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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this guideline.
Deviations:
yes
Remarks:
Individual body weights at study start were not indicated; some parameters, as age of animals at study start and humidity level during study, were not reported; sampling time was only 6 hours following end of treatment.
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test was performed with dibenzyltoluene, new name: dibenzylbenzene, ar-methyl derivative

Test animals

Species:
mouse
Strain:
other: COBS CD-1 (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: no data
- Weight at study initiation: 18-21 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: 2 (preliminary study) or 5 (main study) animals/sex/cage
- Diet (e.g. ad libitum): laboratory diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): no ndata
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12


IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: UGILEC 102 was miscible with corn oil
- Concentration of test material in vehicle: 100, 200 and 400 mg/mL
- Amount of vehicle (if gavage or dermal): animals in all groups were dosed with the standard volume of 0.1 ml/10g bodyweight (10 mL/kg bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ugilec 102 was prepared as a solution in corn oil using a high speed mixer.
Duration of treatment / exposure:
24h.
Frequency of treatment:
2 administrations separated by an interval of 24h.
Post exposure period:
6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 2000 and 4000 mg/kg/day for 2 days
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): yes, a known mutagen (recommended by OECD for this specific test)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg

Examinations

Tissues and cell types examined:
Tissues: bone marrow smear from each femur
Cells: erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on the Maximal Tolerated Dose obtained from preliminary toxicity testing.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling was performed on dead animals sacrificed 6 hours after the last administration.

DETAILS OF SLIDE PREPARATION:
Following animal sacrifice, a direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight. After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor; 1 part Giemsa : 9 parts buffered distilled water, pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried and mounted in DPX.

METHOD OF ANALYSIS:
The stained smears were coded and examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.
Evaluation criteria:
No data
Statistics:
No data

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
from a total dose of 4000 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Phase I (2 animals/sex/dose): 2 administrations from 250 to 10400 mg/kg bw/d (from 500 to 20800 mg/kg bw in total). Phase II (5 animals/sex/dose): 2 administrations from 4500 to 6500 mg/kg bw/d (from 9000 to 13000 mg/kg bw in total)
- Mortality: (see Table 1 in the field Remarks on results)
- Clinical signs of toxicity: After administration of corn oil, the vehicle control, no toxic reactions were observed. After administration of Ugilec 102 at a total dosage of 500 mg/kg bodyweight, no toxic reactions were observed. At a total dosage of 5000 mg/kg bodyweight, no toxic reactions were observed after the first dose until the morning of the second day (24 hours after the first dose) when hunched posture, lethargy and pale external extremities were observed. In addition, all animals in this group were observed to be thinner than those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose. At total dosages between 9000 and 20800 mg/kg bodyweight, a toxic reaction consisting of lethargy was observed 1 hour after the first dose and was still present 7 hours later. On the morning of the second day (24 hours after the first dose), the lethargy was still present together with hunched posture, pale external extremities and ptosis. All animals in each group were observed to be thinner thon those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose.



RESULTS OF DEFINITIVE STUDY (see Table 2 in the field Remarks on results)
- Mortality: no mortality was observed in the low dose animal group. Two females treated at 2000 mg/kg bw/d (4000 mg/kg bw in total) were found dead 6 hours after the second treatment. Macroscopic examination at post mortem of these two unexpected deaths, failed to reveal any abnormalities. Four animals (two females and two males) treated at 4000 mg/kg bw (8000 mg/kg bw in total) were found dead 5 or 6 hours respectively, after the second dose. Macroscopic examination at post mortem of all four animals, failed to reveal any abnormalities.
- Clinical signs of toxicity: After administration of corn oil, the vehicle control, no toxic reactions or deaths were observed.
After administration of Ugilec 102 at a total dosage of 2000 mg/kg bw/d, no toxic reactions were observed. At a total dosage of 4000 mg/kg bodyweight, no toxic reactions were observed after the first dose until the morning of the second day (24 hours after the first dose) when hunched posture, lethargy and pale external extremities were observed. In addition, aIl animals in this group were observed to be thinner thon those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose. At a total dosage of 8000 mg/kg bw/d, a toxic reaction consisting of lethargy was observed 1 hour after the first dose and was still observed 7 hours later. On the morning of the second day (24 hours after the first dose) the lethargy was still present together with hunched posture, pale external extremities and ptosis. All animals in this group were observed to be thinner than those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose.

- Induction of micronuclei (for Micronucleus assay): after administration of Ugilec 102 at all total dosages, the group mean micronucleated cell counts were comparable with the concurrent control value.
- Ratio of NCE/PCE (for Micronucleus assay): After administration of Ugilec 102 at all total dosages, the group mean normochromatic to polychromatic cell ratios were comparable with the concurrent control value.
- Appropriateness of dose levels and route: toxic reactions observed in the micronucleus test as expected as they were similar to the reactions observed in preliminary toxicity study at the same dose levels.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
UGILEC 102 (Dibenzylbenzene, ar-methyl derivative) did not present any in vivo genotoxic activity in the micronucleus test conducted in mice orally gavaged twice at 1000, 2000 and 4000 mg/kg bw.
Executive summary:

The genotoxicity of the test substance, Dibenzylbenzene, ar-methyl derivative (UGILEC 102) was determined in a micronucleus test conducted in vivo with COBS CD-1 (ICR) BR mice using a similar method to the OECD Guideline 474.

Solutions were administered by gavage at a constant volume of 10 mL/kg bw. Following preliminary toxicity assay, 4000 mg/kg bw/d treatment for 2 days was selected as the maximum tolerated dose, based on high rates of mortality and severe clinical signs observed at higher dosages.

Doses of 1000, 2000, and 4000 mg/kg bw/d (2000, 4000 and 8000 mg/kg bw in total) were administered by oral gavage to groups of 10 mice (5 males and 5 females), in two equal dosages, separated by an interval of 24 hours. Mitomycin C, administered intraperitoneally at a total dose level of 8 mg/kg, served as positive control.

Animal were sacrificed 6 hours after the second administration, and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse and for the ratio NCE/PCE. No significant increased mortality was observed in any treated groups.

Signs of toxicity were only observed at the dose of 2000 mg/kg bw/d (4000 mg/kg bw in total, respectively), consisting of hunched posture, lethargy, pale external extremities and also ptosis at the dose-level of 8000 mg/kg bw in total. The NCE/PCE ratio obtained in all treated animals was similar to that of control animals. The number of micronucleated polychromatic cells in animals given dibenzyltoluene was similar to that obtained in control animals. Conversely, animals treated with Mitomycin C presented a highly significant increase in the number of micronucleated polychromatic cells. The test substance was concluded to be negative in the micronucleus test with mice.