Trinickel disulphide

Genetic toxicity: in vitro

Administrative data

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:

19 December 2008 - 16 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed OECD Guideline 476 (In vitro mammalian cell gene mutation test)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

Trinickel disulphide

EC Number:

234-829-6

EC Name:

Trinickel disulphide

Cas Number:

12035-72-2

Molecular formula:

Ni3S2

IUPAC Name:

(trinickel-1-ylidene)-1λ⁴-disulfene

Details on test material:

- Name of test material (as cited in study report): Nickel subsulfide (N19A)
- Molecular weight (if other than submission substance): 240.1
- Physical state: grey powder
- Analytical purity: not provided
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 3% (4 hr exposure), 7.5% (24 hr exposure), or 15% (complete medium) horse serum, 100U/100 ug/ml penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, and 250 ug/ml amphotericin B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction prepared at BSL using Male Wistar rats orally induced with phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Experiment I, with metabolic activation: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mM
Experiment I, without metabolic activation: 0.01, 0.03, 0.05, 0.2, 0.4, 0.6, 0.8, 1.0 mM
Experiment II, with metabolic activation: 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7 mM
Experiment II, without metabolic activation: 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium (RPMI 1640 + 3% horse serum or + 7.5% horse serum for long term exposure)

Controls

Untreated negative controls:

yes

Remarks:

vehicle alone

Negative solvent / vehicle controls:

yes

Remarks:

vehicle alone

True negative controls:

not specified

Positive controls:

yes

Remarks:

multiple positive controls

Positive control substance:

other: With metabolic activation - 3.5 ug/ml benzo[a]pyrene; without metabolic activation - 200 and 500 ug/ml ethylmethanesulfonate, and 10 ug/ml methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- Exposure duration: 4 hrs for all Experiments, except Experiement II without metabolic activation (24 hrs)
- Expression time (cells in growth medium): 72 hrs for all Experiments, except Experiement II without metabolic activation (48 hrs)
- Selection time (if incubation with a selection agent): 11 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable


SELECTION AGENT (mutation assays): 200 uL RPMI 1640 complete medium with 5 ug/ml trifluorothymidine


NUMBER OF CELLS EVALUATED: 2000 cells/well, 4 wells per experimental group


DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency

Evaluation criteria:

There are 4 criteria for considering a mutation assay acceptable:
1. At least three out of four 96-well-plates from the TFT resistance-testing portion of the experiment are scorable.
2. The absolute cloning efficiency: ([number of positive cultures x 100] / total number of seeded cultures) of the negative and/or solvent controls is > 50%.
3. The spontaneous mutant frequency in the negative and/or solvent controls is in the range of BSL historical control data: about 84-245 mutants per 10^6 cells.
4. The positive controls (EMS, MMS and B[a]P) induce significant increases (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.

There are 3 criteria for determining a positive result:
1. clear and dose-related increase in the mutant frequency,
2. biologically relevant response (at least a 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
3. combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Statistics:

Mutation frequency = [-ln(NC/TC(selective medium))]/[-ln(NC/TC(non-selective medium))] x 800, where NC = number of negative cultures, and TC = total number of cultures seeded.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A statistical evaluation of
the results is not regarded as necessary. A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

highest concentration (1.4 mM) resulted in mutation factor >2, and reduction in colony size similar to BaP

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest concentration resulted in severe inhibition of relative growth suspension (11.05%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

highest concentration (1.0 mM) resulted in mutation factor >2; reduction in colony size was greater than vehicle control, but less than MMS

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest concentration resulted in severe inhibition of relative growth suspension (13.95%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

3 highest concentrations (1.3, 1.5, 1.7 mM) resulted in mutation factor >2; reduction in colony size was similar to vehicle control except for 1.3 mM (between vehicle control and BaP).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3 highest concentrations (1.3, 1.5, 1.7 mM) resulted in severe inhibition of relative growth suspension (23.11%, 15.74%, and 10.07%, respectively).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

no dose resulted in mutation factor greater than 2; reduction in colony size was between vehicle control and MMS for 3 highest concentrations (0.3, 0.4, and 0.5 mM).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3 highest concentrations (0.3, 0.4, 0.5 mM) resulted in severe inhibition of relative growth suspension (22.52%, 14.77%, 10.96%, respectively).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, noted in experiments I and II, with and without metabolic activation, at dose 0.05 mM Ni3S2 and higher
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: Three range finding studied were performed.
1. Pre-test for toxicity with metabolic activation (4 hr exposure), using doses of 0.05, 0.1, 0.25, 0.5, 1, and 2 mM Ni3S2. Relative suspension growth began to decrease at 0.5 mM Ni3S2.
2. Pre-test for toxicity without metabolic activation (4 hours exposure), using doses of 0.05, 0.1, 0.25, 0.5, 1, and 2 mM Ni3S2. Relative suspension growth began to decrease at 0.25 mM Ni3S2.
3. Pre-test for toxicity without metabolic activation (24 hr exposure), using doses of 0.02, 0.1, 0.25, 0.5, 0.75, 1, and 1.25 mM Ni3S2. Relative suspension growth began to decrease at 0.02 mM Ni3S2.

COMPARISON WITH HISTORICAL CONTROL DATA:
1. In experiment I with metabolic activation most mutant values found were within the historical control data of the test facility BSL (about 93-245 mutants per 10^6 cells). In three dose groups (0.6, 1.2 and 1.4 mM) the mutant values exceeded the historical control data range.
2. In experiment I without metabolic activation nearly all mutant values found were within the historical control data of the test facility BSL (about 84-218 mutants per 10^6 cells). Only in the highest dose group tested (1 mM) did the number of mutants/10^6 cells exceed the range of the historical control data.
3. In experiment II with metabolic activation the mutant values of the negative controls and of the dose groups up to 1.1 mM were within the historical control data of the test facility BSL (about 93-245) mutants per 10^6 cells). In the three highest dose groups (1.3-1.7 mM) the range of the historical control data range was slightly exceeded.
4. In experiment II without metabolic activation all mutant values found were within or slightly above the historical control data of the test facility BSL (about 84-218 mutants per 10^6 cells).

Remarks on result:

other: other: Experiement I - 4 hr exposure

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Report contains 16 Data Tables.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous The threshold value of 2 was exceeded in at least one dose group for all short term exposure experiements (4 hr). However, no clear dose-response relationships were noted and, in addition, the positive finding in the experiment without metabolic activatio

The authors conclude that the test item Ni subsulfide (N19A) showed inconclusive mutagenicity results in the mouse lymphoma thymidine kinase locus using the cell line L5178Y under the experimental conditions reported. If followed strictly the international relevant guidelines the test item would have to be considered as mutagenic since in the experiments with metabolic activation the threshold value of 2 was exceeded in at least one dose group each. But since the positive finding in the first main experiment without metabolic activation could not be verified in the independent repetition and the hint for clastogenicity could not be verified as well in the independent repetition of the experiment with metabolic activation, the evaluated hint for mutagenicity might be the result of high cytotoxicity with accompanying precipitation of the test item. Therefore the test item is considered to be equivocal in this test system.

Executive summary:

BSL Bioservice conducted an in vitro mammalian cell gene mutation bioassay in mouse lymphoma thymidine kinase locus using the cell line L5178Y according to OECD Test #476 guidelines and using GLP standards. Testing was completed on 2009-05-04 and the final report issued on 2009-07-16. Pre-experiments were performed to determine test concentrations: Experiment I 1.4 mM (with metabolic activation) and 1 mM (without metabolic activation) were selected as the highest concentrations; Experiment II 1.7 mM (with metabolic activation) and 0.5 mM (without metabolic activation) were selected as the highest concentrations. Four hour Ni3S2 exposures were run in Experiment I (with and without metabolic activation) and Experiment II (without metabolic activation only); Experiment II without metabolic activation was performed as a 24 hr long-term exposure assay. The pH-value of the test item was adjusted to 7.0.

Precipitation of the test item was noted in Experiments I and II (with and without metabolic activation) at doses from 0.05 mM and higher. Cytotoxicity in the form of growth inhibition relative to vehicle control was noted in Experiment I (with and without metabolic activation) at 0.4 mM and higher concentrations. Most of the doses utilized in Experiment II (with and without metabolic concentrations) resulted in cytotoxicity. The degree of relative growth inhibition increased with dose, with the most severe cytotoxicity observed at the highest doses in both Experiments I and II (with and without metabolic activation).

Three criteria needed to be met to conclude that a result was positive: 1) a clear and dose-related increase in mutant frequency; 2) at least a 2-fold increase in mutation frequency related to vehicle control for at least one dose; 3) an increased occurrence of slow growth (small) colonies (considered a clastogenic effect). In Experiment I (with metabolic activation), the highest concentration (1.4 mM) resulted in mutation factor >2, and reduction in colony size similar to positive control (BaP). However, this dose corresponded with severe cytotoxicity (total growth relative to negative control of 11.05%). Similar mutation frequency and cytotoxicity results were observed in Experiment I without metabolic activation: mutation factor >2 and relative total growth of 13.95% at the highest dose (1 mM). However, colony size was not considered biologically different relative to vehicle control. Experiment II (with metabolic activation) demonstrated mutagenic frequencies >2 at the three highest doses (1.3, 1.5, 1.7 mM), though the slightly reduced colony size ratios relative to vehicle control were not considered to be indicative of clastogenicity (as demonstrated by reduced colony sizes induced by BaP). Cytotoxicity was apparent at all three doses (relative total growths of 23.11%, 15.74%, and 10.07%, respectively). Finally, the long-term assay (Experiment II without metabolic activation) produced no increases in mutagenic frequency at any dose, and colony size ratios were not considered biologically significant. Though these results contain elements of the necessary criteria for positive mutagenicity results, there were no results from any of the experimental conditions that satisfied all three criteria for a positive result. In addition, the doses at which some criteria were met coincided with high cytotoxicity. Combined with the fact that precipitation of the item was found in all these dose groups, those positive results observed across experiments may be the result of side effects. Based on these facts the final estimation of this assay is equivocal. STUDY RATED BY AN INDEPENDENT REVIEWER