Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

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:

weight of evidence

Study period:

04 December 2015-29 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

EC Number:

944-232-9

Molecular formula:

Not available - UVCB substance

IUPAC Name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Test material form:

solid: particulate/powder

Method

Target gene:

The test item Vat Black 25 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from induced rat ( rat mixed induction)

Test concentrations with justification for top dose:

Main Assay I was performed at the following dose levels:
TA1535, -S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate
TA 1535, +S9: 250, 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +/- S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
WP2 uvrA +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA98, -S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate
TA98, +S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 300, 150, 75.0, 37.5, 18.8, 9.38 mcg/plate

Main Assay II was performed at the following dose levels:
TA1537, -S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +S9: 250, 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA98, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 300, 150, 75.0, 37.5, 18.8, 9.38 mcg/plate

An additional experiment (Main Assay III) was performed using the following dose levels: 250, 125, 62.5, 31.3, 15.6 µg/plate.

Main Assay IV was performed at the following dose levels:
TA1535, -S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA1535, +S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, -S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +S9: 250, 125, 62.5, 31.3, 15.6 mcg/plate
WP2 uvrA, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA98, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Methylmethanesulphonate; sodium azide;2 aminoanthracene

Details on test system and experimental conditions:

Toxicity, Main assay I, II and III were perfomed using the plate incorporation method.Main assay IV was perfomed using the pre-incubation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981).Regression line

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item induced reproducible and dose related increases in the number of revertant colonies with TA1537 and TA100 tester strains in the absence of S9 metabolism, and with TA1537 and TA98 tester strains in its presence. These increases were greater than twice the concurrent negative control value and so can be considered a clear evidence of mutation induction.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item Vat Black 25 induces reverse mutation in Salmonella typhimurium under the reported experimental conditions.

Executive summary:

The test item Vat Black 25 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone. The test item was used as a suspension in dimethylsulfoxide (DMSO).

Toxicity test: Based on results obtained in a preliminary solubility trial, the test item Vat Black 25 was assayed in the toxicity test at the maximum concentration of 500 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 158, 50.0, 15.8 and 5.00 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. Toxicity was observed at the highest or two highest dose levels both in the absence and presence of S9 metabolic activation with all tester strains with the exception of WP2 uvrA and TA98 which showed slight toxicity only in the absence of S9 metabolism. Dose related increases in revertant colonies were observed with TA100 tester strain in the absence and presence of S9 metabolism.

Main Assay I: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535: (- S9) 500, 250, 125, 62.5, 31.3 , 15.6 µg/plate.

(+S9) 250, 125, 62.5, 31.3, 15.6, 7.81 µg/plate.

TA1537: (± S9) 125, 62.5, 31.3, 15.6, 7.81 µg/plate.

WP2 uvrA: (± S9) 500, 250, 125, 62.5, 31.3 µg/plate.

TA98: (- S9) 500, 250, 125, 62.5, 31.3, 15.6 µg/plate.

(+S9) 500, 250, 125, 62.5, 31.3 µg/plate.

TA100 : (± S9) 300, 150, 75.0, 37.5, 18.8, 9.38 µg/plate.

Toxicity was observed with TA1537 and TA100 at the highest and two highest dose levels, respectively, in the absence of S9 metabolic activation and with TA1535 at the two highest dose levels in the presence of S9 metabolic activation. Dose related increases in revertant numbers up to 9.5-fold at the highest concentration were observed at all dose levels with TA1537 in the absence of S9 metabolism and at the three highest dose levels with TA98 up to 5.7 fold in the presence of S9 metabolism.

Mutation results obtained in Main Assay I with TA1537, TA98 and TA100 were not coherent with the results obtained in the preliminary toxicity test. An increase in revertant colonies not indicated in the toxicity test was observed, with TA1537 and TA98, while a lower increase was noticed with TA100 tester strain. Based on these results an additional experiment (Main Assay II), using the plate incorporation method, was performed with these tester strains, both in the absence and presence of S9 metabolic activation. The same concentrations were used as in the first assay except in TA1537 with S9, where concentrations of 250,125, 62.5, 31.3, 15.6,7.81 µg/plate were used, because no toxicity was seen in the first assay.

Dose related increases in revertant numbers were observed with TA1537 both in the absence and presence of S9 metabolism (10.5 fold and 5.1-fold respectively) and TA98 in its presence (4.5-fold). Mild increase (2-fold the concurrent negative control) were observed with TA98 and TA100 in the absence of S9 metabolic activation at the highest concentration. In order to clarify inconsistent results obtained with TA1537 tester strain in the presence of S9 metabolic activation, an additional experiment (Main Assay III) was performed with this tester strain, using the plate incorporation method and once again test item treatments yielded a 2-, 2.2- and 3.6-fold dose related increases in revertant numbers in the three highest concentrations. In order to confirm the results observed using the plate incorporation method, an additional experiment, using the pre-incubation method (Main Assay IV) was performed with all tester strains. Dose levels, displayed in the following table, were selected based on toxicity results obtained in all the previous experiments.

TA1535: (- S9) 500, 250, 125, 62.5, 31.3

(+S9) 125, 62.5, 31.3, 15.6, 7.81

TA1537: (- S9) 125, 62.5, 31.3, 15.6, 7.81

(+S9) 250, 125, 62.5, 31.3, 15.6

WP2 uvrA: (± S9) 500, 250, 125, 62.5, 31.3

TA98: (±S9) 500, 250, 125, 62.5, 31.3

TA100: (±S9) 500, 250, 125, 62.5, 31.3, 15.6

Slight toxicity was observed at the highest dose level with TA1535, both in the absence and presence of S9 metabolic activation, and at the two highest dose levels with TA100 in the presence of S9 metabolism. Dose related increases in revertant numbers were observed with TA1537 and TA100 tester strains in the absence of S9 metabolism up to 5- and 2- fold at the highest dose levels, respectively. In the presence of S9, an increase of mutation frequency of 2.45 was observed at the highest concentration with TA1537 whereas TA98 showed increases in revertant number of 3- and 4 fold in the two highest concentrations.

Conclusion: It is concluded that the test item Vat Black 25 induces reverse mutation in some strains of Salmonella typhimurium under the reported experimental conditions.