4-amino-6-chlorotoluene-3-sulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to the procedure prescribed by Ames et al. (Mut. Res. 21: 347-364, 1975) and was in principle similar to the OECD TG 471, however with following main deviations: neither TA102 nor a DNA repair-proficient E.coli WP2 strain was included as tester strain for detection of cross-linking mutagens. Furthermore, the evaluation of revertants was based on extinction measurement by means of an automatic analyser for microbiological purposes COBAS Bact designed by ROCHE, in conjunction with a Computer Hewlett-Packard 9816. Test considered sufficiently validated.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(No strain for detection of cross-linking mutagens such as TA102 or the DNA repair-proficient E.coli WP2 strain as recommended by the OECD TG was tested.)

Principles of method if other than guideline:

The test was conducted according to the Ames procedure as described by Ames et al. (Mut. Res. 21: 347-364, 1975).
For analysis, the automatic analyser for microbiological purposes COBAS Bact designed by ROCHE was used, in conjunction with a Computer Hewlett-Packard 9816.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-6-chlorotoluene-3-sulphonic acid

EC Number:

201-837-6

EC Name:

4-amino-6-chlorotoluene-3-sulphonic acid

Cas Number:

88-51-7

Molecular formula:

C7H8ClNO3S

IUPAC Name:

2-amino-4-chloro-5-methylbenzene-1-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): 4-Amino-2-chlortoluol-5-sulfonsäure
- Analytical purity: No details on purity given in the study report
- Lot/batch No.: BASF

Method

Target gene:

All strains are mutants from histidine auxotrophy (his) to histidine prototrophy (his+).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: defect in the excision repair system (uvrB)

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from the liver of Sprague-dawley rats treated with Aroclor 1254 mixed with a series of appropriate cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.4 to 2500 µg/mL
Main test, with and without S9 mix: 2.44, 9.77, 39.1, 156, 625, 2500 µg/mL

Vehicle / solvent:

The substance was suspended in DMSO; the solvent alone was used for the negative controls.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(see solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

other: Daunorubicin-HCl 0.5 µg/mL PBS

Remarks:

TA 98

Controls 2

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100

Migrated to IUCLID6: 0.083 µg/mL PBS

Controls 3

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Migrated to IUCLID6: 5µg/mL DMSO

Controls 4

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

other: 2-Aminoanthracene 0.17 µg/mL DMSO

Remarks:

TA 98 and TA 100

Controls 5

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

cyclophosphamide

Remarks:

TA 100 (because of inconsistent findings obtained for this strain when tested with 2-Aminoanthracene, testing with TA 1535 was repeated using Cyclophosphamide for positive control)

Migrated to IUCLID6: 50 µg/mL PBS

Controls 6

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

cyclophosphamide

Remarks:

TA 1535

Migrated to IUCLID6: 133.3 µg/mL PBS

Controls 7

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

other: Acridine orange 3.33 µg/mL DMSO

Remarks:

TA 1537

Details on test system and experimental conditions:

The histidine-auxotrophic bacteria were incubated in minimum medium in measurement chambers with a volume of 0.3 mL together with the test material at 37 °C. In the experiments in which the substance was metabolically activated, 20% of S9 mix was added to the minimum medium. At test initiation the medium contained 1 to 5x10E+6 bacteria/mL.
The minimum medium was Vogel-Bonner medium E, supplemented with 20 g/l glucose, 0.5 mg/l biotin and 0.4% nutrient broth (Difco Laboratories, Detroit, Michigan, U.S.A). The test material in the vehicle was added to the chambers in a volume of 10 µL.
For analysis, the automatic analyser for microbiological purposes COBAS Bact designed by ROCHE was used, in conjunction with a Computer Hewlett-Packard 9816.
Turbidity of the content of the chambers was measured automatically at the wave length of 546 nm at intervals of 20 min over a period of about 46 hours. The cultures were shaken prior to each measurement. Four measurement chambers were incubated per strain and per group (test and control group). In the first experiment for technical reasons the values for the first 6 hours were not registered.
For evaluation, the revertant growth in each of the chamber was determined by measuring the extinction at different time points during incubation (10, 20, 30 and 40 hours).

Evaluation criteria:

A test substance is considered to be mutagenic in this test system if a dose-dependent increase in the number of chambers with revertant growth occurs. The time of the beginning of revertant growth gives an indication of the number of back-mutant bacteria induced. Growth-inhibiting effects can be observed simultaneously. Revertant growth appearing later than after 40 hours was considered to be spontaneous in origin and thus were interpreted as negative result.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(comparison of the number of back-mutant colonies in controls and treated cultures with the various concentrations of test item revealed no marked deviations)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(tested up to 2500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of main test results expressed in revertant growth (+) or none (-):

Mutagenicity Test with COBAS BACT: Summary of Results in absence of S9 mix
Strain	Test group	Chamber
		1	2	3	4
TA 98	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	-
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	+	-
	156 µg/mL	-	-	-	-
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+
TA 100	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	+	+
	9.77 µg/mL	+	-	-	-
	39.1 µg/mL	-	-	-	-
	156 µg/mL	+	-	-	-
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+
TA 1535	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	-
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	-	-
	156 µg/mL	-	-	-	-
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+
TA 1537	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	-
	9.77 µg/mL	-	-	-	+
	39.1 µg/mL	-	-	-	+
	156 µg/mL	-	+	-	+
	625 µg/mL	+	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+

Mutagenicity Test with COBAS BACT: Summary of Results in presence of S9 mix
Strain	Test group	Chamber
		1	2	3	4
TA 98	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	+
	9.77 µg/mL	+	-	-	-
	39.1 µg/mL	+	-	-	-
	156 µg/mL	-	-	-	+
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+
TA 100	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	+
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	-	-
	156 µg/mL	-	-	-	-
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control (2-Aminoanthracene!)	-	+	-	-
TA 1535	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	+
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	-	-
	156 µg/mL	-	-	-	-
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+
TA 1537	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	-
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	-	-
	156 µg/mL	-	-	-	+
	625 µg/mL	-	-	-	-
	2500 µg/mL	-	-	-	-
	Pos. Control	+	+	+	+

Additional testing with TA 100, this time using Cyclophosphamide as positive control substance:

Mutagenicity Test with COBAS BACT: Summary of Results in presence of S9 mix
TA 100	Neg. Control	-	-	-	-
	2.44 µg/mL	-	-	-	-
	9.77 µg/mL	-	-	-	-
	39.1 µg/mL	-	-	-	+
	156 µg/mL	+	+	-	-
	625 µg/mL	-	-	-	+
	2500 µg/mL	-	-	-	-
	Pos. Control (Cyclophosphamide)	+	+	+	+

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative