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Toxicological information

Genetic toxicity: in vitro

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Macrolex Blau 3R was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects, dissolved in DMSO, in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat experiment was performed as preincubation modification for 20 minutes at 37 °C in the absence of S9 mix in doses of up to and including 1600 µg per plate and in the presence of S9 mix of up to and including 5000 µg per plate.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test substance : Macrolex Blau 3R
Further name of test substance: Reinblau RLW
Batch number : CHN 23196
Content : 99.5 %
Visual appearance : blue powder
Chemical name : 1,4-Bis[(2-ethyl-6-methylphenyl)amino]-9,10-anthracenedione
Molecular weight : 474.6 g/mol
Molecular formula : C32H30N2O2
CAS-No. : 41611-76-1

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: Firstly, they are deep rough since certain lipopolysaccharide side chains are missing in the bacterial cell wall. Secondly, their reduced ability to repair damage from UV light (e.g. thymidine dimers) allows the phenotypic detection of mutation events
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Plate incorporation method: with and without S9 mix: 0, 50, 160, 500, 1600, 5000 µg/plate
Preincubation method: with S9 mix: 0, 50, 160, 500, 1600, 5000 µg/tube; without S9 mix 0, 16, 50, 160, 500, 1600 µg/tube
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, mitomycon c, cumene hydroperoxide, 2-aminoanthracene

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The Salmonella/microsome plate incorporation test, employing doses of up to 5000 µg per plate, showed Macrolex Blau 3R not to produce bacteriotoxic effects. Substance precipitation occurred without S9 mix at 500 µg per plate and above and with S9 mix only at 5000 µg per plate.

Evaluation of individual dose groups, with respect to relevant assessment parame-ters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls.

In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity.

Despite this sensitivity, no indications of mutagenic effects of Macrolex Blau 3R could be found at assessable doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

Macrolex Blau 3R was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects, dissolved in DMSO, in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat experiment was performed as preincubation modification for 20 minutes at 37 °C in the absence of S9 mix in doses of up to and including 1600 µg per plate and in the presence of S9 mix of up to and including 5000 µg per plate. Other conditions remained unchanged.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred without S9 mix at the dose of 500 µg per plate and above and with S9 mix at 5000 µg per plate.

Evidence of mutagenic activity of Macrolex Blau 3R was not seen. No biologically relevant increase in the mutant count, in comparison to the negative controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well as in the preincubation modification, under the experimental conditions applied.

The positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluorene, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, Macrolex Blau 3R is negative in the Salmonella/microsome test.