Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-11 until 2012-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male and female animals: 71 - 76 days old

- Weight at study initiation:
Male animals: 299 - 338 g
Female animals: 173 – 202 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet:
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water:
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 2 mg/mL, 6 mg/mL and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 3 days before the administration.
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study.

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle :
A constant treatment volume of 5 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 were cohabited with the same male until copulation occurred. One pair was changed within the 30 mg/kg bw/day and 5 pairs within the high dose group after 14 days of 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. Seven male animals – not selected for toxicity examinations – were mated with non-treated females with respect to low number of females achieving pregnancy at the high dose group from Day 47 to Day 60.
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. 1,1`-(1,1,2,2 -tetramethylethylene)dibenzene was stable in sunflower oil at concentrations of 1 and 200 mg/mL at room temperature for one day and if refrigerated (at 5 ±3 °C) for 3 days. Concentration of the test item in the dosing formulations varied within the range of 93 – 107 % in comparison to the nominal values, thereby confirming proper dosing.
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 47 or 63 days and then they were subjected to necropsy one day after the last treatment. Seven male animals administered with 100 mg/kg bw/day were retained for a second mating with non-treated females of a new batch (n=7). Treatment and observations of these male animals were continued up to day 62 and necropsy was conducted on day 63. Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-5 (for 42 – 58 days, depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant animals were treated up to and including the day before necropsy (for 42-63 days).
Control animals were handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw.
Frequency of treatment:
Animals were treated once per day.
Details on study schedule:
Males: 6 days acclimatisation period, 14 days pre-mating period, 27 days mating period, 6 and 22 days post mating (including 14 days second mating), Day 47 and Day 63 necropsy

Females: 6 days acclimatisation period, 14 days pre-mating period, 27 days mating period, 22 days gestation period, 4-6 days lactation period, Day 42 to 63 necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals/sex in the control and dose groups. 7 animals randomization reserve – these served for base level measurements of clinical pathology. 7 female animals from a different batch were involved in the study for a second mating with 7 high dose treated animals.
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 (14-day oral gavage dose range finding study). The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS:
General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs were recorded including onset, degree and duration of signs.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

BODY WEIGHT:
All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. Body weight of the female animals were additionally weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data were reported individually for adult animals. Individual body weight changes were calculated.
Body weight was measured on day of necropsy for each animal.

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period (pre-mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4) except during mating phase.

EXAMINATION OF PLACENTAL SIGN:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

OBSERVATION OF THE DELIVERY PROCESS:
Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 post-partum with an accuracy of 0.1 g.
In addition to the observations on parent animals, any abnormal behavior of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all dead pups to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology and clinical chemistry were conducted in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 mL 9NC Microtube, 0.106 mol/L, Greiner Bio-One International AG, or equivalent), one for hematology (MiniCollect® EDTA tubes, spray-dried, 0.25 mL, Greiner Bio-One International AG, or equivalent), and the third one (VACUETTE® Serum Tube, 2.5 mL, Greiner Bio-One International AG, or equivalent) to obtain serum samples for clinical chemistry.
Tubes for hematology and coagulation should be filled up to the final volume (marked on the tubes) and at least 1.0 mL blood should be collected, if possible into clinical chemistry tubes.

HEMATOLOGY:
The hematology parameters were measured in reserve animals of randomization on day 0 (base level) and in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by SYSMEX XT-2000iV.
- Parameters checked:
White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential white blood cell count, Activated partial Thromboplastin Time, Prothrombin Time.

CLINICAL CHEMISTRY:
The clinical chemistry measurement was 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.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.
Postmortem examinations (parental animals):
Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

PATHOLOGY:
Gross necropsy was performed on each animal one day after the last treatment. Animals were euthanized by exsanguination after verification of an Isofluran-narcosis.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality were recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions of all adult animals were preserved. Kidneys of all parental male and female animals were also preserved due to macroscopic findings in male animals dosed with 600 mg/kg bw/day. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected for blood collection from each group:
adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with vagina), gross lesions, heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve,
seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid, trachea, urinary bladder, Pups euthanized at day 4 post-partum, or shortly thereafter, were carefully examined for gross abnormalities externally.

ORGAN WEIGHT:
At the time of termination, body weight and weight of the testes, epididymides of all parental animals were determined with an accuracy of 0.01 g. Kidneys were also weighed in all male and female animals due to macroscopic findings in male animals dosed with 600 mg/kg bw/day.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
Paired organs were weighed individually; absolute organ weight was reported. Relative organ weight (to body and brain weight) was calculated and reported.

HISTOPATHOLOGY:
Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histological examination of kidneys was also performed in all animals because test item related changes were observed in the high dose treated male animals.


Postmortem examinations (offspring):
GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)


Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables".
Offspring viability indices:
The offspring viability indices were calculated: survival index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables".

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily Observations
Test item related salivation was observed in animals of all dosed groups in a dose related manner (12/12 male and 11/12 female at 100 mg/kg bw/day; 11/12 male and 7/12 female at 30 mg/kg bw/day and 7/12 male at 10 mg/kg bw/day, including premating, gestation and lactation periods.
The frequency of these observations was the highest in 100 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 30 mg/kg bw/day and salivation did not appear at the low dose treated females. Alopecia was observed in three male and in two female animals: on the abdomen, thigh and scrotum for one male animal at 100 mg/kg bw/day (1/12) between Days 7 and 46; on the forelimbs in one male animal (1/12) dosed with 10 mg/kg bw/day from Day 35 up to the termination; on both sides of the body in one control male animal (1/12) from Day 7 up to Day 46; on the abdomen in two female rats (2/12) at 30 mg/kg bw/day from gestation days 29 and 41 up to termination of observation period (lactation days 5 and 4, respectively). Alopecia is a common finding in this strain of experimental rats and was present in the control and test item treated animals with similar incidence 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 detected at the weekly observations, too.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality in parental animals during the course of study at any dose level (100, 30 and 10 mg/kg bw/day). One dam dosed with 100 mg/kg bw/day became in moribund condition in the course of prolonged parturition and was euthanized for humane reason on lactation day 0 (Day 40). Piloerection, decreased muscle tone and body temperature (cold), paleness and sanguineous vaginal aperture were observed before the necropsy. Histopathological examinations revealed moderate pulmonary emphysema, moderate focal hepatocellular necrosis in the liver and proteinaceous casts in the lumina of glomerular capillaries and in the Bowmann’s capsule in the kidneys as individual lesions causing the death. No similar findings were observed in any of the other treated animals, and, thus, these effects were not considered to be related to the test item exposure.
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 in a dose related manner at 100, 30 and 10 mg/kg bw/day with respect to controls during the entire observation period.
The slight changes in body weight at 10 mg/kg w/day remained well below 10 % with respect to the controls and were not considered to be toxicologically relevant.

The mean body weight of male animals was less than in the control group at 100, 30 mg/kg bw/day from day 7 up to the termination and at 10 mg/kg bw/day from day 27 up to the end of observation due to the less mean body weight gain in all test item treated groups during the entire observation period with respect to control. Statistical significances were noted for less mean body weight gain on weeks 1, 2 and 5 at 100 and 30 mg/kg bw/day, on week 4 at 30 mg/kg bw/day and on weeks 2 and 5 at 10 mg/kg bw/day.

The summarized body weight gain remained significantly below the control value at 100, 30 and 10 mg/kg bw/day (-85 %; -63 % and -23 %, respectively). Similar findings were observed in female animals during the pre-mating period. The mean body weight gain was significantly less at 100, 30 and 10 mg/kg bw/day on week 1 which resulted in less body weight at 100 and 30 mg/kg bw/day on days 7 and 13. The summarized mean body weight gain also remained below the control value at 100, 30 and 10 mg/kg bw/day.
Although no statistical evaluation was performed on data of 100 mg/kg bw/day dosed animals from gestation, the body weight and body weight gain remained below the control values with biological significance at body weight on gestation day 21 and at body weight gain during the entire gestation period.
A less body weight was also observed at 30 mg/kg bw/day during the entire gestation period with respect to control and the body weight gain was below the control value on gestation week 1 and if summarized (between gestation days 0 and 21) with statistical significances. The body weight gain of dams administered with 10 mg/kg bw/day was significantly less on gestation week 1 and these changes resulted in a slightly less body weight on gestation day 7, with respect to control. Slight, but statistically significant less mean body weight was detected at 30 mg/kg bw/day on lactation days 0 and 4 and at 10 mg/kg bw/day on lactation day 4. However the summarized body weight gain was not influenced during the lactation period.
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 100 and 30 mg/kg bw/day doses. The mean daily food consumption of male animals was less than in the control group at 100 and 30 mg/kg bw/day in during the entire observation period with statistical significance on weeks 1, 2, 5 and 6.
The mean daily food intake was also reduced in female animals administered with 100 or 30 mg/kg bw/day during the premating (weeks 1 and 2) and during the first two weeks of gestation and between lactation day 0 and 4. (No statistical evaluation on food consumption of female animals at 100 mg/kg bw/day.) At 10 mg/kg bw/day, the mean daily food consumption remained below the control value during the lactation period.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related changes in the examined hematological parameters in male and female animals at any dose level.

A slight but significant reduction in red blood cell count (RBC) causing a reduction in hemoglobin concentration (HGB) was observed for the male animals at 100 mg/kg bw/day. However, these effects were not considered to be of toxicological significance as they were with low magnitude and differed only slightly from the historical control values.
Further statistically significant differences between the male control and dosed groups in some hematological parameters were not considered toxicologically relevant as these values remained well within the historical control ranges: hematocrit value (HCT), mean corpuscular hemoglobin concentration (MCHC) and platelet count (PLT) at 100 mg/kg bw/day; HGB and MCHC at 30 mg/kg bw/day. In the female animals administered with 100 mg/kg bw/day, the white blood cell count (WBC), percentage of neutrophil granulocytes (NEU) and monocytes (MONO), and mean corpuscular hemoglobin (MCH) were less than in the control group, while the percentage of lymphocytes (LYM) and eosinophil granulocytes (EOS), red blood cell count and prothrombin time (PT) was higher than in the control group. The hemoglobin concentration (30 and 10 mg/kg bw/day), hematocrit value and prothrombin time (both latters at 10 mg/kg bw/day) were less than in the control group. All these differences with respect to control were with low magnitude and were within or marginal to the historical control ranges therefore were not considered to be biologically or toxicologically significant.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related alterations occurred in the examined clinical chemistry parameters. Statistically significant differences in values of some parameters were judged to be of little or no biological significance as the mean values were well within the historical control ranges.
In the male animals, slightly less mean activity alkaline phosphatase (ALP; at 100 mg/kg bw/day), concentration of cholesterol (CHOL; at 100, 30 and 10 mg/kg bw/day) and bile acids (BAC at 30 mg/kg bw/day) were detected with respect to control. In female animals, statistical significances were noted for higher mean total bilirubin (TBIL) and mean total protein (TPROT) concentrations as well as lower albumin-globulin ratio at 100 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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 (100, 30 and 10 mg/kg bw/day, control). Alopecia was detected in three male animal at 100 mg/kg bw/day (1/5), 10 mg/kg bw/day 1/5) and in the control group (1/5), and in one female (1/5) at 30 mg/kg bw/day. Positional struggle in a marked degree was noted for two male rats administered with 30 mg/kg bw/day (1/5) or 10 mg/kg bw/day (1/5). Two control females (2/5) was not able perform the equilibrium test. The variation in positional struggle was within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations, it occurred in one low dose treated animal thus was without any toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The body weight was significantly less with respect to control in all test item treated groups (male and female animals at 100 and 30 mg/kg bw/day and male animals at 10 mg/kg bw/day) therefore absolute organ weights and organ weights relative to body weight were not suitable for a correct evaluation.
In the male animals selected for further examinations, statistical significances were noted for less mean absolute weights of kidneys, heart (100 and 30 mg/kg bw/day) and thymus (30 mg/kg bw/day). Organ weights relative to the body weight exceeded the control value in case of the brain, kidneys, testes and adrenal glands at 100 and 30 mg/kg bw/day, spleen at 100 mg/kg bw/day, liver and epididymides at 30 mg/kg bw/day and testes at 10 mg/kg bw/day. The mean of body weight and heart weight (100, 30 and 10 mg/kg bw/day), epididymides weight (100 mg/kg bw/day) each relative to brain weight were significantly less with respect to control.
In the female animals, absolute weight of kidney at 100 mg/kg bw/day, heart at 100, 30 and 10 mg/kg bw/day, thymus at 30 mg/kg bw/day and spleen at 100 and 10 mg/kg bw/day were significantly less with respect to control. The weight of kidneys (100 mg/kg bw/day) and spleen (100 and 10 mg/kg bw/day) both relative to body weight was below the control and the brain weight relative to body weight (100 and 30 mg/kg bw/day) and thymus weight relative to body weight exceeded the appropriate values of control group. The organ weights relative to brain weight were less with respect to control in case of kidneys at 100 mg/kg bw/day, heart at 100, 30 and 10 mg/kg bw/day, thymus at 30 mg/kg bw/day and spleen at 100 and 10 mg/kg bw/day.
The epididymides weights (absolute and relative to brain weight) were significantly less than in the control group in remaining animals (not selected for further examinations) at 100 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No specific macroscopic alterations related to the test item effect were observed at the gross necropsy of animals.

Dam subjected to early necropsy due to moribund condition at 100 mg/kg bw/day, showed sanguineous vaginal aperture before the euthanasia. Macroscopic observations of organs and tissues revealed pale visceral organs, sanguineous content in the stomach and clotted blood in the right side uterine horn.

Smaller than normal seminal vesicles (4/12) and prostate (1/12), as well as alopecia on the abdomen, thigh and scrotum (1/12) were observed at the necropsy of male animals administered with 100 mg/kg bw/day. There were no macroscopic findings in the male animals of control, 10 and 30 mg/kg bw/day groups.
Congenital absence of left side kidney and uterine horn and compensatory enlargement of the right side kidney were observed in one female animal at 100 mg/kg bw/day, which was judged to be an individual alteration commonly occurring also in untreated experimental rats of this strain.
Hydrometra (indicative of sexual cycle of female animals) is a frequent observation in experimental rats, and has no toxicological meaning without pathological changes, was present in two 100 mg/kg bw/day dose treated animal (2/8 of non-pregnant animals). Alopecia was an individual change occurring in single animals of 100 mg/kg bw/day (1/12, male), 10 mg/kg bw/day (1/12) and control (1/12 groups) and in two dams (2/12) of 30 mg/kg bw/day group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The test item did not cause any toxic or other test item related lesions detectable by histological examination in the genital and other organs of the experimental animals.
One animal administered with 100 mg/kg bw/day was euthanized in moribund condition. Histological examination revealed focal hepatocellular necrosis in the liver and proteinaceous casts in the lumina of glomerular capillaries and in the Bowmann’s capsule in the kidneys as cause of death. In addition acute alveolar emphysema in the lung was observed too. These lesions probably were in connection with an individual disease of endogenous origin in this animal. No similar lesions were detected in the other animals of the study belonging to the high dose group (male and female).
In male animals, the investigated organs of reproductive system (testes, epididymides), were histologically normal and characteristic on the sexually mature organism in all cases of control and high dose treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. The histological picture of epididymides was normal in all cases as well. In the female animals the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation, the epithelial capsule and ovarian stroma was normal in all cases as well, with the exception of two female animals (2/12). In these individuals lack of corpora lutea in the ovaries was detected indicating the delay of ovulation in connection with a possible hormonal disorder.
The uterus, cervix and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. In four cases dilatation of uterus was observed. This finding - without inflammation or other pathological lesion - is a physiological phenomenon in connection with the normal sexual cycle. The histological picture of pituitary was normal as well in the case of male and female treated and control animals.

In the organs of selected animals subjected to a full histopathological examination, no test item related histopathological changes were detected. The focal alveolar emphysema in the lungs in minimal or mild degree, (control: 1/5 male and 1/5 female; 100 mg/kg bw/day: 1/5 female), occurred sporadically and was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. The “foamy cells” in the lung of one control (1/5 male) and in one treated animal (1/5 female at 100 mg/kg bw/day) are common findings in rats in connection with the “alveolar lipidosis”, without toxicological significance. The hyperplasia of bronchus associated lymphoid tissue (BALT) in some control and treated animals (control: 2/5 male, 1/5 female; 100 mg/kg bw/day: 1/5 male, 1/5 female) is a physiological phenomenon.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, kidney, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed. The structure and the cell morphology of the endocrine glands were the same at the control and treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproductive functionality was decreased at 100 mg/kg bw/d.
The number and percentage of pregnant females was significantly less than in the control although the number of mated animals and copulatory indices were within the normal range for males and females. The number and percentage of fertile males and females and fertility indices were significantly less than in the control group.

There were no significant differences between the control and test item treated male animals at 30 and 10 mg/kg bw/day in the examined parameters of reproductive performance. The copulatory and fertility indices were similar at these doses (30 and 10 mg/kg bw/day) with respect to control.
There were no significant differences between the control and test item treated groups in the mean number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices at 30 or 10 mg/kg bw/day. 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 30 and 10 mg/kg bw/day treated groups.

The second mating of 7 male animals at 100 mg/kg bw/day with not treated females revealed reduced ability to mate. The copulatory and fertility indices were low.

Delivery Data of Dams:
The number of females delivered was low (n=2) and not suitable for statistical evaluation at 100 mg/kg bw/day, although the mean number of corpora lutea and implantations, the mean of total births per litter, mean number of live-borns per litter and viable pups per litter were less with respect to control due to test item effect. The duration of pregnancy and parturition was prolonged (>23 days, >4 hours, respectively) for both dams. There were no significant differences between the control and other test item treated groups (30 and 10 mg/kg bw/day) in the examined parameters: mean number of corpora lutea, percent and mean of implantations, pre and post-implantation loss, total intrauterine mortality, as well as duration of pregnancy, percentage and mean of total births per litter and viable pups per litter.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: salivation, reduced body weight development and reduced food consumption
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: low fertility indices, prolonged pregnancy and parturition and changes in delivery data

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Test item related clinical signs did not appear in the pups. The number and percent of cold pups were slightly higher than in the control group at 30 mg/kg bw/day but these were considered to be independent from the treatment as this was observed in pups of one litter only.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of litter was low (n=2) and was not suitable for statistical evaluation at 100 mg/kg bw/day; however the number and percentage of dead pups was high for the only dam evaluated on lactation day 4.
The number and percentage of extra uterine mortality was similar in the control and 30 and 10 mg/kg bw/day groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The number of litter and pups per litter was low (n=2) and not suitable for statistical evaluation at 100 mg/kg bw/day. The mean pup weight was significantly less with respect to control in the observed litter.
The mean litter weight was significantly less than in the control group at 30 and 10 mg/kg bw/day on postnatal day 4, and the litter weight gain was also less than in the control at 30 and 10 mg/kg bw/day between days 0 and 4. These changes were slight and were not considered toxicologically relevant as there were no significant differences in the mean pup weight and weight gain. More specifically, the mean pup weight and the mean weight gain were similar in the control and 30 and 10 mg/kg bw/day groups on days 0 and 4 as well as between days 0 and 4.
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
Description (incidence and severity):
Sex distribution:
The number of litter and pups per litter was low (n=2) and not suitable for evaluation at 100 mg/kg bw/day. Differentiation of gender was not possible for one litter because pups were missing (cannibalized) before weighing and determining the gender.
There were no significant differences between control and test item treated groups (30 and 10 mg/kg bw/day) in the ratio or in the litter means of genders on postnatal days 0 or 4.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: reduced number of litter and pubs, reduced mean pub weight

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The reprotoxic properties of 1,1`-(1,1,2,2 -tetramethylethylene)dibenzene were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:  
NOAEL for male and female rats rats: 10 mg/kg bw/day; NOAEL for reproductive performance of the male and female rats: 30 mg/kg bw/day; NOAEL for F1 Offspring: 30 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 1,1'-(1,1,2,2-tetramethylethylene)dibenzene and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as 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), 100, 30 or 10 mg/kg bw/day at concentrations of 20, 6 and 2 mg/mL corresponding to 5 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. The test item proved to be stable in sunflower oil at concentrations of 1 and 200 mg/mL at room temperature for one day and if refrigerated (at 5 ±3 °C) for 3 days. Concentration of the test item in the dosing formulations varied within the range of 93 – 107 % in comparison to the nominal values, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3 – 5, 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 examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Seven male animals administered with 100 mg/kg bw/day were retained for a second mating with non-treated females of a new batch (n=7). Treatment and observations of these male animals were continued up to day 62 and necropsy was conducted on day 63. The dams were allowed to litter, and rear their young up to day 4 postpartum. Pups were weighed and observed for possible abnormalities and all offspring were euthanized on postnatal day 4 or 5. All dams were subjected to gross pathology one day after the last treatment on postpartal days 4-6. Selected organs were weighed. Full histopathology was performed on the selected animals of control and high dose groups. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle only.

Results

Mortality

There was no test item related mortality at any dose level (100, 30 or 10 mg/kg bw/day). One dam dosed with 100 mg/kg bw/day was euthanized in moribund condition after a prolonged parturition. Histopathological examinations revealed lesions indicative of a possible endogenous individual disease (moderate focal hepatocellular necrosis in the liver and proteinaceous casts in the lumina of glomerular capillaries and in the Bowmann’s capsule in the kidneys, moderate pulmonary emphysema) as cause of death. No similar findings were observed in any of the other treated animals, and, thus, these effects were not considered to be related to the test item exposure.

Clinical observation

Test item related salivation appeared in male and female animals administered with 100, 30 mg/kg bw/day and in males at 10 mg/kg bw/day with a dose related onset and incidence. No toxic signs related to the test item were found at the detailed weekly and terminal functional observations. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).

Body weight and body weight gain

A test item related depression of the body weight development was detected in male and female animals in a dose related manner at 100, 30 and 10 mg/kg bw/day with respect to controls. The slight changes in body weight at 10 mg/kg w/day remained well below 10 % with respect to the controls and were not considered to be toxicologically relevant.

Food consumption

The mean daily food consumption was reduced in male and female animals at 100 and 30 mg/kg bw/day doses during the entire observation period (premating and during the first two weeks of gestation and between lactation day 0 and 4) and at 10 mg/kg bw/day between lactation day 0 and 4.

Clinical pathology (Hematology and clinical chemistry)

Hematology and clinical chemistry examinations did not reveal test item related changes in the examined parameters at any dose level 100, 30 or 10 mg/kg bw/day.

Organ pathology (Necropsy, organ weight and histopathology)

Specific macroscopic alterations related to the test item were not found in male or female animals at the gross necropsy observations, at the organ weight and histopathology examinations. The investigated organs of reproductive system (testes, epididymides, uterus, vagina and ovaries) were histologically normal and characteristic on the sexually matured organism in the control and 100 mg/kg bw/day groups.

Reproduction

Reproductive function was influenced by the treatment with the test item at 100 mg/kg bw/day. Fertility indices were significantly less with respect to controls but copulatory indices were within the normal range for males and females. Females conceived after a longer period of mating than in the control group. The number of females delivered was low (n=2). Also the number of corpora lutea and implantations, the mean of total births per litter, mean number of live-borns per litter and viable pups per litter were less with respect to control. The duration of pregnancy and parturition was prolonged (>23 days, >4 hours, respectively) for both dams of this treatment group. The second mating with not treated females revealed a reduced ability of males to mate.

Offspring

The number of litter and pups per litter were not suitable for evaluation at 100 mg/kg bw/day. The litter weight and litter weight gain were slightly reduced at 30 and 10 mg/kg bw/day, however the changes revealed no dose response relationship and were not considered toxicologically relevant since there were no significant differences in the mean pup weight and weight gain.

Conclusion

Under the conditions of the present study, 1,1`-(1,1,2,2 -tetramethylethylene)dibenzene caused salivation, reduced body weight development and food consumption following an oral administration at 100 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 30 mg/kg bw/day, salivation, changes in body weight and body weight gain and food consumption were (male and female animals) observed. At 10 mg/kg bw/day, salivation (male) and slight changes in body weight and body weight gain (male and female animals) were detected. However the slight changes in body weight at 10 mg/kg w/day remained well below 10 % with respect to the controls and were not considered to be toxicologically relevant. Reproduction function was affected by the test item at 100 mg/kg bw/day as the fertility indices were low for male and female animals. The results of the second mating (untreated females with treated males) indicated that 1,1`-(1,1,2,2 -tetramethylethylene)dibenzene influenced the males reproduction performance in the high treatment group. Females showed slightly prolonged pregnancy and parturition and changes in delivery data (mean number of corpora lutea and implantations, mean of total births per litter, mean number of live-borns per litter and viable pups per litter). Effects on offspring development could not be evaluated at the high dose due to the low fertility index. The variations noted are most likely due to the systemic toxicity noted in the dams.

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

NOAEL for male and female rats: 10 mg/kg bw/day

NOAEL for reproductive performance of male and female rats: 30 mg/kg bw/day

NOAEL for F1 Offspring: 30 mg/kg bw/day