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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Bumetrizole
EC Number:
223-445-4
EC Name:
Bumetrizole
Cas Number:
3896-11-5
Molecular formula:
C17 H18 Cl N3 O
IUPAC Name:
2-tert-butyl-6-(5-chloro-2H-1,2,3-benzotriazol-2-yl)-4-methylphenol
Test material form:
solid
Specific details on test material used for the study:
- Name of test substance (as cited in study report): Bumetrizole
- Analytical purity: 99.9 % w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: stable
- Storage condition of test material: stored sealed in a cabinet at 20.0 - 25.3 °C
- Other: Supplier: Ciba Specialty Chemicals, Osaka, Japan

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: CHL/IU
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced with phenobarbital and 5,6-benzoflavone (see table 1)
Test concentrations with justification for top dose:
Cell growth inhibition test:
Short treatment test (6 hr test/18 hr recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL

Chromosome aberration test:
Short treatment test (6 hr test/18 hr recovery): 150, 300, 600, 1200 or 2400 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 75, 150, 300, 600 or 1200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is commonly used for the reverse mutagenicity test on bacteria and it forms a good suspension.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Pulse treatment: 6 hours; Continuous treatment: 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 in total per dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
The test result was considered negative when the occurrence rate of cells with numerical or structural aberrations was below 5% and false positive for a rate between 5-10 % and positive for above 10 % with a dose related increase. The occurence rate of cells with structural aberrations was calculated with/without gaps and judged by the occurrence rate without gaps.
Statistics:
none

Results and discussion

Test results
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1200 µg/plate (pulse treatment), > 600 µg/plate (continuous treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Any other information on results incl. tables

1. Short Treatment Method (results of the tests are shown in Tables 3 and 4.)

The rate of cells with numerical aberrations was below 1.5% for all concentrations with and without S9 mix. The rate of cells with structural aberrations was below 1.0% in all concentrations with and without S9 mix.

The cell survival rate in plates with S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL, survival rate decreased proportionally to concentrations, reaching 34% at the maximum concentration of 2400 µg/mL. The cell survival rate without S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL the value decreased in proportion to higher concentration, attaining 34% at the maximum concentration of 2400 µg/mL.

A white oil filmy precipitate and white impalpable precipitates were observed in all concentrations.

In the negative control, 0% and 0.5% of cells carried numerical aberrations in the absence of S9 mix and presence of S9 mix, respectively. The occurrence rate of cells with structural aberrations was 0% in the presence of S9 mix and 0.5% in the absence of S9 mix. In the positive controls, the occurrence rate of cells with numerical aberrations with (DMN; 500 µg/mL) and without S9 mix (MMC;0.1 µg/mL) was 0%. The occurrence rate of cells with structural aberrations was 72.5% with S9 mix and 52.5% without S9 mix. In conclusion, the induction rate of chromosomal aberrations in the negative and positive control groups was within the range of background thus satisfying the crieria of a valid test.

2. Continuous Treatment Method (test results are shown in Table 5)

The occurrence rate of cells with numerical aberrations in the bumetrizole treatment group was below 1.5% for all concentrations. Likewise, the occurrence rate of cells with structural aberrations was below 1.0% for all concentrations.

The survival rate of cells was above 90% for 75 and 150 µg/mL. However, above 300 µg/mL, survival decreases in proportion to higher concentration, and was 34% for the maximum concentration of 1200 µg/mL.

White filmy oil precipitates and white impalpable precipitates were observed for all concentrations when dosing preparations were added to culture medium in the beginning and also at the end of treatment.

In the negative control, the percentage of cells with numerical aberrations and structural aberrations was 0% and 0.5%, respectively. In the positive controls, the percentage of cells with numerical aberrations and structural aberrations (MMC: 0.05 µg/mL) was 0%, and 43.5%, respectively. Hence, the induction rate of chromosomal aberrations in negative and positive control groups was within the range of background, thus satisfying the criteria of a valid test.

Table 3: Chromosome aberration test results (short term treatment) -S9

Treatment period (h)

S9 mix

Dosage of test substance (mg/mL)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

6-18

-

Negative control

(DMSO )

200

1

0

0

0

0

1 (0.5)

0

100

200

0

0

0 (0)

6-18

-

0.150 *

200

0

0

0

0

0

0 (0)

0

95

200

1

0

1 (0.5)

6-18

-

0.300 *

200

1

0

0

0

0

1 (0.5)

0

95

200

0

0

0 (0)

6-18

-

0.600 *

200

0

0

0

0

0

0 (0)

0

89

200

0

0

0 (0)

6-18

-

1.200 *

200

1

0

0

0

0

1 (0.5)

0

48

200

3

0

3 (1.5)

6-18

-

2.400 *

200

0

1

0

0

0

1 (0.5)

0

34

200

2

0

2 (1.0)

6-18

-

Positive control (MMC)

0.0001

200

53

75

0

0

0

105 (52.5)

0

88

200

0

0

0 (0)

*: Precipitate observed

Table 4: Chromosome aberration test results (short term treatment) +S9

Treatment period (h)

S9 mix

Dosage of test substance (mg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

6-18

+

Negative control

(DMSO )

200

0

0

0

0

0

0 (0)

0

100

200

1

0

1 (0.5)

6-18

+

0.150 *

200

0

0

0

0

0

0 (0)

0

96

200

1

0

1 (0.5)

6-18

+

0.300 *

200

1

1

0

0

0

2 (1.0)

0

91

200

0

0

0 (0)

6-18

+

0.600 *

200

1

0

0

0

0

1 (0.5)

0

85

200

0

0

0 (0)

6-18

+

1.200 *

200

0

0

0

0

0

0 (0)

0

51

200

3

0

3 (1.5)

6-18

+

2.400 *

200

0

0

0

0

0

0 (0)

0

34

200

3

0

1 (1.5)

6-18

+

Positive control (DMN)

0.500

200

66

124

0

2

0

145 (72.5)

0

85

200

0

0

0 (0)

*: Precipitate observed

Table 5: Chromosome aberration test results (continuous treatment)

Treatment period (h)

Dosage of test substance (mg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell growth index

(%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of aberrations (%)

Number of cells observed

Polyploid

Others

Total numberof aberrant cells (%)

24-0

Negative control

(DMSO )

200

0

1

0

0

0

1 (0.5)

0

100

200

0

0

0 (0)

24-0

0.075*

200

0

0

0

0

0

0 (0)

0

97

200

0

0

0 (0)

24-0

0.150*

200

1

0

0

1

0

2 (1.0)

0

92

200

2

0

2 (1.0)

24-0

0.300*

200

0

1

0

0

0

1 (0.5)

0

88

200

1

0

1 (0.5)

24-0

0.600*

200

1

0

0

0

0

1 (0.5)

0

62

200

3

0

3(1.5)

24-0

1.200*

200

0

0

0

0

0

0 (0)

0

34

200

2

0

2 (1.0)

24-0

Positive control (MMC)

0.00005

200

40

56

0

0

0

87 (43.5)

0

88

200

0

0

0 (0)

*: Precipitate observed

Applicant's summary and conclusion

Conclusions:
In conclusion, the in vitro chromosome aberration assay resulted in the test substance being negative (with and without metabolic activation) for inducing genotoxicity and thus, is considered to be non-clastogenic.