Trisodium [3-[[4-[[6-anilino-1-hydroxy-4-sulpho-2-naphthyl]azo]-5-hydroxy-o-tolyl]azo]naphthalene-1,5-disulphonato(5-)]cuprate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic to bacteria of S. typhimurium strains.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The complete read across justification is detailed in section 13. Test substance is an isomer of the substance under registration; structural difference is not expected to significantly impact the genotoxic potential. Source study has reliability 2: only limited information available from a migrated NONS file, as per Article 25(3) request.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The method used was that of Prival and Mitchell (Mutat Res 97; 103-116:1982), using a metabolising system favourable to azo reduction (uninduced hamster liver S9, a modified list of co-factors, and a pre-incubation period of 30 minutes).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

uninduced hamster liver S9

Test concentrations with justification for top dose:

10-5000 µg per plate

Vehicle / solvent:

Distilled water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Congo Red

Remarks:

the positive control substance used in the presence of metabolic activation gave a satisfactory positive response

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

not specified

Genotoxicity:

other: n.a.

Cytotoxicity / choice of top concentrations:

other: n.a.

Vehicle controls validity:

other: n.a.

Untreated negative controls validity:

other: n.a.

True negative controls validity:

other: n.a.

Positive controls validity:

other: n.a.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

possible effects starting at 1000 µg/plate (details reported below)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

possible effects starting at 1000 µg/plate (details reported below)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Toxic concentrations

Weak toxic effects were seen in TA 98 at 1000 µg/plate with S9 in one of two experiments and in TA 1537 at 1000 µg/plate without S9 in one of three experiments. The substance was also toxic to TA 1537 in one out of three experiments at 5000 µg/plate with and without S9.

Observations

In one experiment a slight increase in revertants in strain TA 1537 was observed after 100 µg/plate without S9 and at 10, 100, 1000 and 5000 µg/plate with S9. These increases were not observed in two subsequent repeat experiments. No other increases in revertants were observed.

Conclusions:

Non mutagenic with and without the metabolic activation.

Executive summary:

Method

The test has been conducted according to the Prival and Mitchell method (Mutat Res 97; 103-116:1982). Information derived from migrated NONS file, as per Article 25(3) request with permission to refer granted by ECHA.

Strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were used with and without S9 metabolic activation.

Results

In one experiment a slight increase in revertants in strain TA 1537 was observed at 100 µg/plate without S9 and at 10, 100, 1000 and 5000 µg/plate with S9. Such increases were not confirmed by two subsequent experiments. No other increases in revertants were observed.

Overall, test substance was considered as non mutagenic to bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The complete read across justification is detailed in section 13. Test substance is an isomer of the substance under registration; structural difference is not expected to significantly impact the genotoxic potential. Source study has reliability 2: only limited information available from a migrated NONS file, as per Article 25(3) request.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

1.58-5000 µg per plate (8 doses)

Vehicle / solvent:

Distilled water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Precipitation of test compound in first trial at 5000 µg/plate.

Bacterial toxicity above 5000 µg/plate.

Conclusions:

Non mutagenic with and without the metabolic activation.

Executive summary:

Method

The test has been conducted according to EU method B.14. Information from derived migrated NONS file, as per Article 25(3) request with permission to refer granted by ECHA.

Five strains of S. typhimurium were used, i.e. TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without the metabolic activation S9 (Aroclor 1254-induced rat liver S-9).

 

Results

Test substance did not show a mutagenic potential to bacteria with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No information on genotoxicity of Direct Blue 273 was available. Therefore, a read across approach was used for the assessment, using available data on Similar Substance 01, i.e. an isomer of Direct Blue 273.

In two available tests, genetic toxicity on bacteria (Ames test) was examined.

The first test was conducted according to the Prival and Mitchell method (Mutat Res 97; 103 -116:1982). This information derived from migrated NONS file, as per Article 25(3) request, with permission to refer to it granted by ECHA. Under test conditions, in one experiment a slight increase in revertants in strain TA 1537 was seen at 100 µg/plate without S9 and at 10, 100, 1000 and 5000 µg/plate with S9. These increases were not observed in two subsequent repeated experiments. No other increases in revertants were seen.

The second test was conducted according to EU method B.14, using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (Aroclor 1254 -induced rat liver S-9). No mutagenic effects were reported.

Justification for classification or non-classification

According to the CLP Regulation a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

Based on negative results, Similar Substance 01 is considered as not mutagenic to bacteria.

Due to the structural similarity between Direct Blue 273 and the Similar Substance 01, Direct Blue 273 is expected to be non mutagenic. On these bases, Direct Blue 273 is not classified under the CLP Regulation (EC 1272/2008).