[N,N,N',N',N'',N''-hexaethyl-29H,31H-phthalocyaninetrimethylaminato(2-)-N29,N30,N31,N32]copper tris(dodecylbenzenesulphonate)

Endpoint summary

Additional information

Bacterial reverse mutation assay (Ames test)

In a reliable key reverse gene mutation assay in bacteria (BASF SE, 2012), strains TA1535, TA 1537, TA100, TA 98 of Salmonella typhimurium and WP2 uvrA of Escherichia coli were exposed to the test item (CAS number 75247 -18 -6, batch: Ho 35761 -124a) at up to 5000 µg/plate in the presence and absence of mammalian metabolic activation system according to OECD guideline 471. The test substance did not lead to a relevant increase in the number of revertant colonies without or with mammalian metabolic activation system (S9 mix) in two experiments carried out independently in the standard plate test and preincubation assay. The positive controls induced the appropriate responses in the corresponding strains. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. Therefore, the test item was considered to be non-mutagenic under the conditions of this bacterial reverse mutation assay.

 

In a supporting reverse gene mutation assay in bacteria (BASF AG, 2001; 40110319/004080), strains TA1535, TA 1537, TA100, TA 98 of Salmonella typhimurium and WP2 uvr A of Escherichia coli were exposed to a powder containing 42.7% of CAS number 75247 -18 -6 (batch: Ablauf 14 -4704) at up to 12000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix according to OECD guideline 471. The test substance was weakly positive only in TA 1537 without metabolic activation in concentrations of 2000 - 4000 µg/plate in two of three experiments. The increase of revertants was 2.5 to 3.4-fold. Here, cytotoxicity appeared in concentrations > 4000 µg/plate. According to the authors impurities could not be ruled out as cause for these findings, because the portion of impurities was more than 50%. All other strains and TA 1537 with metabolic activation showed negative reactions.

In a supporting gene mutation assay in bacteria (BASF SE, 2001, 40110319/004188) the Salmonella typhimurium strain 1537 (only one strain tested) was exposed to CAS number 75247 -18 -6 (batch: P.31 v. 19.04.200, purity ca. 98%) or to a powder containing 42.7% of CAS number 75247 -18 -6 (batch: Ablauf 14-4704) in the presence and absence of S9 -mix in concentrations up to 5000 µg/plate. Information on exposure duration or bacterial toxicity was not cited in the report. Precipitation occurred from 1000 µg/plate onwards. Positive genotoxicity was observed at 2000 - 4000 µg/plate (concentration-dependent) without metabolic activation only after exposure with Ablauf 14 -4704 (purity: 42.7%). This weak positive result demonstrated that the increase in revertants was impurity-related, since it was seen only following exposure to Ablauf 14 -4704 (purity: 42.7%), but not following exposure to CAS number 75247 -18 -6 (purity: ca. 98%).

 

In conclusion, in the key gene mutation assay (BASF SE, 2012) and the supporting study (BASF AG, 2001; 40110319/004080), both conducted according to OECD 471, the test item (CAS number 75247 -18 -6, batch: Ho 35761 -124a) was shown to be non-mutagenic.

 

In vitro mammalian cell gene mutation test (HPRT test)

CAS number 75247 -18 -6 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD guideline 476 (BASF SE, 2012). The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 0.8; 1.5; 3.0; 6.0 and 9.0 µg/mL

with S9 mix: 3.0; 6.0; 12.0; 24.0 and 48.0 µg/mL

Experiment II:

without S9 mix: 0.5; 1.0; 2.0; 3.0 and 4.0 µg/mL

with S9 mix: 0.8; 1.5; 3.0 and 6.0 µg/mL

 

Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% in both parallel cultures occurred in the first experiment at 6.0μg/mL and above without metabolic activation and at 48.0μg/mL with metabolic activation. In the second experiment cytotoxic effects occurred at 3.0μg/mL and above with metabolic activation. Precipitation of the test item was noted at 6.0μg/mL and above in both experiments with metabolic activation. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in both experiments up to and including the maximum concentration.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the first experiment with metabolic activation. This trend however, was judged as irrelevant as the mutation frequency remained within the historical range of solvent controls at all of the data points and there was no significant trend in the parallel culture under identical conditions.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.8 up to 35.0 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.9 up to 35.5 mutants per 10E6 cells. EMS (150μg/mL) and DMBA (1.1μg/mL) were used as positive controls and showed a distinct increase in the number of induced mutant colonies. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

In vitro cytogenicity study in mammalian cells (Chromosome aberration) (waiving)

In accordance with column 2 of REACH Annex VIII, the in vitro cytogenicity study in mammalian cells does not need to be conducted if adequate data from an in vivo cytogenicity test are available. Since an in vivo mammalian erythrocyte micronucleus test is available for a structurally similar substance no test on cytogenicity on mammalian cells in vitro has to be conducted.

 

Mammalian erythrocyte micronucleus test (CAS number 81457 -65 -0) (read across)

The test item (CAS number 81457 -65 -0) was assessed in a nucleus anomaly test (BASF AG, 1982; equivalent to OECD 474) for its potential to induce clastogenic effects in the bone marrow of the Chinese hamster. The test item, dissolved in peanut oil, was administered orally to 3 Chinese hamsters/sex at dose levels of 116, 234 or 468 mg/kg bw once a day for 2 days. The post exposure period was 24 hours. Cyclophosphamide was used as positive control at 128 mg/kg bw. After sacrifice, bone marrow cells were harvested, mixed and transferred on slides. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. The positive control yielded a significant increase of the percentage of cells with anomalies. In conclusion of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

The read-across substance is also a high molecular weight modified copper phthalycyanine colorant of good solubility/dispersibility in octanol. It is also a salt formed by ammonium and sulfonate functions which is of very low solubility in water. In regard to genotoxicity it covers the same functional groups. In regard to systemic uptake, it has the same challenges (low water solubility, solubility/dispersibility in octanol) but is of lower molecular weight. It is therefore considered to give an adequate prediction of in-vivo genotoxicity in the micronucleus assay.

Justification for selection of genetic toxicity endpoint
Two in vitro tests (Ames test, HPRT assay) and one in vivo test (micronucleus test) are used for assessment of genotoxicity. In accordance with column 2 of REACH Annex VIII, the in vitro cytogenicity study in mammalian cells does not need to be conducted if adequate data from an in vivo cytogenicity test are available. Since an in vivo mammalian erythrocyte micronucleus test is available for the structurally similar substance CAS no. 81457-65-0 (read across approach, please refer to chapter 13 of the IUCLID, Assessment reports) no test on cytogenicity on mammalian cells in vitro has to be conducted.

Short description of key information:
The absence of a mutagenic potential was demonstrated for gene mutations in bacteria (Ames test) and in mammalian cells (HPRT assay). Both tests were conducted with the target substance CAS no. 75247-18-6. A mammalian erythrocyte test with the structural similar substance CAS no. 81457-65-0 demonstrated the absence of clastogenicity in vivo.

In conclusion, based on negative results in all relevant in vitro and in vivo tests, the test substance is considered as non mutagenic.

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 third time in Directive (EC 618/2012).