4'-aminoazobenzene-4-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not iinduce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration assay:

The test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration assay:

The test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

WoE derived based on the experimental data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

10% and 30% HLI and RLI S-9 (9,000 g supernatant) fractions were prepared from Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

1. 0, 33, 100, 333, 1000 or 3333 µg/plate
2. 10-250 mg

Vehicle / solvent:

1./2.
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9) and 2-aminoanthracene (all strains; +S9)

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

2. METHOD OF APPLICATION: Plate incorporation assay

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. Evaluations were made at both the individual trial and overall chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two fold over background for a chemical to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.
Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.

2. Material which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, was denoted as mutagens. Those which reduced the number of revertants were considered inhibitory.

Statistics:

1. Mean ± SD
2. No data

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA1537, TA100, TA1535

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The test chemical was tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

2. No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not iinduce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 33, 100, 333, 1000 or 3333 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

Based on the observations made, the test chemical did not induce gene mutation in the Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL / 1

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster V79 cells

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 fractions (livers of Aroclor 1254-treated male Sprague-Dawley rats.)

Test concentrations with justification for top dose:

1. -S9 (Harvest time: 10 hrs): 0, 2500, 3850, 5000 µg/mL
+S9 (Harvest time: 12 hrs): 0, 2500, 3850, 5000 µg/mL

2. Experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix

Experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix

Experiment IB: 31.3, 62.5, and 125.0 without S9-mix

Experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix

Experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix

Experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix

Experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

2. - Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The test chemical is soluble in Deionized water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

1

Untreated negative controls:

yes

Remarks:

as per OECD

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

as per OECD

Positive control substance:

not specified

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration:
- S9: 8 hrs
+ S9: 2 hrs
- Expression time (cells in growth medium): 8 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): -S9: 10 hrs, +S9: 12 hrs

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: One hundred to 200 cells from each of the three highest scorable doses were analyzed

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration:
Experiment I (without & with S9-mix): 4 hr
Experiment II (with S9-mix): 4 hr
Experiment II (without S9-mix): 20 hrs

- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: For assessment of cytotoxicity a XTT test was performed

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding.

Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture.

For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test.

Rationale for test conditions:

No data

Evaluation criteria:

1. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation. Gaps and endoreduplications were recorded but were not included in the statistical analyses.

2. The cell line was observed for micronucleated cells

Statistics:

1. Trend test.

2. No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL / 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

mammalian cell line, other: Chinese hamster V79 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 – 7.5
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Chemicals were tested up to 5 mg/ml or as limited by solubility and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent (water, dimethyl sulfoxide, acetone, or ethanol, in that order of preference). In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). The doses used were generally the TD, 0.75 TD, 0.50 TD, 0.25 TD, 0.1 TD, 0.075 TD, 0.05 TD, and 0.025 TD. The highest three doses with a sufficient number of cells were analyzed for chromosomal aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively.

Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation.

In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant.

The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity.

Other: In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose. The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data from various test chemicals was reviewed to determine the mutagenic nature of the test chemicals. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells to determine the mutagenic nature of the test chemical.

Different conc. of test chemical was used in the various experiment. They are-

Experiment IA:128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix

Experiment IA:128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix

Experiment IB:31.3, 62.5, and 125.0 without S9-mix

Experiment IIA:128.1, 256.3 and 512.5 μg/ml without S9-mix

Experiment IIA:128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix

Experiment IIB:100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix

Experiment IIB:100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix

 

Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.

Based on the data available for the various test chemicals, the test chemical did not induce chromosomal aberrations in the mammalian cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian cell gene mutation assay

Target gene:

1. Thymidine kinase
2. hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y TK+/- 3.7.C / 1

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

1. 1642-3680 µg/mL
2. 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix.
0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix

Vehicle / solvent:

1. No data available
2. - Vehicle(s)/solvent(s) used: Phosphate buffered saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in Phosphate buffered saline

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Remarks:

1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

Cells at start: 6000000 cells

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

2. METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data available
- Exposure duration: 2 h treatment with S9-mix or 20 h without S9-mix
- Expression time (cells in growth medium): 5 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: Cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations.

Rationale for test conditions:

No data

Evaluation criteria:

1. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

2. The cell line was observed for gene mutation at the hprt locus

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: No data available

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.

Executive summary:

Data available for the various test chemicals were reviewed to determine the mutagenic nature. The studies are as mentioned below:

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature ofthe test chemical. The cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 1642-3680 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

In an in vitro mammalian cell gene mutation assay, the test chemical was exposed to the V79 cells in the concentration of 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix and 0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix in phosphate buffered saline without and with S9-mix. Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment. In the main tests, cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations. Data on toxicity were restricted to those on cloning efficiency after the selection period. Negative and positive controls were included. The data on clonings efficiency after the selection period did not indicate strong cytotoxicity; the required 10-20% survival after the highest dose was not reached at any concentration. In both experiments a biologically relevant and dose dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation. Occasionally, an increase in mutant frequency was found; these were not reproducible and considered not biologically relevant. Since, biologically relevant and concentration dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation, hence, the test chemical was considered to be negative for mammalian cell gene mutation assay in vitro (with and without) when tested on V79 cells.

Based on the observations made, the test chemical did not induce mammalian cell gene mutation assay both in the presence and absence of S9 activation system in the mammalian cell line used and hence is not likely to be gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the various test chemicals was reviewed to determine the mutagenic nature. The studies are as mentioned below:

Ames assay:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 33, 100, 333, 1000 or 3333 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

In vitro chromosome aberration assay:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO-WBL cells in the presence and absence of exogeneous metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels upto 5 mg/mL. In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay in most instances when cell cycle delay was observed in the SCE assay. In these tests the cell growth period was extended to about 20 hr. Cells were harvested. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed and the chromosomal aberrations were scored. The test chemical did not induce chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250µg/ml and above. Based on the observations made, the test chemical did not induce chromosome aberrations in the CHO-WBL cells in the presence and absence of exogeneous metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells to determine the mutagenic nature of the test chemical.

Different conc. of test chemical was used in the various experiment. They are-

Experiment IA:128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix

Experiment IA:128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix

Experiment IB:31.3, 62.5, and 125.0 without S9-mix

Experiment IIA:128.1, 256.3 and 512.5 μg/ml without S9-mix

Experiment IIA:128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix

Experiment IIB:100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix

Experiment IIB:100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix

 

Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.

In vitro mammalian chromosome aberration assay:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 1642-3680 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

In yet another in vitro mammalian cell gene mutation assay, the test chemical was exposed to the V79 cells in the concentration of 0, 5, 19, 20, 50 and 100 μg/ml without S9-mix and 0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix in phosphate buffered saline without and with S9-mix. Test concentrations were based on the results of an initial toxicity test measuring cell survival 5 days after treatment. In the main tests, cells were treated for 2h with S9-mix or for 20h without S9-mix followed by an expression period of 5 days to fix the DNA damage into stable hprt mutations. Data on toxicity were restricted to those on cloning efficiency after the selection period. Negative and positive controls were included. The data on clonings efficiency after the selection period did not indicate strong cytotoxicity; the required 10-20% survival after the highest dose was not reached at any concentration. In both experiments a biologically relevant and dose dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation. Occasionally, an increase in mutant frequency was found; these were not reproducible and considered not biologically relevant. Since, biologically relevant and concentration dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation, hence, the test chemical was considered to be negative for mammalian cell gene mutation assay in vitro (with and without) when tested on V79 cells.

In vitro mammalian cell transformation assay:

In vitro transformation study in mammalian cells was performed to evaluate the mutagenic nature of the test chemical. The study was performed using BHK21/C13 in the presence of aroclor 1245 rat liver induced S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 0.025, 0.25, 2.5, 25 or 250µg/mL. The methods employed when testing a compound for potential carcinogenicity using growth of mammalian cells in semi-solid agar. The experiments were conducted in small groups using duplicate plates at each dose level, and on each occasion 4-dimethylaminoazobenzene and its 3-methyl analogue were used as the appropriate chemical-class-positive and -negative controls. A positive result is recorded when the transformation frequency per 10⁶survivors at the LD50 exceeds 5X the control frequency. Based on the observations made, thetest chemical does not exhibit in vitro mammalian cell transformation frequency in the presence of S9 metabolic activation system and thus proves to be non-mutagenic and non-carcinogenic. There are at least 2 possible explanations for the non-carcinogenicity of the test chemical.First, it could be suggested that the addition of a sulphonic-acid group to the test chemical will reduce its lipid solubility and thereby prevent, or inhibit, its transport in vivo to critical intracellular sites. Second, it is possible that the marked negative inductive effect exerted by the sulphonic-acid group might critically affect both the electronic resonance of the NMe2 group with the aromatic system and the metabolism of the molecule, which would lead to the test chemical being inactive both in vivo or in vitro.

Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This is further supported by the negatiive in vitro cell tranformation ability of the test chemical. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This is further supported by the negatiive in vitro cell tranformation ability of the test chemical. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.