Nickel(2+) diammonium 2,2',2'',2'''-(ethane-1,2-dinitrilo)tetraacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

1983

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:

other: bacterial reverse mutation assay

Specific details on test material used for the study:

Stock solutions of chemicals were prepared at 0.1 or 0.01 M concentrations in double glass-distilled water, sterilized by membrane filtration, and held in sealed glass bottles at room temperature. Aliquots of these stock solutions were used for mutagenicity assays at nontoxic concentrations. At the time of assay, serial half-log dilulions of each test chemical were prepared in distilled water.

Target gene:

Reverse mutation

Species / strain / cell type:

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

Remarks:

Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538 wete obtained from Professor B. N. Ames and The E. coli strain (DG1153) was obtained from Dr. D. G. MacPhee (La Trobe University)

Vehicle / solvent:

Double distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

PLATE INCORPORATION ASSAY: Plastic petri dishes were filled with 25 ml autoclave sterilised minimal glucose agar medium. lnto each 2 ml of top agar was added 100 uL of the appropriate Salmonella strain from an ovemight broth culture, followed by 100 uL of test chemical, prepared in serial half·log worklng dilutions. The top agar was mixed and poured over the surface of a minimal agar plate. After the agar had set, plates were inverted and incubated at 37'C for 3-5 days. Unless stated otherwise, assay results ate those from plates incubated for 3 days. Colony numbers were determined manually and plates were examined microscopically for evidence of lawn toxicity and for colony appearance. Each assay was repeated at least once.
FLUCTUATION ASSAY: The microtiter fluctuation test was petformed according to the rnethod of Gatehouse (1978). Test chemical dilutions were added to 20-ml aroounts of prepared medium, to give the required final dilutlon. Then 1 ml of a 1/10 dilution of 3- to 4-hr broth cultures of bacteria was added in each 20-ml test or control bottle, which was mixed and dispensed in 0.2-ml amounts into the 96 wells of Linbro/Titertek trays (Flow Laboratoties). Trays with Iids were tightly wrapped in plastlc to minimize evaporation and incubated at 37°C for 3 days. To determine bacterial growth, 20 uL of bromothymol blue (600 ug/ml) was added to each well: negative wells appeared blue-green and positive wells appeared ye!low. Where metals affected the color of the indicator dye, wells were assessed for turbidity, based on absorbance measurements in a MicroELISA Auto Reader (Dynatech Model MR580) at 630 nm.

Evaluation criteria:

The criteria for positive results were based on Ames, et al. (1975) and de Serres and Shelby (1979) and 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:

x2 test

Key result

Species / strain:

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

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.

Remarks on result:

other: all strains tested

Conclusions:

Nickel sulphate was found not to be mutagenic in the reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and E.Coli strain (DG1153).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

Justification for type of information:

The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be not genetic toxic only the EDTA-Ni complex must be regarded as toxic. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their toxicity. EDTA and its sodium salts were found to have a low mutagenic potential at extremely high doses and, on the basis of the various negative findings, it can be concluded that EDTA and its sodium salts are not mutagenic for humans (source: EU SUMMARY RISK ASSESSMENT REPORT prepared in 2004 by BAuA in Germany). In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-(NH4)Na2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.

Reason / purpose for cross-reference:

read-across source

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:

other: bacterial reverse mutation assay

Specific details on test material used for the study:

Stock solutions of chemicals were prepared at 0.1 or 0.01 M concentrations in double glass-distilled water, sterilized by membrane filtration, and held in sealed glass bottles at room temperature. Aliquots of these stock solutions were used for mutagenicity assays at nontoxic concentrations. At the time of assay, serial half-log dilulions of each test chemical were prepared in distilled water.

Target gene:

Reverse mutation

Species / strain / cell type:

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

Remarks:

Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538 wete obtained from Professor B. N. Ames and The E. coli strain (DG1153) was obtained from Dr. D. G. MacPhee (La Trobe University)

Vehicle / solvent:

Double distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

PLATE INCORPORATION ASSAY: Plastic petri dishes were filled with 25 ml autoclave sterilised minimal glucose agar medium. lnto each 2 ml of top agar was added 100 uL of the appropriate Salmonella strain from an ovemight broth culture, followed by 100 uL of test chemical, prepared in serial half·log worklng dilutions. The top agar was mixed and poured over the surface of a minimal agar plate. After the agar had set, plates were inverted and incubated at 37'C for 3-5 days. Unless stated otherwise, assay results ate those from plates incubated for 3 days. Colony numbers were determined manually and plates were examined microscopically for evidence of lawn toxicity and for colony appearance. Each assay was repeated at least once.
FLUCTUATION ASSAY: The microtiter fluctuation test was petformed according to the rnethod of Gatehouse (1978). Test chemical dilutions were added to 20-ml aroounts of prepared medium, to give the required final dilutlon. Then 1 ml of a 1/10 dilution of 3- to 4-hr broth cultures of bacteria was added in each 20-ml test or control bottle, which was mixed and dispensed in 0.2-ml amounts into the 96 wells of Linbro/Titertek trays (Flow Laboratoties). Trays with Iids were tightly wrapped in plastlc to minimize evaporation and incubated at 37°C for 3 days. To determine bacterial growth, 20 uL of bromothymol blue (600 ug/ml) was added to each well: negative wells appeared blue-green and positive wells appeared ye!low. Where metals affected the color of the indicator dye, wells were assessed for turbidity, based on absorbance measurements in a MicroELISA Auto Reader (Dynatech Model MR580) at 630 nm.

Evaluation criteria:

The criteria for positive results were based on Ames, et al. (1975) and de Serres and Shelby (1979) and 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:

x2 test

Key result

Species / strain:

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

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.

Remarks on result:

other: all strains tested

Conclusions:

Nickel sulphate was found not to be mutagenic in the reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and E.Coli strain (DG1153).

Reference 3

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:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No information on replication. Purity of test substance not reported.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Modification of the method of Nakamura et al. (1983)

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay using modification of the method of Nakamura et al. (1983)

Specific details on test material used for the study:

Purchased from Wako Pure Chemical Industries ( Osaka)

Species / strain / cell type:

other: Mouse mammary carcinoma cells (FM3A)

Details on mammalian cell type (if applicable):

FM3A cells, a C3H mouse mammary carcinoma cell line, were grown at 37 °C in suspension in Eagle's minimum essential medium (MEM) containing 3% fetal bovine serum (FBS) and 1% non-essential amino acids ( Gibco, NY) in a humidified atmosphere containing 5% C02.For the mutation assay, the cells were recloned, exposed to HAT medium (hypoxanthine, 1 X 10e-4 M; aminopterin, 4 X 10e-7 M; thymidine, 1.6 x 10e-5 M) for 1 week, and then cultured in normal medium supplemented with hypoxanthine (1 X 10e-4 M) and thymidine (1.6 X 10e- 5 M) to recover from the HAT treatment.

Metabolic activation:

with and without

Metabolic activation system:

cells were exposed to a test compound for 5 h in mixture consisting of 2.5% S15 (15,000 x g microsome fraction prepared from the livers of PCB-treated Sprague Dawley rats), 6 mM MgCI2,1 mM NADPH,5 mM glucose-6-phosphate,and 20 mM HepesHanks solution

Test concentrations with justification for top dose:

0, 1.0 x 10e-4, 2.0 x 10e-4, 3.0 x 10e-4, and 4.0 x 10e-4 M

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

FM3A cells were treated with various concentrations of lest compounds for 3-48 h. After the cells had been washed with Hanks' balanced salt solution, they were cultured for 7 days for the expression of mutation. During this time, the cells were subcultured twice, and the total number of cells was maintained above 1 x 10^6 cells).Then, 2 X 10^6 cells were inoculated into 40 ml of selective agar medium containing 10 ug/ml 6TG. After 14 days of incubation, the number of 6TG colanies was counted and the mutation frequency per 106 surviving cells was calculated.

Statistics:

Statistical evaluations were done with the Welch test.

Key result

Species / strain:

other: Mouse mammary carcinoma cells (FM3A)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Additional information on results:

FM3A cells were treated for 48 h with the test item. All 5 compounds tested showed dose-dependent cytotoxicity at concentrations of over 1 X 10e-4 M Ni(CH 3C00)2. In the presence of a 4 X 10-4 M concentration of the above compounds, the average numbers of 6TG colanies decreased. Concentration of 3.0 x 10e-4 M of the test item showed significant increases in mutant colonies. At 0 M treatment, survival was 71+/-12%, with 100% relative plating efficiencies; at 4 M treatment, survival was 13+/-1%.The number of mutants per 10e6 surviving cells increased in with Ni exposure.

Conclusions:

In the test conditions described above, the test item Nickel di(acetate) showed weak mutagenicity in mouse mammary carcinoma cells (FM3A).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No information on replication. Purity of test substance not reported.

Justification for type of information:

The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be not genetic toxic only the EDTA-Ni complex must be regarded as toxic. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their toxicity. EDTA and its sodium salts were found to have a low mutagenic potential at extremely high doses and, on the basis of the various negative findings, it can be concluded that EDTA and its sodium salts are not mutagenic for humans (source: EU SUMMARY RISK ASSESSMENT REPORT prepared in 2004 by BAuA in Germany). In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-(NH4)Na2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Modification of the method of Nakamura et al. (1983)

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay using modification of the method of Nakamura et al. (1983)

Specific details on test material used for the study:

Purchased from Wako Pure Chemical Industries ( Osaka)

Species / strain / cell type:

other: Mouse mammary carcinoma cells (FM3A)

Details on mammalian cell type (if applicable):

FM3A cells, a C3H mouse mammary carcinoma cell line, were grown at 37 °C in suspension in Eagle's minimum essential medium (MEM) containing 3% fetal bovine serum (FBS) and 1% non-essential amino acids ( Gibco, NY) in a humidified atmosphere containing 5% C02.For the mutation assay, the cells were recloned, exposed to HAT medium (hypoxanthine, 1 X 10e-4 M; aminopterin, 4 X 10e-7 M; thymidine, 1.6 x 10e-5 M) for 1 week, and then cultured in normal medium supplemented with hypoxanthine (1 X 10e-4 M) and thymidine (1.6 X 10e- 5 M) to recover from the HAT treatment.

Metabolic activation:

with and without

Metabolic activation system:

cells were exposed to a test compound for 5 h in mixture consisting of 2.5% S15 (15,000 x g microsome fraction prepared from the livers of PCB-treated Sprague Dawley rats), 6 mM MgCI2,1 mM NADPH,5 mM glucose-6-phosphate,and 20 mM HepesHanks solution

Test concentrations with justification for top dose:

0, 1.0 x 10e-4, 2.0 x 10e-4, 3.0 x 10e-4, and 4.0 x 10e-4 M

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

FM3A cells were treated with various concentrations of lest compounds for 3-48 h. After the cells had been washed with Hanks' balanced salt solution, they were cultured for 7 days for the expression of mutation. During this time, the cells were subcultured twice, and the total number of cells was maintained above 1 x 10^6 cells).Then, 2 X 10^6 cells were inoculated into 40 ml of selective agar medium containing 10 ug/ml 6TG. After 14 days of incubation, the number of 6TG colanies was counted and the mutation frequency per 106 surviving cells was calculated.

Statistics:

Statistical evaluations were done with the Welch test.

Key result

Species / strain:

other: Mouse mammary carcinoma cells (FM3A)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Additional information on results:

FM3A cells were treated for 48 h with the test item. All 5 compounds tested showed dose-dependent cytotoxicity at concentrations of over 1 X 10e-4 M Ni(CH 3C00)2. In the presence of a 4 X 10-4 M concentration of the above compounds, the average numbers of 6TG colanies decreased. Concentration of 3.0 x 10e-4 M of the test item showed significant increases in mutant colonies. At 0 M treatment, survival was 71+/-12%, with 100% relative plating efficiencies; at 4 M treatment, survival was 13+/-1%.The number of mutants per 10e6 surviving cells increased in with Ni exposure.

Conclusions:

In the test conditions described above, the test item Nickel di(acetate) showed weak mutagenicity in mouse mammary carcinoma cells (FM3A).

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Detailed description of materials and methods.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was undertaken to examine whether Nickel chloride was internalized by Salmonella tester strains by measuring the association of the metal ion with cells, and to assess the mutagenesis activity of Nickel in bacteria. Mutation studies utilized TA102, TA1535, TA1975, TA1538, TA1978. These 4 strains were chosen to determine whether DNA-repair capability had any differential effect on survival or mutagenesis induced by Nickel Chloride.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purchased from Sigma Chemical Co. (St. Louis, MO).

Species / strain / cell type:

other: S. typhimurium TA102, TA1535, 1538, 1975,1978

Details on mammalian cell type (if applicable):

Cell lines were purchased from B. Ames, University of Califomla, Berkeley and maintained and tested as recommended (Maron and Ames, 1983)

Metabolic activation:

without

Test concentrations with justification for top dose:

NiCl2 conc: 0, 1.0, 5.0, 10 mM

Positive controls:

yes

Positive control substance:

other: Na2CrO4

Remarks:

Conc.: 0, 0.1, 0.5, 2.5 mM

Details on test system and experimental conditions:

A!iquots of a log phase culture of TA102 were preincubated with varying concentrations of NiCI2 or Na2CrO4 for 1 h pr!or to plating.
For TA1535/TA1975 and TA1538/ TA1978, 2 ml of eilher minimal medium supplemented with 1.0 mM histidine or nutrient broth were inoculated with 100 ul of late log-phase cultures and NiCl2 was added at the concentrations ind!cated. The cultures were incubated ca. 16 h at 37'C with shaking. The degree of growth was evaluated by absorbance at 550 nm.
To examine the uptake of nickel and correlate this with mutagenicity, a 16-h culture of each bacterial strain was diluted 1: 10 into nutrient broth and incubated for 2 h at 37°C with shaking. 2-ml aliquots were removed and preincubated with varying concentrations of NiC2 for 2 h at 37"C with shaking.

Key result

Species / strain:

other: S. typhimurium TA102, TA1535, 1538, 1975,1978

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

survival 38-20% (for TA102)

Positive controls validity:

valid

Treatment of all the strains with NiCl2 did not induce an increase in revertant colonies compared with untreated cells.

The growth in the 4 strains TA1535/TA1975 and TA1538/ TA1978 was markedly inhibited by NiCi2 in nutrient broth, but there was very little inhibition by NiCl2 in minimal medium supplemented with histidine.

For all strains tested, there was a substantial increase in intracellular Ni associated wlth increasing dose and decreasing survival when cells were treated with NiC2 in nutrient broth media.

Nickel entered bacterial cells when cultured in nutrient broth but not when cultured in minimal medium supplemented with histidine.

Conclusions:

negative without metabolic activation.

NiCl2 does not induce detectable base-pair substitution mutations or frameshift mutations in repair-proficient or repair-deficient strains of Salmonella at nontoxic and cytotoxic concentrations as determlned by the Iack of an increased number of revertant colonies in the treated samples compared to the untreated samples.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Detailed description of materials and methods.

Justification for type of information:

The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be not genetic toxic only the EDTA-Ni complex must be regarded as toxic. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their toxicity. EDTA and its sodium salts were found to have a low mutagenic potential at extremely high doses and, on the basis of the various negative findings, it can be concluded that EDTA and its sodium salts are not mutagenic for humans (source: EU SUMMARY RISK ASSESSMENT REPORT prepared in 2004 by BAuA in Germany). In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-(NH4)Na2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was undertaken to examine whether Nickel chloride was internalized by Salmonella tester strains by measuring the association of the metal ion with cells, and to assess the mutagenesis activity of Nickel in bacteria. Mutation studies utilized TA102, TA1535, TA1975, TA1538, TA1978. These 4 strains were chosen to determine whether DNA-repair capability had any differential effect on survival or mutagenesis induced by Nickel Chloride.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purchased from Sigma Chemical Co. (St. Louis, MO).

Species / strain / cell type:

other: S. typhimurium TA102, TA1535, 1538, 1975,1978

Details on mammalian cell type (if applicable):

Cell lines were purchased from B. Ames, University of Califomla, Berkeley and maintained and tested as recommended (Maron and Ames, 1983)

Metabolic activation:

without

Test concentrations with justification for top dose:

NiCl2 conc: 0, 1.0, 5.0, 10 mM

Positive controls:

yes

Positive control substance:

other: Na2CrO4

Remarks:

Conc.: 0, 0.1, 0.5, 2.5 mM

Details on test system and experimental conditions:

A!iquots of a log phase culture of TA102 were preincubated with varying concentrations of NiCI2 or Na2CrO4 for 1 h pr!or to plating.
For TA1535/TA1975 and TA1538/ TA1978, 2 ml of eilher minimal medium supplemented with 1.0 mM histidine or nutrient broth were inoculated with 100 ul of late log-phase cultures and NiCl2 was added at the concentrations ind!cated. The cultures were incubated ca. 16 h at 37'C with shaking. The degree of growth was evaluated by absorbance at 550 nm.
To examine the uptake of nickel and correlate this with mutagenicity, a 16-h culture of each bacterial strain was diluted 1: 10 into nutrient broth and incubated for 2 h at 37°C with shaking. 2-ml aliquots were removed and preincubated with varying concentrations of NiC2 for 2 h at 37"C with shaking.

Key result

Species / strain:

other: S. typhimurium TA102, TA1535, 1538, 1975,1978

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

survival 38-20% (for TA102)

Positive controls validity:

valid

Treatment of all the strains with NiCl2 did not induce an increase in revertant colonies compared with untreated cells.

The growth in the 4 strains TA1535/TA1975 and TA1538/ TA1978 was markedly inhibited by NiCi2 in nutrient broth, but there was very little inhibition by NiCl2 in minimal medium supplemented with histidine.

For all strains tested, there was a substantial increase in intracellular Ni associated wlth increasing dose and decreasing survival when cells were treated with NiC2 in nutrient broth media.

Nickel entered bacterial cells when cultured in nutrient broth but not when cultured in minimal medium supplemented with histidine.

Conclusions:

negative without metabolic activation.

NiCl2 does not induce detectable base-pair substitution mutations or frameshift mutations in repair-proficient or repair-deficient strains of Salmonella at nontoxic and cytotoxic concentrations as determlned by the Iack of an increased number of revertant colonies in the treated samples compared to the untreated samples.

Reference 7

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:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

Qualifier:

according to guideline

Guideline:

other: Maron and Ames 1983

Principles of method if other than guideline:

Study performed according to: Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Compound was dissolved and/or diluted either in bidistilled water or in dimethyl sulfoxide (DMSO)

Species / strain / cell type:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97 and E.Coli WP 2, WP67 and CM871

Remarks:

Salmonella strains were provided from Prof. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, CA, U.S.A.). E. coli strains were provided from Prof. C. Monti-Bragadin (Institute of Microbiology, University of Trieste, ltaly).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared according to Ames et al. (1975), contained 10% liver S9 fractions from Aroclor- treated Sprague-Dawley rats, whose protein concentration had been adjusted to 30 mg/mL

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dissolved or diluted in either bidistilled water or dimethyl sulfoxide (DMSO).

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:

The compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates).

Key result

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97 and E.Coli WP 2, WP67 and CM871

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In both the presence and absence of a metabolic activation system (i.e., S9 mix), nickel acetate was found not to be mutagenic under the conditions tested. The potency on the Salmonella strains (revertants/nmole) was < 0.007. For the E.coli strains, the potency (sum of minimal inhibitory concentrations/nmole) was 0.

Remarks on result:

other: all strains/cell types tested

The density of bacteria is not a critical factor in

influencing the results of this test.

Conclusions:

In both the presence and absence of a metabolic activation system (i.e., S9 mix), nickel acetate was found not to be mutagenic under the conditions tested.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

Justification for type of information:

The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be not genetic toxic only the EDTA-Ni complex must be regarded as toxic. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their toxicity. EDTA and its sodium salts were found to have a low mutagenic potential at extremely high doses and, on the basis of the various negative findings, it can be concluded that EDTA and its sodium salts are not mutagenic for humans (source: EU SUMMARY RISK ASSESSMENT REPORT prepared in 2004 by BAuA in Germany). In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-(NH4)Na2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Maron and Ames 1983

Principles of method if other than guideline:

Study performed according to: Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Compound was dissolved and/or diluted either in bidistilled water or in dimethyl sulfoxide (DMSO)

Species / strain / cell type:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97 and E.Coli WP 2, WP67 and CM871

Remarks:

Salmonella strains were provided from Prof. Bruce N. Ames (Department of Biochemistry, University of California, Berkeley, CA, U.S.A.). E. coli strains were provided from Prof. C. Monti-Bragadin (Institute of Microbiology, University of Trieste, ltaly).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared according to Ames et al. (1975), contained 10% liver S9 fractions from Aroclor- treated Sprague-Dawley rats, whose protein concentration had been adjusted to 30 mg/mL

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dissolved or diluted in either bidistilled water or dimethyl sulfoxide (DMSO).

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:

The compound was tested with each strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates).

Key result

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97 and E.Coli WP 2, WP67 and CM871

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In both the presence and absence of a metabolic activation system (i.e., S9 mix), nickel acetate was found not to be mutagenic under the conditions tested. The potency on the Salmonella strains (revertants/nmole) was < 0.007. For the E.coli strains, the potency (sum of minimal inhibitory concentrations/nmole) was 0.

Remarks on result:

other: all strains/cell types tested

The density of bacteria is not a critical factor in

influencing the results of this test.

Conclusions:

In both the presence and absence of a metabolic activation system (i.e., S9 mix), nickel acetate was found not to be mutagenic under the conditions tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In 3 of the 4 studies considered, Ni2+ was found to be non mutagenic. Only in one study Nickel di(acetate) showed weak mutagenicity in mouse mammary carcinoma cells (FM3A) without being sufficient for a classification.