2-{[(2-{3-[(4-{Ethyl[3-(vinylsulfonyl)phenyl]amino}-6-fluoro-heteromonocycle-2-yl)amino]-2- (hydroxy-O)phenyl}hydrazono-N2)(phenyl)methyl]diazenyl-N1}benzoato(6-)-O]cuprate(4-) sodium salt, polysulfo

Endpoint summary

Additional information

Genetic toxicity

- Gene mutation in bacteria

To characterize the genotoxic potential of the test substance in vitro, an Ames assay was conducted equivalent to OECD 471 with a standard battery of Salmonella typhimurium tester strains including TA 98, TA 100, TA 102, TA 1535 and TA 1537 in concentrations ranging from 31.6 to 5000 µg/plate with and without metabolic activation in a plate incorporation and a pre-incubation assay (pre-incubation for 30 min) (Donath, 2011). The test substance did not exhibit biologically relevant increases in revertant colony numbers of any tester strain either in the absence or presence of metabolic activation. Further, no cytotoxicity was observed. In conclusion, the test substance is considered as non-mutagenic in the Ames test.

 

- Chromosome aberration in mammalian cells

Further, a recent GLP-guideline study according to OECD guideline 473 was performed to determine clastogenic properties of the test substance (Oppong-Nketiah, 2012). Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation for 4 hours in duplicates. For analyses, 100 metaphases per culture were scored. the test substance induced cytotoxicity at 125 µg/mL without and at 600 µg/mL with metabolic activation which was determined by a reduced mitotic index (125 µg/mL: 62%, 600 µg/mL:44% of controls, respectively). Moreover, the aberration rate increased at the same concentrations (without S9, 125 µg/mL: 2.5 vs 7% (excl. gaps); with S9, 600 µg/mL: 1 vs. 4% for controls and test plates, respectively (excl. gaps). As cytotoxicity and clastogenicity occurred at the same concentrations, the induction of chromosomal aberrations due to cytotoxicity cannot be excluded. Further, false-positive results due to methodological reasons cannot be obviated as control cells revealed a relatively high aberration rate in the main experiment (controls: without S9: 4.5% (incl. gaps) and 2.5% (excl. gaps); with S9: 4% (incl. gaps) and 1% (excl. gaps)) which partially exceeded the amount of aberrant cells exposed to the lowest dose concentrations applied (without S9: 31.3 µg/mL: 1.5% (incl. gaps) and 1% (excl. gaps), 62.5 µg/mL: 3.5% (incl. gaps) and 1.5% (excl. gaps); with S9: 500 µg/mL: 2% (incl. gaps) and 1.5% (excl. gaps)). Thus, the results of the conducted mammalian chromosome aberration test are inconclusive and are therefore not taken into account for assessment.

 

-Genetic toxicity in vivo

Moreover, a mammalian erythrocyte micronucleus test according to OECD guideline 474 was performed (Hofman-Hüther, 2012). 5 NMRI mice per sex and dose group received a single intraperitoneal injection of 20, 50 and 100 mg/kg bw to cover a range from the maximum tolerable dose to moderate systemic toxicity as determined in a previous range finding study. Control animals received either 0.9% NaCl or 40 mg/kg bw cyclophosphamide as negative or positive control substances, respectively. Peripheral blood samples obtained from the tail vein were collected 44 hours and 68 hours after test substance administration. For evaluation, immature erythrocytes were labelled with anti-CD27 antibodies and were counted in a flow cytometer (10 000 immature erythrocytes per animal). Further, the ratio between immature and mature erythrocytes was calculated to detect toxic effects to the bone marrow. Low- and mid-dose animals showed slight or moderate signs of systemic toxicity whereas strong signs of toxicity were determined in all animals of the high-dose group including reduced spontaneous activity, constricted abdomen, piloerection (determined only in males), bradykinesia, catalepsis, recumbency opisthotonos, half eyelid closure and eye closure thereby proving systemic availability of the test substance. Moreover, a slight decrease in the ratio of immature and mature erythrocytes among the groups was determined which indicates toxic effects on the bone marrow and therefore availability of the test substance in the target tissue. Further, skin and urine of high-dose animals were blue coloured attributable to the test item. Mean values of micronuclei frequency were comparable among the different treatment groups after 44 hours. In contrast, positive control animals showed a statistically significant increase in the micronuclei frequency about 7- and 9-fold in males and females, respectively, thereby validating the study. In conclusion, the conducted mammalian micronucleus test did not reveal an indication that the test substance induces cytogenetic damage in vivo. Thus, the test substance did not show clastogenic properties in the conducted micronucleus test in mice.

 

Taken all these data together, the test substance did not show mutagenic properties in the bacterial reverse mutation test nor did it induce cytogenetic damage in vivo. Therefore, the criteria for classification according to GHS are not met.

Justification for selection of genetic toxicity endpoint
Hazard assessment is based on 3 available genotoxicity studies.

Short description of key information:
In vitro:
- Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537: negative with and without metabolic activation
- Cytogenicity in mammalian cells (OECD 473, chromosome aberration assay): inconclusive in V79 cells

In vivo:
-Chromosome aberration (OECD 474, micronucleus assay): negative (maximum tolerable dose: 100 mg/kg bw orally administered to mice; post exposure period: 44 and 68 h)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.