Calcium bis[2-[(2-hydroxynaphthyl)azo]naphthalenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are only very limited data on genotoxicity of the test item available. Since all of the substances are metal laked salts with comparable structures and similar solubility, information on mutagenicity and cytogenicity were also derived from experimental data of a structural analogue. In several Ames test with and without prival modification conducted equivalent or according to OECD 471 no genotoxicity was seen. Also a mouse lymphoma assay and an UDS assay in rat hepatocytes yielded negative results. In two in vitro chromosome aberration assasy conducted equivalent or according to OECD guideline 473 no increase in structural chromosome aberrations was seen. An in vivo Micronucleus test according to OECD guideline 475 as well as a Drosophila SLRL and an UDS in vivo gave also no positive results. Based on the data of these studies the test substance was not considered to be genotoxic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Prival modification

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Test concentrations with justification for top dose:

3 - 5000 microgramm/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene

Remarks:

with metabolic activation (Hamster liver S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 48h, 37°C
- The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.

NUMBER OF REPLICATIONS: two independent experiments, in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertants

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experirnent.
A dose dependent increase in the number of revertant colanies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: from 800ug/plate onward

COMPARISON WITH HISTORICAL CONTROL DATA: yes, historical control data from January 2011 until December 2011, WP2 uvrA the historical data are based on approx. 200 experiments, TA 98 positive control with congo red historical data are based on approx. 50 experiments (2007 - 201 0)

Conclusions:

non mutagenic in the Ames (Prival)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Read across justification

Only limited data on mutagenicity of the test item are available. The chemical shares structural similarity to analogue substances. All of them are metal laked salts and include 1 -amino-2 -naphthol which is connected via azo bond with an aromatic sulfonic acid. The salts are solid pigments and poor soluble in water and octanol. Therefore, it is acceptable to derive information on genotoxicity from experimental data of the analogue substance. A detailed read across justification is given in Annex I of the CSR.

Procedure and observations key study Ames assay This study was performed to investigate the potential of the test item to induce gene mutations according to the pre-incubation test with and without hamster liver S9 (both experiments) at concentrations of 3 - 5000 ug/plate using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA. No toxic effects occurred in the test groups. No substantial increase in revertant colony numbers was observed following treatment with the test item at any dose Ievel. read across data and supporting studies

The test material and the analogue pigment red 53:1, respectively, have been tested for genotoxicity in a series of Ames tests with and without metabolic activation including the Prival test (Ciba 1985, Muzzall et al 1979, Myagoshi et al 1983, Hoechts 1989), in the Cytogenetic assay with V79 cells and with ovary cells of Chinese hamsters (CHO) (Ivett et al 1989, Hoechst 1989), in the Mouse lymphoma assay (Myhr et al 1991), in the Sister chromatid exchange assay with ovary cells of Chinese hamsters (CHO) (Ivett et al 1989) and the Unscheduled DNA synthesis in rat hepatocytes (Kornbrust et al 1985). In all these in vitro studies the test substance as well as the analogue gave negative results. Pigment red 53:1 was also assayed for genotoxicity in vivo using the rat micronucleus test (Westmoreland and Gatehouse 1992), a rat ex vivo liver UDS assays and the SLRL-test in Drosophila. Uniformly negative results were obtained in all assays, even though large oral doses were used (up to 2 g/kg).

Discussion

The test item as well as analogue substances were found to be non-mutagenic in several ames test with and without prival modification. In various in vitro and in vivo studies the analogue substance, pigment red 53:1, was proved to be non-genotoxic. Due to the similarities of both substances in structure and toxicological profile, the test item is not considered to be genotoxic.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

 

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).