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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
Study period:
From December 13, 1989 to April 20, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to guideline study with acceptable restrictions: - Test was performed on males only instead of males and females, without any justification, - 5 rats were used but one gave a slide with insufficient cell density, leading to conclude with only 4 animals, - Positive control was not concurrently tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Study conducted prior to the adoption of the most
Deviations:
yes
Remarks:
Animals were treated for 4 months; only one dose was tested: no cytotoxicity was elicited at this only tested dose level; only male animals were used; 1000 PCE were scored for micronuclei instead of at least 2000. 5 rats were used but one gave a slide wi
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzylbenzene, ar-methyl derivative
EC Number:
258-649-2
EC Name:
Dibenzylbenzene, ar-methyl derivative
Cas Number:
53585-53-8
Molecular formula:
C21H20
IUPAC Name:
dibenzylbenzene, ar-methyl derivative
Test material form:
liquid
Details on test material:
Test was performed with dibenzyltoluene, new name: dibenzylbenzene, ar-methyl derivative
Specific details on test material used for the study:
- Name of the test material (as cited in study report): JARYLEC DBT
- Stability under test conditions: found to be stable in dosing solutions (suspensions in 10% aqueous gum arabic solutions) by analysis of content in solutions at approximately 3 months, 4 months, and 4 months and 2 weeks after the start of the treatment.
- Batch number: 5807
- Composition: Dibenzyltoluenes (97%), tribenzyltoluenes (1.9%), benzyltoluene (< 0.05%).
- Impurities: dibenzylxylenes (0.9%), dibenzylbenzenes (0.2%), anthracene (95 ppm), benzene (35 ppm), toluene (35 ppm), xylenes (< 10 ppm).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: Mean initial bodyweight for males was 185 g
- Fasting period before study: not reported
- Housing: individually; space allocated: 345 square cm x 17 cm
- Diet: a complete commercial diet from U..A.R, ad libitum
- Water: tap-water through automatic waterers, ad libitum
- Acclimation period: at least 1 week before beginning treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 60 ± 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 August To: 13 December 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
10% aqueous gum arabic solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
They were prepared just before administration as suspensions in 10% aqueous gum arabic solution.
Duration of treatment / exposure:
At least 120 days
Frequency of treatment:
daily
Post exposure period:
no
Doses / concentrations
Remarks:
Doses / Concentrations:
500 mg/kg/day for 4 months
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control test was designed for the validation of the purification method, but not for the validation of the specific assay with test substance. Cyclophosphamide was administred intraperitoneally as a single dose at 15 mg/kg bw. Test with positive control was conducted separately from the main study with test substance, but at the same period, i.e. January 3, 1990.
- Justification for choice of positive control(s): no
- Route of administration: intraperitoneally
- Doses / concentrations: 15 mg/kg bw

Examinations

Tissues and cell types examined:
Smears of bone marrow of each femur were prepared. Erythrocytes were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The in vivo genotoxic potential of Jarylec DBT was assessed in the male rat as part of a 4-month toxicity study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Necropsy was performed from Day 134 to 143 (final kill, all surviving animais).

DETAILS OF SLIDE PREPARATION:
Rat bone marrow slides were prepared after purification and enrichment of polychromatic erythrocyte fractions according to Romagna and Staniforth's method (1989).
At the scheduled sacrifice times (from days 134 to 143), five mice per treatment were anaesthetized by pentobarbital and exsanguinated.
Immediately following sacrifice, the femurs were removed and the bone marrow was flushed with newborn calf serum (NCS) supplemented with 25 mM EDTA. The bone marrow cell suspension was purified in a column filled with a mixture of microcrystalline cellulose and alpha-cellulose fibres (50/50).
Elution was performed using 25 ml HBSS (calcium- and magnesium-free). The eluate was filtered at the column exit and centrifuged for 10 min
at 1700 rpm. The supernatant was drawn off and the remaining cell pellet was resuspended in 500 µl NCS. A step-gradient separation was performed using Percoll solutions (at 80% and 30%). After centrifugation for 10 min at 1700 rpm, the sharp band
at the interface of the 30/80% gradient was collected and diluted with HBSS. After an additional centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded and cells were resuspended in 500 µl NCS. Purified cell suspension were diluted in NCS (1:28 or 1:40) and 50 µl were spread onto clean glass slides. The slides were stained with Wright using an automatic Hemostain equipment (Geometric Data Corporation). - PCE/NCE ratio was determined until a total of at least 1000 cells (PCE+NCE) were counted - A total of 1000 PCE was examined for micronuclei.

METHOD OF ANALYSIS:
Reading was performed using a microscope (immersion lens x 100). Evaluation of bone marrow differential cell counts involved:
¿ the number of micronucleated polychromatic cells per 1000 polychromatic erythrocytes,
¿ the number of micronucleated normochromatic cells during the readings;
¿ the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes. Both cellular types were counted until the first 1000 PCE were numbered.
¿ the increase in micronucleated polychromatic cells indicates a possible genotoxic effect whereas the decrease in PCE/NCE ratio shows medullary toxicity.
Evaluation criteria:
Criteria for recognizing the micronuclei are given below:
¿ shape and color giving them the aspect of small nuclei, with well defined outlines, 1/20th to 1/50th the size of an erythrocyte;
¿ structure identical to that of a nucleus (no refraction at focus);
¿ difference from an artefact which is an element appearing indifferently in all types of cells, and sometimes outside cells.
Statistics:
The mean and standard error of the mean were calculated, and treated groups were compared with the control group using the Kruskal-Wallis non parametric test.When the test was significant (non homogeneous means), this method was used for group comparisons; when not, means were considered as homogeneous.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY (see table 1 in the field Remarks on results)
- Induction of micronuclei (for Micronucleus assay): no increase in the number of micronucleated, normochromatic or polychromatic cells was noted in animals treated with Jarylec DBT at 500 mg/kg/d.
- Ratio of PCE/NCE (for Micronucleus assay): no significant decrease in the PCE/NCE ratio was noted, indicating the absence of medullary toxicity of Jarylec DBT.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Dibenzylbenzene, ar-methyl derivative did not induce any increase in the proportion of micronuclei per cell after a 4-month treatment in the rat at 500 mg/kg bw/day by gavage.
Executive summary:

As part of the 4-month toxicity study (see section 7.5.1 Repeated dose toxicity oral) with Dibenzylbenzene, ar-methyl derivative (JARYLEC DBT) administered orally by gavage (0, 5, 50, and 500 mg/kg/day) to Sprague-Dawley rats, bone marrow slides were collected in male animals to evaluate the number of micronuclei per 1000 polychromatic erythrocytes. Finally, only slides of control and high dose treated groups were read.

Bone marrow slides were prepared from 5 male rats/group after purification and enrichment of polychromatic fractions on Percoll gradient. Scoring of the number of micronucleated polychromatic cells per 1000 polychromatic erythrocytes, the number of micronucleated normochromatic cells during the reading, and the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes was performed on control and high dose treated groups.

No increase in the number of micronucleated, normochromatic or polychromatic cells, and no significant decrease in the PCE/NCE ratio were noted in rats treated at 500 mg/kg bw/d for approximately 4 months.

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