N-butylbenzenesulphonamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and documented study, according to GLP and guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-butylbenzenesulphonamide

EC Number:

222-823-6

EC Name:

N-butylbenzenesulphonamide

Cas Number:

3622-84-2

Molecular formula:

C10H15NO2S

IUPAC Name:

N-butylbenzenesulfonamide

Details on test material:

- Common name: BBSA
- Appearance: colourless clear liquid
- Purity: 99.94 %
- Lot nr: 200911030035
- Date of manufacture: 03.11.2009
- Shelf life: 2 years
- Date of receipt: 03.12.2009
- Storage: required quantity of the test substance was received from the test substance controller (TSC, IIBAT) and stored in a dry and cool place
- Handling: test substance was handled with necessary protective clothing and all recommended safety measures were followed.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. An aliquot of frozen cells from storage was revived and washed off the freezing medium (SOP/GTX/012). Cells were handled with care giving appropriate time to recover before use in the lymphima assay, and subsequently were checked for contamination. No contamination was recorded.
- Source: American Type Culture Collection, ATCC, USA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (cofactor supplemented post mitochondrial fraction, prepared for the liver of rats treated with enzyme inducers)

Test concentrations with justification for top dose:

0.079 - 0.157 - 0.313 and 0.625 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 0.1 ml

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: with S9 - DMSO (2 and 3 ùg/L)

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without S9 - DMSO (10 and 20 ùg/L)

Details on test system and experimental conditions:

METHOD OF APPLICATION: collected in sterile centrifuge tube and than plated into duplicate microtitre plates

DURATION
- Preincubation period: cultures maintained in flask for 2 days
- Incubation period: 2 weeks
- Exposure duration: 3h with and without S9, 24h without S9
- in humidified 5% CO2 atmosphere at 37 +/- 1°C

NUMBER OF CELLS EVALUATED: 1.6 cells/well in a total of 192 wells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; mutation efficiency, relative total growth, relative suspension growth

Evaluation criteria:

the assay was considered valid as it met the following criteria:
- the spontaneous mutant frequency of the solvent control was within 50 to 170 TFT resistant mutants per 10^6 viable cells. The cloning efficiency of the solvent control group was greater than 50%
- both the concentrations employed for each of the positive control exhibited mutant frequencies of >200 mutants per 10^6 cells. The colony size distribution for MMS showed an increase in bith small and large colonies.

Statistics:

As unequivocal results were confronted and were comparable to the historical data, statistical treatment of data was not done.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.625 ùl/ml in 3h and 24h treatments

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.313 ùl/ml in the 24h treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTATION FREQUENCY
- experiment 1 ranged 69.48 to 93.34 ùg/L with S9 and 68.14 to 92.84 without S9.
- a similar trend was observed in the duplicates (67.87 to 97.86 with S9, 69.79 to 92.06 without S9)
- solvent control: 63.01 to 63.57 with S9, 64.49 to 60.56 without S9

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The test substance BBSA did not induce an elevated (significant) mutagenic reponse under the experimental conditions employed in L5178Y TK+/- cell line -mouse lymphoma assay.

Executive summary:

An in vitro mammalian cell gene mutation assay was conducted N-n-butylbenzenesulphonamide in the L5178Y TK+/- mouse lymphoma cell line with and without metabolic fraction at concentrations of 0.079 - 0.157 - 0.313 and 0.625 µl/ml. The test substance BBSA did not induce an elevated (significant) mutagenic reponse under the experimental conditions employed in L5178Y TK+/- cell line -mouse lymphoma assay.