Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(1-methylethoxy)propyl]amino]sulfonyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in bacteria (in four tester strains and in cultivated mammalian cells (OECD 476). It is not genotoxic in the micronucleus assay in Chinese Hamster (similar to OECD 474).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guidleine compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hgprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Before freezing, the level of spontaneous mutants was depressed by treatment with HAT.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

19.5 to 2500 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate and 7,12-dimethylbenz(a)anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 3-4 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 19 days

SELECTION AGENT (mutation assays): 6-thioguanine


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen¬trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not examined

Positive controls validity:

valid

Additional information on results:

The highest concentration applied in the pre-experiment (2500.0 µg/mL) was limited by the solubility properties of the test item in DMSO and aqueous medium. The concentration range of the main experiments was limited by precipitation of the test item.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The Ames test was performed in a procedure similar to OECD guideline 471. Only 4 tester strains were used(E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 missing). No independent repeat experiment was performed. The efficacy of the S-9 mix was confirmed in only one tester strain (i.e. cyclophosphamide in TA1535). The study was performed prior to the introduction of GLP, but performance and reporting details are adequate for evaluation.

The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA 100, TA 1535, TA 1537.

The tests were performed with the following concentrations

without and with microsomal activation: 25, 75, 225, 675 and 2025 ug/O.l ml. Precipitation in the softagar occurred at doses of 75 ug/plate and above. The substance was dissolved in acetone. S9 fraction of liver from rats induced with Aroclor 1254 was used for metabolic activation. When the colonies had been counted, the arithmetic mean was calculated.

The test substance was considered to be nonmutagenic if the colony count in relation to the negative control had not doubled at any concentration.

No increase in mutant frequency was observed for the test substance. Incubation with the control substances (vehicle or positive controls) confirmed the functioning of the assay.

In conclusion, the substance is not mutagenic in bacteria.

An OECD 476 study was performed to investigate the potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (BASF 2012). The study was performed under GLP and is valid without restrictions.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration applied in the pre-experiment (2500.0 µg/mL) was limited by the solubility properties of the test item in DMSO and aqueous medium. The concentration range of the main experiments was limited by precipitation of the test item.No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

A study for clastogenicity in Chinese Hamster is available (Ciba-Geigy Ltd. 1982). The study is titled to be a nucleus anomaly test as the term micronucleus assay and its OECD testing guideline were not yet established. The design of the study is however comparable to this guideline with deviations that only 1000 bone marrow cells were scored and that evaluation criteria were given merely as a statistically significant increase in the number of cells with anomalies. The doses of 116, 234 and 468 mg/kg bw given by gavage in peanut oil were based on incidences of mortality in a range-finder study.

The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo. Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells. These anomalies occur in interphase cells as a consequence of damage during the mitotic process. The increase in anomalies shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. Treatment consisted of one daily dose on two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made.

The percentage of anomalies in bone marrow smears from animals treated with various doses of the test substance (range 0 - 0.3) showed no significant difference from the control (range 0.1- 0.2).

The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide

(128 mg/kg) yielded 5.6% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle alone.

In conclusion, the test substance did not cause clastogenicity in Chinese Hamsters in vivo. Therefore, no experimental data on clastogenicity in vitro was retrieved.

Justification for selection of genetic toxicity endpoint
Study in mammalian cells, representative for all three studies.

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. No mutagenicity was observed in bacteria and cultivated mammalian cells. No genotoxicity was observed in a micronucleus assay in vivo. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

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.

No mutagenicity was observed in bacteria and cultivated mammalian cells. No genotoxicity was observed in a micronucleus assay in vivo.

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 EC 944/2013.