Nickel difluoride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data on Nickel Sulphate is used in the endpoint coverage for the in vitro gene mutation study in bacteria and in vitro cytogenicity study in mammalian cells. A reliable study conducted with Nickel Difluoride is available to cover the endpoint on in vitro gene mutation study in mammalian cells, although this endpoint was not mandatory to cover given the positive results obtained in other in vitro studies.   

 

In vitro gene mutation study in bacteria: In an Ames test conducted in tester strains S. typhimurium TA98, TA100, TA1535, TA1537, and TA1538 and E. coli strain (DG1153)(Arlauskas et al., 1985, K2), nickel sulphate was found not to be mutagenic. 

In vitro cytogenicity study in mammalian cells: Nickel Sulphate was positive for chromosomal aberrations in Syrian hamster embryo cells when tested at concentrations up to 5 µg/mL (Larramendy et al., 1981, K2). 

In vitro gene mutation study in mammalian cells: A suspension of nickel fluoride tetrahydrate (N111) is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y (Kraft, 2008, K1, OECD 476).

 

 

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

Qualifier:

according to guideline

Guideline:

other: Ames Test

Principles of method if other than guideline:

According to Ames et al. 1975

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Reverse mutation

Species / strain / cell type:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and E.Coli strain (DG1153)

Details on mammalian cell type (if applicable):

S. typhimurium strains were obtained from Prof. B.N. Ames, University of California (Berkeley).  

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations with justification for top dose:

not reported

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Double distilled water
- Justification for choice of solvent/vehicle: not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: The plate incorporation assay was used, plastic petri dishes were filled with 25 ml autoclave
sterilised minimal glucose agar medium.

DURATION
- Preincubation period: not reported
- Exposure duration: Plates were incubated at 37 deg. C for 3-5 days.    
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not reported

SELECTION AGENT (mutation assays): not reported

NUMBER OF REPLICATIONS: The assay was repeated at least once.

NUMBER OF CELLS EVALUATED: not reported

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other: Plates were microscopically examined and the numbers of revertant colonies were counted.

Evaluation criteria:

The criteria for positive results were based on Ames, et al. (1975) and de Serres and Shelby (1979).  
Criteria included (1) a reproducible, dose-related increase in the number of revertant colonies and 
(2) a  doubling of colony numbers on test plates compared with background control plates.

Statistics:

Chi square test

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and E.Coli

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not reported

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Nickel sulfate was found not to be mutagenic.

TEST-SPECIFIC CONFOUNDING FACTORS: No data
RANGE-FINDING/SCREENING STUDIES: No data
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: all strains/cell types tested

Conclusions:

Nickel sulphate was found not to be mutagenic in the reverse mutation assay using S. typhimurium strains.

Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER.

ROBUST SUMMARY DEVELOPED BY AN INDEPENDENT REVIEWER.

Robust summary for Arlauskas et al. (1985):

S. typhimurium strains were obtained from Prof. B.N. Ames, University of California (Berkeley).  The plate incorporation assay was used.  Plates  

were incubated at 37 deg. C for 3-5 days.  Plates were microscopically examined and the numbers of revertant colonies were counted.  The assay  

was repeated at least once.

The criteria for positive results were based on Ames, et al. (1975) and de Serres and Shelby (1979).  Criteria included (1) a reproducible,  

dose-related increase in the number of revertant colonies and (2) a doubling of colony numbers on test plates compared with background control plates.

Nickel sulphate was found not to be mutagenic in the reverse mutation assay using S. typhimurium strains.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Meets generally accepted scientific standards with acceptable restrictions Study lacks information on replication. No positive control group included. No information on a vehicle (acetone) control. Only a single dose level used for chromosome aberration tests; no dose-response can be determined. No statistical evaluation of the data. Non-standard cell line used (embryo cells).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

No standard guideline followed. Sister chromatid exchange assay and Chromosomal aberration test.

GLP compliance:

not specified

Type of assay:

other: Sister chromatid exchange assay and Chromosomal aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Syrian hamster embryo cells and human lymphocyte cultures

Details on mammalian cell type (if applicable):

- Type and identity of media: Hamster embryo cells were collected after trypsinization of the embryos minus the liver and plated (density of 10e7 cells/100 mm dish) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. 
Other details not reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 1.0, 2.5, 5.0 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Sister chromatid exchange (SCE) and chromosomal aberration tests were conducted with Syrian hamster embryo cells and human lymphocyte 
cultures.   Hamster embryo cells were collected after trypsinization of the embryos minus the liver and plated (density of 10e7 cells/100 mm dish) 
in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.  

DURATION
Hamster embryo cells were incubated at 37 deg. C in an 11% CO2 air incubator.  24 hours later, monolayer cultures (10e6 cells/100 mm dish) were treated with nickel sulfate (prepared in acetone) and 10 ug BrdUrd/ml medium and incubated for 24 hours.  Four hours prior to  harvest, cells were treated with Colcemide (0.13 ug/ml), then collected, centrifuged, suspended in KCl, and fixed in methanol:glacial acetic acid.   

For SCE tests, slides were prepared and stained with 4% Giemsa in Gurr's buffer solution.  A minimum of 30 metaphases were scored.  For 
chromosome aberration tests, preparations were air dried and stained with Gurr's buffer solution and 5% Giemsa.  At least 125 metaphases were 
examined.  Aberrations were scored per the International system for human cytogeneic nomenclature (1978).

For chromosome aberration tests, preparations were air dried and stained with Gurr's buffer solution and 5% Giemsa.  At least 125 metaphases 
were examined. 

Evaluation criteria:

Aberrations were scored per the International system for human cytogeneic nomenclature (1978).

Statistics:

Not reported

Species / strain:

other: Syrian hamster embryo cells and human lymphocyte cultures

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

NiSO4 increased the frequency of SCEs in hamster embryo cells in a dose dependent manner:

Hamster embryo cells [frequency of SCEs/metaphase (standard error)]:
BrdUrd control: 11.55 (0.84)
1.0 ug/ml: 15.95 (0.92)
2.5 ug/ml: 17.25 (1.44)
5.0 ug/ml: 21.25 (1.13)

NiSO4 also increased the number of chromosomal aberrations relative to the control:

Hamster embryo cells [percent cells with aberrations and mean aberrations/metaphase (standard error)]:
Control: 1.50%, 0.01 (0.01)
5.0 ug/ml: 16.50%, 0.16 (0.03) (Chromosomal aberrations noted in embryo cells included gaps, breaks, exchanges, and minutes)

Remarks on result:

other: all strains/cell types tested

Conclusions:

NiSO4 was positive for chromosomal aberrations in Syrian hamster embryo cells.

Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER.

ROBUST SUMMARY DEVELOPED BY AN INDEPENDENT REVIEWER.

Robust Summary for Larramendy et al.(1981):

Sister chromatid exchange (SCE) and chromosomal aberration tests were conducted with Syrian hamster embryo cells and human lymphocyte cultures.   

Hamster embryo cells were collected after trypsinization of the embryos minus the liver and plated (density of 10e7 cells/100 mm dish) in   

Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.  Hamster embryo cells were incubated at 37 deg. C in an 11% CO2  

air incubator.  24 hours later, monolayer cultures (10e6 cells/100 mm dish) were treated with nickel sulfate (prepared in acetone) and 10 ug  

BrdUrd/ml medium and incubated for 24 hours.  Four hours prior to harvest, cells were treated with Colcemide (0.13 ug/ml), then collected,  

centrifuged, suspended in KCl, and fixed in methanol:glacial acetic acid.  For SCE tests, slides were prepared and stained with 4% Giemsa in Gurr's  

buffer solution.  A minimum of 30 metaphases were scored.  For chromosome aberration tests, preparations were air dried and stained with Gurr's  

buffer solution and 5% Giemsa.  At least 125 metaphases were examined.  Aberrations were scored per the International system for human cytogeneic 

nomenclature (1978).

Human blood cultures were collected from healthy donors and diluted with Hank's balanced salt solution (without Ca and Mg).  0.5 ml of heparinized  

blood was mixed with 9.5 ml of RPMI 1640 medium supplemented with 20% fetal bovine serum and 0.25 ml phytohemagglutinin M.  Lymphocytes were  

collected by centrifugation (400 rpm for 40 minutes), resuspended in complete medium, and then washed three times with Hank's balanced salt  

solution.  Lymphocytes were cultured in flasks at a density of 1.5 x 10e6 cells in 5 ml RPMI 1640 medium supplemented with 20% fetal bovine serum  

and 0.1 ml phytohemagglutinin.  24 hours later, lymphocytes were treated with nickel sulfate (prepared in acetone) and 30 ug BrdUrd/ml medium and  

incubated for 48 hours.  Two hours prior to harvest, cells were treated with Colcemide (0.13 ug/ml), then collected, centrifuged, suspended in  

KCl, and fixed in methanol:glacial acetic acid.  For SCE tests, slides were prepared and stained with 4% Giemsa in Gurr's buffer solution.  A  

minimum of 30 metaphases were scored.  For chromosome aberration tests, preparations were air dried and stained with Gurr's buffer solution and  

5% Giemsa.  At least 125 metaphases were examined.  Aberrations were scored per the International system for human cytogeneic nomenclature (1978).

NiSO4 increased the frequency of SCEs in human lymphocytes and hamster embryo cells in a dose dependent manner:

Human lymphocytes [frequency of SCEs/metaphase (standard error)]:
BrdUrd control: 11.30 (0.60)
2.5 ug/ml: 17.20 (0.90)
5.0 ug/ml: 18.95 (1.52)

Hamster embryo cells [frequency of SCEs/metaphase (standard error)]:
BrdUrd control: 11.55 (0.84)
1.0 ug/ml: 15.95 (0.92)
2.5 ug/ml: 17.25 (1.44)
5.0 ug/ml: 21.25 (1.13)

NiSO4 also increased the number of chromosomal aberrations relative to the control:

Human lymphocytes [percent cells with aberrations and mean aberrations/metaphase (standard error)]:
Control: 1.50%, 0.01 (0.01)
5.0 ug/ml: 11.20%, 0.07 (0.02)
(Chromosomal aberrations noted in human lymphocytes included gaps, breaks, rings, and minutes)

Hamster embryo cells [percent cells with aberrations and mean aberrations/metaphase (standard error)]:
Control: 1.50%, 0.01 (0.01)
5.0 ug/ml: 16.50%, 0.16 (0.03)
(Chromosomal aberrations noted in embryo cells included gaps, breaks, exchanges, and minutes)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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

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:

Thymidine Kinase Locus/TK +/-

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

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolica activation

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

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

EVALUATION OF RESULTs:

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 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.

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 performed 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 experiments 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 10^6 cells). 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/10^6 cells and in the range of 55.70 to 128.73 mutants/10^6 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 10^6 cells). 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/10^6 cells and in the range of 53.47 to 101.97 mutants/10^6 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 10^6 cells). 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/10^6 cells and in the range of 62.98 to 134.08 mutants/10^6 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 10^6 cells). 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/10^6 cells and in the range of 38.62 to 205.52 mutants/10^6 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 of 2 (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.

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 metabolic 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 experiments 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 efficiency of the test system to indicate potential clastogenic effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Nickel Sulphate Hexahydrate was evaluated as negative in the rat bone marrow micronucleaus assay (Oller and Erexson, 2007, K2, OECD 474). 

A number of supporting and less reliable studies are described in section Additional information to provide further data on the genetic toxicity of Nickel Fluoride. 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC, USA.
- Age at study initiation: 8 weeks
- Weight at study initiation: 239-267 g
- Fasting period before study: not reported
- Housing: 2 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64 - 79 deg F
- Humidity (%): humidity 30-70%
- Air changes (per hr): 10 per hr
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle.

IN-LIFE DATES: From: April 21, 2003 To: Aug. 2003

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Cell culture control water
- Lot No: 017156 and 01100526

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: each day the test article was prepared by adding to the vehicle. Formulations held at room temperature.
Animals were dosed by oral gavage once daily for three consecutive days to six males per dose level. The dose levels were 125, 250, or 500 mg/kg/day.

DIET PREPARATION
- not applicable

Duration of treatment / exposure:

3 days

Frequency of treatment:

Once daily

Post exposure period:

24 hours after 3rd dose

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide was used as the positive control administered by oral gavage dissolved in deionized water at a dose of 60 mg/kg

Tissues and cell types examined:

Animals were euthanized approximately 24 hours after the third dose for extraction of bone marrow.
Blood was also collected prior to euthanization.
Nickel in bone marrow and blood was analyzed by AAS.

Details of tissue and slide preparation:

Following centrifugation to pellet the marrow, the supernatant was spread on slided fixed with methanol and stained in acridine orange, dried, and analyzed under fluorescent microscopy.

Evaluation criteria:

Slides were scored for micronuclei and to determine the PCE to NCE cell ratio. The percent micronucleated cells was determined by analyzing micronuclei from at least 2000 PCEs per animal. The criteria were those of Schmid 1976.

Statistics:

Data analysis was conducted using ANOVA

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 125-1750 mg/kg-day
- No cytotoxicity observed at 750 or 1000 mg/kg-day. Mean PCE:NCE ratios were 0.32 and 0.64 compared to 0.81 (control)
- Other: the maximum tolerated dose was estimated at 500 mg/kg-day

RESULTS OF DEFINITIVE STUDY
Clinical signs of toxicity were noted in all treatment animals including hypoactivity, salivation, black feces, irregular respiration, squinted/closed eyes.

No mortality occurred.

Nickel did not significantly increase micronucleated PCEs at any dose level. Nickel was not significantly cytotoxic to bone marrow at any dose level.
Dose-dependent nickel concentrations were detected in plasma and bone marrow samples.

The author's suggest the results support the non-genotoxic mode of action for soluble nickel.

Conclusions:

The test article, nickel sulfate hexahydrate, was evaluated as negative in the rat bone marrow micronucleaus assay under the conditions of this assay.

Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Genetic toxicity - in vitro:

Nickel difluoride tetrahydrate was assessed for its potential to induce mutations at mouse lymphoma thymidine kinase locus using the cell line L5178Y (Kraft, 2008, K1).

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 0.3mM (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
- 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
- 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 experiments 1 and 2 with and without metabolic activation.
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 experiments 1 and 2, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
Nickel Fluoride is concluded to be mutagenic in the in vitro gene mutation study in mammalian cells.

 

No further data on Nickel Difluoride is available on the other genetic toxicity endpoints; therefore data from the Nickel Sulphate dossier were used to cover these endpoints.

The in vitro gene mutation study in bacteria with nickel sulphate is negative (Arlauskas et al. 1985, K2). Bacterial reverse mutation study with S. typhimurium is negative for all the strains tested at all doses of nickel sulphate using a modified media that prevented precipitation of the nickel ions. In vitro cytogenicity study (chromosomal aberrations) in mammalian cells or in vitro micronucleus study with nickel sulphate have been positive. 

Genetic toxicity - in vivo:
The in vivo studies with nickel sulphate have produced mixed results. Two studies (a K1 and a K2) looking at micronucleus in bone marrow of rats (oral) and mice (intraperitoneal) exposed repeatedly to nickel sulphate were negative (Oller and Erexson, 2007; Morita et al., 1997); two K3 studies looking at the oral induction of micronucleus in mice indicated positive results (Sharma et al., 1987; Sobti and Gill, 1989). A study by Benson et al. (2002) showed that nickel sulphate given by inhalation seemed to induce genotoxicity (DNA damage) in lung cells at the same or higher concentrations at which it induces inflammation after repeated exposures. Evidence from human studies is limited. There are no definitive tests of nickel compounds on the germ cells but evidence for a possible effect is limited. Whilst there is evidence that the nickel ion reaches the testes, no effect on spermatogonial cells was seen in the Mathur et al. (1978) study with nickel sulphate. The effects seen in spermatozoa in the Sobti & Gill (1989) study with several water-soluble Ni compounds may reflect toxic effects on germ cells rather than chromosomal damage. In addition, a dominant lethal test (Deknudt & Léonard, 1982) with water soluble Ni compounds, was negative and these results are relevant for nickel sulphate. Whilst some effects are seen in males (e.g. sperm abnormalities) there is little evidence for inheritable effects on the germ cells. Taken together, the in vitro and in vivo studies indicate that nickel sulphate is a weak genotoxicants with thresholds.

In April 2004, the Specialised Experts concluded that nickel sulphate, nickel chloride and nickel nitrate should be classified as Muta. Cat. 3; R68 (now Muta. 2: H341 under CLP classification). This conclusion was based on evidence of in vivo genotoxicity in somatic cells, after systemic exposure (the 2007 negative oral MN study was not available at that time). Hence the possibility that the germ cells are affected could not be excluded (European Commission, 2004).

The following information is taken into account for any hazard / risk assessment:

For water soluble nickel compounds like nickel fluoride and nickel sulphate, there is evidence indicating that they are weak genotoxicants in vitro and may exhibit clastogenic activity. Some in vivo studies with nickel sulphate have been positive while two recent micronucleus studies via oral and intraperitoneal injection were negative. Evidence from human studies is limited. There are no definitive studies on germ cells, and little evidence concerning hereditable effects. Nickel fluoride and other water-soluble nickel compounds carry a harmonized Muta. 2: H341 CLP classification. Recently, nickel compounds have been recognized as genotoxic carcinogens with threshold mode of action in ECHA RAC opinion on nickel and nickel compounds OELs (see discussion of ECHA 2018 report in Appendix C2 of the NiSO4 CSR).

K1 and K2 studies included in the current version of the nickel sulphate dossier were reviewed and included. K3 and K4 studies from the NiSO4 dossier were not included in the NiF2 dossier if new studies were included in the last update of the NiSO4 dossier.

Justification for classification or non-classification

Nickel difluoride is classified as Muta. 2:H341 in the 1st ATP to the CLP Regulation.