N-(2-hydroxypropyl)benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key studies were available for the evaluation of bacterial reverse mutation, and mammalian cell gene Mutation Assay demonstrating tthat there is no genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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- (base –pair substitutions)
S. typhimurium TA 1537 his C3076 rfa- Δ uvr B- ( frame shift mutations)
S. typhimurium TA 98 his D3052 rfa- Δ uvr B- R+ (frame shift mutations)
S. typhimurium TA 100 his G46 rfa- Δ uvr B- R+ (base –pair substitutions)
E.coli: WP2 uvrA:trp;uvrA- (base –pair substitutions and others)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Tester strain of TA 98,TA 1535, and E coli were obtained from MOLTOX, INC NC 28607 USA.
Test strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9Tester

Test concentrations with justification for top dose:

31.6,100,316,1000,2500,and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

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):

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:

other: S.typhimurium TA 98,TA 100,TA 1535,TA 1537 and E.coli WP2.

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

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N-(2-hydroxypropyl)benzenesulfonamide did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, N-(2-hydroxypropyl)benzenesulfonamide is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item N-(2-hydroxypropyl)benzenesulfonamide was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-(2-hydroxypropyl)benzenesulfonamide at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus of L5178Y TK+/- mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen, the original cultures were obtained from American Type Culture Collection (ATCC), U.S.A. Prior to setting up the assay, the cells were cleansed of the spontaneous TK-/- cells by growing it in the selective medium (24-hour in the presence of Thymidine, Hypoxanthine, Methotrexate and Glycine (THMG) in DMEM A medium and 2 days in the presence of THG in DMEM A medium). Cells were screened for mycoplasma using the Mycoplasma detection kit (119K0675-1KT, Sigma) and no contamination was observed. For each experiment, one or more vials were thawed rapidly, the cells diluted in DMEM 10 and incubated in a humidified atmosphere of 5% (v/v) CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks. Cell characterization was performed upon receipt and once in 6 months.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/ml. Range finding study: 10,50,150,500,1000,and 2000 μg/mL were tested without and with metabolic activation. The selection of the concentration used in the main experiment was based on the data from the pre-experiment. 2000 µg/ml( without and with metabolic activation) was selected as the highest concentration.
Main study: 50,100,250,500,1000,1500,2000 μg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

RPMI+5 %HS

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

For a short-term exposure experiment 1 x 107 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 105 cells/mL in a total culture volume of 20 mL, if necessary.
After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium (see below) with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 106 viable cells”
Mutant frequency = -ln [NW/TW (selective medium)] x 800
-ln [NW/TW (non-selective medium)]
NW: number of negative wells
TW: number of total wells
Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100 .
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Evaluation criteria:

The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Statistics:

Statistical analysis was performed

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

nterpretation of results (migrated information):
negative

It is concluded, that under the conditions of the test, N-(2-hydroxypropyl)benzenesulfonamide did not induce mutations in L5178Y TK+/- cells when tested up to concentrations inducing acceptable levels of cytotoxicity.

Executive summary:

The test item N-(2-hydroxypropyl)benzenesulfonamide was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations (without and with metabolic activation): 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL No precipitation of the test item was noted in the experiment. No growth inhibition was observed without and with metabolic activation. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Additionally, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item N-(2-hydroxypropyl)benzenesulfonamide is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The predicted value for N-n-(2-hydroxypropyl)benzenesulphonamide for the in vivo chromosome aberration test was negative, using the OECD QSARtoolbox .

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Type of information:

(Q)SAR

Remarks:

QSAR Toolbox gives a Qualitative prediction of in vivo Chromosome aberration .

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

according to guideline

Guideline:

other: Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals, May 2008

Deviations:

no

Principles of method if other than guideline:

The data are taken from the: ECHA CHEM database on Genetic Toxicity.

GLP compliance:

no

Type of assay:

other: QSAR/ read across modeled data

Details on species / strain selection:

Experimantal data were retrieved from differnt test animas ( Chinese hamster, mouse, rat)

Route of administration:

oral: unspecified

Key result

Genotoxicity:

negative

Remarks on result:

other: in vivo chromosome aberration based on QSARtoolbox

Additional information on results:

-prediction approach: read across among category members Experimental values for the target substance (if any) were not used in prediction calculations
- calculation approach: Taking highest mode value from the 5 nearest neighbours
- descriptor: log Kow N-n-(hydroxylpropyl) benzenesulphoneamide : 0.280
-Prediction confidence: 100.0%; 20 of 20 neighbours' data points have experimental value equal to prediction "Negative"
- predicted value "in vivo chromosome aberration= negative" corresponds to "negative " on the scale of "negative, positive and there were no statistical significant changes”.
- Profiling results for the target substance: DNA binding by OASISv 1.2: No alert found, DNA binding by OECD: No alertfound, Estrogen Receptor Binding: No binder, without OH or NH2 group, OECD HPV Chemical Categories: Not categorized, Protein binding by OASIS v1.2: No alert found, Protein binding by OECD; No alert found, Protein Binding Potency: Not possible to classify according to these rules, Superfragments: No superfragment, Toxic hazard classification by Cramer (original) High( classIII), Toxic hazard classification by Cramer (with extension)High (class III), US-EPA New Chemical Categories: Not categorized

	Endpoints	Descriptor
	in vivo chromosome aberration	log Kow
Target chemical		0.280
chemical No 1 cas 79 -10 -7	negative	0.440
chemical No 2 cas 7237 -83 -4	negative	0.120
chemical No 3 cas 872 -50 -4	negative	-0.110
chemical No 4 cas 7195-44 -0	negative	0.740
chemical No 5 cas 110 -71 -4	negative	-0.210
chemical No 6 cas 122 -99 -6	negative	1.10
chemical No 7 cas 15214 -89 -8	negative	-0.610
chemical No 8 cas 57 -55 -6	negative	-0.780
chemical No 9 cas 109 -86 -4	negative	-0.910
chemical No 10 cas 55 -63 -0	negative	1.51

Conclusions:

Interpretation of results (migrated information):
negative

The predicted value for N-n-(2-hydroxypropyl)benzenesulphonamide for the in vivo chromosome aberration test was negative, using the OECD QSARtoolbox .

Executive summary:

For the determination of the in vivo chromosome aberration potential of N-n-(2 -hydroxypropyl)benzenesulphonamide the OECD QSAR toolbox was used. For the prediction read-across among category members was applied and 5 nearest neighbours (compounds with CAS n° 79 -10 -7, 7237 -83 -4, 872 -50 -4, 7195 -44 -0,110 -71 -4) were taken. Log Kow was taken into account as descriptor. For the target compound, log Kow is 0.280 and the 5 neighbours were within the log Kow range of -0.211 -0.741.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The predicted value for N-n-(2-hydroxypropyl)benzenesulphonamide for the in vivo chromosome aberration test was negative, using the OECD QSARtoolbox .

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-(2 -hydroxypropyl) benzenesulfonamide does not have to be classified and has no obligatory labelling requirement for genotoxicity.