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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 September 2018 - 11 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in publication): Nickel metal
Specific details on test material used for the study:
CAS No. 7440-02-0, Batch No. 10206

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
ATCC, CCL-93
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
With metabolic activation:
0.05, 0.10, 0.25, 0.50, 1.0 mM

Without metabolic activation:
0.10, 0.25, 0.50, 1.0, 2.5 mM

Highest doses selected based on data from pre-experiments and the dose at which precipitation is observed
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
Treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
ethylmethanesulfonate (without metabolic activation); 7,12-dimethylbenz(a)anthracene (with metabolic activation)
Remarks:
Final concentration: EMS 300 ug/mL; DMBA 1.0 ug/mL
Details on test system and experimental conditions:
In the pre-experiment for toxicity, the exposure concentrations without metabolic activation were 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5 and 5.0 mM and with metabolic activation were 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 2.5 and 5.0 mM. The experiments with and without metabolic activation were performed as 4 h short-term exposure assay.
Approximately 10 x 10e6 cells per concentration, solvent/negative and positive controls were seeded in complete culture medium. About 24 h after seeding, the cells were exposed to the selected test concentrations of Ni metal powder with or without S9. After 4 h exposure, the cultures were checked for precipitation prior to removing the treatment medium.
Cytotoxicity was evaluated by relative survival.
Evaluation criteria:
A mutation assay was considered acceptable if it meets the following criteria: 1) Negative and/or solvent controls fall within the 95th control limits of the historical data of the performing laboratory, 8.5-40.2 mutants/10e6 cells (without metabolic activation) and 9.6-44.0 mutants/10e6 cells (with metabolic activation). 2) The absolute cloning efficiency of the negative and/or solvent controls is >50%. 3) The positive controls induce statistically significant increase compared with the concurrent negative controls and are compatible with the laboratory historical data base, and 4) Two experimental conditions (with and without metabolic activation) are tested unless one results in a positive response.

According to OECD 476, a test is negative if 1) none of the test concentrations exhibits statistically significant increase compared with the concurrent solvent control, 2) there is no concentration-related increase when evaluated with an appropriate trend test, and 3) all results are inside the distribution of the historical negative control data.
Similarly, a test is considered clearly positive if 1) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control, 2) the increase is concentration-related when evaluated with an appropriate trend test, and 3) any of the results are outside the distribution of the historical negative control data.
Statistics:
Non-parametric Mann-Whitney test was used to check the concentration groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequences of the negative/solvent controls were used as reference.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Without metabolic activation With metabolic activation
Relative survival Mutant Frequency/10e6 cells Relative surivival Mutant Frequency/10e6 cells
Negative control 1 126 24.2 99 19.7
Negative control 2 117 31.8 101 27.0
Solvent control 1 100 27.2 100 20.2
Solvent control 2 100 23.9 100 12.0
EMS (positive control) 71 166.1
DMBA (positive control) 81 565.8
0.05 mM 100 17.9
0.10 mM 115 36.8 106 14.5
0.25 mM 100 31.9 88 31.8
0.50 mM 93 56.0 102 25.0
1.0 mM 59 36.9 92 31.2
2.5 mM 38 25.5

Remarks on result:
other: Nickel metal is non-mutagenic at the HPRT locus in V79 cells

Applicant's summary and conclusion

Conclusions:
In the described, guideline-compliant mutagenicity study, nickel metal powder (N36F-PTL) is non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

The test item Nickel metal was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments. The experiment with and without metabolic activation were performed as a 4 h short-term exposure assay. The test item was investigated in the main experiment at the following concentrations for evaluating mutagenicity:

without metabolic activation:

0.10, 0.25, 0.50, 1.0 and 2.5 mM

and with metabolic activation:

0.05, 0.10, 0.25, 0.50 and 1.0 mM

Precipitation of the test item was noted in the main experiment at concentrations of 2.5 mM (without metabolic activation) and 1.0 mM (with metabolic activation).

Biologically relevant growth inhibition (relative survival < 70%) was observed in the experiment without metabolic activation. The relative survival was 38% for the highest concentration (2.5 mM) evaluated.

No growth inhibition was observed in the experiment with metabolic activation.

In the experiments no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). All mutant values are within the historical data base of the test facility.

A statistical analysis displayed that at one concentration (0.25 mM) the mutant frequency of the experiment with metabolic activation was significantly increased over those of the solvent controls and a concentration-response relationship was determined in the χ² test for trend. However, since the mutant frequencies at all concentrations were within the historic control range, the statistically significant increase at 0.25 mM was not regarded as biologically relevant.

In the experiment without metabolic activation some of the mutant frequencies were significantly increased over those of the solvent controls, but no concentration-response relationship was determined in the χ² test for trend. However, as the highest evaluated concentrations were within the historic control range, the statistically significant increases in the mid-range were not regarded as biologically relevant.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

THIS STUDY WAS REVIEWED BY AN INDEPENDENT EXPERT