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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test conduct was not GLP conform but was in accordance with the current requirements for the UDS assay as mentioned in the OECD TG 482 (1986).

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 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

(1986)

Deviations:

no

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

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-methylbenzenesulfonic acid

Details on test material:

- Name of test material (as cited in study report): 4-Amino-2-chlortoluol-5-sulfonsäure
- Analytical purity: commercial grade
- Lot/batch No.: BASF
- Stability under test conditions: stability was ensured by the sponsor

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

hepatocytes: isolated from a male Sprague-Dawley obtained from CIBA-GEIGY Tierfarm, Sisseln

Details on mammalian cell type (if applicable):

The freshly isolated hepatocytes from the male rat weighing about 194 g were cultivated in WILLIAMS' Medium E containing 10% fetal bovine serum

Test concentrations with justification for top dose:

Preliminary toxicity test: 15.625 - 1000 µg/mL
UDS assay: 8, 40, 200 and 1000 µg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

(medium)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Migrated to IUCLID6: (100 µM)

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
This test was performed to determine the highest concentration to be used in the DNA-repair assay. The suitable concentration
as the highest to be used in the DNA-repair test is determined according to following three criteria:
- a sufficiently large number of cells must adhere to the cover-slips;
- at least 25% of the cells must show viability upon examination by means of the vital-staining technique;
- a corresponding percentage of the cells must be in good condition upon morphological examination.
From the results of the toxicity test, the highest suitable concentration for the DNA repair assay was set at 500 µg/mL.

UDS ASSAY
First Experiment:
A series of compartments in Multiplates containing gelatinized THERMANOX cover-slips was seeded with 4 x 10E+5 cells per compartment (density 10 E+5 cells/mL; 4 mL/compartment).The cells were allowed to attach to the cover-slips during an attachment period of 1.5-2 hours. They were then washed and cultivated overnight in renewed medium (adhesion period). The compartments were filled with 4 mL of culture medium during the attachment period and with 2 mL during the adhesion period.
From the test item and from the positive control substance stock solutions were prepared, from each of which 20 µL were added to four compartments containing 2 mL medium. In the case of negative controls corresponding volumes of the vehicle and of the culture medium were added.
Immediately after addition of the test substance, 3H-thymidine was added (specific activity 20 Curies/mmol, obtained from the Radiochemical Centre, Amersham, England, Batch Nr. 135). 8 µCi in 8 µL was added to the 2 mL medium in each compartment. At the end of the incubation period of 5 hours the cells were washed twice with BSS and fixed with ethanol/acetic acid (3:1 v/v). The cover-slips were mounted on microscope slides and prepared for autoradiography. The exposure time was 6 days. The autoradiographs were stained with hematoxylin-eosine.

EVALUATION OF THE SLIDES
The background in the autoradiographs was determined in cell-free areas microscopically and was found to be negligibly low.
From each of the treatment groups and from the positive and the negative controls 150 nuclei in altogether three slides (50 cells/slide) were scored, the number of silver grains counted, the mean values and the standard deviations calculated. Counting of silver grains over the nuclei of the hepatocytes was carried out by means of an electronic counter (ARTEK Model 982) attached to a ZEISS microscope. Cells which were in the DNA-synthesis phase showed more than 120 silver grains/nucleus (0.1% of cells concerned); these cells were excluded from the determination of the silver grain/nucleus count.

Evaluation criteria:

The test substance is generally considered to be mutagenic or carcinogenic if the mean number of silver grains per nucleus in relation to the negative controls is more than doubled at any concentration.

Results and discussion

Test results

Species / strain:

hepatocytes: isolated from a male rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

Comparison of the mean number of silver grains per nucleus in the negative controls and after treatment with the test item in the various concentrations revealed no marked differences.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(up to 1000 µg/mL, no cytotoxicity was evident.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST
The toxicity test was performed with concentrations of 15.625 to 1000 µg/mL. None of the tested concentrations proved toxic. Therefore the concentration of 1000 µg/mL was selected as the highest for the DNA-repair assay.

UDS ASSAY
The negative controls (medium and solvent) gave mean values of 1.65 +/- 1.17 and 1.54 +/- 0.97 grains per nucleus, respectively.
In the treated groups, the mean number of silver grains per nucleus were as follows:
8 µg/mL: 1.83 +/- 1.42
40 µg/mL: 1.89 +/- 1.36
200 µg/mL: 1.98 +/- 1.32
1000 µg/mL: 2.09 +/-1.47
Thus, the evaluation criteria for a positive result, that the mean number of silver grains per nucleus in relation to the negative controls has to be more than doubled at any concentration, was not fulfilled.
The positive control substance DMN showed a mean value of 22.2 +/- 9.75 silver grains per nucleus, thus having produced a marked increase when compared to negative control.

Remarks on result:

other: strain/cell type: see above

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative