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EC number: 204-485-1 | CAS number: 121-60-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Reverse Mutation Assay:
The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in bacterial reverse mutation test (according to OECD TG 471) in S. typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA strains at concentrations up to 5000µg/plate both in the presence and absence of S9 metabolic activation system.
Chromosomal Aberration Study:
The registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8), was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cells and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.
In vitro mammalian cell gene mutation Study:
The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) was tested non-mutagenic (negative) in a mammalian cell gene mutation assay (according to OECD TG 476) when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from an authoritative database.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- According to OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Purity:99.7%
- Target gene:
- His and Trp
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 microsomal fraction was prepared from phenobarbital and 5,6-bezoflavone-induced male Sprague Dawley rats.
- Test concentrations with justification for top dose:
- i) Plate Incorporation Method:
Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
ii) Pre-incubation Method
Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains) - Vehicle / solvent:
- N,N-dimethylformamide
- Justification for choice of solvent/vehicle: The test chemical was soluble in N,N-dimethylformamide - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- N,N-dimethylformamide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- furylfuramide
- other: 2- aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Triplicate per test
- Number of independent experiments : Two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): NA
- Test substance added in medium; in agar (plate incorporation)
- Rationale for test conditions:
- A confirmatory test was carried out in pre-incubation method, as negative mutagenic effects was observed in plate incorporation method.
- Evaluation criteria:
- The test chemical was adjudged as a postive mutagen when the number of the revertant colonies in the test chemical treated increased dose dependently and became two-fold or more than that of the negative control and significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system and is considered as negative when any of the above criteria was not fulfilled.
- Statistics:
- No statistical analysis was performed.
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 1000 ug/plate concentration without S9, above 2500 ug/plate with S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At and above 2500 ug/ml concentration with and without S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 1000 ug/plate concentration without S9 and at 2500 ug/plate with S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 1000 ug/plate concentration without S9, at 2500 ug/plate with S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 2500 ug/ml concentration with and without S9.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: No mutagenic potential of the test chemical was observed.
- Conclusions:
- The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in bacterial reverse mutation test (according to OECD TG 471) in S. typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA strains, both in the presence and absence of S9 metabolic activation system.
- Executive summary:
A GLP-compliant bacterial reverse mutation study (OECD TG 471) was performed to assess the mutagenic potential of the registered substance in S. typhimurium and E.coli strains. The following strains were used: Salmonella typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA.
The following test concentrations were used:
i) Plate Incorporation Method:
Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
ii) Pre-incubation Method
Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
N,N-dimethylformamide was used as a vehicle. A confirmatory test according to the pre-incubation method was carried out, as the test chemical was found non-mutagenic in the first experiment performed according to the plate incorporation method. The test chemical was considered positive (mutagenic) when the number of the revertant colonies in treated cultures increased dose-dependently and became two-fold or more than that of the vehicle negative control, and a significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system. The test chemical was considered negative (non-mutagenic) when any of the above criteria were not fulfilled. No statistical analysis was performed. Results: The test substance did not induce a two-fold and/or biologically relevant increase in revertant colony counts compared with the vehicle control when it was tested up to 5000µg/plate neither in the presence nor the absence of S9 metabolic activation in S. typhimurium and E. coli strains used. Conclusion:The test substance was considered non-mutagenic (negative) in the bacterial reverse mutation test (OECD TG 471).
- 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
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Datais from an authoritative database.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Principles of method if other than guideline:
- According to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Purity:99.7%
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- No Data Available
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 microsomal fraction was obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats.
- Test concentrations with justification for top dose:
- -S9: 0, 0.065, 0.130, 0.260 mg/mL (short term treatment)
+S9: 0, 0.065, 0.130, 0.260 mg/mL (short term treatment)
24 hrs: 0, 0.065, 0.130, 0.260 mg/ml (continous treatment)
48 hrs: 0, 0.065, 0.130, 0.260 mg/ml (continous treatment) - Vehicle / solvent:
- A solution of 1% carboxymethyl cellulose sodium salt was used.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- A solution of 1%carboxymethyl cellulose sodium salt was used.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- mitomycin C
- Details on test system and experimental conditions:
- In the case of short term treatment without (-S9) and with (+S9), the cells were treated for 6 hrs without and with S9 and cultivated with fresh medium for 18 hrs. In the case of continuous treatment, the cells were treated for 24 and 48 hrs without S9.
In the case of the confirmation test, the cells were treated for 6 hrs with S9 and cultivated with fresh medium for 24 hrs.
Frequency of dosing time: One time
Plates/dose: 2
Negative control: solution of 1% carboxymethyl cellulose sodium salt
Positive controls:
-S9: Mitomycin
+S9: Benzo(a)pyrene
Number of metaphases analyzed;100 metaphases/plate or specimen - Rationale for test conditions:
- A confirmation test was conducted in the short term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance is the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time.
- Evaluation criteria:
- The test chemical was judged to be a positive mutagen when the incidence of cells with chromosomal aberration increased dose-dependently as compared with those of concurrent negative controls or reproducible increase in the incidence of cells with chromosomal aberration at one or more concentrations, while others were judged as negative mutagens.
- Statistics:
- No statistical method was followed
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Short term treatment (-S9, +S9): at 0.2 mg/ml >50% cell growth inhibition Continuous treatment (24, 48 krs): at 0.2 mg/ml >50% cell growth inhibition
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: No mutagenic potential of the test chemical was observed.
- Conclusions:
- The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell both in the presence and absence of S9 metabolic activation.
- Executive summary:
A GLP-compliant in vitro cytogenicity study according to OECD TG 473 was performed with the registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) to assess its clastogenic potential in Chinese Hamster Lung (CHL/IU)cells.
The concentrations of the test chemical used in the study were the follows:
-S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)
+S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)
24 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)
48 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)
The test was performed both in the presence and absence of liver S9 microsomal fraction obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats. A solution of 1% carboxymethyl cellulose sodium salt was used as a vehicle. Mitomycin C (-S9) and benzo(a)pyrene (+S9) were used as positive controls.
A confirmation test was carried out in short-term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance in the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time. Two plates per dose and 100 metaphases per plate were analyzed.
Results:More than 50% cell growth inhibition was observed at≥0.2 mg/ml in both short and continuous treatment protocols. The test chemical did not increase the incidence of cells with chromosomal aberration compared to the vehicle control when tested up to a cytotoxic concentration in short or continuous treatment, neither in the presence nor in the absence of S9 metabolic activation. The percent of aberrant cells were the follows: 1% (VC), 0.5% (at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.0% ( at 0.260 mg/ml) and 0.5% (VC), 0.5% (at 0.065 mg/ml), 1.0% (at 0.130 mg/ml), 0.5% (0.260 mg/ml) in short-term treatment I the absence and in the presence of S9 metabolic activation, respectively.In continues treatment the percentages of cells with chroosaml aberrations were 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 1.0% (at (0.260 mg/ml) and 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.5% (at (0.260 mg/ml) at 24 and 48 hours in absence and in the presence of S9 metabolic activation, respectively. Positive control substances induced significant increase in percent aberrant cells; MMC 15% (short term -S9), 41% (24 hrs) and Benzo(a)pyrene 47% (short term, +S9), 62% (48hrs).Conclusion:The registered substance was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study provides experimental data on the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- Adopted July 29, 2016
- Principles of method if other than guideline:
- The ability of the registered substance to induce gene mutation in the hprt gene was tested in CHO cell line.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- Purity: 98.8%
- Target gene:
- Hprt gene
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: NCCS, Pune, India
- Suitability of cells: As per recommendations specified in OECD 476
- Normal cell cycle time (negative control): No data
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: CHO cells were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml, and incubated at 37±2 °C, 5% CO2 in a CO2 incubator. - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital and β-naphthoflavone-induced rat liver microsomal fraction (S9 homogenate) was used.
- Test concentrations with justification for top dose:
- 0 mg/ml (solvent control)
0 mg/ml (negative control)
0.25 mg/ml
0.5 mg/ml
1 mg/ml
2 mg/ml
Justification: No limiting cytotoxicity was observed in the preliminary cytotoxicity assay as the relative survival values were ≥ 65 at 2 mg/ml (-S9, +S9). - Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle:The Test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml). Hence, dimethyl sulfoxide (DMSO) was selected as a vehicle for the study. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Cultures of CHO cells were grown in a complete RPMI-1640 medium; cells at a density of 10 x 106/25 cm2 were used for cytotoxicity measurement and mutation frequency calculation. Before the experiment, spontaneous mutant CHO cells were cleansed by the treatment with 100 μM hypoxanthine, 400 µM aminopterin and 16 μM thymidine (HAT medium) for 3 days at 37 ±2°C in a CO2 incubator.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: One experiment was performed.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 106 /cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Test substance was added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): NA
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):7 days
- Selection time (if incubation with a selective agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):7 days of expression period +9 day mutant selection period =16 days
- Method used: agar or microwell plates for the mouse lymphoma assay: agar plates were used
- Selective agent used: 2x 105cells / 10 ml of cloning medium were seeded with10 µl/ml 6-thioguanine (6TG)in triplicates.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for Cloning efficiency 1 (CE1): 10 ml of cloning media containing 100 cells were dispensed in 60 mm culture plates in triplicates. Plates were incubated at 37±2°C, 5 % CO2, in a CO2 incubator for 7 days.
Plating for expression: 3x105 cells / 5 ml cells were seeded and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 7 days to allow phenotypic expression of the induced mutation.
Plating for Cloning efficiency 2 (CE2): At the end of the expression period, cells were trypsinized and counted. Cells at the density of 100 cells / 10 ml of cloning media were plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 9 days.
Plating for Mutation Frequency: 2x10 5 cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C in a CO2 incubator for 9 days for mutation frequency.
- Fixation and staining: At the end of the incubation, cells in the culture plates were fixed with 2.5 % and 10 % of formaldehyde in water for 10 minutes each. After fixation, colonies were stained with 5 % Giemsa stain for 10 minutes, followed by washing with distilled water.
- Colony counting: Colonies in all the plates for CE1, CE2 and Mutation Frequency (MF) was counted manually and recorded in the raw data. - Rationale for test conditions:
- - Solubility:The test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml).
- Precipitation check:Precipitation check was performed by adding 50 µl of the test Item (200 mg/ml) to 4.950 ml of culture media to attain a concentration of 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.
- pH check:50 µl of test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final concentration of 2 mg/ml in the medium. Changes in the pH were measured at 0 and 4th hours.
- Preparation of the test item solution:200.3 mg of test item was dissolved in 1 ml of DMSO to achieve a final concentration of 200 mg/ml. From this stock, subsequent serial dilutions with DMSO were made using spacing factor 2 to obtain concentrations of 1, 0.5 and 0.25 mg/ml.
- Preliminary cytotoxicity assay:Cytotoxicity was assessed at concentrations of 0, 0.125, 0.5, 1 and 2 mg/ml in both presence and absence of metabolic activation using triplicate cultures. - Evaluation criteria:
- Criteria of acceptance of the test:
- The concurrent negative control is considered acceptable for addition to the literature
negative control database.
- Concurrent positive controls induce responses compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested
unless one resulted in positive results.
- Adequate number of cells and concentrations are analyzable.
- The criteria for the selection of top concentration are consistent.
- The spontaneous mutant frequency of vehicle control should be between 5 and 20 x10-6.
Evaluation criteria
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the literature negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the literature negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Fisher´s exact test (NCSS statistical software) was used to assess the dose-dependency upon comparing the mutation frequencies of the test-item-treated and control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2mg/ml 72.16% (-S9), 64.93% (+S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:To determine the changes in the pH of the medium, 50 µl of the Test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final Test Item concentration of 2 mg/ml in the medium. Changes in the pH are as mentioned in any other information on materials and methods
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: The chemical was insoluble in distilled water
- Precipitation and time of the determination: Precipitation of Test Item was performed at the start of the experiment by adding 50 µl of the Test Item (200 mg/ml) to 4.950 ml of culture media to attain 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.
RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary cytotoxicity test:
Based on solubility and precipitation test, the preliminary cytotoxicity testing was performed with concentrations of 0 (VC), 0 (NC), 0.125, 0.25, 0.5, 1 and 2 mg/ml and both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation system. Cytotoxicity was assessed by the relative survival (RS, that is, cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls) values. No cytotoxicity (<60% RS ) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation.
Main study:
In the main study, CHO cells were exposed to the test item at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. There was no significant reduction in RS (cytotoxicity) or increase of mutation frequency at any concentrations tested neither in the presence nor in the absence of S9 metabolic activation.No significant reduction in RS (cytotoxicity) and no increase in MF was observed in vehicle control (DMSO) when compared to the negative control (distilled water) either in the presence or absence of S9 metabolic activation. The positive controls (Ethylmethanesulfonate (-S9) and Beno[a]pyrene (+S9) produced statistically significant increases in mutation frequency as compared to the vehicle control.
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
There was no significant reduction in RS (cytotoxicity), and no increase in MF was observed in vehicle control (dimethyl sulfoxide) when compared to the negative control (distilled water) either in the presence or absence of metabolic activation.
The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (201.18x10-6 [Ethylmethanesulfonate], 216.46x10-6 [Benzo(a)pyrene]indicating the sensitivity of the test system to specific mutagens and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Kindly refer to the tables as mentioned in any other information on results including tables
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Prior to treatment (24 hours), CHO cells were prepared with a density of 10 × 106 cells /flask.
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency - Conclusions:
- The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.
- Executive summary:
A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 96.76% (vehicle control), 93.37% (at 0.25 mg/ml), 91.56% (0.5 at mg/ml), 77.29% (at 1 mg/ml) and 72.16% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.57% (vehicle control), 90.78% (0.25 mg/ml), 89.98% (at 0.5 mg/ml), 77.63% (at 1 mg/ml) and 64.93% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence (8.71 x 10-6, 8.76 x 10-6, 10.02 x 10-6 and 10.08 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation (7.93 x 10-6, 9.04 x 10-6, 8.80 x 10-6 and 9.40 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control (8.90 x 10-6, 7.85 x 10-6, absence and presence of S9 metabolic activation, respectively). No significant reduction in RS values (cytotoxicity) was observed in vehicle control (DMSO) (RS:97.76% (-S9), 98.57% (+S9) when compared to the negative control (distilled water). Similarly, no increase in MF values was noted in vehicle control (MF: 8.90 x 10-6, -S9; 7.85 x 10-6, +S9) when compared to the negative control (MF:7.14 x 10-6, S9; 6.15 x 10-6, +S9) neither in the presence nor in the absence of S9 metabolic activation. The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS:201.18 x 10-6; Beno[a]pyrene 216.46 x 10-6 p<0.05) as compared to the vehicle control (8.90 x 10-6, -S9; 7.85 x 10-6, +S9). Conclusion: The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) did not induce gene mutation in the Hprt locus of CHO cells up to the highest test concentration of 2 mg/ml neither in the presence nor in the absence of S9 metabolic activation system in under the experimental conditions described.
Referenceopen allclose all
Short term treatment method
Processing time (h) |
S9 mix |
Conc.{mg/mL) |
No. of cells observed |
Chromatid break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total aberrant cells in (%) |
Gap |
Cell growth (%) |
no. of cells |
Polyploid cells |
other |
Total polyploid cells(%) |
||||
|
|
VC |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
— |
(CMC-Na) |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
100 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
1 |
1 |
0 |
0 |
0 |
2 |
( |
1.0) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
|
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
— |
0.065 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
88 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
0 |
1 |
0 |
0 |
0 |
1 |
( |
0.5) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
_ |
0.130 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
72 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
0 |
0 |
0 |
0 |
0 |
0 |
( |
0.0) |
2 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
100 |
0 |
0 |
0 |
||||
6 -18 |
一 |
0.260 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
38 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
0 |
0 |
0 |
0 |
0 |
0 |
( |
0.0) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
PC |
100 |
6 |
10 |
1 |
2 |
0 |
17 |
1 |
100 |
1 |
0 |
1 |
|||||
6 -18 |
一 |
(MMC) |
100 |
7 |
7 |
0 |
1 |
0 |
13 |
3 |
100 |
0 |
0 |
0 |
|||||
|
|
0.0001 |
200 |
13 |
17 |
1 |
3 |
0 |
30 |
( |
15.0 ) |
4 |
|
200 |
1 |
0 |
1 |
( |
0-5) |
|
|
VC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
+ |
(CMC-Na) |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
100 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
1 |
0 |
0 |
0 |
0 |
1 |
( |
0.5) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
+ |
0.065 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
94 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
1 |
0 |
0 |
0 |
0 |
1 |
( |
0.5) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0) |
|
|
|
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
+ |
0.130 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
69 |
100 |
0 |
0 |
0 |
||||
|
|
|
200 |
2 |
0 |
0 |
0 |
0 |
2 |
( |
1.0) |
0 |
|
200 |
0 |
0 |
0 |
( |
0.0 ) |
|
|
|
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
|
100 |
0 |
0 |
0 |
||||
6-18 |
十 |
0.260 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
41 |
100 |
1 |
0 |
1 |
||||
|
|
|
200 |
1 |
0 |
0 |
0 |
0 |
1 |
( |
0.5) |
0 |
|
200 |
1 |
0 |
1 |
( |
0-5) |
|
|
PC |
100 |
15 |
34 |
0 |
4 |
0 |
44 |
5 |
100 |
0 |
0 |
0 |
|||||
6-18 |
+ |
(B[a]P) |
100 |
17 |
40 |
1 |
4 |
0 |
50 |
4 |
100 |
1 |
0 |
1 |
|||||
|
|
0.02 |
200 |
32 |
74 |
1 |
8 |
0 |
94 |
|
47.0 ) |
9 |
200 |
1 |
0 |
1 |
( |
0,5 ) |
Continous treatment method
Processing Time (h) |
Doses (mg/mL) |
No. of cells with chromosome aberration |
gap |
Cell growth |
No. of cells with numerical chromosome aberration |
|||||||||
No. of cells observed |
Chromatid break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total aberrant cells(%) |
No. of cells |
Polyploid cells |
Other |
Total polyploid cells(%) |
||||
24 |
VC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
100 |
100 |
0 |
0 |
0 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
1 |
0 |
0 |
0 |
0 |
1(0.5 ) |
1 |
200 |
0 |
0 |
0 ( 0.0) |
|||
24 |
0.065 |
10D |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
89 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0' |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
0 |
0 |
0 ( 0.0 ) |
1 |
200 |
0 |
0 |
0 ( 0.0 ) |
|||
24 |
0.130 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
73 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
0 |
0 |
0C0,0) |
0 |
200 |
0 |
0 |
0 ( 0.0 ) |
|||
24 |
0.260 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
32 |
100 |
0 |
0 |
0 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
100 |
D |
0 |
0 |
|||
200 |
2 |
0 |
0 |
0 |
0 |
2 (1.0) |
0 |
200 |
0 |
0 |
0 { 0.0) |
|||
24 |
PC |
100 |
14 |
31 |
0 |
3 |
0 |
46 |
3 |
100 |
0 |
0 |
0 |
|
100 |
16 |
23 |
1 |
3 |
0 |
37 |
7 |
100 |
1 |
0 |
1 |
|||
200 |
30 |
54 |
1 |
6 |
0 |
83 (0.40) |
10 |
200 |
1 |
0 |
1(0,5) |
|||
48 |
VC |
100 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
100 |
10D |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
1 |
0 |
1{ 0,5 ) |
0 |
200 |
0 |
0 |
0 ( 0.0) |
|||
48 |
0.065 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
89 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
0 |
0 |
0 ( 0.0 ) |
0 |
200 |
0 |
0 |
0 ( 0.0) |
|||
48 |
0.130 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
64 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
0 |
0 |
0 ( 0.0 ) |
1 |
200 |
0 |
0 |
0C0.0) |
|||
48 |
0.260 |
100 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
36 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
100 |
0 |
0 |
0 |
|||
200 |
0 |
0 |
0 |
1 |
0 |
1(0.5 ) |
1 |
200 |
0 |
0 |
0 ( 0.0) |
|||
48 |
PC |
100 |
26 |
41 |
0 |
4 |
1 |
57 |
6 |
100 |
1 |
0 |
1 |
|
100 |
29 |
52 |
1 |
3 |
2 |
68 |
6 |
100 |
1 |
0 |
1 |
|||
200 |
55 |
93 |
1 |
7 |
3 |
125 (0.62) |
12 |
|
200 |
2 |
0 |
2 (1.0 ) |
Short term treatment method, confirmation test
Processing Time |
S9 mix |
Conc. (mg/mL) |
No. of cells with chromosome aberration |
Gap |
Cell growth {%) |
No. of cells with numerical chromosome aberration |
|||||||||
No. of cells observed |
Chromatod break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total aberrant cells (%) |
No. of cells |
Polyploid cells |
Other |
Total polyploid cells(%) |
|||||
6-18 |
+ |
VC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
||||
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
200 |
0 |
0 |
0 ( 0.0 ) |
||||
6-18 |
+ |
0.065 |
100 . |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
95 |
100 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
||||
200 |
0 |
0 |
0 |
0 |
0 |
0 ( 0.0 ) |
0 |
200 |
0 |
0 |
0 ( 0.0 ) |
||||
6-18 |
+ |
0,130 |
100 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
77 |
100 |
0 |
0 |
0 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
100 |
1 |
0 |
1 |
||||
200 |
0 |
1 |
0 |
1 |
0 |
2 (1.0 ) |
0 |
200 |
1 |
0 |
1 (0.5 ) |
||||
6-18 |
+ |
0,260 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
35 |
100 |
0 |
0 |
D |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
100 |
0 |
0 |
0 |
||||
200 |
1 |
0 |
0 |
0 |
0 |
1(0.5 ) |
1 |
200 |
0 |
0 |
0 (0.0) |
||||
6-18 |
+ |
PC |
100 |
16 |
39 |
1 |
5 |
0 |
52 |
6 |
|
100 |
1 |
0 |
1 |
10D |
19 |
29 |
1 |
5 |
0 |
45 |
4 |
100 |
0 |
0 |
0 |
||||
200 |
35 |
68 |
2 |
10 |
0 |
97 { 4.5 ) |
10 |
200 |
1 |
0 |
1 (0.5) |
Table 1. Relative Survival – Preliminary
Cytotoxicity Assay: Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23850000 |
241 |
235 |
230 |
235.33 |
100 |
2.353 |
2.806 |
100 |
VC |
Dimethylsulfoxide |
20000000 |
23400000 |
230 |
241 |
227 |
232.67 |
100 |
2.327 |
2.722 |
97.00 |
T1 |
0.125 mg/ml |
20000000 |
23200000 |
224 |
214 |
211 |
216.33 |
100 |
2.163 |
2.509 |
92.19 |
T2 |
0.25 mg/ml |
20000000 |
23200000 |
220 |
219 |
204 |
214.33 |
100 |
2.143 |
2.486 |
91.33 |
T3 |
0.5 mg/ml |
20000000 |
23000000 |
214 |
208 |
213 |
211.67 |
100 |
2.117 |
2.434 |
89.42 |
T4 |
1 mg/ml |
20000000 |
22600000 |
191 |
187 |
197 |
191.67 |
100 |
1.917 |
2.166 |
79.56 |
T5 |
2 mg/ml |
20000000 |
21400000 |
181 |
184 |
174 |
179.67 |
100 |
1.797 |
1.922 |
70.62 |
Table 2. Relative survival - Preliminary Cytotoxicity Assay: Presence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23600000 |
237 |
233 |
241 |
237.00 |
100 |
2.370 |
2.797 |
100 |
VC |
Dimethylsulfoxide |
20000000 |
23500000 |
224 |
234 |
247 |
235.00 |
100 |
2.350 |
2.761 |
98.74 |
T1 |
0.125 mg/ml |
20000000 |
23300000 |
231 |
224 |
221 |
225.33 |
100 |
2.253 |
2.625 |
95.07 |
T2 |
0.25 mg/ml |
20000000 |
23100000 |
217 |
214 |
221 |
217.33 |
100 |
2.173 |
2.510 |
90.91 |
T3 |
0.5 mg/ml |
20000000 |
22800000 |
217 |
227 |
211 |
218.33 |
100 |
2.183 |
2.489 |
90.14 |
T4 |
1 mg/ml |
20000000 |
22400000 |
204 |
187 |
184 |
191.67 |
100 |
1.917 |
2.147 |
77.74 |
T5 |
2 mg/ml |
20000000 |
21200000 |
168 |
174 |
164 |
168.67 |
100 |
1.687 |
1.788 |
64.95 |
Table 3. Relative Survival – Main Study: Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
24850000 |
224 |
230 |
233 |
229.00 |
100 |
2.290 |
2.845 |
100 |
VC |
Dimethylsulfoxide |
20000000 |
23940000 |
231 |
228 |
231 |
230.00 |
100 |
2.300 |
2.753 |
96.76 |
T1 |
0.25 mg/ml |
20000000 |
23440000 |
221 |
218 |
219 |
219.33 |
100 |
2.193 |
2.571 |
93.37 |
T2 |
0.5 mg/ml |
20000000 |
23340000 |
214 |
210 |
224 |
216.00 |
100 |
2.160 |
2.521 |
91.56 |
T3 |
1 mg/ml |
20000000 |
22840000 |
178 |
187 |
194 |
186.33 |
100 |
1.863 |
2.128 |
77.29 |
T4 |
2 mg/ml |
20000000 |
22240000 |
187 |
174 |
175 |
178.67 |
100 |
1.787 |
1.987 |
72.16 |
PC |
400 µg/ml |
20000000 |
23450000 |
182 |
191 |
194 |
189.00 |
100 |
1.890 |
2.216 |
80.49 |
Table 4. Relative Survival – Main Study: Presence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
24860000 |
230 |
224 |
235 |
229.67 |
100 |
2.297 |
2.855 |
100 |
VC |
Dimethylsulfoxide |
20000000 |
24540000 |
230 |
230 |
228 |
229.33 |
100 |
2.293 |
2.814 |
98.57 |
T1 |
0.25 mg/ml |
20000000 |
23800000 |
219 |
214 |
211 |
214.67 |
100 |
2.147 |
2.555 |
90.78 |
T2 |
0.5 mg/ml |
20000000 |
23480000 |
217 |
214 |
216 |
215.67 |
100 |
2.157 |
2.532 |
89.98 |
T3 |
1 mg/ml |
20000000 |
23240000 |
187 |
191 |
186 |
188.00 |
100 |
1.880 |
2.185 |
77.63 |
T4 |
2 mg/ml |
20000000 |
22840000 |
158 |
162 |
160 |
160.00 |
100 |
1.600 |
1.827 |
64.93 |
PC |
30 µg/ml |
20000000 |
23240000 |
172 |
164 |
170 |
168.67 |
100 |
1.687 |
1.960 |
69.65 |
Table 5. Cloning Efficiency (Non-selective medium) Main Study: Absence of metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
214 |
204 |
211 |
210 |
2.10 |
VC |
Dimethylsulfoxide |
100 |
210 |
207 |
202 |
206 |
2.06 |
T1 |
0.25 mg/ml |
100 |
187 |
194 |
191 |
191 |
1.91 |
T2 |
0.5 mg/ml |
100 |
186 |
194 |
191 |
190 |
1.90 |
T3 |
1 mg/ml |
100 |
180 |
174 |
194 |
183 |
1.83 |
T4 |
2 mg/ml |
100 |
174 |
184 |
188 |
182 |
1.82 |
PC |
400 µg/ml |
100 |
164 |
171 |
171 |
169 |
1.69 |
Table 6. Cloning Efficiency (Non-selective medium) Main Study: Presence of metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
216 |
216 |
220 |
217 |
2.17 |
VC |
Dimethylsulfoxide |
100 |
211 |
214 |
210 |
212 |
2.12 |
T1 |
0.25 mg/ml |
100 |
194 |
191 |
182 |
189 |
1.89 |
T2 |
0.5 mg/ml |
100 |
182 |
174 |
197 |
184 |
1.84 |
T3 |
1 mg/ml |
100 |
184 |
194 |
190 |
189 |
1.89 |
T4 |
2 mg/ml |
100 |
172 |
181 |
177 |
177 |
1.77 |
PC |
30 µg/ml |
100 |
184 |
167 |
170 |
174 |
1.74 |
Table 7. Cloning Efficiency (Selective medium): Absence of metabolic activation
Dose level |
Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
4 |
3 |
2 |
3.00 |
0.00001500 |
VC |
Dimethylsulfoxide |
200000 |
3 |
4 |
4 |
3.67 |
0.00001835 |
T1 |
0.25 mg/ml |
200000 |
3 |
3 |
4 |
3.33 |
0.00001665 |
T2 |
0.5 mg/ml |
200000 |
4 |
3 |
3 |
3.33 |
0.00001665 |
T3 |
1 mg/ml |
200000 |
4 |
2 |
5 |
3.67 |
0.00001835 |
T4 |
2 mg/ml |
200000 |
3 |
5 |
3 |
3.67 |
0.00001835 |
PC |
400 µg/ml |
200000 |
67 |
74 |
63 |
68.00 |
0.00034000 |
Table 8. Cloning Efficiency (Selective medium): Presence of metabolic activation
Dose level |
Selective medium |
|
|||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
2 |
4 |
2 |
2.67 |
0.00001335 |
VC |
Dimethylsulfoxide |
200000 |
4 |
3 |
3 |
3.33 |
0.00001665 |
T1 |
0.25 mg/ml |
200000 |
4 |
3 |
2 |
3.00 |
0.00001500 |
T2 |
0.5 mg/ml |
200000 |
4 |
2 |
4 |
3.33 |
0.00001665 |
T3 |
1 mg/ml |
200000 |
3 |
4 |
3 |
3.33 |
0.00001665 |
T4 |
2 mg/ml |
200000 |
2 |
4 |
4 |
3.33 |
0.00001665 |
PC |
30 µg/ml |
200000 |
79 |
71 |
76 |
75.33 |
0.00037665 |
Table 9. Mutation Frequencies
Dose level |
Absence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000714 |
7.14 |
VC |
Dimethylsulfoxide |
0.00000890 |
8.90 |
T1 |
0.25 mg/ml |
0.00000871 |
8.71 |
T2 |
0.5 mg/ml |
0.00000876 |
8.76 |
T3 |
1 mg/ml |
0.00001002 |
10.02 |
T4 |
2 mg/ml |
0.00001008 |
10.08 |
PC |
400 µg/ml |
0.00020118 |
201.18 |
Dose level |
Presence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000615 |
6.15 |
VC |
Dimethylsulfoxide |
0.00000785 |
7.85 |
T1 |
0.25 mg/ml |
0.00000793 |
7.93 |
T2 |
0.5 mg/ml |
0.00000904 |
9.04 |
T3 |
1 mg/ml |
0.00000880 |
8.80 |
T4 |
2 mg/ml |
0.00000940 |
9.40 |
PC |
30 µg/ml |
0.00021646 |
216.46 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Bacterial reverse mutation assay
A GLP-compliant bacterial reverse mutation study (OECD TG 471) was performed to assess the mutagenic potential of the registered substance in S. typhimurium and E.coli strains. The following strains were used: Salmonella typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA.
The following test concentrations were used:
i) Plate Incorporation Method:
Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
ii) Pre-incubation Method
Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
N,N-dimethylformamide was used as a vehicle. A confirmatory test according to the pre-incubation method was carried out, as the test chemical was found non-mutagenic in the first experiment performed according to the plate incorporation method. The test chemical was considered positive (mutagenic) when the number of the revertant colonies in treated cultures increased dose-dependently and became two-fold or more than that of the vehicle negative control, and a significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system. The test chemical was considered negative (non-mutagenic) when any of the above criteria were not fulfilled. No statistical analysis was performed.Results:The test substance did not induce a two-fold and/or biologically relevant increase in revertant colony counts compared with the vehicle control when it was tested up to 5000µg/plate neither in the presence nor the absence of S9 metabolic activation in S. typhimurium and E. coli strains used.Conclusion:The test substance was considered non-mutagenic (negative) in the bacterial reverse mutation test (OECD TG 471).
Chromosomal Aberration Study:
A GLP-compliant in vitro cytogenicity study according to OECD TG 473 was performed with the registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) to assess its clastogenic potential in Chinese Hamster Lung (CHL/IU)cells.
The concentrations of the test chemical used in the study were the follows:
-S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)
+S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)
24 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)
48 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)
The test was performed both in the presence and absence of liver S9 microsomal fraction obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats. A solution of 1% carboxymethyl cellulose sodium salt was used as a vehicle. Mitomycin C (-S9) and benzo(a)pyrene (+S9) were used as positive controls.
A confirmation test was carried out in short-term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance in the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time. Two plates per dose and 100 metaphases per plate were analyzed.
Results:More than 50% cell growth inhibition was observed at≥0.2 mg/ml in both short and continuous treatment protocols. The test chemical did not increase the incidence of cells with chromosomal aberration compared to the vehicle control when tested up to a cytotoxic concentration in short or continuous treatment, neither in the presence nor in the absence of S9 metabolic activation. The percent of aberrant cells were the follows: 1% (VC), 0.5% (at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.0% ( at 0.260 mg/ml) and 0.5% (VC), 0.5% (at 0.065 mg/ml), 1.0% (at 0.130 mg/ml), 0.5% (0.260 mg/ml) in short-term treatment I the absence and in the presence of S9 metabolic activation, respectively.In continues treatment the percentages of cells with chroosaml aberrations were 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 1.0% (at (0.260 mg/ml) and 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.5% (at (0.260 mg/ml) at 24 and 48 hours in absence and in the presence of S9 metabolic activation, respectively. Positive control substances induced significant increase in percent aberrant cells; MMC 15% (short term -S9), 41% (24 hrs) and Benzo(a)pyrene 47% (short term, +S9), 62% (48hrs).Conclusion:The registered substance was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.
In vitro mammalian cell gene mutation assays:
A GLP-compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 96.76% (vehicle control), 93.37% (at 0.25 mg/ml), 91.56% (0.5 at mg/ml), 77.29% (at 1 mg/ml) and 72.16% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.57% (vehicle control), 90.78% (0.25 mg/ml), 89.98% (at 0.5 mg/ml), 77.63% (at 1 mg/ml) and 64.93% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence (8.71 x 10-6, 8.76 x 10-6, 10.02 x 10-6 and 10.08 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation (7.93 x 10-6, 9.04 x 10-6, 8.80 x 10-6 and 9.40 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control (8.90 x 10-6, 7.85 x 10-6, absence and presence of S9 metabolic activation, respectively). No significant reduction in RS values (cytotoxicity) was observed in vehicle control (DMSO) (RS:97.76% (-S9), 98.57% (+S9) when compared to the negative control (distilled water). Similarly, no increase in MF values was noted in vehicle control (MF: 8.90 x 10-6, -S9; 7.85 x 10-6, +S9) when compared to the negative control (MF:7.14 x 10-6, S9; 6.15 x 10-6, +S9) neither in the presence nor in the absence of S9 metabolic activation. The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS:201.18 x 10-6; Beno[a]pyrene 216.46 x 10-6 p<0.05) as compared to the vehicle control (8.90 x 10-6, -S9; 7.85 x 10-6, +S9). Conclusion:The registered substance, did not induce gene mutation in the Hprt locus of CHO cells up to the highest test concentration of 2 mg/ml neither in the presence nor in the absence of S9 metabolic activation system in under the experimental conditions described.
Justification for classification or non-classification
The registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8), was tested non-mutagenic (negative) in both bacterial and mammalian cells in GLP-compliant and OECD guideline studies performed according to OECD TGs 471 and 473. The registered substance was non-clastogenic in mammalian cells in a GLP-compliant in vitro cytogenicity study conducted according to OECD TG 473. Overall, considering the results of the genotoxicity testing battery, the registered substance is regarded to be classified as Not Classified for genetic toxicity according to Regulation EC 1272/2008.
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