N-butylbenzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No in vitro mutagenicity response has been observed in following test methods with and without metabolic activation: Bacterial reverse mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and Escherichia coli WP2 uvrA strain; Cytogenetic Chromosomal Aberration test in human lymphocytes; Mammalian cell gene mutation assay in the L5178Y TK+/- mouse lymphoma cell line.

Link to relevant study records

Reference

Reference 1

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

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

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

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)

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2018 - 27 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Additional strain tested to Salmonella typhimurium strains tested in UCB 180/83524 study (1983).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

only one strain was used as requested by the Sponsor

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No.440/2008

Deviations:

yes

Remarks:

only one strain was used as requested by the Sponsor

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, 1998

Deviations:

yes

Remarks:

only one strain was used as requested by the Sponsor

Principles of method if other than guideline:

Only 1 strain was tested as other strains were already tested.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 201705080023
- Expiration date of the lot/batch: 08 May 2019
- Purity test date: 99.93%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%), protected from humidity (tightly closed container)
- Stability under test conditions: Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was soluble in DMSO at 100 mg/mL concentration. Therefore, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The obtained stock solution (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Final dilution of a dissolved solid, stock liquid or gel: All dilutions in the main tests of test item were made in the testing laboratory using Dimethyl sulfoxide (DMSO). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation.

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: Production date: 08 May 2017

Target gene:

tryptophan

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: defective in DNA excision repair

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (from phenobarbital/ β-naphthoflavone-induced rats)

Test concentrations with justification for top dose:

Examined concentrations (a total of eight concentrations) were selected according to the results of the already performed study with Salmonella typhimurium strains provided by the Sponsor and the results of the experiments.
Experiment I: 5000, 1500, 500, 150 and 50 μg/plate
Experiment III: 1500, 500, 150, 50, 15, 5 and 1.5 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Dimethyl sulfoxide (DMSO). The test item was soluble in DMSO at 100 mg/mL concentration. Therefore, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Distilled water

True negative controls:

yes

Remarks:

Untreated

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation (invalid)
Experiment III: preincubation

DURATION
Experiment I:
- Preincubation period: No
- Exposure duration: The plates were incubated at 37°C for approximately 48 hours.
- Selection time (if incubation with a selection agent): approximately 48 hours.

Experiment II:
- Preincubation period: 20 minutes at 37ºC in a shaking incubator
- Exposure duration: The plates were incubated at 37°C for approximately 48 hours.
- Selection time (if incubation with a selection agent): approximately 48 hours.

Experiment III:
- Preincubation period: 20 minutes at 37ºC in a shaking incubator
- Exposure duration: The plates were incubated at 37°C for approximately 48 hours.
- Selection time (if incubation with a selection agent): approximately 48 hours.

SELECTION AGENT (mutation assays): 2.5 mLTryptophan solution (2 mg/mL)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduced or absent background lawn

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in the main tests;
- at least five analysable concentrations were presented in the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control.

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

borderline cytotoxicity was detected with and without metabolic activation at 5000 µg test item/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment III

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absent/reduced background lawn was noted in the Experiment III with and without metabolic activation at 1500 µg test item/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed on the plates in the experiments with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The reference mutagens showed the expected increase in the number of revertant colonies.
Positive reference control data
-S9:
Positive reference control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E. coli TA98 TA100 TA1535 TA1537 E. coli
Mean 363.9 1216.4 1167.3 447.3 1028.0 2409.2 2423.8 230.9 219.7 255.1
St. dev. 105.6 193.5 188.7 155.7 133.5 290.5 267.0 123.9 51.7 104.1
Range 152-2336 536-2120 208-2440 149-2104 488-1708 312-4918 1192-5240 101-2216 117-838 125-2512


- Negative (solvent/vehicle) historical control data: The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range.
Untreated control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E. coli TA98 TA100 TA1535 TA1537 E. coli
Mean 22.5 102.3 12.0 7.7 35.2 29.0 109.7 11.5 9.4 40.4
St. dev. 5.6 20.3 4.8 3.5 10.8 6.8 19.2 3.7 3.9 10.4
Range 9-50 54-210 1-46 1-26 11-82 10-56 65-204 1-39 1-29 16-89

DMSO control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E. coli TA98 TA100 TA1535 TA1537 E. coli
Mean 21.5 98.0 12.1 7.6 34.0 28.1 107.3 11.3 9.1 39.4
St. dev. 5.5 19.7 4.7 3.4 10.5 6.9 20.2 3.6 3.8 10.3
Range 6-55 40-217 1-43 1-27 7-81 11-67 53-229 2-33 1-29 9-85

Distilled water control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E. coli TA98 TA100 TA1535 TA1537 E. coli
Mean 23.3 101.7 12.1 8.5 36.2 29.7 109.7 11.3 9.9 41.5
St. dev. 5.7 21.3 4.6 3.5 10.7 6.9 21.1 3.5 3.8 10.3
Range 11-45 45-215 2-47 2-24 12-84 10-53 64-222 3-39 1-24 13-91

Conclusions:

The test item N-n-butylbenzenesulphonamide (Batch Number: 201705080023) had no mutagenic activity on Escherichia coli WP2 uvrA strain under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay using tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included an Experiment I (Plate Incorporation Method), an Experiment II* (Pre-incubation Method) and an Experiment III (Pre-incubation Method).

*Note: In the performed Experiment II using the pre-incubation method, excessive cytotoxicity was observed in the examined Escherichia coli WP2 uvrA strain with and without metabolic activation at two concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines, thus it was considered invalid. Therefore, an additional experiment (Experiment III) was performed in this strain using the pre-incubation method to complete the data. The data of the invalid experiment is not reported, but all the relevant documentation is kept and archived in the raw data binder.

Based on the results of the Compatibility Test, the test item was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. The examined concentrations were in the Experiment I 5000, 1500, 500, 150 and 50 µg/plate. The examined concentrations were in the Experiment III 1500, 500, 150, 50, 15, 5 and 1.5 µg/plate.

In the experiments the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect

No precipitate was observed on the plates in the experiments with and without metabolic activation.

Absent/reduced background lawn was noted in the Experiment III with and without metabolic activation at 1500 µg test item/plate concentration.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented of the experiments, the examined concentration range was considered to be adequate. The study was therefore, considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes in the genome of the strain used.

In conclusion, the test item N-n-butylbenzenesulphonamide had no mutagenic activity on Escherichia coli WP2 uvrA strain under the test conditions used in this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/06/1983 - 13/07/1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

the following strains will be used in the Ames test:
S. typhimurium TA 1535 his G46 rfa- Δ uvr B-
S. typhimurium TA 1537 his C3076 rfa- Δ uvr B-
S. typhimurium TA 1538 his D3052 rfa- Δ uvr B-
S. typhimurium TA 98 his D3052 rfa- Δ uvr B- R+
S. typhimurium TA 100 his G46 rfa- Δ uvr B- R+
All 5 strains are defective in DNA repair capacity (Δ uvr B-) and have a defective lipopolysaccharide barrier on the cell wall (rfa-). These 2 properties confer extra sensitivity to DNA damage and also greater permeability of larger molecules into the cell. Strains TA98 and TA 100 also contain a resistance transfer factor (plasmid pKM101). This factor, which confers resistance to ampicillin, enhances the operation of an error-prone repair system.

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: 0,1 ml of an overnight bacterial culture containing approximately 2*10E9 cells/mland 0,5 ml S-9 mix or 0,5 ml 0,1 M sodium phosphate buffer (pH 7,4) will be placed in glass bijou bottles. 0,1 ml of the test solution will be added followed by 2 ml histidine deficient agar. The mixture will be thoroughly shaken and overlaid on to previously prepared plates containing 15 ml minimal agar. Single petri dishes will be used for each dose level. They will be incubated at 37°c for 72 hrs.. After this period the plates will be examined for the appearance of a complete bacterial lawn. Revertant colonies will be counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance will be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

20, 150, 500 and 5000 µg/plate.

Vehicle / solvent:

- solvent(s) used: dimethylsulphoxide
- Justification for choice of solvent:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

2-nitrofluorene (10µg) (TA1538,98), 9-aminoacridine (20µg) (TA1537), sodium azide (5µg) (TA1535, 100)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2µg) (TA1535, 1537, 1538, 98 and 100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
bacterial suspension and sodium phosphate buffer added to sterile bijou bottles.
the test compound added to cultures at 5 concentrations separated by half-log 10 intervals.
histidine-deficient agar added to each bottles, thoroughly mixed and overlaid onto previously prepared plates containing minimal agar.

DURATION
- Preincubation period: 72 hours
- Exposure duration: 72 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): no selection agent
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

colonies will be counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.

Statistics:

NO DATA

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

BBSA was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.
No substantial increases in the revertant colony numbers of any of the 5 strains were observed following treatment with BBSA at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).
It is concluded that no evidence of mutagenic potential of BBSA was obtained in this bacterial test system at the dose levels used.

Executive summary:

A bacterial reverse muatation test was conducted in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation to assess the potential mutagenic effect of N-butylbenzenesulphonamide. BBSA was not toxic towards the tester strains, therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in the revertant colony numbers of any of the 5 strains were observed following treatment with BBSA at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29/06/2006 - 29/07/2006

Reliability:

1 (reliable without restriction)

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Lymphocytes in peripheral blood obtained from a healthy adult male non-smoking donor without any recent history of illness and under no medication was used for this study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background:no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Main study: 550, 275, 138, 69 and 35µgBBSA/ml of lymphocyte culture

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

cultures with 0.1ml of DMSO

True negative controls:

yes

Remarks:

1 µg/ml Mitomycin C

Positive controls:

yes

Remarks:

20µg/ml 20 µg/ml

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
Cultures were incubated with PHA-M for 48h @ 37°c +-0.5°c. Following this, testsubstance BBSA was added to the culture at 550, 275, 138, 69 and 35µg/ml with and without S9.

DURATION
- Preincubation period: 4h
- Exposure duration: subsequently, cultures were washed free of test substance and incubated in freshly prepared medium with all aditions (except PHA-M and S9). Cultures were incubated at 37+-0.5°c for 32h.
- Expression time (cells in growth medium): Cultures were incubated at 37+-0.5°c for 32h.
- Selection time (if incubation with a selection agent): /
- Fixation time (start of exposure up to fixation or harvest of cells): one hour prior to harvest


SELECTION AGENT (mutation assays): /
SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: thousand consecutive cells were examined.


NUMBER OF CELLS EVALUATED:thousand consecutive cells were examined and cells in metaphase were enumerated. Two hundred well spread, intact metaphases were analysed.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: chromosomal aberrations


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: /
- Other: /


OTHER:

Evaluation criteria:

- the overall aberration frequencies
- the percentage of cells with aberrations
- the percentage of cells with more than one aberration
- any evidence for increased amounts of damage with increasing dose, (i.e.) a positive dose response
- the estimated number of breaks involved in production of the different types of aberrations observed (i.e.,) complex aberrations, may have more significance than simple breaks.

Statistics:

The percent frequencies of numerical aberrations, mean frequency of total structural aberrations per metaphases and mitotic indices were statistically compared between the various treatment groups, using Student 't',test for significance.
The type of aberation, its frequency, the statistical significance of any increase and its correlation to concentration in a given time period were all considered in the evaluation of the mutagenic potential of the test substance.
The criteria for a positive response are either a statistically significance increase in the number of metaphases with structural aberrations at one concentration, or a statistically significant, concentration-related increase in the number of metaphases with structural aberrations. The final decision was based upon scientific judgement.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

700 ug/ml or higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not determined
- Effects of osmolality: not determined
- Evaporation from medium: no
- Water solubility: moderately soluble
- Precipitation:no
- Other confounding effects: no


RANGE-FINDING/SCREENING STUDIES:
Pre test, 3 concentrations of BBSA: 2000, 1000 and 500 µg/ml of culture was tested on human lymphocytes with and without metabolic activation system S9 to test cytotoxicity. Cytotoxicity was observed at 2000 and 1000 µg of test substance with and without metabolic activation system. The percent mean mitotic index of 500µg/ml of culture without S9 was 6,8 and with S9 6,5.


COMPARISON WITH HISTORICAL CONTROL DATA:
no data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:


COMPARISON WITH HISTORICAL CONTROL DATA:


ADDITIONAL INFORMATION ON CYTOTOXICITY:mitotic index for different concentrations between 500-700 ug/ml

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

Under the conditions of the above experiment, no evidence of induction of numerical and structural aberrations was observed at the test dose levels, however, at 550 µg/ml concentration of the test substance, numerical aberration (polyploidy) was recorded. The percentage of polyploidy recorded at this concentration falls within the limit of the historical data.
Hence, N-n-benzenesulphonamide was considered as non-mutagenic in in vitro chromosomal aberration assay using human lymphocytes.

Executive summary:

An in vitro Cytogenetic Assay measuring chromosomal Aberration test was conducted with N-n-Benzenesulphonamide (BBSA) in human lymphocytes with and without metabolic activation at concentrations of 69, 138, 275 and 550 µg BBSA/mL. Under the conditions of the above experiment, no evidence of induction of numerical and structural aberrations was observed at the test dose levels, however, at 550 µg/mL concentration of the test substance, numerical aberration (polyploidy) was recorded. The percentage of polyploidy recorded at this concentration falls within the limit of the historical data. Hence, N-n-benzenesulphonamide was considered as non-mutagenic in in vitro chromosomal aberration assay using human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro: 

No in vitro mutagenicity response has been observed in following test systems:

- A bacterial reverse mutation test was conducted in S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation to assess the potential mutagenic effect of N-butylbenzenesulphonamide (Richold et al, 1983). BBSA was not toxic towards the tester strains, therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in the revertant colony numbers of any of the 5 strains were observed following treatment with BBSA at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).

- A bacterial reverse mutation test was also conducted in Escherichia coli WP2 uvrA with and without metabolic activation to assess the potential mutagenic effect of N-butylbenzenesulphonamide (Orosz, 2018). BBSA was tested up to 5000 µg/plate in the incorporation method and up to 1500 µg/plate in the preincubation method due to excessive cytotoxicity. The test item did not induce gene mutations by base pair changes in the genome of the strain.

- An in vitro Cytogenetic Assay measuring chromosomal Aberration test was conducted with N-n-Benzenesulphonamide (BBSA) in human lymphocytes with and without metabolic activation at concentrations of 69, 138, 275 and 550 µg BBSA/mL (IIBAT, 2006). Under the conditions of the above experiment, no evidence of induction of numerical and structural aberrations was observed at the test dose levels, however, at 550 µg/mL concentration of the test substance, numerical aberration (polyploidy) was recorded. The percentage of polyploidy recorded at this concentration fell within the limit of the historical data. Hence, N-n-benzenesulphonamide was considered as non-mutagenic in in vitro chromosomal aberration assay using human lymphocytes.

- 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 (IIBAT, 2010). The test substance BBSA did not induce an elevated (significant) mutagenic response under the experimental conditions employed in L5178Y TK+/- cell line -mouse lymphoma assay.

 

Justification for selection of genetic toxicity endpoint: Although all three studies were assigned as key studies, the in vitro mammalian gene mutation study was considered as more relevant to mutagenicity for human species.

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), N-butylbenzenesulphonamide does not have to be classified and has no obligatory labelling requirement for genetic toxicity.