2-phenyl-1H-benzimidazole-5-sulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from July 18, 2002 to Oct. 07, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

EC Number:

248-502-0

EC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Cas Number:

27503-81-7

Molecular formula:

C13H10N2O3S

IUPAC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Method

Target gene:

Chromosomal aberations in human peripheral blood lymphocytes

Species / strain

Species / strain / cell type:

other: human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

a liver homogenate fraction (S9) from Aroclor 1254 treated male rats

Test concentrations with justification for top dose:

0.33 mM, 3.3 mM, and 10 mM in the tests with 24 hours of treatment without a metabolic activation system,
1.0 mM, 3.3 mM, and 10 mM in the tests with 3.5 hours of treatment with and 4 hours of treatment without a metabolic activation system.

Vehicle / solvent:

distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/ml

Positive control substance:

mitomycin C

Remarks:

without s9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

25 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

with s9

Details on test system and experimental conditions:

Test system:
Lymphocyte cultures
Samples of human peripheral blood were obtained by venipuncture from a male healthy donor, who was not undergoing any drug treatment and collected in heparinised vessels. Lymphocyte cultures were established by addition of small inocula of whole blood (0.4 ml) to each culture tube containing 5 ml of Ham's F10 complete medium, to which 0.1 ml of phytohaemagglutinin was added. The tubes were sealed and incubated at 37 °C.
Metabolic Activation System
The rat liver homogenate fractions used were obtained from King & Hamasch GmbH (Lot KH1501)S Kirchzarten. The preparation of the 9000g supernatant of liver homogenates (S9) from Sprague Dawley male rats (8-10 weeks old, Harlan Winkelman, D-33176 Borchen) induced with Aroclor 1254 (500 mg/kg body weight) is in accordance with the method recommended by Ames et al. (1975).
The S9 preparations (in 0.15 M KC1) were stored in liquid nitrogen.
S9-mix containing 25% S9 was freshly prepared for each mutagenicity assay. The concentrations of the cofactors in the S9-mix are: NADP, 4 mM; glucose-6-phosphate, 25 mM, MgCl2, 8 mM; KC1, 33 mM; phosphate buffer pH 7*4, 100 mM. This solution is filter sterilized by passage through a 0.2 µm filter. S9-mix was kept on ice.

Evaluation criteria:

A significant increase of mutant cells by the test compound will be stated, when the frequency of cells with aberrations in the concurrent negative controls and the frequency of cells with aberrations at each dosage level differ significantly.

Statistics:

The statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Results and discussion

Test results

Key result

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The highest concentration tested induced 69% (1st exp., 24h) and 27 % cytotoxicity (2nd exp., 4h) in the absence and 58 % (1st exp.) and 52 % (2nd exp.) cytotoxicity in the presence of S9-mix.
Test substance did not induce any increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system.

Remarks on result:

other:

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, test article did not cause clastogenic effects in human peripheral blood lymphocytes with and without metabolic activation system.

Executive summary:

The chromosome aberration study was undertaken to examine the mutagenic effect of test article for its capability to induce chromosome damage in human peripheral blood lymphocytes in vitro. All tests were carried out both in the presence and absence of metabolic activation system. Two independent experiments were performed. A sampling time at 1.5 times the normal cell-cycle length from the beginning of the treatment (24 h) was used in the first and second experiment. The two experiments without S9-mix differ in the duration of the exposure to the test substance. In the first experiment the cells were treated in the absence of S9 for 24 hours with the test compound, whereas in the second experiment without S9 the test compound was washed away 4 hours after the start of the treatment. In the test with a metabolic activation system, the cells were always exposed to the test substance for 3.5 hours. The following concentrations of test article were tested: 0.33 mM, 3.3 mM, and 10 mM in the tests with 24 hours of treatment without a metabolic activation system 1.0 mM, 3.3 mM, and 10 mM in the tests with 3.5 hours of treatment with and 4 hours of treatment without a metabolic activation system.

At the concentrations tested, the test article did not induce any increase in the number of chromosome aberrations in cultured human blood lymphocytes in the absence and presence of a metabolizing system. In conclusion, the results indicate that, under the experimental conditions described, the test article was not mutagenic in the in vitro mammalian chromosome aberration test with human peripheral blood lymphocytes.