(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

Exp. I:

10.0; 100.0; 333.3; 1000.0; and 5000.0; µg/plate.

Exp. II:

10.0; 100.0; 333.3; 1000.0; 2500.0; and 5000.0; µg/plate,

Toxic effects, evidenced by a reduction in the number of revertants, occurred in nearly all strains with and without metabolic activation in both experiments.

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of 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 point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Micronucleus assay:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse

The test article was dissolved in aqua des t. This solvent was used as negative control. The volume administered orally was 20 ml/kg b. w. . 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei, with the exception of the test article groups at preparation intervals 24 and 48 hours in which only nine animals could be investigated because of lethalities. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval; 5000 mg/kg b. w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions .

However, in the larger sample of the mutagenicity study 7 out of 36 treated animals died.

After treatment with the test article the ratio between PCEs and NCEs was not substantially affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuc lei at any preparation interval after application of the test article .

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse .

Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

The test item is not classified for genetic toxicity according to the CLP Regulation (EC.n.1272/2008). For justification see the results of available studies.