4-nitrotoluene-2-sulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration 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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene-2-sulphonic acid

EC Number:

204-445-3

EC Name:

4-nitrotoluene-2-sulphonic acid

Cas Number:

121-03-9

Molecular formula:

C7H7NO5S

IUPAC Name:

2-methyl-5-nitrobenzenesulfonic acid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from sprague Dawlay rat livers, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

without S9-mix
0, 550, 1100, 2200 µg/ml (short-term and continuous exposure)
with S9-mix
0, 550, 1100, 2200 µg/ml (short-term exposure)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Two independent experiments were performed. In each experimental group two parallele cultures were set up.

Preliminary test (cell growth inhibition test)
The cells were disseminated in cell culture multi-plates, and the test substance fluid was treated on the 3rd day after the culture. In case of continuous treatment, the treatment was carried out continuously for 24 or 48 hours, and in the short-term treatment, the fluid was treated for 6 hours in the absence (-S9 mix) or presence (+S9 mix) of S9 mix, then with fresh culture fluid replaced, the culture was continued for further 18 hours.

100 metaphase images per each plate, namely 200 metaphase images per dose were observed microscopically, were classified, in terms of chromosomal morphological change, into gap (gap), chromatid break (ctb), chromosome break (csb), chromatid exchange (cte), chromosome exchange (cse) and other (oth) structural abnormalities. The incidence of ploidy cells was recorded at the same time. The chromosomal analysis was carried out in accordance with the classification by Japanese Environmental Mutagen Society, Mammal Test Subcommittee 2).

Positive control
mitomycin C (MMC: Kyowa Hakko Kogyo Co., Ltd.) was tested in the dose of 0.05 μg/mL for 24 hours treatment, and 0.025 μg/mL for 48 hours treatment, in case of continuous treatment, and cyclophosphamide (CP: Shionogi & Co., Ltd.) was tested in the dose of 12.5 μg/mL in case of short-term treatment.

Evaluation criteria:

The frequency of appearance of cells having structural abnormalities or ploidy cells in each test group was determined according to the criteria of Ishidate et al. 3). The frequency of appearance of cells having structural abnormalities was qualified as negative (-) for that less than 5%, pseudo-positive (±) for that not less than 5% and less than 10%, and positive (+) for that not less than 10%. In case a reproducibility or dose-dependency is recognized, it was finally determined as being positive.

Statistics:

Further, no test using statistical methods was carried out.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Additional information on results:

In case of 2-methyl-5-nitrobenzenesulfonic acid treatment, no tendency of inducing the chromosomal structural aberration and ploidy cells was observed in either dose. On the other hand, a remarkable induction of chromosomal structural aberration was recognized in cells treated with MMC, positive control material. The test results for short-term treatment group were shown in Table 2. In case of test substance treatment group, no tendency of inducing the chromosomal structural aberration and ploidy cells was observed in either dose in the absence and presence of S9 mix. On the other hand, a remarkable induction of chromosomal structural aberration was recognized only in the presence of S9 mix for the cells treated with CP, positive control material. Further, in each test system of continuous treatment and short-term treatment, a decrease in pH value for the culture fluid was recognized in all test doses, however, it has returned to the neutral range at the exchange of culture fluid or the completion of culture. Taking the above test results into consideration, the chromosomal aberration inducibility of 2-methyl-5-nitrobenzenesulfonic acid to cultured Chinese hamster cells was determined as negative in the present test conditions.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

(Table 1) Chromosomal aberration test on CHL cells treated with 2-methyl-5-nirobenzenefulfonic acid

 [continuous exposure]

Compound	(µg/ml)	Exposure (h)	Of cells analyzed	Structural aberrations						[+gap] (%)	[-gap] (%)	Cells (%)	judgement
				Gap	Ctb	Csb	Cte	Cse	Oth				SA	Pol
Test sub.	0	24	200	0	1	0	0	0	0	0.5	0.5	1.0	-	-
	550	24	200	0	0	0	1	0	0	0.5	0.5	0.5	-	-
	1100	24	200	3	0	0	1	0	0	2.0	0.5	1.5	-	-
	2200	24	200	3	0	1	2	0	0	3.0	1.5	1.0	-	-
MCC*	0.05	24	200	23	39	2	74	1	0	50.5	47.5	0.5	+	-
Test sub.	0	48	200	1	0	0	0	1	0	1.0	0.5	0.0	-	-
	550	48	200	1	0	0	0	0	0	0.5	0.0	1.0	-	-
	1100	48	200	1	0	0	0	0	0	0.5	0.0	0.0	-	-
	2200	48	200	1	2	0	1	0	0	2.0	1.5	1.0	-	-
MMC*	0.025	48	200	13	34	0	68	1	0	46.0	43.0	0.5	+	-

*: Positive Contol (mitomycin C)

Ctb: chromatid break  csb: chromosome break cte: chromatid exchange  cse: chromosome exchange   oth: others

SA: Structural aberration   Pol: polyploid cell

(Table 2) Chromosomal aberration test on CHL cells treated with 2-methyl-5-nirobenzenefulfonic acid

 [short-term exposure]

Compound	(µg/ml)	Mix	Exposure (h)	Of cells analyzed	Structural aberrations						[+gap] (%)	[-gap] (%)	Cells (%)	judgement
					Gap	Ctb	Csb	Cte	Cse	Oth				SA	Pol
Test sub.	0	-	6	200	1	1	0	1	0	0	1.0	1.0	0.5	-	-
	550	-	6	200	1	0	1	1	0	0	1.5	1.0	0.0	-	-
	1100	-	6	200	1	2	0	0	0	0	1.5	1.0	0.5	-	-
	2200	-	6	200	1	0	0	0	0	0	0.5	0.0	1.0	-	-
CP*	12.5	-	6	200	1	1	0	0	0	0	1.0	0.5	0.0	-	-
Test sub.	0	+	6	200	1	0	0	1	0	0	0.5	0.5	0.0	-	-
	550	+	6	200	0	2	0	3	0	0	2.0	2.0	1.5	-	-
	1100	+	6	200	1	0	1	2	0	0	2.0	1.5	0.0	-	-
	2200	+	6	200	2	0	0	2	0	0	2.0	1.0	0.0	-	-
CP*	12.5	+	6	200	18	43	2	145	1	0	79.0	78.0	0.0	+	-

*: Positive Contol (cyclophosphamide)

Ctb: chromatid break  csb: chromosome break cte: chromatid exchange  cse: chromosome exchange   oth: others

SA: Structural aberration   Pol: polyploid cell

Applicant's summary and conclusion

Conclusions:

negative with and without metabolic activation