Registration Dossier

Administrative data

Description of key information

Based on the results of the 90-day repeated dose toxicity study, the NOAEL was determined to be 10 mg/kg bw/day for male and female Wistar rats.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2015-05-19 and 2016-03-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
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 animals: 35 – 38 days old Female animals: 35 – 38 days old
- Weight at study initiation: Male animals: 156 – 188 g Female animals: 114 – 130 g
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 – 15 / hour
- Photoperiod: 12 hrs dark / 12 hours light

IN-LIFE DATES: From: 2015-05-20 To: 2015-09-15
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil in a frequency based on the stability features of the test item in the vehicle (stable at room temperature at least for one day and at 5 ± 3 °C for 3 days).

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 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 1.5, 5, 15 mg/mL
- Amount of vehicle: 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored in a refrigerator (at 5 ± 3 °C) until analysis. Formulation samples were diluted with Tetrahydrofuran and with Acetonitrile and analysed by HPLC. Measured concentrations varied between 95 and 106 % of the nominal concentrations and all formulations were considered to be homogeneous.

HPLC conditions:
Detector: 220 nm
Column: Purospher STAR RP-18e 30-2 mm, 3 μm No.: 014664
Mobil Phase: Acetonitrile : water = 60 : 40 (v/v)
Flow Rate: 0.5 mL/min
Injection volume: 5 μL
Temperature: 25 °C
Retention time of CUROX®CC-DC: 4 min
Run time: 6 minutes
Duration of treatment / exposure:
90 or 91 days (depending on the day of necropsy)
Frequency of treatment:
7 days/week basis, every day at a similar time (± 2 hours)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals/sex in the control and dose groups; 5 animals/sex in the control and high dose groups for recovery observations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 30, 10 and 3 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity studies with the test item in the rat (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat OECD 422; GLP, study no. 552.422.2950). Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for selecting satellite groups: five animals per sex in the control and in the high dose group (according to guideline)
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter.
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighing was performed on day 0, then weekly. Fasted body weight was measured on the day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION:
- Food consumption was determined on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatization period
- Dose groups that were examined: all control and high dose test animals prior to test termination (day 85)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on main group animals one day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of the recovery period (Day 118)
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on main group animals one day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of the recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 84)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: yes
- Time schedule for examinations: during the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy)
- Dose groups that were examined: all female animals, with exception of the recovery group
- Examinations: A vaginal smear was prepared from each female. The type of cycle (regular or irregular), number of pro-estrous, estrous and diestrous days , number of cycle during the two weeks, number of animals with prolonged diestrous intervals, number of animals with prolonged estrous intervals were evaluated.

SPERM EXAMINATION: yes
- Time schedule for examinations: at necropsy
- Dose groups that were examined: 10 male animals of the control and in 10 male animals of the high dose group
- Quantitative examinations: Testes and epididymides were frozen at necropsy and enumeration was performed on the same animals used later for qualitative examinations. One testis was homogenized for total sperm count determination.
- Qualitative examinations: Sperm motility was determined in a sample of the ductus deferens at necropsy. For the evaluation of the sperm motility, the mean percentage of motile and immotile sperms was determined. The total sperm count and number of immotile sperms were recorded.
A morphological evaluation of ductus deferens sperm sample was performed in the same animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, including organ weights (see table No. 4); All animals were necropsied one day after the last treatment (main groups) or after four weeks of recovery (recovery groups).
HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose (30 mg/kg bw/day) groups, including recovery groups. In addition, thymus was also processed histologically in a single animal of the 3 mg/kg bw/day dose group due to macroscopic observations at necropsy.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data: body weight, food consumption, estrous cycle, haematology, blood coagulation, clinical chemistry, organ weight data and sperm parameters.
The heterogeneity of variance between groups was checked by Bartlett’s 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.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of the distribution was not normal, we applied the non-parametric method of Kruskal-Wallis one-way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the two-sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison to the values of the control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in the section “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p <0.05 and <0.01. Male and female rats were evaluated separately.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group (control, 30, 10 or 3 mg/kg bw/day) during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item effect on body weight development was observed in male and female animals at 30 mg/kg bw/day. The body weight changes were only reversible in male animals but not in females. In female animals at 10 mg/kg bw/day, a slight reduction of the mean body weight was also detected. However, the difference was lower than 10 % with respect to the control group. Therefore, it was not considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item influence on the mean daily food consumption was detected at 30, 10 or 3 mg/kg bw/day (male or female).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities in the eyes of all animals were detected before the treatment and in animals of the high dose group (30 mg/kg bw/day) at termination of the treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no pathological changes in the examined hematological parameters in any of the groups (male and female, 30, 10 or 3 mg/kg bw/day).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters at 30, 10 or 3 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any treatment-related difference in any of the animals in the behaviour or in reactions to different types of stimuli at the end of the treatment period with respect to the controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects on the examined organ weights in male or female animals at 30, 10 or 3 mg/kg bw/day. Statistical differences of some organ weights at 30 mg/kg bw/day in males and females are considered to be partially or fully due to body weight changes in these groups. As these changes are not supported by histopathological alterations, these were considered to be of no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings related to the effect of the test item were not detected in organs or tissues of male or female animals at 30, 10 or 3 mg/kg bw/day at the necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination did not reveal alterations related to the test item in the organs or tissues in male or female animals at 30 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
ESTROUS CYCLE
A test item influence on the estrous cycle was not detected at any dose level.

SPERM ANALYSIS
Sperm examinations on sperm cells from the 30 mg/kg bw/day group did not show any test item related effects.
Key result
Dose descriptor:
NOAEL
Effect level:
10 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:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) was determined to be 10 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The objective of this study was to obtain information on the possible health hazards likely to arise from repeated exposure to the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

 

The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 30, 10 and 3 mg/kg bw/day corresponding to concentrations of 0, 15, 5 and 1.5 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were handled identically up to Day 89 and then observed without administration for another four weeks (recovery observations).

The suitability of the chosen vehicle for the test item and sufficient stability of the test item in the vehicle was analytically verified up front. The test item was stable in the applied concentrations in sunflower oil at room temperature for one day and in a refrigerator (5 ± 3°C) for 3 days.

Concentrations of the test item in the dosing formulations varied from 95 % to 106 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing.

Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at the end of the treatment period. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups, including recovery groups. In addition, the thymus was also processed histologically in single male animals of 3 mg/kg bw/day dose group based on macroscopic observation at necropsy.

The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. Toxic signs related to the test item were not detected at any dose level (30, 10 or 3 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery.

The body weight development of male and female animals was reduced in male and female animals at 30 mg/kg bw/day by, which was reversible only in male animals but not in females. Slight reduction of body weight development in female animals at 10 mg/kg bw/day occurred at < 10%. Hence, it was not considered to be toxicologically relevant.

The mean daily food consumption was comparable in animals of the control and test item treated groups (30, 10 and 3 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 30 mg/kg bw/day).

A test item influence on the estrous cycle was not detected (30, 10 and 3 mg/kg bw/day).

Haematology examinations did not reveal test item related toxic changes in the evaluated parameters (30, 10 and 3 mg/kg bw/day). Slight elongation of blood coagulation times (PT, APTT) in male and female animals at 30 mg/kg bw/day were judged to be toxicologically not relevant due to the low degree (mean values remained well within the historical control ranges).

Pathological changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 30, 10 or 3 mg/kg bw/day. Slightly elevated activity of gamma glutamyl transferase at 30 and 10 mg/kg bw/day (mainly in females) were indicative of a test item influence on the hepatic function as an adaptation response to the altered demand. However, there were no related histopathological changes to substantiate their toxicological relevance.

Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period.

A test item influence on the examined organ weights was not detected.

Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 30 mg/kg bw/day.

There were no histological lesions related to the test item effect.

 

Under the conditions of the present study, a dose of 30 mg/kg bw/day the test item reduced the body weight development in male and female Hsd.Han:Wistar rats after the consecutive 90-day oral (gavage) administration. Bodyweight changes were reversible in male animals but not in the female animals.

There were no toxicologically relevant changes of the examined parameters in male or female animals at 10 mg/kg bw/day or 3 mg/kg bw/day.

Based on these observations, the No Observed Adverse Effect Level (NOAEL) was determined to be 10 mg/kg bw/day for male and female Hsd.Han:Wistar rats.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2011-11-16 to 2011-11-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP 14 day dose range finding study for 28 day repeated dose toxicity study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 3 October 2008
Deviations:
yes
Remarks:
14 day dose range finder
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Regulation (EC) No 440/2008
Deviations:
yes
Remarks:
14 day dose range finder
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, July 2000
Deviations:
yes
Remarks:
14 day dose range finder
GLP compliance:
no
Remarks:
The principles of GLP were followed and all data were recorded and retained.
Limit test:
no
Species:
rat
Strain:
Wistar
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: 6 –8 weeks old
- Weight at study initiation: Male animals: 181 – 198 g; Female animals: 125 – 144 g
- Fasting period before study: no
- Housing: 2 or 3 animals sex/cage in Type III polypropylene/polycarbonate cages.
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimatisation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 8 - 12 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil, in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility 2 or 3 days before administration.

A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week, animals received volumes based on the actual body weight on day 0.
The corresponding testing-doses administered to the animals were 1000 mg/kg bw/day, 300 mg/kg bw/day and 100 mg/kg bw/day.
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 once during the study. Samples were taken on study day 0 and measurement was conducted the following day (November 16 and 17, 2011, respectively). The measured concentrations ranged from 101 % to 107 % of nominal concentrations.
The suitability of the chosen vehicle for the test item was analytically proven. 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.
Duration of treatment / exposure:
14 days
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 (± 2 hours). 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 6 days acclimatisation and was continued up to and including the day before necropsy.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in vehicle
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in vehicle
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in vehicle
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in a previously performed acute oral toxicity study with 1,1'-(1,1,2,2-tetramethylethylene)dibenzene in rats (LD50 >2000 mg/kg bw). 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. The mid dose was interpolated geometrically.
Positive control:
none
Observations and examinations performed and frequency:
Clinical Examination:
Detailed clinical observations were made on all animals once a day, after 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 behaviour 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.

Weight Assessment:
All animals were weighed with an accuracy of 1 g on days 0, 7 and 13. Individual body weight changes were calculated between days 0 - 7, 7 - 13 and 0 - 13, latter for the total body weight gain. Fasted body weight was measured on day of necropsy (day 14).

Food Consumption:
The food consumption was determined with an accuracy of 1 g on days 7 and 13 by reweighing the non-consumed diet.

Clinical Pathology
Clinical pathology examinations including haematology and clinical chemistry were conducted one day after the last treatment. Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anaesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 ml 9NC Greiner Bio-One International AG), one for haematology (MiniCollect® K3EDTA, 0.25 mL, Greiner Bio-One International AG), and the third one (Vacuette® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.

Hematology:
- Time schedule for collection of blood: before sacrifice
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- 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:
- Time schedule for collection of blood: before sacrifice
- Animals fasted: Yes
- How many animals: all
- 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.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Pathology:
Gross necropsy was performed on each animal. Animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® (details are presented in "Details of Other Materials") one day after the last treatment. 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, colour, shape and size.

Organ Weight:
The following organ weights were determined and recorded:
- With precision of 0.01 g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands
- With precision of 0.001 g: adrenals, ovaries
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- haematology
- clinical chemistry
- organ weight data
The heterogeneity 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. Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The frequency of clinical symptoms and pathologic findings were calculated. Results were evaluated in comparison with values of control group (term “control value” is used in the text). Parameters stared with statistical significances were listed as deviations from control value in paragraph “Results”.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day
Salivation was noted for all male (5/5, slight degree) and female animals (5/5, slight or moderate degrees) and with variable occurrence from day 5 up to the end of study.
300 mg/kg bw/day
Slight or moderate salivation was observed in all male (5/5) and in two female (2/5) rats with variable occurrence from day 5.
100 mg/kg bw/day
Slight or moderate salivation was detected in one male (1/5) and in one female (1/5, slight) rats on days 8, 13 and on day 13, respectively.

Control
Alopecia and exfoliation on the skin was detected in one male (1/5) rat on day 13. No clinical signs were found in female animals.

In summary, test item related salivation appeared in a dose related manner from 100 mg/kg bw/day dose in male and female animals. Skin alteration of one control male animal was considered to be an individual sign, which occurs frequently in experimental rats.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 100, 300 or 1000 mg/kg bw/day groups during the course of 14-day treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000, 300 and 100 mg/kg bw/day
The mean body weight was significantly lower (p <0.01 in the most cases) than in the control group (male and female animals) on days 7 and 13 due to the significantly reduced body weight gain during weeks 1 and 2. This resulted in a significantly less total body weight gain (determined between days 0 and 13) with respect to controls in all test item treated groups.

In summary, a dose related test item influence on the body weight development was found at 1000, 300 and 100 mg/kg bw/day for male and female animals during the entire observation period. The slightly lower body weight gain resulted in lower body weight in all dose groups with respect to controls on weeks 1 and 2.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000, 300 and 100 mg/kg bw/day
The mean daily food consumption was significantly less than in the control group in all test item treated groups during the entire study.

In summary, the test item caused reduced food consumption at 1000, 300 and 100 mg/kg bw/day (male and female) during the first and second weeks of treatment period in accordance with the body weight values.
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):
1000 mg/kg bw/day
In the male animals, statistical significances were noted for the slightly lower mean values of hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH). The mean value obtained for the percentage of reticulocytes (RET) was significantly reduced. The mean value of corpuscular hemoglobin concentration (MCHC) was slightly but significantly elevated.
In the female animals, the mean values of white blood cell count (WBC), the mean corpuscular hemoglobin concentration and platelet count were above and the per cent of eosinophil granulocytes (EOS) and mean corpuscular volume were below the appropriate control values.
300 mg/kg bw/day
In the male animals, the values for hemoglobin concentration, hematocrit value, and mean corpuscular volume were slightly but significantly reduced. The mean corpuscular hemoglobin content and the values obtained from the platelet count (PLT) were slightly elevated. The mean value obtained for the percentage of reticulocytes was significantly reduced.
In the female animals, the mean white blood cell count and the mean platelet count exceeded the control values, while the per cent of eosinophil granulocytes was lower comparing to the control group.
100 mg/kg bw/day
In the male animals, the mean values of white blood cell count (WBC), the mean corpuscular volume, mean corpuscular hemoglobin content, per cent of reticulocytes were lower and the mean red blood cell count (RBC), the mean of corpuscular hemoglobin concentration were higher than in the control group. The prothrombin time (PT) slightly exceeded the control value.
In the female animals, the per cent of neutrophil granulocytes (NEU) and eosinophil granulocytes was less and the per cent of lymphocytes (LYM) and the prothrombin time was higher than in the control group.

In summary, statistical significances noted for red blood cell parameters (RBC, HGB, HCT, MCV, MCH, MCHC), reticulocytes and white blood cell parameters (WBC, NEU, LYM, EOS), and blood coagulation time (PT) and platelet count were with low degree, not related to doses and were within or marginal to historical control ranges so these were judged to be without toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg /kg bw/day
In the male animals, significant differences with respect to controls were noted for higher activity of alanine aminotransferase (ALT), and gamma glutamyltransferase, higher concentrations of total bilirubin (TBIL), creatinine (CREA), urea and total protein (TPROT). The values obtained for the activity of aspartate aminotransferase (AST) and alkaline phosphatase (ALP), serum concentrations of cholesterol (CHOL), bile acids (BAC), sodium (Na+), potassium (K+), as well as albumin–globulin ratio (A/G) were lower than the appropriate control values.
In the female animals, statistical significances were observed in activity of alanine aminotransferase and gamma glutamyltransferase, in concentrations of total bilirubin, creatinine, urea, bile acids, inorganic phosphorous (Pi), calcium (Ca2+), sodium, chloride (Cl-), albumin (ALB) and total protein and in albumin–globulin ratio.
300 mg/kg bw/day
In the male animals, the mean activity of alanine aminotransferase and gamma glutamyltransferase and mean concentrations of total bilirubin, creatinine, urea and total protein were higher than in the control group. The glucose, cholesterol, bile acids and sodium concentrations in serum and A/G ratio were below the control value.
In the female animals, activity of alanine aminotransferase and gamma glutamyltransferase and concentrations of total bilirubin, creatinine, urea, inorganic phosphorous, calcium, and total protein were higher than in the control group. The mean chloride concentration and albumin –globulin ratio were lower than in the control group.
100 mg/kg bw/day
In the male animals, statistically significant differences were found in the higher mean activity of alanine aminotransferase and mean concentrations of total bilirubin and creatinine, as well as in the lower concentrations of glucose, cholesterol and bile acids when compared to the relevant values of control group.
In the female animals, mean concentrations of total bilirubin and calcium exceeded the control value, while mean activity of aspartate aminotransferase, concentration of cholesterol and albumin-globulin ratio were below the values of control group.

In summary, a test item influence on renal and hepatic function seemed to be likely because of the higher mean activity of alanine aminotransferase and gamma glutamyltransferase and mean concentrations of total bilirubin, creatinine and urea in male and female animals treated with 1000 and 300 mg /kg bw/day. In male animals administered with 100 mg/kg bw/day, similar changes were induced in the activity of alanine aminotransferase and mean concentrations of total bilirubin and creatinine but to a smaller degree. Slightly elevated total protein concentrations and lower values of A/G ratio (along with normal albumin levels) were due to a slightly higher concentration of globulins at 1000 and 300 mg /kg bw/day (male and female).
Some statistically significant differences in aspartate aminotransferase and alkaline phosphatase activity, in levels of glucose, cholesterol, bile acid, phosphorous, calcium, sodium, potassium, chloride and albumin were judged to be of little or no biological significance as the mean values were within or marginal to the historical control ranges or these were without any dose-response relationship.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute organ weights and organ weights relative to the body weight were not considered to be relevant for evaluation because of the significantly lower mean body weight of male and female animals administered with 1000, 300 and 100 mg/kg bw/day.
In the male animals, significantly lower organ weights relative to brain weight were noted for body weight, heart, thymus, spleen and seminal vesicles at 1000, 300 and 100 mg/kg bw/day, for prostate at 1000 and 300 mg/kg bw/day and for testes and epididymides at 1000 mg/kg bw/day. The liver weight and weight of adrenal glands both relative to brain weight, exceeded the control value at 1000 and 300 mg/kg bw/day, and at 100 mg/kg bw/day, respectively.
In the female animals, body weight relative to brain weight was less than in the control group at 1000, 300 and 100 mg/kg bw/day. The weights of thymus and ovaries both relative to brain weight were below the control value, while the liver weight relative to the brain weight was above the mean value of control group at 1000 and 300 mg/kg bw/day. The spleen weight relative to brain weight was slightly less than in the control group at 300 mg/kg bw/day.

In summary, higher liver weights relative to brain weight reflected a test item influence at 1000 and 300 mg/kg bw/day both in male and female animals. Changes noted for the seminal vesicles weights relative to brain weight at 1000, 300 and 100 mg/kg bw/day, for prostate weights relative to the brain weight at 1000 and 300 mg/kg bw/day, for testes and epididymides weights relative to brain weight at 1000 mg/kg bw/day were also considered to be related to test item. The significantly less organ weights relative to the brain weight (heart, thymus, spleen in male animals at 1000, 300 and 100 mg/kg bw/day; thymus, ovaries in female animals at 1000 and 300 mg/kg bw/day; spleen in female animals at 300 mg/kg bw/day) with respect to controls were indicative of the test item influence probably in accordance with the body weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day
Smaller than normal seminal vesicles (5/5 male), enlargement (1/5 female) and mottled surface of kidneys (4/5 male and 4/5 female) were observed.
300 mg/kg bw/day
Smaller than normal seminal vesicles (5/5 male), paleness (1/5 female) and mottled surface of kidneys (4/5 male and 3/5 female) were detected at the necropsy.
100 mg/kg bw/day
Congenital absence of one side testis (1/5), smaller than normal seminal vesicles (5/5 male) and moderate hydrometra (1/5) were noted for animals of this group.

Control group
There were no macroscopic findings in male animals (5/5) and slight hydrometra (1/5) occurred in one female rat (1/5).

In summary, mottled surface of the kidneys at 1000 and 300 mg/kg bw/day (male and female) and smaller than normal seminal vesicles at 1000, 300 and 100 mg/kg bw/day were considered to be test item related necropsy findings. A test item effect cannot be excluded in development of enlargement and paleness of kidneys in single female rats at 1000 and 300 mg/kg bw/day.
Congenital absence of one side testis is a common finding also in untreated rats and occurred in the low dose group in this study. Hydrometra indicative of sexual cycle of female animals is a frequent observation in experimental rats, which has no toxicological meaning.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Critical effects observed:
not specified
Conclusions:
Based on this 14 day dose range finder study, the doses for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study) were determined as follows: 10, 30 and 100 mg/kg bw/day.
Executive summary:

The aim of this 14-Day toxicity study was to obtain first information on the toxic potential of 1,1´-(1,1,2,2-tetramethylethylene)dibenzene in rats at three dose levels. The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n = 5 animals/sex/dose) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses applied in concentrations of 20, 60 and 200 mg/mL, corresponding to a dose volume of 5 mL/kg bw for 14 days. Stability and concentration of test item in sunflower oil were analytically confirmed. 1,1´-(1,1,2,2 -tetramethylethylene)dibenzene proved to be stable at 5 ± 3 °C for 3 days (recovery was 102 % of starting concentrations at 1 mg/mL and 104 % at 200 mg/mL). The test item content of formulations administered to animals ranged from 101 % to 107 % of nominal concentrations. Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.  

No mortality occurred during the observation period. Test item related salivation appeared in a dose related incidence, degree and occurrence in male and female animals at 1000, 300 and 100 mg/kg bw/day doses. Individual clinical signs (slight alopecia and exfoliation on the skin) were noted for single control male animal which are common in experimental rats. The body weight gain was reduced at 1000, 300 and 100 mg/kg bw/day (male and female) during weeks 1 and 2 resulting in lower body weight values on days 7 and 13 and total body weight gain at the end of study with respect to controls. The mean daily food consumption was significantly less comparing to control group at 1000, 300 and 100 mg/kg bw/day (male and female) during the first and second weeks of treatment period in full accordance with the body weight development.

There were no test item related alterations in the examined haematology and blood coagulation parameters. Statistical significances noted for some parameters were with low degree, not related to doses and were within or marginal to historical control ranges so these were judged to be without toxicological significance.

The higher mean activity of alanine aminotransferase and gamma glutamyltransferase and mean concentrations of total bilirubin, creatinine and urea referred to a test item influence on renal and/or hepatic function in male and female animals dosed with 1000 and 300 mg/kg bw/day. Most of these changes occurred in a lower degree in male animals dosed with 100 mg/kg bw/day. Slightly elevated protein concentrations and lower values of A/G ratio (along with normal albumin levels) were due to a slightly higher concentration of globulins at 1000 and 300 mg/kg bw/day (male and female). Necropsy Gross pathology revealed test item related changes in the kidneys (mottled surface) at 1000 and 300 mg/kg bw/day in male and female animals and in seminal vesicles (smaller than normal) at 1000, 300 and 100 mg/kg bw/day. Enlargement and paleness of kidneys were noted for single female rats at 1000 and 300 mg/kg bw/day, respectively.

The absolute organ weights and organ weights relative to the body weight were not considered to be relevant for evaluation because of the significantly lower mean body weight of male and female animals administered with 1000, 300 and 100 mg/kg bw/day. Organ weights relative to brain weight reflected a test item influence on the liver at 1000 and 300 mg/kg bw/day both in male and female animals and on seminal vesicles at 1000, 300 and 100 mg/kg bw/day, on prostate at 1000 and 300 mg/kg bw/day, and on testes and epididymides at 1000 mg/kg bw/day.

 

Based on these observations the doses for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study) were determined as follows:

 

Group 1: Vehicle control

Group 2: 10 mg/kg bw/day

Group 3: 30 mg/kg bw/day

Group 4: 100 mg/kg bw/day

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2012-01-11 to 2012-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species:
rat
Strain:
Wistar
Remarks:
Hsd.Brl.Han:Wist
Details on species / strain selection:
- Strain: 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)
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
- Humidity (%): 30 - 70
- Air changes (per hr): 8 - 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on oral 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.
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. The test item 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.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
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
Observations and examinations performed and frequency:
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 behavioural 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 was 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 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 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 haematology 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 haematology 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.

HAEMATOLOGY:
The haematology 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.
Sacrifice and pathology:
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, colour, 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, colour, 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.
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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.

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.

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 (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.
Mortality:
mortality observed, 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).
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
Water consumption and compound intake (if drinking water study):
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 haematological 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 haematological 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):
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.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
1,1'-(1,1,2,2-tetramethylethylene)dibenzene 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 sub-acute 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
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, reduced food consumption
Key result
Critical effects observed:
not specified
Conclusions:
The subacute toxicity oral of 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was 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: 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 behaviour, 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, haematology, 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.

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.

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).

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.

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.

Haematology 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.

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.

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.

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 the male and female rats: 30 mg/kg bw/day

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline and GLP compliant study. Reliable without restriction.
System:
other: unspecific systemic toxicity
Organ:
other: body weight development

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study

Repeated dose toxicity: oral (90 days)

The objective of this study was to obtain information on the possible health hazards likely to arise from repeated exposure to the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The study was performed according to OECD 408.

 

The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 30, 10 and 3 mg/kg bw/day corresponding to concentrations of 0, 15, 5 and 1.5 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were handled identically up to Day 89 and then observed without administration for another four weeks (recovery observations).

The suitability of the chosen vehicle for the test item and sufficient stability of the test item in the vehicle was analytically verified up front. The test item was stable in the applied concentrations in sunflower oil at room temperature for one day and in a refrigerator (5 ± 3°C) for 3 days.

Concentrations of the test item in the dosing formulations varied from 95 % to 106 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing.

Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at the end of the treatment period. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups, including recovery groups. In addition, the thymus was also processed histologically in single male animals of 3 mg/kg bw/day dose group based on macroscopic observation at necropsy.

The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

No animal died during the course of the study. Toxic signs related to the test item were not detected at any dose level (30, 10 or 3 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery.

The body weight development of male and female animals was reduced in male and female animals at 30 mg/kg bw/day by, which was reversible only in male animals but not in females. Slight reduction of body weight development in female animals at 10 mg/kg bw/day occurred at < 10%. Hence, it was not considered to be toxicologically relevant.

The mean daily food consumption was comparable in animals of the control and test item treated groups (30, 10 and 3 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 30 mg/kg bw/day).

A test item influence on the estrous cycle was not detected (30, 10 and 3 mg/kg bw/day).

Haematology examinations did not reveal test item related toxic changes in the evaluated parameters (30, 10 and 3 mg/kg bw/day). Slight elongation of blood coagulation times (PT, APTT) in male and female animals at 30 mg/kg bw/day were judged to be toxicologically not relevant due to the low degree (mean values remained well within the historical control ranges).

Pathological changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 30, 10 or 3 mg/kg bw/day. Slightly elevated activity of gamma glutamyl transferase at 30 and 10 mg/kg bw/day (mainly in females) were indicative of a test item influence on the hepatic function as an adaptation response to the altered demand. However, there were no related histopathological changes to substantiate their toxicological relevance.

Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period.

A test item influence on the examined organ weights was not detected.

Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 30 mg/kg bw/day.

There were no histological lesions related to the test item effect.

 

Under the conditions of the present study, a dose of 30 mg/kg bw/day the test item reduced the body weight development in male and female Hsd.Han:Wistar rats after the consecutive 90-day oral (gavage) administration. Bodyweight changes were reversible in male animals but not in the female animals.

There were no toxicologically relevant changes of the examined parameters in male or female animals at 10 mg/kg bw/day or 3 mg/kg bw/day.

Based on these observations, the No Observed Adverse Effect Level (NOAEL) was determined to be 10 mg/kg bw/day for male and female Hsd.Han:Wistar rats.

Supporting study

Combined repeated dose and reproduction/developmental toxicity study

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 behaviour, 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, haematology, 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.

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.

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).

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.

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.

Haematology 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.

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.

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.

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 the male and female rats: 30 mg/kg bw/day

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

Repeated dose toxicity: inhalation

In accordance with column 2 of REACH Regulation (EC) No 1907/2006, Annex IX, the test repeated dose toxicity after inhalation (required in section 8.6) does not need to be conducted as a repeated dose toxicity study for oral application is available. In addition, the volatility of the substance is very low and inhalation exposure is in conclusion expected unlikely. Thus, no test on repeated dose inhalation toxicity has to be conducted and risk

assessment is based on the oral study.

Repeated dose toxicity: dermal

In accordance with column 2 of REACH Regulation (EC) No 1907/2006, Annex IX, the test repeated dose toxicity after dermal application (required in section 8.6) does not need to be conducted as a repeated dose toxicity study for oral application is available. Furthermore, an acute dermal toxicity study was conducted and did not reveal any systemic alterations. Thus, no test on repeated dose dermal toxicity has to be conducted and risk assessment is based on the oral study.

Justification for classification or non-classification

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. The NOAEL in a 90-d oral repeated dose study in rats was 10 mg/kg bw/d for males and females, respectively, based on depressed body weight development. Based on pathological and histopathological examinations there are no indications that certain target organs were specifically affected by the test item treatment. Moreover, no mortalities were observed in the course of thes subchronic study. As a result the substance is not considered to require classification as STOT RE under Regulation (EC) No. 1272/2008, as amended for the twelfth time in Regulation (EU) No. 2019/521.