Nickel difluoride

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:

June 30,2008 to September 28,2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report Date:

2008

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. certificate)

Type of assay:

mammalian cell gene mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Target gene:

Thymidine Kinase Locus/TK +/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:Male Wistar rats
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Exposure concentrations
The selection of the concentrations was based on data from the pre-experiment . In experiment 1 1,6 mM (with metabolic activation) and 1.2 mM (without metabolic activation) were selected as the highest concentrations. In experiment 2 1,9 mM (with metabolic activation) and
0.3 mM (without metabolic activation) were selected as the highest concentration. Experiment 2 without metabolic activation was performed as a 24 h long-term exposure assay, The test item was investigated at the following concentrations:
with metabolic activation: 0.2 ; 0.4 ; 0.6 ; 0.8 ; 1.0 ; 1.2; 1.4 ; 1.6 ; mM
and without metabolic activation: 0.08 ; 0.1 ; 0.2 ; 0.4 ; 0.6 ; 0.8 ; 1.0 ; 1.2 mM
Experiment 2
with metabolic activation:
0.3 ; 0.5 ; 0.7 ; 0.9 ; 1.1 ; 1.3 ; 1.5 ; 1.9 mM
and without metabolic activation:
0.02 ; 0.04 ; 0.07 ; 0.1 ; 0.15 ; 0.2 ; 0.25 ; 0.3 mM
According to OECD Guidelines at least 8 concentrations of the test item were set up in the experiments with and without metabolic activation.

Vehicle / solvent:

Medium RPMI 1640

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With Metabolic activation, Dissolved in DMSO

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolica activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
-cell concetration: 1x10^7 cells/mL
DURATION
- Preincubation period:
- Exposure duration:4 hours
- Expression time (cells in growth medium):1x10^7 cells/mL

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type:

Any other information on results incl. tables

EVALUATION OF RESULT:

There are several criteria for determining a positive result:

-Clear and dose-related increase in the mutant frequency,

-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.

-Combined with a positive effect in the inutant 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.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A stalistical 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.

Discussion

The test item Nickel fluoride tetrahydrate (N111) was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The main experiments were carried out with and without metabolic activation. The experiments with metabolic activation were perfonned by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. Since the L5178Y cells do not have the cytochrome-based P450 metabolic oxidation system, an exogenous metabolic activation system, the S9 microsomal fraction was added.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 476. In experiment I 1.6 mM (with metabolic activation) and 1.2 mM (without metabolic activation) were selected as the highest concentrations. In experiment II 1.9 mM (with metabolic activation) and 0.3 mM (without metabolic activation) were selected as the highest concentrations.

Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The pH-value detected with the test item was within the physiological range.

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

0,2, 0.4, 0,6, 0.8, 1.0, 1.2, 1.4, 1.6 mM

and without metabolic activation:

0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mM

Experiment II

with metabolic activation:

0.3, 0.5, 0.7, 0.9, 1.1, 1.3, L5,.1.9 mM

and without metabolic activation:

0.02, 0.04, 0.07, 0.1, 0.15, 0,2, 0.25, 0.3 mM

Toxicity:

Growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 12.39% for the highest concentration (1.6 mM) evaluated. The highest concentration evaluated without metabolic activation was 1.2 mM with a RTG of 11.53 %.

In experiment II with metabolic activation the relative total growth (RTG) was 12.08% for the highest concentration (1.9 mM) evaluated. The highest concentration evaluated without metabolic activation was 0.3 mM with a RTG of19.55%.

Mutagenicity:

In experiment I with metabolic activation, all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 34-161 mutants per 106 cells, Table 16). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies of the negative controls were found to be 49,91 and 79.73 mutants/106 cells and in the range of 55.70 to 128.73 mutants/106 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.99 was found at a concentration of 1.6 mM with a RTG of 12.39%.

Without metabolic activation, all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 33-153 mutants per 106 cells, Table 16). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the

negative controls. Mutation frequencies of the negative controls were found to be 52.76 and 65.23 mutants/106 cells and in the range of 53.47 to 101.97 mutants/106 cells with the test item, respectively. The highest mutation factor (compared to the .negative control values) of 1.81 was found at a concentration of 1.2 mM with a RTG of 11.53%.

In experiment II with metabolic activation, all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 34-161 mutants per 106 cells, Tablo 16). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative controls were found to be 66,28 and 70.00 mutants/106 cells and in the range of 62.98 to 134.08 mutants/10' cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.97 was found at a concentration of 1.5 mM with a RTG of 19.01%.

In experiment II without metabolic activation, the mutant values of the negative control were within the historical control data of the test facility BSL BIOSERVICE (about 33-154 mutants per 106 cells, Table 16), Most mutant values of the dose groups evaluated were within the historical control data, too. At concentrations of 0.25 mM and higher the mutant values exceeded the historical control data. In addition, a dose-response relationship could be observed. Mutation frequencies of the negative controls were found to be 51.78 and 56,23 mutants/106 cells and in the range of 38.62 to 205.52 mutants/106 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 3.81 was found at a concentration of 0.25 mM with a RTG of 21.61%. In addition, at concentrations of 0.25 mM and 0.3 mM the mutation factor exceeded the threshold value of2 (3.81 resp. 2.89). EMS (500 µg/mL and 200 µg/mL), MMS (10 µg/mL) and B[a}P (3.5 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Relationship of large to small colonies:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony 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. In experiment I with metabolic activation, the quotients of large/small colonies of the negative controls were found to be 1.65 and 1.62. The quotient of large/small colonies of the positive control was found to be 0.53. The quotients of the highest dose groups were found to be 1.54

(1.2 mM), 0.98 (1.4 mM) and 1.56 (1.6 mM). Without metabolic activation, the quotients of large/small colonies of the negative controls were found to be 2.47 and 1.64. The quotient of large/small colonies of the positive control was found to be 0.40. The quotients of the highest dose groups were found to be 1.68 (0.8 mM), 1.74 (1.0 mM) and 1.56 (1.2 mM).

All dose groups were considered as not clastogenic. In experiment II with metabolic activation, the quotients of large/small colonies of the negative controls were found to be 1.45 and 1.66, the quotient of large/small colonies of the positive control was found to be

0.61. The quotients of the highest dose groups were found to be 1.23 (1.3 mM), 1.13 (1.5 mM) and 1.36 (1.9 mM). Without metabolic activation, the quotients of large/small colonies of the negative controls were found to be 2.00 and 0.91, the quotient of large/small colonies of the positive control was found to be 0.30. The quotients of the highest dose groups were found to be 1.61 (0.2 mM), 0.57 (0.25 mM) and 0.65 (0.3 mM). All dose groups were considered as not clastogenic.

The positive controls MMS (10 µg/mL) and B{a]P (3.5 µg/mL) induced a significant increase of the mutant frequency and a biologically significant increase of small colonies, thus proving the ability of the test system to indicate potential clastogenic effects.

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under experimental conditions reported , a suspension of nickel fluoride tetrahydrate (N111) is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Executive summary:

The test item Nickel difluoride tetrahydrate (N111) was assessed for its potential to induce mutations at mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations was based on data from the pre-experiment. In experiment 1 1.6 mM (with metabolic activation) and 1.2 mM (without matabiloc activation) were selected as the highest concentrations. In experiment 2 1.9 mM (with metabolic activation) and 0.3mM (without metabolic activation) were selected as the highest concentration. Experiment 2 without metabolic activation was performed as a 24h long-term exposure assay.

The test item was investigated at the following concentration:

Experiment 1:

With metabolic activation: 0.2 ; 0.4 ;0.6 ; 0.8 ; 1.0 ; 1.2 ; 1.4 ; 1.6 mM

and without metabolic activation: 0.08 ;0.1 ;0.2 ; 0.4 ; 0.6 ;1.0 ;1.2 mM

Experiment 2:

with metabolic activation: 0.3 ; 0.5 ; 0.7 ; 0.9 ; 1.1 ; 1.3 ; 1.5 ; 1.9 mM

and without metabolic activation: 0.02 ; 0.04 ; 0.07 ; 0.1 ; 0.15 ; 0.2 ; 0.25 ; 0.3 mM

Growth inhibition was observed in experiment 1 and 2 with and without metabolic activation.

In experiment 1 with metabolic activation the relative total growth (RTG) was 12.39% for the highest concentration (1.6mM) evaluated. The highest concentration evaluated without metabolic activation was 1.2 mM wita a RTG of 11.53%. In experiment 2 with metabolic activation the relative total growth (RTG) was 12.08% for the highest concentration (1.9mM) evaluated. The highest concentration evaluated without metabolic activation was 0.3 mM with a RTG of 19.55%.

In experiment 2 a biologically relevant increase of mutant was found after treatment with the test item(without metabolic activation). A dose-response relationship was observed.

In experiment 1 and 2 colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

EMS , MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally , MMS and B[a]P significantly increased the number of small colonies, thus proving the effeiciency of the test system to indicate potential clastogenic effects.