownish-red thymus (1/20), one or both sided pyelectasia in kidneys, (4/20), slight, moderate or marked hydrometra (4/20)
-50 mg/kg bw/day: right or both sided pyelectasia in the kidneys (7/20), moderate or marked hydrometra (9/20), yellowish deposition on left lobe of thymus, on lungs, liver, spine and adhesion of liver to diaphragm in one animal (1/20)
-150 mg/kg bw/day: brownish-red thymus (1/19), both sided pyelectasia in the kidneys (6/19), slight, moderate or marked hydrometra (7/19), alopecia on the fore-limbs (1/19), pea-sized erosion on the fundic part of the stomach (1/19)
-450 mg/kg bw/day: right or both sided pyelectasia in the kidneys (5/20), slight, moderate or marked hydrometra (10/20), Hernia diaphragmatica with a part of liver (1/20),
Yellow deposition caused adhesion of organs in the thoracic cavity in one male animal at 450 mg/kg bw/day and in one female animal at 50 mg/kg/bw/day the latter extending to surface of liver.
Pyelectasia, hydrometra and alopecia are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with clear liquid content) related to the female sexual cycle, is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item-related as no dose response was noted.
Brownish-red colored thymus is also frequently observed in experimental rats in connection with the exsanguination procedure.
Hernia diaphragmatica is a developmental disorder occurring commonly in untreated experimental rats, too.
Erosion of the stomach mucosa and smaller than normal testes and epididymides (supported by organ weights) were considered to individual findings. - Histopathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathological examinations revealed signs of chronic progressive nephropathy (distended tubules with hyaline casts, tubular basophilia or lymphocytic and histiocytic infiltration) in male animals at 150 and 450 mg/kg bw/day and accumulation of brown fatty tissue with mineral deposits in male and female animals at 50, 150 and 450 mg/kg bw/day in Cohort 1A.
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands, ovaries, uterus, vagina) were histologically normal and characteristic for the sexually mature organism in all of examined male and female animals in Cohort 1A.
Quantitative analysis of ovaries verified normal structure and function of the organ (primordial, primary, secondary and tertiary follicles, follicular atresia and corpora lutea) in the control and high dose treated animals in F1 Cohort 1A.
The following histological findings were detected in male and female animals per group:
Male animals:
-control group: one or both sided pyelectasia (9/20)
-50 mg/kg bw/day: one or both sided pyelectasia (8/20), accumulation of brown fatty tissue with mineral deposits (2/2)
-150 mg/kg bw/day: distended renal tubules with hyaline casts (6/20), one or both sided pyelectasia (7/20), accumulation of brown fatty tissue with mineral deposits (1/1), dermoid cyst at sternum region (1/1)
-450 mg/kg bw/day: distended renal tubules with hyaline casts (18/20), tubular basophilia (16/20), lymphocytic and histiocytic infiltration (14/20), one or both sided pyelectasia (11/20), accumulation of brown fatty tissue with mineral deposits (2/2)
Female animals:
-control group: one or both sided pyelectasia (6/20), dilatation of uterine horns (6/20)
-50 mg/kg bw/day: one or both sided pyelectasia (5/5), accumulation of brown fatty tissue with mineral deposits (2/2), dilatation of uterine horns (6/6)
-150 mg/kg bw/day: one or both sided pyelectasia (10/10), accumulation of brown fatty tissue with mineral deposits (2/2), dilatation of uterine horns (8/8)
-450 mg/kg bw/day: one or both sided pyelectasia (3/20), accumulation of brown fatty tissue with mineral deposits (3/3), splenic hyperplasia (1/20), thymic congestion (1/20), dilatation of uterine horns (6/20)
Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ in the control and high dose treated animals. The mean number of primordial and primary follicles and corpora lutea was similar in the control and at 450 mg/kg bw/day. Slight but statistically significant difference with respect to the control was detected at the slightly lowered mean number of secondary and tertiary follicles and follicular atresia at 450 mg/kg bw/day.
These findings were considered to be variation and not related to the test item. Individual values were comparable in the control and high dose treated animals.
theThe histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
The severity and incidence of renal lesions (distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat) were related to doses of 150 and 450 mg/kg bw/day in male animals and were not present in male animals in control or low dose group or in female animals at any dose level in F1 Cohort 1A. CPN is a spontaneous renal disease of laboratory rat with similar age, therefore 150 and 450 mg/kg bw/day doses of test item were assumed to be a predisposing/ accelerating factor in the development of pathogenesis of CPN in male animals. Accumulation of brown fatty tissue with mineral deposits were detected in some male and female animals in each dose group independently from doses with different localization, mainly on the surface of serous membranes of thoracic organs (thymus, lung, pharynx, thoracic pleura, sternum, diaphragm, bifurcation region of the lungs). The appearance was demarcated from the surrounding organs or tissues, and the accumulated adipose tissue was demarcated with serous membrane as well. The structure was built by brown fat cells, smaller than white fat cells, and the intercellular connective tissue consisted of fibrocytes, collagen and reticular fibers and capillaries. Scattered basophilic mineral deposits occurred in the intercellular connective tissue. Mononuclear cells and separately giant cells were seen around the deposits. No necrosis, fibrosis, abscess formation or malignant tumor cells with high mitotic activity were observed. The focal accumulation of brown adipose tissue with mineral deposits – regarding its macroscopic appearance, and the presence of mineral deposits, the mononuclear cell infiltrates and the giant cells, - is considered as a pathophysiologic phenomenon, in contrast with the normal brown adipose tissue. The pathogenesis of this phenomenon may be accidental, but the predisposing role of test item cannot be excluded in the development of this lesion. Dilatation of uterine horns is a slight neuro-hormonal phenomenon in connection with sexual function – pro-estrous phase – of the inner genital organs. Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is common in laboratory rats of this strain and is considered to be an individual disorder without pathological significance.
Some spontaneous individual disorders were also detected at the histopathology investigation – splenic hyperplasia, thymic congestion – which are well-known lesions and commonly observed in experimental rats of this strain and not related to the test item: There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system. The cytomorphology of endocrine glands were the same in the control and treated animals. - Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Estrous Cycle: The estrous cycle was not affected in the F1 Cohort 1A female animals at 50, 150 or 450 mg/kg bw/day.The examined parameters of the estrous cycle – percentage of animals with regular or irregular cycles, number of cycles, length of cycles, number of days in pro-estrous, percentage of animals in prolonged estrous/ diestrus – were comparable in the control and 50, 150 or 450 mg/kg bw/day groups. The mean number of irregular cycles was slightly higher in the 450 mg/kg bw/day group but this value lies within the historical control data. The mean number of days in estrous was slightly lower and the mean number of days in diestrus was slightly higher than in the control group at 450 mg/kg bw/day. These minor differences from control were judged to be biological variation and lie in the range of the historical control data and independent from the test item.
The estrous cycle was not adversely affected in the F1 Cohort 1B female animals at 50, 150 or 450 mg/kg bw/day.Statistical significances were observed at the lower mean number of estrous cycle at 150 and 450 mg/kg bw/day and at the lower mean number of days in estrous in female animals 150 mg/kg bw/day when compared to control. These minor differences with respect to the control in the mid and high dose groups were independent from doses and were considered to be indicative of biological variation as values were close or within the historical control. Therefore, these changes have no toxicological relevance.
Sperm Examinations: Sperm examinations did not reveal any test item-related influence on the sperm cells at 450 mg/kg bw/day.
The mean percentage of motile, immotile sperms, sperms with normal morphology and separated head and tail were comparable in the control and 450 mg/kg bw/day groups. Since no effects have been observed, the low and mid dose groups were not analyzed. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Splenic Lymphocyte Subpopulation Analysis: There were no test item-related changes in the splenic lymphocyte subpopulations in male or female animals in F1 Cohort 1A animals in 50, 150 or 450 mg/kg bw/day groups. The percentage of T lymphocytes (T cells), B lymphocytes (B cells), Natural killer (NK) cells, helper T cells and cytotoxic cells were predominantly comparable in male and female animals in the control, 50, 150 and 450 mg/kg bw/day groups. Statistical significance with respect to the control was only detected at the slightly elevated mean number of NK cells in male animals at 450 g/kg bw/day.
This minor change was considered to be indicative of biological variation as all mean values met well or were close to the historical control ranges. Some outlier individual vales (1-3 animals/ group) were probably individual variations.
Cohorte 3: No anti-IgM antigen peak response after immunization with KLH (Keyhole limpet hemocyanin) was detected on day 6 in the control animals due to technical reasons. A validation study was performed for the ELISA Assay (Study number 392-504-6274), which showed that the assay as such detected standard rat KLH IgM according to the established protocol. The same ELISA protocol was followed for the rat samples in the main study. In a pre-liminary study the antibody peak was observed 6 days after subcutaneous immunization in Han:WIST rats (no report available). A different batch of KLH was used for immunization in the main study for Cohort 3 animals, which might be the reason for the non-response which
was observed. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- development
- Generation:
- F1
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- other: pinna detachment
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Generation:
- F1
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Generation:
- F1
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The main study according to OECD TG 443 was performed in Han:Wist rats including Cohort 3 (ECHA request, CCH-D-2114493102-56-01/F). Based on the results of the pre-study the parent (P) generation was dosed 10 weeks prior to mating with dosages of 50, 150 or 450 mg/kg bw/day. The reproductive performance was not impaired in male or female rats after administration of TBPND via oral gavage. No treatment-related changes were noted in any of the reproductive parameters investigated (i.e. mating and fertility indices, sperm parameters, precoital time, number of implantations, estrous cycle). Test-item related systemic effects in the body weight, food consumption and in the kidneys- related parameters (urine and clinical chemistry, organ weights, macroscopic and microscopic findings) were observed in high dose parental male animals. Signs of systemic effect manifested in changes and of clinical chemistry parameters and increased liver weight in parental female animals in the high dose group.
In F1 offspring (pre-weaning), depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. No treatment-related effect on anogenital distance, areola/nipple retention, surface righting reflex and thyroid hormone levels (FT4 and TSH in PND 22 pups) were detected in F1 offspring.
In F1 adult (F1 Cohort 1A, Cohort 1B, Cohort 3, post-weaning) at 450 mg/kg bw/day reduction in body weight and food consumption (Cohorts 1A, 1B and 3) in male or female animals and kidneys related parameters (urine and clinical chemistry parameters, organ weight, macroscopic and microscopic findings) in male animals of F1 generation (Cohorts 1A) was detected. No treatment-related effects were recorded for developmental parameters in F1-animals, including balano-preputial separation, vaginal opening, occurrence of first estrus, sperm parameters, ovarian follicle counts and histopathology of reproductive organs.
At 150 mg/kg bw/day of male animals in F1 generation (Cohorts 1A and B), body weight and kidney-related findings were observed in a lesser degree. No test item-related adverse effects were detected at 50 mg/kg bw/day in F1 adults (Cohorts 1A, 1B and 3).
No treatment-related immunotoxic effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day - Executive summary:
The subject of this study was to investigate the reproductive toxicity of TBPND by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for using the rat, which is the preferred rodent species for reproduction toxicity testing. The aim of the extended one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item TBPND on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered via oral gavage to animals at doses of 50, 150 and 450 mg/kg bw/day compared to control animals. The effect of the test item on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) was investigated. Furthermore, the effect on offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A, 1B and Cohort 3) to adulthood following oral (by gavage) administration was the main focus of this study. Four groups of Han:WIST rats (n= 26/sex/group) were administered with the test item via oral gavage once a day at 0 (vehicle), 50, 150 and 450 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 225 mg/mL in parental generation in a volume of 2 mL. Control animals received only vehicle, sunflower oil, in an identical manner. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Analysis of formulations used for administration were performed six times during the study. TBPND concentrations in the formulations varied within the range of 98 % and 109 % in comparison to the nominal values and samples were homogenous.
All animals of the parent (P) generation were dosed for 10 weeks prior to mating and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 112, 113 or 114 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 114-125 days). Not delivered, not mated and non-pregnant females were administered for 99 or 104 days.
Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Clinical pathology examinations - urinalysis, hematology, blood coagulation, clinical chemistry - were conducted in randomly selected ten male and ten female animals from each group (parental animals and Cohort 1A). Estrous cycle was monitored in parental animals by examining vaginal smears before the mating for two weeks and during the mating period until evidence of copulation and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum. All F1 offspring were observed individually for health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention). Twenty F1 animals/sex/group were randomly selected for Cohort 1A, Cohort 1B and Cohort 3 in the control, low, mid and high dose groups on post-natal day 21 for follow-up examinations. Dosing of F1 offspring selected for follow-up examinations begun on post-natal day 22 and treatment was continued up to the day before the necropsy. F1 adult animals in Cohort 1A and Cohort 1B were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology (Cohort 1A only) and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputial separation, vaginal patency (Cohort 1A, Cohort 1B, Cohort 3) and appearance of first cornified vaginal smear (Cohort 1A).
Cohort 1A animals were subjected to necropsy, organ weighing, sperm analysis and immunotoxic examinations – one day after the termination of the exposure – on PND92-98.
Cohort 1B animals were subjected to necropsy and organ weighing on PND97-106.
Cohort 3 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen were administered and observed up to and including the day before blood sampling and euthanasia on PND61. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P, male and female) animals/sex/ group at termination, in surplus pups at PND4 (pooled by litters), from F1 pups not selected for cohorts on PND22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring. All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter. Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals at necropsy. Selected organ weights were determined in adult animals (P, F1). Sperm parameters were determined in all control and high dose male animals in P generation and in all control and high dose male animals F1 generation (Cohort 1A). At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group. Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. The kidneys were also examined histologically in all parental and F1 Cohort 1A male animals (control, low, mid and high dose group) on the basis of necropsy observation. In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A).Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups. A quantitative evaluation of follicles (primordial, primary, secondary and tertiary follicles, follicular atresia) as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 450 mg/kg bw/day groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.
Results
Analytical control of dosing formulations
TBPND concentrations in the samples varied within the range of 98 % to 109 % in comparison to the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.
Parental (P) generation
Mortality
There was no test item related fatal death in parental animals at any dose level (50, 150 or 450 mg/kg bw/day) during the course of the observation period.
One male animal at 450 mg/kg bw/day was found dead presumably due to suffocation and shock on Day 64.
Clinical observation
Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day). Salivation followed test item administration was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration. Therefore, salivation was judged to be toxicologically not relevant in this study.
The behavior and physical condition of all male and female animals was normal in each group at the detailed weekly observations.
Body weight and body weight gain
The body weight development was depressed in a dose related manner in parental male animals at 150 and 450 mg/kg bw/day during the entire treatment period. The body weight reduction was less than 5 % relative to control in male animals at 150 mg/kg bw/day, therefore, was judged to be toxicologically not relevant.
Food consumption
The mean daily food consumption was not adversely affected in parental male or female animals at 50, 150 or 450 mg/kg bw/day during the entire treatment period.
Estrous cycle
The estrous cycle was similar in the control and 50, 150 and 450 mg/kg bw/day groups during the two weeks observation period.
Delivery data of dams
There were no toxicologically relevant differences in the evaluated parameters of pregnancy and delivery in female animals between the control and 50, 150 or 450 mg/kg bw/day groups.
Reproductive performance
The reproductive performance of male and female animals was normal (the same as in not treated animals of this strain of experimental animals) in control, 50, 150 and 450 mg/kg bw/day groups.
Urinalysis
Urinalysis revealed changes in male urine (pH, protein and ketone body), which were probably related to renal tubular changes in male animals at 150 and 450 mg/kg bw/day.
Hematology and blood coagulation
There were no toxic changes in the examined hematology and blood coagulation or clinical pathology parameters in parental male or female animals at 50, 150 or 450 mg/kg bw/day.
Clinical chemistry
Minor changes in creatinine (male) and cholesterol (female) concentrations at 150 mg/kg as well as elevated activity of alanine aminotransferase (male and female), higher concentrations in creatinine (male), cholesterol (female) and urea (male and female) at 450 mg/kg bw/day referred to a test item influence on renal and/or hepatic function.
Thyroid hormones
The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.
Necropsy
Gross necropsy observations revealed test item related changes in the kidneys (pale, enlarged, soft) in majority of parental male animals at 450 mg/kg bw/day.
The findings of dead animal refer to systemic changes caused by suffocation and acute stress supported by histopathological findings.
Organ weight
Elevated weights of kidneys in male animals refer to test item influence in accordance with macroscopic or histopathology findings at 450 mg/kg bw/day and in a lesser degree at 150 mg/kg bw/day. Test item related changes were observed in the elevated weights of the liver in female animals at 450 mg/kg bw/day.
Sperm examinations
Sperm examinations did not reveal any test item-related damage in the sperm cell morphology and motility, and total sperm count in parental male animals at 450 mg/kg bw/day.
Histopathology
The cell morphology of examined reproductive organs (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental animals in control and 450 mg/kg bw/day including not mated, non-pregnant and not delivered female animals and their mating partners and in mid dose, too. Test item related renal changes – distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat – were detected only in male animals at 150 and 450 mg/kg bw/day (50 and 100% of examined animals, respectively) in a dose related manner.
Offspring
In F1 offspring pre-weaning, depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. However, this is considered secondary to maternal toxicity and as a nonspecific effect. No treatment-related effect on anogenital distance, areola/nipple retention, surface righting reflex and thyroid hormone levels (FT4 and TSH in PND 22 pups) were detected in F1 offspring.
F1 adult generation (Cohort 1A, Cohort 1B and Cohort 3)
Mortality
There was no test item related mortality in F1 Cohort 1A, Cohort 1B or Cohort 3 animals in control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).
In F1 Cohort 1B, one female animal at 150 mg/kg bw/day (1/20) died due to a probably mis-gavage during the administration with gastric tube on post-natal day 76.
Clinical observation
The behavior and physical condition of all animals was normal at each dose level (50, 150 or 450 mg/kg bw/day) in F1 Cohort 1A, Cohort 1B and Cohort 3 based on the daily and weekly detailed clinical observations during the entire treatment period.
Body weight and body weight gain
The body weight development was depressed in F1 Cohort 1A, Cohort 1B and Cohort 3 (male and female animals) at 450 mg/kg bw/day during the entire treatment period.
Food consumption
The mean daily food consumption of male animals was reduced at 450 mg/kg bw/day F1 Cohort 1A, Cohort 1B Cohort 3.
Sexual maturity
The sexual maturity was similar to control in male or female animals in F1 Cohort 1A, 1B and Cohort 3 at 50, 150 and 450 mg/kg bw/day.
Estrous cycle
The estrous cycle was comparable with their control in the F1 Cohort 1A, and Cohort 1B female animals at 50, 150 and 450 mg/kg bw/day.
Urinalysis
The pH of urine was slightly lowered and ketone bodies were present in urine in male animals at 150 and 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations in F1 Cohort 1A.
Hematology and blood coagulation, clinical chemistry
Pathologic alterations were not detected at the evaluation of hematology and blood coagulation parameters in F1 Cohort 1A male or female animals at 50, 150 or 450 mg/kg bw/day.
Clinical chemistry
A slightly higher mean creatinine level may be related to the renal changes in male animals at 450 mg/kg bw/day in F1 Cohort 1A.
Thyroid hormones
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in male or female animals in the F1 Cohort 1A at 50, 150 or 450 mg/kg bw/day.
Necropsy
Macroscopic alterations related to the effect of the test item were detected in the kidneys in male animals at 450 mg/kg bw/day in Cohort 1A, Cohort 1B (paleness, white area between the cortex and medulla) at the necropsy.
A test item effect may be supposed in development of yellowish deposition on serous membrane of several organs in the thoracic cavity in some male or female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1A and Cohort 1B.
Organ weight
Test item related changes were observed in the weights of kidneys in male animals and in the liver in female animals at 450 mg/kg bw/day in F1 Cohort 1A. The weights of the examined organs were not adversely affected in male or female animals at any dose level in F1 Cohort 1B.
Sperm examinations
Sperm morphology and motility, and total sperm count were similar to their control in male animals at 450 mg/kg bw/day in F1 Cohort 1A.
Histopathology
Histopathological examinations revealed chronic progressive nephropathy (distended tubules with hyaline casts, tubular basophilia or lymphocytic and histiocytic infiltration) in male animals at 150 and 450 mg/kg bw/day and accumulation of brown fatty tissue with mineral deposits in male and female animals at 50, 150 and 450 mg/kg bw/day in Cohort 1A.
Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ in the control and high dose treated animals.
Splenic lymphocyte subpopulation analysis
There were no test item-related changes in the splenic lymphocyte subpopulations in male or female animals in F1 Cohort 1A animals in 50, 150 or 450 mg/kg bw/day groups.
Developmental immunotoxicity testing
No anti-IgM antigen peak response after immunization was detected on day 6 in the control animals due to technical reasons. A validation study was performed for the ELISA Assay, which showed that the assay as such detected standard rat KLH IgM according to the established protocol. The same ELISA protocol was followed for the rat samples in the main study. In a pre-liminary study the antibody peak was observed 6 days after subcutaneous immunization in Han:WIST rats (no report available). A different batch of KLH was used for immunization in the main study for Cohort 3 animals, which might be the reason for the non-response which was observed. No treatment-related immunotoxic effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day
Referenceopen allclose all
There was no test item related mortality during the course of study.
Clinical Observations
Daily Observations
Test item related salivation was observed in animals of all dosed groups (12/12 male and 12/12 female at 600 mg/kg bw/day; 12/12 male and 12/12 female at 200 mg/kg bw/day and 11/12 male, 5/12 female at 60 mg/kg bw/day) in a dose related manner regarding the degree, onset and frequency. Marked salivation only occurred in the high dose treated animals and only slight degree of salivation was noted for the low dose treated animals. The frequency of observation was the highest in 600 mg/kg bw/day group. Male animals proved to be more sensitive than females as number of animals showing salivation and frequency of observation were higher in males than in females at 60 mg/kg bw/day.
Alopecia was noted on the left forelimb for one male animal (1/12) dosed with 60 mg/kg bw/day from day 13 up to the termination; on the left side of the back for one female (1/12) at 60 mg/kg bw/day and on the limbs and abdomen for single control female animal (1/12) during the gestation and lactation period. Alopecia is a common finding in this strain of experimental rats and was present in the control and low dose only therefore had no toxicological meaning in this study. For detail please refer to tables in the attachment.
Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period (pre-mating, mating and post-mating).
Alopecia as described above was observed at detected at the weekly observations, too.
Functional Observations
Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (600, 200 and 60 mg/kg bw/day, control).
Body Weight
A test item related depression of the body weight development was detected in male and female animals at 600 and 200 mg/kg bw/day with respect to controls.
In the male animals dosed with 600 mg/kg bw/day, the body weight gain was less than in the control group with statistical significances on weeks 1, 2 and 6, thus the total body weight gain remained below control value, too. The reduced body weight gain resulted in a slightly but statistically significantly lower body weight values during the study from day 13 (days 13, 20, 27, 34 and 40) with respect to controls.
In the females, a less body weight gain was observed during the premating and gestation period with respect to control with statistical significances at the summarized body weight gain during the premating, on gestation weeks 2 and 3, and the total body weight gain during the gestation period. The body weight remained below the control value on gestation day 21 and during lactation period (lactation days 0 and 4).
At 200 mg/kg bw/day, in the male animals the mean body weight gain was significantly less than in the control between day 27 and 40 and if summarized (between day 0 and 40).
In the females, significantly less body weight gain was found during the premating period (no statistical significance on week 1) and the mean body weight was also lower than in the control on lactation days 0 and 4.
The body weight development was slightly less than in the control in male animals at 60 mg/kg bw/day between days 27 and 34 however there was no significant difference in the body weight and in the mean summarized body weight gain comparing to the control.
In the females, the mean body weight and body weight gain was similar to that of the control group during the entire observation period (premating, gestation and lactation). For detail please refer to tables in the attachment.
Food Consumption
A test item influence on the mean daily food consumption was observed in male and female animals at 600 and 200 mg/kg bw/day. The statistical significances indicated only slight differences between the control and 200 mg/kg bw/day groups and were not considered to be biologically significant. The mean daily food consumption was lower than in the control group at 600 and 200 mg/kg bw/day doses, during the premating (male), during first week of premating (female), on gestation weeks 2 and 3.
In male animals, the higher mean food consumption at 600 mg/kg bw/day between days 27 and 34 and the less mean food consumption at 60 mg/kg bw/day between days 7 and 14 were not judged to be toxicologically significant.
Hematology
There were no test item related changes in the examined hematological parameters in male animals at any dose level. Statistically significant differences between the control and dosed groups in some hematological parameters (neutrophil granulocytes (NEU), lymphocytes (LYM), monocytes (MONO), eosinophil granulocytes (EOS) and activated partial thromboplastin time (APTT) were not considered toxicologically relevant as these were with low magnitude and values remained well within the historical control ranges.
In the female animals, the hemoglobin concentration (HGB) and hematocrit vale (HCT) were slightly below the control value at 600 mg/kg bw/day and the percentage of reticulocytes (RET) was higher than in the control group at 600, 200 and 60 mg/kg bw/day (latter without statistical significance).
For detail please refer to tables in the attachment.
Clinical chemistry
In the male animals dosed with 600 mg/kg bw/day, a slightly higher mean activity of alanine-aminotransferase (ALT) and higher mean concentration of creatinine (CRE), inorganic phosphorous (Pi) and potassium (K+), less activity of aspartate-aminotransferase (AST) and concentration of total bilirubin (TBIL), calcium (Ca2+) were observed. No statistical significance was noted for the mean urea concentrations, although it was higher than the control value and also exceeded the historical control range.
In the female animals of this group, significantly higher activity of alanine-aminotransferase and alkaline phosphatase (ALP), higher concentration of urea and potassium were detected.
At 200 mg/kg bw/day, in male animals a less mean concentration of total bilirubin and potassium, in female animals a higher activity of alanine-aminotransferase and higher concentration of urea and cholesterol (CHOL) was observed.
In group of 60 mg/kg bw/day, a slightly less activity of aspartate-aminotransferase and less concentration of total bilirubin was noted for male animals, while in female there were no statistically significant changes with respect to controls, however the alanine-aminotransferase activity slightly exceeded the control value (no statistical significance).
In summary, the higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and concentration of creatinine and urea were also indicative of the test item effect.
The statistically significant differences in levels of some parameters were judged to be of little or no biological significance as the mean values were within or marginal to the historical control ranges (inorganic phosphorous, potassium, alkaline phosphatase) or these were without any dose-response relationship (total bilirubin, calcium, cholesterol and aspartate aminotransferase). For detail please refer to tables in the attachment.
Necropsy
Enlarged (5/12) and pale (3/12) kidneys reflected a test item influence in male animals dosed with 600 mg/kg bw/day. In one male animal (1/12) pyelectasia occurred.
Hydrometra (indicative of sexual cycle of female animals) is a frequent observation in experimental rats, was present in one high dose treated animal (1/12) and has no toxicological meaning without pathological changes.
Alopecia was an individual change occurring in single animals of 60 mg/kg bw/day (1/12, male, 1/11 dams) and control (1/12) groups. For detail please refer to tables in the attachment.
Organ weight
In the male animals, the liver weights were higher than in the control at 600 mg/kg bw/day (absolute and relative to body and brain weights) and at 200 mg/kg bw/day (relative to body weight). The kidney weights (absolute and relative to body and brain weights) also exceeded the control value at 600 and 200 mg/kg bw/day. The fasted body weight, the thymus weights (absolute and relative to body and brain weights) and adrenal weights (absolute and relative to brain weight) were less than in the control at 600 and 60 mg/kg bw/day, respectively. Spleen (200 mg/kg bw/day) and testes (600 and 200 mg/kg bw/day) weights relative to body weigh were above the control value.
In the female animals, liver weights were higher than the control at 600 and 200 mg/kg bw/day (absolute and relative to body and brain weights) and at 60 mg/kg bw/day (relative to body weight). Statistical significances were also noted for less fasted body weight, body weight relative to brain weight, and brain weight relative to body weight, kidney weights relative to body weight at 600 and 200 mg/kg bw/day and heart weight relative to body weight at 600 mg/kg bw/day.
In summary, a test item related higher liver and kidney weights were found at 600 and 200 mg/kg bw/day with a difference between the genders and in compliance with changes in clinical chemistry parameters. Changes noted for the liver weight were present both in males and females at 600 mg/kg bw/day and in females at 200 mg/kg bw/day. Alterations in kidney weights at 600 and 200 mg/kg bw/day were pronounced in male animals, however it was only present in the kidney weight relative to body weight in females in which the fasted body weight was slightly (but statistically significantly) less with respect to controls, therefore a test item influence in females may be questionable.
The liver weight relative to body weight of female animals administered with 60 mg/kg bw/day was slightly above the control value in compliance with the slight changes of alanine aminotransferase activities.
The lower thymus weights (absolute and relative to body and brain weights) at 600 mg/kg bw/day might be also related to test item.
The significantly higher organ weights of heart, spleen and testes relative to body weight with respect to controls were considered to be independent from the test item, these were probably due to the slightly lower body weight values moreover no supporting findings were found in clinical pathology, necropsy or histopathology observations. For detail please refer to tables in the attachment.
Histopathology
Hyaline-like droplets in the epithelial cells of some proximal convoluted tubules (12/12, 12/12, 11/12), segmental tubular basophilia (10/12, 10/12, 3/12) accompanied with slight inter-tubular lymphocytic infiltration and dilatation of tubuli in the distal area at the border of cortical - medullary region (12/12, 8/12, 1/12) were observed in male animals in all dose groups (600, 200 and 60 mg/kg bw/day, respectively). Minimal alveolar emphysema (2/5, 2/5) and mild hyperplasia of bronchus associated lymphoid tissue (BALT; 0/5, 2/5) were detected in the lungs of examined male animals in 600 mg/kg bw/day and control group, respectively.
The evaluated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all investigated male animals of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. The histological picture of epididymides and pituitary was normal in all cases as well.
Minimal alveolar emphysema (2/5) and mild hyperplasia of bronchus associated lymphoid tissue (1/5) in the lungs and dilatation of uterus (1/12) were detected in female animals of the control group.
The ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation, the epithelial capsule and ovarian stroma were normal in all cases as well.
The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. The histological picture of pituitary was normal as well in the case of treated and control animals.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed (male and female).
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals (male and female). For detail please refer to tables in the attachment.
In summary, test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group as compared with the middle or high dose groups.
Hyaline droplet nephropathy and “hydrocarbon nephropathy” are terms that describe a spectrum of morphologic changes in the kidneys of male rats induced by a variety of structurally related and unrelated compounds. There is abnormal accumulation of α-2µ-globulin phagolysosomes of tubular epithelium. One proposed mechanism suggests that the chemical or a metabolite binds with the α-2µ-globulin - which is only present in the male species - or alters the structure of the protein so that the tubular cell lysosomal enzymes cannot degrade the protein complex. In this case the observed nephropathy is specific to the male rat and has no relevance to humans. Other proposed mechanisms include a direct cytotoxic effect. It is unlikely that the various chemicals associated with hyaline droplet nephropathy in the male rat act by the same mechanism. Some chemicals which produce hyaline droplet nephropathy in male rats produce renal toxicity (unassociated with α-2µ-globulin) in female rats, whereas other chemicals produce no effects in the kidney of female rats.
The focal alveolar emphysema in the lungs occurred sporadically and was considered as consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. The hyperplasia of bronchus associated lymphoid tissue is a physiological phenomenon occurred in some control and treated animals. Uterus dilatation – without inflammation or other pathological lesion – is a physiological phenomenon in connection with the normal sexual cycle.
Delivery Data of Dams
The number and percentage of post-implantation loss and stillborns, duration of pregnancy was higher and the number of live-born pups was less than the appropriate values of control group at 600 mg/kg bw/day. Although there were no statistically significant differences with respect to control, the mean of post-implantation loss and total intrauterine mortality, mean number of stillborns per litter, number of dams with prolonged pregnancy were higher, and the mean of total births per litter, mean number of live-borns per litter and viable pups per litter, were less than in the control at 600 mg/kg bw/day.
Statistical significances noted for the higher number and percent of implantations and less number and percent of pre-implantation loss were not relevant toxicologically in the 200 and 60 mg/kg bw/day groups and for total intrauterine mortality in the 200 mg/kg bw/day group.
Reproductive Performance
There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance. The copulatory and fertility indices were similar at each dose level. Spermatogenesis in the rate male appeared unaffected by the test item.
There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval were similar to or were the same as in the control group in all test item treated groups.
However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns, increased extra uterine mortality and higher numbers of dams with prolonged duration of pregnancy.
The number and percentage of extra uterine mortality was slightly higher than in the control group in 600 mg/kg bw/day group between postnatal days 0 and 4. The mean number of dead pups was also higher than in the control group. The survival indices were similar between all groups (600, 200 and 60 mg/kg bw/day and control groups).
The higher mean of female viable pups at 60 mg/kg bw/day and litter mean has no toxicological significance.
Sex Distribution
There were no significant differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4
Clinical Observations
Test item related clinical signs did not appear in the pups. The number and percent of cold and not suckled pups were slightly higher than in the control group at 600 and 200 mg/kg bw/day but these were considered to be independent from the treatment as these signs occur in not treated animals with similar percentage. For detail please refer to tables in the attachment.
Body Weight
A test item influence on the litter weight and litter weight gain and pup’s mean weight and weight gain were detected at 600 and 200 mg/kg bw/day.
The mean litter weight was significantly less than in the control group at 600 mg/kg bw/day on postnatal day 4, and the litter weight gain between days 0 and 4 was also less than in the control at 600 and 200 mg/kg bw/day. The mean pup weight on days 0 and 4 as well as the mean weight gain of pups between days 0 and 4 were significantly less than in the control group at 600 and 200 mg/kg bw/day.
The mean litter weight and mean pup weight were similar in the control and test item treated groups, no test item related changes were found. For detail please refer to tables in the attachment.
Necropsy
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination. More specifically, no signs of a teratogenic effect of the test item were noted and no structural or visceral malformations were observed in the offspring during the macroscopic examination. For detail please refer to tables in the attachment.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 450 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- OECD TG 443/GLP
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
OECD TG 422 (2012)
The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the toxic potential of TBPND and on its possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 60, 200 and 600 mg/kg bw/day at concentrations of 30, 100 and 300 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 8, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups.
The first five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, haematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on days 4 – 9 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. The kidneys of all animals were also processed and evaluated histologically due to macroscopic observations at the necropsy. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The reproductive organs and pituitary of non-pregnant female animal and male cohabited with in the low dose group were also processed and evaluated histologically.
The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.
There was no test item related mortality at any dose level (600, 200 and 60 mg/kg bw/day). Test item related salivation appeared in male and female animals administered with 600, 200 and 60 mg/kg bw/day groups in all with a dose related degree, onset and incidence. No toxic signs related to the test item were found at the detailed weekly and terminal functional observations. The behaviour and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods). The body weight gain was reduced with respect to controls in male and female animals at 600 and 200 mg/kg bw/day resulting in a slightly less body weight at 600 mg/kg bw/day in male animals from day 13 up to the termination and in female animals on gestation day 21 and lactation days 0 and 4. The summarized body weight gain also remained below the control value in male animals administered with 600 and 200 mg/kg bw/day and in females at 600 and 200 mg/kg bw/day during the premating and at 600 mg/kg bw/day during the gestation period. The mean daily food consumption was less comparing to the control group at 600 and 200 mg/kg bw/day doses during the premating (male), during first week of premating (female), on gestation weeks 2 and 3. Hematology examinations revealed test item related higher percentage of reticulocytes in female animals administered with 600, 200 and 60 mg/kg bw/day and less hemoglobin concentration and hematocrit value at 600 mg/kg bw/day with respect to controls. A higher mean activity of alanine aminotransferase and mean concentrations of urea referred to a test item influence on renal and/or hepatic function in female animals dosed with 600 mg/kg bw/day and 200 mg/kg bw/day. In male animals dosed with 600 mg/kg bw/day, the slightly elevated mean activity of alanine aminotransferase and higher concentration of creatinine and urea were also indicative of the test item
Test item related renal changes (enlarged and pale kidneys) were observed in male animals dosed with 600 mg/kg bw/day. Specific macroscopic alterations related to the test item were not found in female animals during the necropsy. Higher kidneys weights of male animals administered 600 and 200 mg/kg bw/day and higher liver weights in males at 600 mg/kg bw/day, in females at 600, 200 and 60 mg/kg bw/day reflected a test item influence on renal and hepatic functions in accordance with clinical chemistry and necropsy findings. Test item related renal lesions (hyaline-like droplets in the epithelial cells of some proximal convoluted tubules, segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration and dilatation of tubuli in the distal part of tubuli) resembling on the “hyaline droplet nephropathy of male rats” were observed in all test item treated male groups (600, 200 and 60 mg/kg bw/day). The severity of lesions was less in the low dose group with respect to the middle or high dose groups. Hyaline droplet nephropathy was associated with interference to α-2µ-globulin. In this case the observed nephropathy is specific to the male rat and has no relevance to humans.
There were no differences between the control and test item treated groups in the reproductive ability of male and female animals. Spermatogenesis in the rat male appeared unaffected by the test item. However, a test item influence on the dam’s delivery appeared with respect to the controls at 600 mg/kg bw/day with a higher percentage of post-implantation loss and stillborns and higher numbers of dams with prolonged duration of pregnancy
A test item effect on the offspring development was observed in the higher number and percentage of extra uterine mortality in 600 mg/kg bw/day group between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight and weight gain at 600 and 200 mg/kg bw/day.
Under the conditions of the study, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected. Dam’s delivery was affected by the test item at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed. At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day.
Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:
NOEL for male rats: < 60 mg/kg bw/day
NOAEL for male rats: 60 mg/kg bw/day
NOAEL for female rats: 60 mg/kg bw/day
NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day
NOAEL for F1 Offspring: 60 mg/kg bw/day
Modified OECD 421 (2021)
A modified OECD 421 study in rats was performed. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 150 and 300 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.
All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.
There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behaviour and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.
The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.
The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.
The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats:150 mg/kg bw/day
NOAEL for F1 Offspring: 50 mg/kg bw/day
OECD TG 443 (2022)
The subject of this study was to investigate the reproductive toxicity of TBPND by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for using the rat, which is the preferred rodent species for reproduction toxicity testing. The aim of the extended one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item TBPND on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered via oral gavage to animals at doses of 50, 150 and 450 mg/kg bw/day compared to control animals. The effect of the test item on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) was investigated. Furthermore, the effect on offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A, 1B and Cohort 3) to adulthood following oral (by gavage) administration was the main focus of this study. Four groups of Han:WIST rats (n= 26/sex/group) were administered with the test item via oral gavage once a day at 0 (vehicle), 50, 150 and 450 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 225 mg/mL in parental generation in a volume of 2 mL. Control animals received only vehicle, sunflower oil, in an identical manner. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Analysis of formulations used for administration were performed six times during the study. TBPND concentrations in the formulations varied within the range of 98 % and 109 % in comparison to the nominal values and samples were homogenous.
All animals of the parent (P) generation were dosed for 10 weeks prior to mating and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 112, 113 or 114 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 114-125 days). Not delivered, not mated and non-pregnant females were administered for 99 or 104 days.
Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Clinical pathology examinations - urinalysis, hematology, blood coagulation, clinical chemistry - were conducted in randomly selected ten male and ten female animals from each group (parental animals and Cohort 1A). Estrous cycle was monitored in parental animals by examining vaginal smears before the mating for two weeks and during the mating period until evidence of copulation and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum. All F1 offspring were observed individually for health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention). Twenty F1 animals/sex/group were randomly selected for Cohort 1A, Cohort 1B and Cohort 3 in the control, low, mid and high dose groups on post-natal day 21 for follow-up examinations. Dosing of F1 offspring selected for follow-up examinations begun on post-natal day 22 and treatment was continued up to the day before the necropsy. F1 adult animals in Cohort 1A and Cohort 1B were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology (Cohort 1A only) and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputial separation, vaginal patency (Cohort 1A, Cohort 1B, Cohort 3) and appearance of first cornified vaginal smear (Cohort 1A).
Cohort 1A animals were subjected to necropsy, organ weighing, sperm analysis and immunotoxic examinations – one day after the termination of the exposure – on PND92-98.
Cohort 1B animals were subjected to necropsy and organ weighing on PND97-106.
Cohort 3 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen were administered and observed up to and including the day before blood sampling and euthanasia on PND61. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P, male and female) animals/sex/ group at termination, in surplus pups at PND4 (pooled by litters), from F1 pups not selected for cohorts on PND22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring. All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter. Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals at necropsy. Selected organ weights were determined in adult animals (P, F1). Sperm parameters were determined in all control and high dose male animals in P generation and in all control and high dose male animals F1 generation (Cohort 1A). At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group. Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. The kidneys were also examined histologically in all parental and F1 Cohort 1A male animals (control, low, mid and high dose group) on the basis of necropsy observation. In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A).Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups. A quantitative evaluation of follicles (primordial, primary, secondary and tertiary follicles, follicular atresia) as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 450 mg/kg bw/day groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.
Results
Analytical control of dosing formulations
TBPND concentrations in the samples varied within the range of 98 % to 109 % in comparison to the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.
Parental (P) generation
Mortality
There was no test item related fatal death in parental animals at any dose level (50, 150 or 450 mg/kg bw/day) during the course of the observation period.
One male animal at 450 mg/kg bw/day was found dead presumably due to suffocation and shock on Day 64.
Clinical observation
Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day). Salivation followed test item administration was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration. Therefore, salivation was judged to be toxicologically not relevant in this study.
The behavior and physical condition of all male and female animals was normal in each group at the detailed weekly observations.
Body weight and body weight gain
The body weight development was depressed in a dose related manner in parental male animals at 150 and 450 mg/kg bw/day during the entire treatment period. The body weight reduction was less than 5 % relative to control in male animals at 150 mg/kg bw/day, therefore, was judged to be toxicologically not relevant.
Food consumption
The mean daily food consumption was not adversely affected in parental male or female animals at 50, 150 or 450 mg/kg bw/day during the entire treatment period.
Estrous cycle
The estrous cycle was similar in the control and 50, 150 and 450 mg/kg bw/day groups during the two weeks observation period.
Delivery data of dams
There were no toxicologically relevant differences in the evaluated parameters of pregnancy and delivery in female animals between the control and 50, 150 or 450 mg/kg bw/day groups.
Reproductive performance
The reproductive performance of male and female animals was normal (the same as in not treated animals of this strain of experimental animals) in control, 50, 150 and 450 mg/kg bw/day groups.
Urinalysis
Urinalysis revealed changes in male urine (pH, protein and ketone body), which were probably related to renal tubular changes in male animals at 150 and 450 mg/kg bw/day.
Hematology and blood coagulation
There were no toxic changes in the examined hematology and blood coagulation or clinical pathology parameters in parental male or female animals at 50, 150 or 450 mg/kg bw/day.
Clinical chemistry
Minor changes in creatinine (male) and cholesterol (female) concentrations at 150 mg/kg as well as elevated activity of alanine aminotransferase (male and female), higher concentrations in creatinine (male), cholesterol (female) and urea (male and female) at 450 mg/kg bw/day referred to a test item influence on renal and/or hepatic function.
Thyroid hormones
The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.
Necropsy
Gross necropsy observations revealed test item related changes in the kidneys (pale, enlarged, soft) in majority of parental male animals at 450 mg/kg bw/day.
The findings of dead animal refer to systemic changes caused by suffocation and acute stress supported by histopathological findings.
Organ weight
Elevated weights of kidneys in male animals refer to test item influence in accordance with macroscopic or histopathology findings at 450 mg/kg bw/day and in a lesser degree at 150 mg/kg bw/day. Test item related changes were observed in the elevated weights of the liver in female animals at 450 mg/kg bw/day.
Sperm examinations
Sperm examinations did not reveal any test item-related damage in the sperm cell morphology and motility, and total sperm count in parental male animals at 450 mg/kg bw/day.
Histopathology
The cell morphology of examined reproductive organs (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental animals in control and 450 mg/kg bw/day including not mated, non-pregnant and not delivered female animals and their mating partners and in mid dose, too. Test item related renal changes – distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat – were detected only in male animals at 150 and 450 mg/kg bw/day (50 and 100% of examined animals, respectively) in a dose related manner.
Offspring
In F1 offspring pre-weaning, depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. However, this is considered secondary to maternal toxicity and as a nonspecific effect. No treatment-related effect on anogenital distance, areola/nipple retention, surface righting reflex and thyroid hormone levels (FT4 and TSH in PND 22 pups) were detected in F1 offspring.
F1 adult generation (Cohort 1A, Cohort 1B and Cohort 3)
Mortality
There was no test item related mortality in F1 Cohort 1A, Cohort 1B or Cohort 3 animals in control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).
In F1 Cohort 1B, one female animal at 150 mg/kg bw/day (1/20) died due to a probably mis-gavage during the administration with gastric tube on post-natal day 76.
Clinical observation
The behavior and physical condition of all animals was normal at each dose level (50, 150 or 450 mg/kg bw/day) in F1 Cohort 1A, Cohort 1B and Cohort 3 based on the daily and weekly detailed clinical observations during the entire treatment period.
Body weight and body weight gain
The body weight development was depressed in F1 Cohort 1A, Cohort 1B and Cohort 3 (male and female animals) at 450 mg/kg bw/day during the entire treatment period.
Food consumption
The mean daily food consumption of male animals was reduced at 450 mg/kg bw/day F1 Cohort 1A, Cohort 1B Cohort 3.
Sexual maturity
The sexual maturity was similar to control in male or female animals in F1 Cohort 1A, 1B and Cohort 3 at 50, 150 and 450 mg/kg bw/day.
Estrous cycle
The estrous cycle was comparable with their control in the F1 Cohort 1A, and Cohort 1B female animals at 50, 150 and 450 mg/kg bw/day.
Urinalysis
The pH of urine was slightly lowered and ketone bodies were present in urine in male animals at 150 and 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations in F1 Cohort 1A.
Hematology and blood coagulation, clinical chemistry
Pathologic alterations were not detected at the evaluation of hematology and blood coagulation parameters in F1 Cohort 1A male or female animals at 50, 150 or 450 mg/kg bw/day.
Clinical chemistry
A slightly higher mean creatinine level may be related to the renal changes in male animals at 450 mg/kg bw/day in F1 Cohort 1A.
Thyroid hormones
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in male or female animals in the F1 Cohort 1A at 50, 150 or 450 mg/kg bw/day.
Necropsy
Macroscopic alterations related to the effect of the test item were detected in the kidneys in male animals at 450 mg/kg bw/day in Cohort 1A, Cohort 1B (paleness, white area between the cortex and medulla) at the necropsy.
A test item effect may be supposed in development of yellowish deposition on serous membrane of several organs in the thoracic cavity in some male or female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1A and Cohort 1B.
Organ weight
Test item related changes were observed in the weights of kidneys in male animals and in the liver in female animals at 450 mg/kg bw/day in F1 Cohort 1A. The weights of the examined organs were not adversely affected in male or female animals at any dose level in F1 Cohort 1B.
Sperm examinations
Sperm morphology and motility, and total sperm count were similar to their control in male animals at 450 mg/kg bw/day in F1 Cohort 1A.
Histopathology
Histopathological examinations revealed chronic progressive nephropathy (distended tubules with hyaline casts, tubular basophilia or lymphocytic and histiocytic infiltration) in male animals at 150 and 450 mg/kg bw/day and accumulation of brown fatty tissue with mineral deposits in male and female animals at 50, 150 and 450 mg/kg bw/day in Cohort 1A.
Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ in the control and high dose treated animals.
Splenic lymphocyte subpopulation analysis
There were no test item-related changes in the splenic lymphocyte subpopulations in male or female animals in F1 Cohort 1A animals in 50, 150 or 450 mg/kg bw/day groups.
Developmental immunotoxicity testing
No anti-IgM antigen peak response after immunization with KLH (Keyhole limpet hemocyanin) was detected on day 6 in the control animals due to technical reasons. A validation study was performed for the ELISA Assay, which showed that the
assay as such detected standard rat KLH IgM according to the established protocol. The same ELISA protocol was followed for the rat samples in the main study. In a pre-liminary study the antibody peak was observed 6 days after subcutaneous immunization in Han:WIST rats. A different batch of KLH was used for immunization in the main study for
Cohort 3 animals, which might be the reason for the non-response which was observed.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day
Effects on developmental toxicity
Description of key information
The test substance was examined for its possible prenatal developmental toxicity in rats and rabbits. Based on these observations in the study with rats, the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60 mg/kg bw/day NOAEL (developmental toxicity): 60 mg/kg bw/day NOAEL (teratogenicity): 200 mg/kg bw/day (high dose). In the study with rabbits, the NOAEL for maternal toxicity was 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day based on the effects (abortions) in the 300 and 100 mg/kg bw/day dose groups.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-05-11 to 2016-09-02
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2001
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: Young adult and nulliparous females, 11-12 weeks of age at start of the mating period
Males: experienced males, 38-39 weeks of age at start of the mating period
- Fasting period before study: None
- Housing: Before mating: 1-3 females per cage 1-2 males per cage
Mating: 1 male and 1-3 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: Animals will receive ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m. - Route of administration:
- oral: gavage
- Vehicle:
- other: sunflower oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 10 mg/mL, 30 mg/mL and 100 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before administration and were stored in the refrigerator (5 +/- 3°C) until application.
VEHICLE
- Justification for use and choice of vehicle: Sunflower oil (Helianthii annui oleum raffinatum), The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of the nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively.
- Lot/batch no.: 1506-4604
- Purity: 98 and 102 % - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility at least twice during the study.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL). A separate analytical report provided this information. - Details on mating procedure:
- - Impregnation procedure: The females will be paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieves at least twenty two.
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male to three females
- Length of cohabitation: two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy - Duration of treatment / exposure:
- The sperm positive females were treated from gestational day 5 to 19. The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
- Frequency of treatment:
- Daily
- Duration of test:
- The sperm positive females were treated from gestational day 5 to 19. Gross pathology afterwards.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Remarks:
- 10 mg/mL
- Dose / conc.:
- 60 mg/kg bw/day (nominal)
- Remarks:
- 30 mg/mL
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Remarks:
- 100 mg/mL
- No. of animals per sex per dose:
- 80 males, 130 females to achieve at least 22 sperm positive females per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose setting is based on findings obtained in a GLP Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422). The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured at least once in the pre-mating period, but will not be statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All sperm positive females were sacrificed by decapitation after anesthetizing with Isofluranum on day 20 of gestation.
- Organs examined: The abdomen was opened, the uterus with cervix and the left ovary was removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Fetal examinations:
- - External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter] - Statistics:
- The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. - Indices:
- Pre-implanation loss:
((number of corpora lutea)-(number of implantations))/(number of corpora lutea)*100
Post-implanation loss:
((number of implantations)-(number of live fetuses))/(number of implantations)*100
Sex distribution:
(number of male (female)fetuses)/(number of fetuses)*100
External abnormalities/ litter:
(number of fetuses with abnormality/(number of fetuses)*100
Visceral abnormalities/litter:
(number of fetuses with abnormality/(number of fetuses examined)*100
Skeletal abnormalities/litter
(number of fetuses with abnormality/(number of fetuses examined)*100 - Historical control data:
- Findings were compared to historical control data.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related clinical observations. Alopecia and small wound on the skin were observed with a low incidence without a relationship to the test item. Piloerection was recorded in one dam in the high dose group on the day before Caesarean section. Considering that this occurred only in one female, this sign was not attributed to the treatment.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no significant differences in the body weight values among the dose groups. Statistically significantly, slight to moderate lower body weight gain was indicated in the 60 (-11%) and 200 (-24%) mg/kg bw/day groups for the days between 17 and 20 (p<0.05) and in the 200 mg/kg bw/day group for between days 0 to 20 (-12%). The differences in the body weight parameters in the 200 mg/kg bw/day dose group were considered as treatment-related as there was also a statistically significant (p<0.05), moderate reduction of the corrected body weight gain as most relevant weight parameter in this group. The slightly lower body weight gain in the 60 mg/kg bw/day group was considered as non-adverse as no other weight parameter was affected accordingly.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Slight but statistically significant reduction of the food consumption was observed (p<0.05, -8%) between gestation days 17 and 20 in the 200 mg/kg bw/day dose group. Considering that this reduction correlated with a moderately lower body weight- and corrected body weight gain, a relationship to the treatment was considered as likely. There were no significant differences indicated in the food consumption of the animals in the 20 and 60 mg/kg bw/day groups relative to the control.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related macroscopic alterations recorded. At necropsy the stomach was found to be empty in case of three dams in the 200 mg/kg bw/day group. Also an empty stomach except some bedding was recorded for two dams in the 60 mg/kg bw/day group. These females were considered to have no macroscopic alterations. There was one female in the 200 mg/kg bw/day group where pin-prick sized dark coloured points were to see in the glandular stomach. Clotted bloody stomach content with intact stomach surface was observed in one dam in the 20 mg/kg bw/day dose group. These findings were judged not to be in relationship with the treatment considering the isolated occurrence .
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Number of abortions:
- not examined
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no effect related to the administration of the test item in the mean percent of pre-implantation loss, early embryonic, late- and fetal death, the mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses in the test item treated groups. Moreover the preimplantation loss as non-relevant finding was statistically significantly lower in the 200 mg/kg bw/day group. Statistically significantly (p<0.05) higher mean number of early embryonic death in all test item treated groups as well as postimplantation loss in the 60 mg/kg bw/day group and in the 200 mg/kg bw/day group both without a dose response-relationship considering the mean percentage values was indicated only by the Chi square test and not in case the mean percentage was calculated. Therefore, the observed differences were finally judged to be not related to the treatment.
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- not examined
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The number of sperm positive females was 25 in the control and high dose as well as 22 both in the low and mid dose groups. There were two non- pregnant females each in the control and 200 mg/kg bw/day and five in the 20 mg/kg bw/day group. In the 60 mg/kg bw/day dose group all females were pregnant. Two females which had less than 3 implantations (one each in the control and 200 mg/kg bw/day group) were excluded from the data evaluation. Data of one litter (with one fetus) were excluded from the data evaluation in the 200 mg/kg bw/day dose group. In total, on gestation day 20 there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 20, 60 and 200 mg/kg bw/day groups respectively.
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weight of the fetuses and absolute placental weight (but not the most relevant relative placental weight) was slightly but statistically significantly (p<0.05 to (p<0.01) lower in the 200 mg/kg bw/day group compared to the control. The reduction of the mean fetal weights was judged to be in relationship with the treatment. In contrast, the observation regarding the reduced mean absolute placental weight was considered as not relevant as the most relevant relative placental weight due to its relation to body weight was not affected and moreover, the mean relative placental weights were slightly higher, although without statistically significance, than the respective, control values.
- Reduction in number of live offspring:
- not examined
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- The number of evaluated fetuses was 253, 198, 225 and 253 in the control, 20, 60 and 200 mg/kg bw/day groups, respectively.
Malformations
One fetus was found with a malformation (umbilical hernia) at external examination in the 200 mg/kg bw/day group. According to the experience with this species in this laboratory, umbilical hernia occurs sporadically without a relationship to the treatment. This is in line with historical control data of other Wistar rats (Crl:WI(Han), Ginkis and Clifford, 2009) and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993). Both are known to have low incidences of umbilical hernia as spontaneous finding. Consequently, this single malformation in the high dose group in this study was judged to be incidental.
Variations
Body weight retardation (below 3.03 g for males and 2.79 g for females) was evaluated as an external variation. The incidence of body weight retarded fetuses increased statistically significantly (p<0.01) in the 200 mg/kg bw/day dose group which was considered to be in a relationship with the treatment of the dams. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The number of examined fetuses was 126, 99, 113 and 128 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of fetuses with abnormalities among the experimental groups.
Malformations:
Malformations were found only in single cases and distributed among allthe experimental groups. Slightly split or split xiphoid process was recorded for one fetus both in the control and 200 mg/kg bw/day group. Split sternum, bent scapula in two separate fetuses in the 20, dumb-bell shaped cartilage of thoracic vertebral centrum in the 60 mg/kg bw/day dose group and a hemicentric thoracic centrum in one control fetus were considered to be without any relationship to the treatment of the dams with the test item.
Variations:
Incompletely ossified skull bones, bipartite supra occipital, unossified hyoid, incompletely or not ossified, misaligned or bipartite sternebra, wavy, interrupted ribs, shorter 13th rib, neck rib anlage, bipartite (with or without asymmetric ossification), incomplete ossification or unossified vertebral arches, incompletely ossified pubic bones, asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination. Slightly but statistically significantly (p<0.05) increased incidence if only 3 or less sternebra were ossified in the high dose group.
Opposed to this the incidence of fetuses with incomplete ossification of the skull bones which was statistically significantly lower in the 200 mg/kg bw/day group as well as the occurrence of wavy ribs was statistically significantly lower in the 60 and 200 mg/kg bw/day groups. In summary the slight increases and decreases in the incidence of skeletal variations were judged to be not related to the treatment. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The number of examined fetuses was 127, 99, 112 and 125 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of variations and malformations among the experimental groups.
Malformations:
Besides the umbilical hernia discussed above at the section external examination, an isolated case of unilateral microphthalmia was found in one fetus in the high dose group. According to the background database of Toxi-Coop Zrt. (Appendix XXIV/A and B) this finding may occur sporadically in control fetuses. Considering the background data and that it was a single fetus with this abnormality in this study, this isolated observation was judged to be not related to the test item. Hydronephrosis was recorded for one fetus in the 20 mg/kg bw/day dose group and none in the higher dose groups hence was judged to be incidental.
Variations:
Hydroureter as variation was found in all groups including control. In addition dilated renal pelvis was found in one single fetus in the high dose group. Considering the low incidence and lack of dose response both variations were found to be without a test item response - Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental toxicity
- Effect level:
- 60 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- fetal/pup body weight changes
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Teratogenicity
- Effect level:
- 200 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- external malformations
- skeletal malformations
- visceral malformations
- Key result
- Abnormalities:
- no effects observed
- Description (incidence and severity):
- Only slightly lower fetal weight was observed at 200 mg/kg bw/day
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 200 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- not specified
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows:
NOAEL (maternal toxicity): 60 mg/kg bw/day
NOAEL (developmental toxicity): 60 mg/kg bw/day
NOAEL (teratogenicity): 200 mg/kg bw/day (high dose) - Executive summary:
The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 July 2020 to 19 August 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment
- Version / remarks:
- 24th July 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: S & K-LAP Kft., Császár út 135, 2173 Kartal, Hungary
- Age at study initiation: young adult animals
- Weight at study initiation: 3407-4011 g
- Housing: individually in metal cages
- Diet: S&K LAP separating rabbit diet produced by Cargill Takarmány Zrt., 5300 Karcag, Madarasi út 0399, Hungary, ad libitum
- Water: tap water from water bottles ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.8- 21.0
- Humidity (%): 42 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):12/12
IN-LIFE DATES: From: 06 July 2020 To: 19 August 2020 - Route of administration:
- oral: gavage
- Vehicle:
- other: Sunflower oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil) in concentrations of 15 mg/mL, 50 mg/mL and 150 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before administration and were stored in the refrigerator (5 ± 3°C) until application.
The suitability of the chosen vehicle and a sufficient stability for the test item at the used concentrations was analytically verified upfront. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of concentrations of the test item in the dosing formulations varied in the acceptable range between 98 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing.
- Details on mating procedure:
- - Impregnation procedure: artificial insemination, receptal hormone preparation with intramuscular injection into the femur region to provoke ovulation
- Duration of treatment / exposure:
- one oral (gavage) dose per day
- Frequency of treatment:
- The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
- Duration of test:
- The inseminated females were treated from gestational day 6 to 27.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- 15 mg/mL
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- 50 mg/mL
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- 150 mg/mL
- No. of animals per sex per dose:
- 25 females per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: based on a preliminary range-finding study
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g). The corrected body weight was calculated for the 28th day of pregnancy (body weight on day 28 minus the weight of the gravid uterus).
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 28 by re-weighing the non-consumed diet (accuracy 1 g).
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: The organs of neck, thorax and abdomen of the does were examined macroscopically. Organ pathological changes which could not be diagnosed macroscopically were fixed in 4 % neutral formaldehyde solution. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter] - Statistics:
- The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance is the result of the Bartlett’s test, the
Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. - Indices:
- Pre-implanation loss:
((number of corpora lutea)-(number of implantations))/(number of corpora lutea)*100
Post-implanation loss:
((number of implantations)-(number of live fetuses))/(number of implantations)*100
Sex distribution:
(number of male (female)fetuses)/(number of fetuses)*100
External abnormalities/ litter:
(number of fetuses with abnormality/(number of fetuses)*100
Visceral abnormalities/litter:
(number of fetuses with abnormality/(number of fetuses examined)*100
Skeletal abnormalities/litter
(number of fetuses with abnormality/(number of fetuses examined)*100 - Historical control data:
- Findings were compared to historical control data.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Small amount of faeces was recorded for several animals across the groups including control with a similar distribution. Lack of faeces was observed in a few animals which had later evaluable litters in all groups including control, however in the 300 mg/kg bw/day dose group lack of faeces was observed in all five females which aborted later. This observation was not recorded for the aborted females in the lower groups. In the 100 mg/kg bw/day group, besides the animals which showed abortion, no dose related clinical signs were observed. There were no effects of treatment with the test item in the dose level of 30 mg/kg bw/day. Other clinical symptoms were observed without a dose response or in that animal excluded from the study for reasons of ill health and were therefore incidental to treatment with the test item.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- None of the pregnant females died due to toxicity or was moribund. Death of one non-pregnant female in the 300 mg/kg bw/day dose group was assumed to be due to toxicity, considering the similar clinical condition of this rabbit before death as the high dose animals were affected by a test item effect (weight loss, lack of faeces) and that it showed no signs for illness. However, the cause of death was not proved. Since this was a non-pregnant animal, it was excluded from the data evaluation. Two does in the control group and one non-pregnant in the 30 mg/kg bw/day group died due to a disease. One pregnant in the 100 mg/kg bw/day died due to misgavage. The animals died before scheduled necropsy were excluded from the data evaluation.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- There was an effect of treatment with 300 and 100 mg/kg bw/day on maternal body weight gain. The mean body weight of the animals in the high dose group was lower versus control on GD 9 (p<0.05). The mean body weight gain was negative in all groups from the first treatment to GD 9, (this was minimal in the control and 30 mg/kg bw/day group) but the mid and high dose animals lost significantly more weight with a dose response (p<0.05 and p<0.01 respectively). According to the individual body weight of the aborted animals in the 300 mg/kg bw/day group, significant weight loss was observed from the first treatment and the condition of these animals became poor (underfeed) before abortion. Weight loss was characteristic from the first treatment (except between GD 9 and 12) up to abortion for both 100 mg/kg bw/day animals, which aborted on GD 24 and 26. Sporadically, statistical significances were revealed in higher body weight at 30 mg/kg bw/day on GD 15, in the higher body weight gain at 100 mg/kg bw/day between g.d 21 and 24, as well as in the lower body weight gain in the 30 mg/kg bw/day group between GD 24 and 27 (weight loss). These differences were not dose related. No adverse effect of treatment with the test item at 30 mg/kg bw/day on maternal body weight was indicated. There were no statistical significances indicated in the corrected body weight and corrected body weight gain.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- There was a dose related effect observed in the treatment groups of 300 and 100 mg/kg bw/day regarding maternal food consumption. Significantly reduced food consumption was observed in the first three days interval of the treatment (from GD 6 to GD 9) in the 300 and in the 100 mg/kg bw/day dose group (p<0.01 and p<0.05 respectively). A notable reduction was seen in the food consumption of the dams aborted at 300 mg/kg bw/day (four animals from GD 6 and one animal from GD 12). Both dams aborted on GD 24 and 26 at 100 mg/kg bw/day, started to reduce food consumption significantly on GD 12. There was sporadically some variation in maternal food consumption in form of higher values at 30, 100 and 300 mg/kg bw/day. No adverse effect of the treatment with the test item at 30 mg/kg bw/day on maternal food consumption was observed.
- 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
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no effects of treatment with the test item on the incidence of macroscopic findings at necropsy at the dose levels of 100 and 30 mg/kg bw/day. The most common observation for the evaluated litters was pinhead-sized or point-like haemorrhages in the lungs of animals from all groups including controls. Small, reddish or brownish or light coloured or corroded-like areas in the stomach mucosa were recorded in six animals in the 300 mg/kg bw/day group (4 out of these 6 aborted) and one in the 30 mg/kg bw/day group (aborted). However also two evaluated control animals had a similar macroscopic finding. The non-pregnant animal died on GD 24 at 300 mg/kg bw/day had no macroscopic alterations. The stomach was fuller than usual in case of one doe in the 30 and one in the 300 mg/kg bw/day (both aborted). The stomach was full to distention (feed and fur) in case of four aborted animals in the 300 mg/kg bw/day group. The poor condition (underfeed) and abortion in this group was considered to be associated with obstruction of gastric outflow. The gall bladder of one doe (aborted) in the 300 mg/kg bw/day was full. Bloody nose and blood in the thoracic cavity was observed for the mis-dosed female (100 mg/kg bw/day). Dark red lung lobe, purulent and reddish mottled lungs were observed in two control rabbits died due to a disease. The reason for death was noted as pneumonia. The non-pregnant rabbit in the 30 mg/kg bw/day group which died on GD 20 had purulent fluid in the thoracic cavity. These animals were excluded due to an intercurrent disease. Pinched spleen was observed in two females in the 100 mg/kg bw/day group (one pregnant and one non-pregnant). This finding was considered to have no association with the treatment.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- effects observed, treatment-related
- Description (incidence and severity):
- Abortions were recorded for one, one, three and five cases in the control, 30, 100 and 300 mg/kg bw/day respectively. Abortions on GD 27 or 28 occurred in one animal each in the control, 30 and 100 mg/kg bw/day dose group. Considering that abortion or early delivery on GD 27 or 28 occurred also in the control group, this was not attributed to the treatment with the test item. Earlier abortions on GD 26 or before at 100 (two dams) and 300 mg/kg bw/day (five dams) were attributed to a secondary effect of weight loss and reduced food consumption, as well as obstruction of gastric outflow (latter at 300 mg/kg bw/day). The majority of the animals in the 300 mg/kg bw/day group started aborting earlier (one on GD 24, one on GD 26 and three on GD 20 and 21) than the ones in the 100 mg/kg bw/day group (one on GD 24 and one on GD 26) which showed an association with the dose level.
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the dams with live fetuses on GD 28 evaluated, there was no difference in the effects between the dose groups on the survival of the fetuses in utero. According to the Ch2 test, statistical significances were indicated in the pre-implantation loss in the 30 and 100 mg/kg bw/day group and consequently in the total intrauterine mortality only at 30 mg/kg bw/day without a dose response and toxicological relevance. There were no significant differences in post implantation loss i.e. mean or early- and late-resorptions or dead fetuses as well as mean number of viable fetuses. There was only one female with total post-implantation loss in the study, in the 30 mg/kg bw/day group.
- Total litter losses by resorption:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was only one female with total post-implantation loss in the study, in the 30 mg/kg bw/day group.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- There were no significant differences in postimplantation loss i.e. mean or early- and late-resorptions or dead fetuses as well as mean number of viable fetuses.
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- not examined
- Changes in number of pregnant:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- number of abortions
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The fetal weight but not the length was slightly lower in the 300 mg/kg bw/day group. The weight and length of fetuses in the 30 and 100 mg/kg bw/day groups was slightly higher than in the control. There was no statistical significance indicated either in the 300 or in the 30 and 100 mg/kg bw/day groups. There were no statistically significant differences revealed at the evaluations of placental weight between dose groups and control.
- Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the dams with live fetuses on GD 28 evaluated, there was no effect on the survival of the fetuses in utero. According to the Ch2 test, statistical significances were indicated in the pre-implantation loss in the 30 and 100 mg/kg bw/day group and consequently in the total intrauterine mortality only at 30 mg/kg bw/day without a dose response and toxicological relevance. There were no significant differences in post-implantation loss i.e. mean or early- and late-resorptions or dead fetuses as well as mean number of viable fetuses. There was only one female with total post-implantation loss in the study, in the 30 mg/kg bw/day group.
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Anogenital distance of all rodent fetuses:
- not examined
- Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The incidence of abnormalities (variations and malformations together) was within the historical control range, however statistically significantly higher (p<0.05) incidence of abnormalities due to variations (p<0.05) in the 300 mg/kg bw/day group (there were no malformations in this group) was observed. Also, in the 100 mg/kg bw/day group the incidence of abnormalities was statistically significantly higher (p<0.05). In both groups statistics revealed significance only by Chi square test and only for the incidence of affected fetuses and not for the litters. The ANOVA test revealed no statistical significance. Moreover, the variations were found with low incidences and only in two and one litter in the 300 and 100 mg/kg bw/day groups respectively. Also, there was no dose related increase in the incidence of malformations, (there were no malformations found in the 300 mg/kg bw/day group). The incidence of externally malformed fetuses/litters was 0/0, 2/1, 2/1 and 0/0 in the control, 30, 100 and 300 mg/kg bw/day groups respectively. Anencephaly was observed in two fetuses in the 30 mg/kg bw/day group in one litter, in addition the forepaws were malrotated (proved by skeletal examination) of one of these fetuses. In the 100 mg/kg bw/day dose group one fetus was multiply malformed (acrania, hyperflexion of forepaws, thoracogastroschisis) and in the same litter open eye (absent cornea and retina fold by Wilson’s section) was found in another fetus. Hyperflexion of the limbs (not proved by skeletal examination, hence categorized as a variation) and neural tube defect in form of spina bifida was observed in control fetuses according to the historical control data. The occurrence of these findings was within or around the historical control range. Considering the low incidences (two and one fetuses affected in the 30 and 100 mg/kg bw/day group and none in the 300 mg/kg bw/day group and one litter was affected each in the 30 and 100 mg/kg bw/day group, the external malformations were not proved to be due to an effect of the test item.
The number of fetuses/litters with external variations was 0/0, 0/0, 2/1 and 4/1 in the control, 30, 100 and 300 mg/kg bw/day groups respectively. There were no significant differences in the incidences of variations according to the ANOVA test, only by Chi square test. Four fetuses were found with slight neck edema in one litter in the 300 mg/kg bw/day group and distended abdomen in two fetuses also only in one litter in the 100 mg/kg bw/day group. Considering that only one litter was affected in both groups, these changes was not proved to be treatment related. There were no variations found in the 30 mg/kg bw/day group. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There was no statistically significant increase in incidence of fetuses with skeletal abnormalities. There was no effect of treatment with the test item on the incidence of skeletal abnormalitites/malformations/variations in the test item treated groups. The mean percentage of litter means was similar in the experimental groups, moreover slightly lower in the control group. The incidence of fetuses/litters with malformations was 12/6, 10/7, 9/7 and 8/6 in the control, 30, 100 and 300 mg/kg bw/day groups respectively. Malformations of the skull, sternebra, ribs and vertebrae were observed with low incidences or in single cases or without a dose response or/and present in the control group. Statistical significance was revealed only in case of fetal incidence of fused and misaligned strenebrae in the 100 mg/kg bw/day dose group without a dose response and only by Chi square test, hence not attributed to an effect of the test item.Fused sternebra in more forms was found in several fetuses in the groups including control. If all types of fusions were summarized, there was no dose related increase observed versus control There was no increase indicated in the different type of malformations.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no significant differences in the incidence of fetuses and litters with visceral abnormalities, malformations and variations in the experimental groups. For the abnormalities investigated (variations plus malformations), the incidence of affected fetuses and litters with variations or malformations was 12 fetuses in 10 litters (56% of the 18 evaluated litters), 7 fetuses in 6 litters (29% of the 21 evaluated litters), 10 fetuses in 7 litters (41% of the 17 evaluated litters) and 12 fetuses in 8 litters (50% of the evaluated 16 litters) for the control, 30, 100 and 300 mg/kg bw/day groups respectively, hence the incidence of affected litters was the highest in the control group. The incidence of malformed fetuses/litters was 3 fetuses in 3 litters (17% of the evaluated litters), 1 fetus in one litter (5% of the evaluated litters), 2 fetuses in one litter (6% of the evaluated litters), 5 fetuses in 4 litters (25% of the evaluated litters) in the control, 30, 100 and 300 mg/kg bw/day groups. The number of fetuses with the final category “variation” was 9, 6, 8 and 7 in 7, 5, 6 and 7 litters, hence 39, 24, 35 and 44% of the evaluated litters was affected in the control, 30, 100 and 300 mg/kg bw/day groups respectively. Brain malformations such as larger perimeningeal space with slightly pushed brain was found in one control fetus. Dilated IIIrd brain ventricle and dilated IVth brain ventricle was recorded for one 300 mg/kg bw/day fetus. The same finding was observed in one fetus in the control group, moreover the incidence in the 300 mg/kg bw/day group (0.64%) was at or below the historical control range (between 0.00 and 1.05%). Dilated (markedly) lateral ventricles with pushed brain was found in one fetus in the 300 mg/kg bw/day dose group. Markedly dilated IIIrd ventricle and lateral ventricles were found in one control fetus. Similar changes in slighter manner were chategorized as variations such as slightly enlarged or enlarged perimeningeal space (without pushed form of the brain), slightly or moderately dilated brain ventricles and were found with low incidences and without statistically significant differences. If these brain variations and malformations are summarized, the number of fetuses with larger perimeningeal space was 5, 3, 2, 5, with dilated brain ventricles 4, 2, 2, 6, with pushed brain 1, 0, 0, 1 in the control, 30, 100 and 300 mg/kg bw/day groups respectively, hence there was no significant dose related increase in these findings. The head of one fetus with anencephaly in the 30 mg/kg bw/day dose group) was examined by Wilson’s section and multiply malformations were recorded. Open eye was proved for one fetus in the 100 mg/kg bw/day dose group, and in addition, the cornea was absent and retina fold was seen in the other eye (slight retina fold as a variation was observed for one control fetus). This malformation was not present in the high dose group. The aortic arch was seen as thicker in one fetus in the 300 mg/kg bw/day dose group, as a single case therefore this was not proved to be test item related. Intermedial lung lobe (variation) was recorded for one fetus in the 30 mg/kg bw/day dose group Two fetuses in the same litter (the ones with distended abdomen at external examination) had fluid filled abdomen in the 100 mg/kg bw/day group. The nasal cavity was observed as slightly asymmetric (variation) only in one fetus in the 300 mg/kg bw/day group. Acut angle lined origin of the ureter was found in one or two fetuses and only in the control, 30 and 100 mg/kg bw/day groups. Considering the lack of dose related increase and/or occurrence in single cases, the findings revealed at visceral examination were not proved to be test item related.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: abortions
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 100 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The dose levels of 300 and 100 mg/kg bw/day induced maternal toxicity manifested as weight loss and reduced body weight gain together with reduced food consumption and consequently abortion. The NOAEL for maternal toxicity was 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day based on the abortions in the 300 and 100 mg/kg bw/day dose groups.
- Executive summary:
Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with TBPND at the dose level of 300 mg/kg bw/day caused no deaths but clinical sign as lack of faeces and necropsy finding as to distention full stomach. The death of one non-pregnant female was assumed to be due to toxicity which was secondary to weight loss and reduced food consumption, however it was not proved. The treatment with the test item caused no clinical signs at 100 and 30 mg/kg bw/day in the does evaluated with live litters. The treatment of the does with the test item caused reduced food consumption and weight loss at 300 and 100 mg/kg bw/day and consequently abortion of 5 of 21 and 2 of 21 females respectively. The treatment of the does with the test item had no effect on the body weight, food consumption at 30 mg/kg bw/day. The dose levels of 300, 100 and 30 mg/kg bw/day in the evaluated does/litters had no effect on the post-implantation loss, number of implantations, sex distribution, fetal growth, placental weight, external, visceral and skeletal development of the fetuses. Neither the type nor incidence of fetal malformations found during external/visceral/skeletal examinations provided evidence for any treatment-related effect; the malformations were considered to be spontaneous in origin and incidental to treatment.
As a conclusion, the dose levels of 300 and 100 mg/kg bw/day induced maternal toxicity manifested as weight loss and reduced body weight gain together with reduced food consumption and consequently abortion. The NOAEL for maternal toxicity was 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day based on the abortions in the 300 and 100 mg/kg bw/day dose groups.
Referenceopen allclose all
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 30 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- OECD 414/GLP
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
OECD 414 rat
The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 106 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).
OECD 414 rabbit
Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with TBPND at the dose level of 300 mg/kg bw/day caused no deaths but clinical sign as lack of faeces and necropsy finding as to distention full stomach. The death of one non-pregnant female was assumed to be due to toxicity which was secondary to weight loss and reduced food consumption, however it was not proved. The treatment with the test item caused no clinical signs at 100 and 30 mg/kg bw/day in the does evaluated with live litters. The treatment of the does with the test item caused reduced food consumption and weight loss at 300 and 100 mg/kg bw/day and consequently abortion of 5 of 21 and 2 of 21 females respectively. The treatment of the does with the test item had no effect on the body weight, food consumption at 30 mg/kg bw/day. The dose levels of 300, 100 and 30 mg/kg bw/day in the evaluated does/litters had no effect on the post-implantation loss, number of implantations, sex distribution, fetal growth, placental weight, external, visceral and skeletal development of the fetuses. Neither the type nor incidence of fetal malformations found during external/visceral/skeletal examinations provided evidence for any treatment-related effect; the malformations were considered to be spontaneous in origin and incidental to treatment. As a conclusion, the dose levels of 300 and 100 mg/kg bw/day induced maternal toxicity manifested as weight loss and reduced body weight gain together with reduced food consumption and consequently abortion. The NOAEL for maternal toxicity was 30 mg/kg bw/day. The NOAEL for developmental toxicity was 30 mg/kg bw/day based on the abortions in the 300 and 100 mg/kg bw/day dose groups.
Justification for classification or non-classification
Final conclusion on fertility and development
In the OECD TG 443 study no toxicologically relevant differences in the evaluated parameters of pregnancy and delivery in female animals between the control and 50, 150 or 450 mg/kg bw/day groups have been reported. Systemic toxicity at 450 mg/kg bw/day referred to a test item influence on renal and/or hepatic function was observed in parental and adult F1 males and females (decreased body weight and weight gain; higher liver and kidney weight, higher concentrations in creatinine, cholesterol and urea, histopathological effects in kidney of males). No histopathological changes have been reported for reproductive organs. No effects in sperm count and sperm parameters were reported. The number of pregnant females, duration of pregnancy, dams delivered, mean number of implantation sites, mean number of post-implantation loss, the mean number of total births, mean number of viable pups and liveborn and the live birth index were similar in all groups. The mean number of stillborn was slightly higher than in the control (but within HCD) however the mean number of viable pups and liveborn and the live birth index were similar in all groups. The reproductive performance of male and female animals was not affected by treatment. The slight effects on fertility, which have been reported in the modified OECD TG 421 study like reduction of mean number of total births, reduced fertility index and higher follicular atresia at 300 mg/kg bw/day were not observed at higher dose of 450 mg/kg bw/day in the OECD TG 443 study.
The F1 offspring’s development (pinna detachment, eye opening, body weight) was delayed at 450 mg/kg bw/day in the OECD TG 443 study, while the mortality, sex distribution, clinical signs, surface righting reflex, anogenital distance, necropsy findings and organ weight were comparable to the control at 50, 150 and 450 mg/kg bw/day. The body weight was reduced in offspring up to -25% in the high dose. However, this can be linked to statistically reduced body weight, body weight gain and food consumption in the dams in the sensitive time period during gestation and lactation. Furthermore, inadequate nursing behaviour in the first days in high dose dams was reported and is associated with the higher number of cold pups and pups with no milk in stomach in mid and high dose in the first few days. Therefore, delayed pinna detachment, eye opening and decreased pup weight are regarded as secondary effects to maternal toxicity and as a rather unspecific effect on development due to inadequate nursing and weakness of the dams, which is considered to be reversible. Sexual maturity was similar to control in F1 post-weaning in all dose groups. No treatment-related immunotoxicological effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.
In the OECD 414 study in rats the substance did not reveal any adverse effect on the pre- and post-implantation loss, number of implantation and the sex distribution of the fetuses. Slightly lower fetal weight was observed at 200 mg/kg bw/day accompanied by maternal effects. The substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. In the OECD 414 study in rabbits no increase of post-implantation loss or increase of fetal variations and malformations was revealed at the examinations of the evaluated does/litters up to the 300 mg/kg bw/day group. Maternal toxicity manifested as weight loss and reduced body weight gain together with reduced food consumption and consequently abortion at mid and high dose. Earlier abortions on GD 26 or before at 100 (two dams) and 300 mg/kg bw/day (five dams) were attributed to a secondary effect of weight loss and reduced food consumption, as well as obstruction of gastric outflow (latter at 300 mg/kg bw/day).
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on reproduction and developmental toxicity screening test the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.
Additional information
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