dipotassium 8-(phenylamino)-5-((E)-(4-((E)-(3-sulfonatophenyl)diazenyl)naphthalen-1-yl)diazenyl)naphthalene-1-sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test was negative.

Micronucleous mutagenicity test (OECD 487): negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental study on similar substance

Adequacy of study:

key study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, test procedure in accordance with OECD 471 methodes, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. The test has been performed on only four Salmonella strains.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats

Test concentrations with justification for top dose:

Main Test: 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

Ta 98 and TA1537 without S9 fraction

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 an TA1535 without S9 fraction

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with S9 fraction

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)PREPARATION OF FRACTION S9: The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats strain Wistar Hanlbm* (BRL, CH-4414 Füllingsdorf; weight approx. 220 - 300 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika,D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously.After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80° C. Smallnumbers of the ampoules are kept at -20° C for only one week before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).The protein concentration in the S9 preparation was 45 mg/ml. PREPARATION OF S9 MIX: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:8 mM MgCl233 mM KCl5 mM glucose-6-phosphate5 mM NADPin 100 mM sodium-ortho-phosphate-buffer, pH 7.4.During the experiment the S9 mix was stored in an ice bath.PREPARATION OF PLATES: For each strain and dose level, incltiding the controls three plates were used as a minimum.The following materials were mixed in a test tube and poured onto the selective agar plates :100 ul Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),500 ul S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),100 ul Bacteria suspension (cf. test system, pre-culture of the strains),2000 ul Overlay agarIn the pre-incubation assay 100 ul test solution, 500 ul S9 mix / S9 mix substitution buffer and 100 ul bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°) was added to each tube.The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 4 8 hours at 37° C in the dark.DURATION- Expression time (cells in growth medium): 48 to 72 hours

Evaluation criteria:

Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Statistics:

Kier, L.E., D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Privai, T.K. Rao and V. Ray (1986)The Salmonella typhimurium/mammalian microsomal assay A report of the U.S. Environmental Protection Agency Gene-Tox Program Mutation Res. 168, 69-240.

Species / strain:

S. typhimurium TA 98

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

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

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

Species / strain:

S. typhimurium TA 97

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:

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ng/plate (active ingredient)Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 5000.0 (ig/plate without S9 mix in experiment I and with S9 mix in experiment II. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix inall strains used.No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the tested substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Conclusions:

negative

Executive summary:

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the tested substance at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore Acid Blue 113 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Bacterial reverse mutation assay, Ames test, showed negative results both with and without metabolic activation.
In vitro micronucleos genotoxicity test gave negative results.

Based on the test results it could be stated that no classification for mutagenicity is warranted under Regulation (CE) 1272/2008 for Acid Blue 113.