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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-05-22 to 2012-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study with well-characterized test material. Based on ECHA-guidance, a default reliability of 2 is assigned to account for read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
[N,N,N',N',N'',N''-hexaethyl-29H,31H-phthalocyaninetrimethylaminato(2-)-N29,N30,N31,N32]copper tris(dodecylbenzenesulphonate)
EC Number:
278-150-3
EC Name:
[N,N,N',N',N'',N''-hexaethyl-29H,31H-phthalocyaninetrimethylaminato(2-)-N29,N30,N31,N32]copper tris(dodecylbenzenesulphonate)
Cas Number:
75247-18-6
IUPAC Name:
75247-18-6
Details on test material:
Please refer to confidential details on test material

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment I:
without S9 mix (4 hours exposure time): 0.8; 1.5; 3.0; 6.0 and 9.0 µg/mL
with S9 mix (4 hours exposure time): 3.0; 6.0; 12.0; 24.0 and 48.0 µg/mL

Experiment II:
without S9 mix (24 hours exposure time): 0.5; 1.0; 2.0; 3.0 and 4.0 µg/mL
with S9 mix (4 hours exposure time): 0.8; 1.5; 3.0 and 6.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO for the test item/ nutrient medium for the positive control substance
- Justification for choice of solvent/vehicle: relative non-toxicity towards the cells and solubility properties of the test item.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I: 4 hours with and without metabolic activation
Experiment II: 4 hours with and 24 hours without metabolic activation.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT: 6-Thioguanine

CYTOTOXIC EFFECTS
Indicated by a relative cloning efficiency or cell density below 50%

Evaluation criteria:
Acceptability of the assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10E6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of results
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-mutagenic 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 frequency at least at one of the concentrations 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 corresponding 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 was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
First experiment: at 6.0 µg/mL and above without metabolic activation and at 48.0 µg/mL with metabolic activation. In the second experiment cytotoxic effects occurred at 3.0 µg/mL and above with metabolic activation.
Additional information on results:
- No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration.
- In both experiments (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.
- Precipitation of the test item was noted at 6.0 μg/mL and above in both main experiments with metabolic activation.
- 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 cultures of 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.
- EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The lowest positive control frequency of EMS failed the range of the historical data in the first culture of experiment I without metabolic activation. However, the induction factor of three times the corresponding solvent control was clearly exceeded and the number of mutant colonies in the parallel culture was acceptable.
- A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. Relevant toxicity effects were observed at 100 µg/mL and above with metabolic activation. In addition, relevant toxicity effects were seen at 6.3 µg/mL and above without metabolic activation after both treatment intervals.

Applicant's summary and conclusion