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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the presence of S9-mix, Disperse Brown 27 induced dose-related in the tester strains TA1535, TA98 and TA100. Although the increase observed in tester strain TA1535 was more than three-fold (3.1 times the concurrent controls), the increase was not outside the laboratory historical control data range and in one experiment only. The increases observed in tester strain TA98 were in two independently repeated experiments, were above the laboratory historical control data range, were dose-related and were already seen at nonprecipitating dose levels. Furthermore the increases were up to 7.4-fold the concurrent controls. Although the increase observed in tester strain TA100 was above the laboratory historical control data range, the increase was only seen in the dose range finding test, not dose related, beyond precipitate and was not more than two times the concurrent control.

Taken together, the increase in tester strain TA98 in the presence of S9-mix is the only increase which is considered to be biological biologically relevant.

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that Disperse Brown 27 is mutagenic in tester strain TA98 of the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. Disperse Brown 27 is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA100) or Escherichia coli reverse mutation assay using strain WP2uvrA.

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.

Disperse Brown 27 is not mutagenic in the TK mutation test system under the experimental conditions described in report

Link to relevant study records

Reference

Reference 1

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

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

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%.

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 / 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)

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).

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

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The batch of Disperse Brown 27 (4852-1501) tested was a brown powder with a purity of 99.7 %

Target gene:

The objective of this study was to evaluate the test item for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.
The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens (1-5).

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Remarks:

rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Disperse Brown 27 was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164 and 512 μg/plate.

In the dose range finding test, precipitation of Disperse Brown 27 on the plates was observed at the start of the incubation period at concentrations of 52 μg/plate and upwards and at 512 μg/plate and above at the end of the incubation period. In the first experiment, precipitation of Disperse Brown 27 on the plates was observed at the start of the incubation period at concentrations of 52 μg/plate and upwards and at the end of the incubation period at 164 μg/plate and above and 512 μg/plate in the absence and presence of S9-mix, respectively.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay have shown to be rapid and adequate indicators for the mutagenic activity of a wide range of chemical compounds. The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay have been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of chemical compounds.
The assay was conducted in the absence and presence of a metabolizing system (S9-mix). The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens (1-5).

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains are regularly checked to confirm their histidinerequirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UVsensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris- EDTA treatment. The strain is regularly checked to confirm the tryptophanrequirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Rationale for test conditions:

Recommended test system in international guidelines (e.g.OECD, EC).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Disperse Brown 27 was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164 and 512 μg/plate.

In the dose range finding, precipitation of Disperse Brown 27 on the plates was observed at the start of the incubation period at concentrations of 52 μg/plateand upwards and at 512 μg/plate and above at the end of the incubation period. In the first experiment, precipitation of Disperse Brown 27 on the plates was observed at the start of the incubation period at concentrations of 52 μg/plate and upwards and at the end of the incubation period at 164 μg/plate and above and 512 μg/plate in the absence and presence of S9-mix, respectively.

To determine the toxicity of Disperse Brown 27, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The bacterial background lawn was not reduced at any of the concentrations tested. No reduction in the number of revertants was observed up to the dose level of 1600 μg/plate. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined

In tester strain TA100, the test item induced a 1.8-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In tester strain TA98, the test item induced up to 4.7 and 7.4-fold dose-related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. All other bacterial strains showed negative responses over the entire dose range, i.e. no doserelated,
increase in the number of revertants.

To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 492 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitation of Disperse Brown 27 on the plates was observed at the start of the incubation period at concentrations of 27 μg/plate and upwards. At the end of the incubation period, precipitation on the plates was observed at 86 μg/plate and above in the absence of S9-mix and at 154 μg/plate and above in the presence of S9-mix.

In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

In tester strain TA1535, the test item induced a 3.1-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In tester strain TA98, the test item induced up to 4.2 and 5.7-fold dose-related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. Although statistical analysis showed potential significance, the figures were lower that would be expected for a true positive.

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, increase in the number of revertants.

Conclusions:

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In the absence of S9-mix, Disperse Brown 27 induced dose-related increases in tester strain TA98. Although the increases observed were in two independently repeated experiments and were up to 4.7-fold the concurrent controls, the increase was above the laboratory historical control data range only in the second experiment and were only seen at or beyond precipitate.

In the presence of S9-mix, Disperse Brown 27 induced dose-related in the tester strains TA1535, TA98 and TA100. Although the increase observed in tester strain TA1535 was more than three-fold (3.1 times the concurrent controls), the increase was not outside the laboratory historical control data range and in one experiment only. The increases observed in tester strain TA98 were in two independently repeated experiments, were above the laboratory historical control data range, were dose-related and were already seen at nonprecipitating dose levels. Furthermore the increases were up to 7.4-fold the concurrent controls. Although the increase observed in tester strain TA100 was above the laboratory historical control data range, the increase was only seen in the dose range finding test, not dose related, beyond precipitate and was not more than two times the concurrent control.

Taken together, the increase in tester strain TA98 in the presence of S9-mix is the only increase which is considered to be biological biologically relevant, but the results are low enough to be considered ambiguous.
All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that Disperse Brown 27 is mutagenic in tester strain TA98 of the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. Disperse Brown 27 is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA100) or Escherichia coli reverse mutation assay using strain WP2uvrA.

Executive summary:

Disperse Brown 27 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments both in the absence and presence of S9 -mix (rat liver S9 -mix induced Aroclor 1254).

Batch 4852-1501 of Disperse Brown 27 was a brown powder with a purity of 99%. The vehicle of the test item was dimethyl sulfoxide.

In the dose range finding study, Disperse Brown 27 was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested. No reduction in the number of revertants was observed up to the dose level of 1600 μg/plate. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 27 to 492 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of

revertants was observed.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

64 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

Mutation frequency of one of the solvent control cultures was recorded to be outside the range of ≥ 50 per 106 survivors and ≤ 170 per 106 survivors. Deviation in mutation frequency had no effect on study.

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

Batch 4852-1501 of Disperse Brown 27 was a brown powder with a purity of 99.7%

Target gene:

The thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate). S9-mix components contained per ml physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose- 6-phosphate, 3.4 mg NADP, 4 μmol HEPES. Concentration of S9-fraction in exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

Upon mixing with exposure medium the test item precipitated at concentrations of 12.5 mg/ml (= 125 μg/ml in the exposure medium) and above. The concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 12.5 to 200 μg/ml in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test, Without and with S9-mix: 0.79, 1.58, 3.15, 6.3, 12.5, 25, 50, 100 and 200 μg/ml exposure medium.
To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 200 μg/ml was not selected for mutation frequency measurement.

Vehicle / solvent:

DMSO (Dimethyl sulfoxide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Disperse Brown 27 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Except the response of one of the solvent control groups in the second experiment, in which the mutation frequency of one of the solvent control cultures was just above the acceptability criteria of this assay.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced

significant increases in the mutation frequency. In addition, the mutation frequency 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.

The suspension growth over the two-day expression period for cultures treated with DMSO was between 10 and 12 (3 hour treatment) and 64 and 67 (24 hour treatment).

 

In the absence of S9-mix, Disperse Brown 27 did not induce a significant increase in the

mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment. In the presence of S9-mix, Disperse Brown 27 did not induce a significant increase in the mutation frequency

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)