[2-(29H,31H-phthalocyaninylmethyl)-1H-isoindole-1,3(2H)-dionato(2-)-N29,N30,N31,N32]copper

Endpoint summary

Additional information

The target substance has been tested for mutagenicity in the Ames test (OECD 471, GLP) (GPT 2004). It was found to be non mutagenic and not cytotoxic. Experimental data on mutagenicity and clastogenicity in mammalian cells in vitro is available for the UVCB analogue with the slightly average substitution grade (2.48 versus 1.4). Both substances share the same components; the analogue UVCB substance contains more of the higher substituted components. All components are poorly soluble and have a molecular weight greater than 500 g/mol. A mode of action for genotoxicity is reactivity with DNA or proteins In this case, the structural features are identical and any potential for reactivity present in the target substance would be identified from the higher substituted analogue. Therefore, it is acceptable to use the data of the analogue for read-across.

A general read-across justification with structures and data matraces has been added to the Chemical Safety Report (toxikokinetic section).

The GLP-study was performed according to the OECD Guideline TG 471 for the bacterial reverse mutation test with Salmonella typh.TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA (GPT 2004).Exposure of the test system was performed using the pre-incubation method. The test item was applied at concentrations of 0.2 mg, 0.1 mg, 0.05 mg, 0.01 mg, and 0.005 mg/plate (main study) and 0.03 mg, 0.01 mg, 0.003 mg, 0.001 mg and 0.0003 mg/plate (repeat study) with and without metabolic activation (S9 rat liver). Concentrations were chosen so low because the OECD guideline recommends testing non-precipitating concentrations. The visual control of the plates showed signs of precipitation at all five test item concentrations applied (main study), and at 0.03 mg/plate and 0.01 mg/plate (repeat study) of test item at all strains with and without metabolic activation. At all tested concentrations no cytotoxic effects on the test systems were observed.

For none of the strains - with and without metabolic activation - a clear increase in the number of revertant colonies was observed. Furthermore, no dose-effect relationship could be determined. The respective negative controls produced a low number of revertants. The strain-specific positive controls were shown to produce the expected increase in revertant colonies, with and without metabohc activation, respectively. The substance was found to be non mutagenic.

Another Ames test with a crude version of the substance (GPT 2004b) also showed absence of mutagenicity.

The following data is available for the substance with the average higher substitution grade:

The blue powder was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA in a reverse mutation assay (OECD 471 adopted 1997, GLP). Doses ranged from 0.02 - 5 mg and 0.004 - 2.5 mg per plate in the standard plate test and preincubation test, respectively. Aroclor-induced rat S9 mix served for metabolic activation.

Precipitation of the test substance was found from about 0.1 mg/plate onward. An increase in mutant frequency was not observed showing that the substance is not mutagenic in bacteria.

The positive and negative control experiments showed the expected results.

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (OECD 476, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period)

0; 3.13; 6.25; 12.5; 25.00; 50.00; 100.00 μg/mL

with S9 mix (4-hour exposure period)

0; 3.13; 6.25; 12.5; 25.00; 50.00; 100.00 μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 5.00; 10.00; 20.00; 40.00 μg/mL

with S9 mix (4-hour exposure period)

0; 5.00; 10.00; 20.00; 40.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa

and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

The test substance was poorly soluble either in organic solvents or culture medium. In the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied test substance concentrations which were at the border of test substance solubility in culture medium. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

The substance was assessed for its potential to induce structural chromosome aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro (OECD 473, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test, the test substance did not exhibit any pronounced toxicity up to the highest applicable concentration of 1500 μg/mL (approx.1.38 mM), at which distinct test substance precipitation was observed. Test concentrations as indicated n the results table were chosen. A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures where only 50 metaphases were scored due to clearly increased aberration rates.

The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosome aberrations.

The test substance was poorly soluble either in organic solvents or culture medium. No clear cytotoxicity was observed up to the highest applied test substance concentrations which were at the border of test substance solubility in culture medium.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases at both sampling times either without S9 mix and/or after adding a metabolizing system.

No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the substance is considered not to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Short description of key information:
The substance is not genotoxic in the Ames test. A related substance was not genotoxic in the HPRT test and in the chromosome abberration test in vitro. All studies were performed with well characterized test material accoring to OECD guidelines and under GLP. There is no concern for genotoxic properties.

Endpoint Conclusion: No adverse effect observed (negative)

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 mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, 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 mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC944/2013.