N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

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

Study period:

41 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

In first cytogenetic assay in one of the duplicate cultures of the positive control only 116 and 128 metaphases were examined for chromosome aberrations. Scoring of additional metaphases would have given limited information, no effect on study outcome.

GLP compliance:

yes

Type of assay:

other: Chromosome aberrations in cultured human lymphocytes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

EC Number:

264-439-1

EC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Cas Number:

63741-10-6

Molecular formula:

C17H17N4O2Cl3

IUPAC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Test material form:

solid: particulate/powder

Details on test material:

Anthraquinone dye, blue powder

Specific details on test material used for the study:

Batch 4852-1501 of Disperse Brown 27 was a brown powder with a purity of 99%.

Method

Target gene:

Whole blood samples obtained from healthy subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals.
The stimulated lymphocytes were exposed to Disperse Brown 27 both in the absence and presence of a metabolic activation system (S9-mix). In combination with this metabolic activation system indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be tested for possible clastogenic effects in vitro.

Species / strain

Species / strain / cell type:

lymphocytes:

Remarks:

Human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In the first cytogenetic assay, Disperse Brown 27 was tested up to 125 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Disperse Brown 27 precipitated in the culture medium at this dose level.
In the second cytogenetic assay, Disperse Brown 27 was tested up to 90 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 62.5 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Disperse Brown 27 precipitated in the culture medium at these dose levels.

Vehicle / solvent:

DMSO (Dimethylsulfoxide)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that Disperse Brown 27 is not clastogenic in human lymphocytes under the experimental conditions described in study report.

Executive summary:

Batch 4852-1501 of Disperse Brown 27 was a brown powder with a purity of 99%. Disperse Brown 27 was dissolved or suspended in dimethyl sulfoxide.

In the first cytogenetic assay, Disperse Brown 27 was tested up to 125 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Disperse Brown 27 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Disperse Brown 27 was tested up to 90 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 62.5 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Disperse Brown 27 precipitated in the culture medium at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Disperse Brown 27 did not induce any statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of Disperse Brown 27 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Disperse Brown 27 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Disperse Brown 27 is not clastogenic in human lymphocytes under the experimental conditions