Benzethonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

A bacterial mutation assay was performed in accordance with OECD test guidelines no. 471 and 472. No evidence of mutagenic activity was seen at any concentration of Benzethonium chloride in this mutation test.

In further in-vitro studies for chromosomal aberration on human lymphocytes (OECD no. 473) and for gene mutation on mouse lymphoma cells (OECD no. 476), neither clastogenic nor mutagenic effects were noted.

Chromosomal aberration in-vitro

A study (OECD 473) was performed to assess the ability of the test item to induce chromosomal aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemag- glutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Concentrations of 3.91 ug/ml up to 31.25 ug/ml (wit and without S9 -mix) were selected for metaphase analysis. In both the absence and presence of S9 mix, Benzethonium chloride caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test. A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No increases in the proportion of polyploid cells were seen in either test. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.

Gene mutation in-vitro

Benzethonium chloride was tested for mutagenic potential in an in vitro mammalian cell mutation assay (OECD 476). This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK +/-) to the thymidine kinase deficient genotype (TK -/-). Two independent tests in the absence of exogenous metabolic activation (S9 mix) and two independent tests in the presence of S9 mix were carried out. Toxicity was observed after treatment with the test item in all tests in the absence and the presence of S9 mix. Exposure to Benzethonium chloride was not associated with significant increases in mutant frequency in any of the tests, either in the absence or the presence of S9 mix. In all tests the positive control substances increased mutant frequencies significantly. It was concluded that Benzethonium chloride did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Source:

GLP-reports (owner: Lonza)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2001 - January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL
- Identity: LZ1442.3, Benzethonium chloride
- Chemical name: Benzethonium chloride
- Appearance: White powder
- Storage conditions: Room temperature
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Batch No.: OC0509
- Expiry date: 7 June2002
- Purity: Responsibility of sponsor
- Date received: 20 August 2001
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Source of cells: National Collection of Type Cultures, London, England (Salm. typh.) and National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland (E.coli)

Additional strain / cell type characteristics:

other: histidine dependent auxotrophic mutants of Salmonella typhimurium, strains; tryptophan dependent mutant of Escherichia coli

Metabolic activation:

with and without

Metabolic activation system:

S9-liver fractions of rats, induced with AROCLOR 1254

Test concentrations with justification for top dose:

Concentrations of LZ1442.3 up to 5000 µg/plate were tested in the first mutation test. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. Because of excessive toxicity observed in this test, it was repeated using concentrations of LZ1442.3 up to 150 µg/plate. The second mutation test employed concentrations of LZ1442.3 up to 150 µg/plate in the presence of S9 mix, or 50 µg/plate in the absence of S9 mix.

Vehicle / solvent:

Water (purified in-house by reverse osmosis) was used as the solvent for this study

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

furylfuramide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Method of application: the first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage

Rationale for test conditions:

The highest concentration of test substance tested was 50 mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows.

Evaluation criteria:

If exposure to a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon eta/ (1989) and will usually be analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

No statistical analysis performed

Key result

Species / strain:

other: Salm. typh. TA1535, TA1537, TA98 and TA100 / E.coli WP2uvrA/pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Because of excessive toxicity observed in the first test up to 5000 ug/plate, it was repeated using concentrations up to 150 ug/plate. The second mutation test employed concentrations up to 150 ug/plate in the presence of S9 mix, or 50 ug/plate in the absence of S9 mix. Toxicity (observed as thinning of the background lawn of non-revertant cells, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test item at one or more concentrations in both tests.

Conclusions:

It is concluded that, under the test conditions employed, the test item showed no evidence of mutagenic activity in this bacterial test system.

Executive summary:

The study was performed in accordance with OECD test guidelines no. 471 and 472. In this in vitro assessment of the mutagenic potential of the test item, Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100 and Escherichia coli, strain WP2uvrA were exposed to the test substance diluted in water. Water was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage. Concentrations up to 5000 ug/plate were tested in the first mutation test. Because of excessive toxicity observed in this test, it was repeated using lower concentrations up to 150 ug/plate. The second mutation test employed concentrations up to 150 ug/plate in the presence of S9 mix, or 50 ug/plate in the absence of S9 mix. Toxicity (observed as thinning of the background lawn of non-revertant cells, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test item at one or more concentrations in both tests. No evidence of mutagenic activity was seen at any concentration of Benzethonium chloride in either mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the GHS criteria, the test item does not require classification as mutagenic or clastogenic.