Nickel difluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

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).

Cross-reference

Reason / purpose:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Induction by inorganic metal salts of sister chromatid exchange and aberrations in human and Syrian hamster cell strains.

Author:

Larramendy, M.L., N.C. Popescu, and J.A. DiPaolo.

Year:

1981

Bibliographic source:

Environmental Mutagenesis. 2:597-606.

Materials and methods

Test guideline

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

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Nickel Sulfate Hexahydrate, 10101-97-0
- Molecular formula (if other than submission substance): not different than submission substance
- Molecular weight (if other than submission substance): not different than submission substance
- Smiles notation (if other than submission substance): not different than submission substance
- InChl (if other than submission substance): not different than submission substance
- Structural formula attached as image file (if other than submission substance): not different than submission substance
- Purity: 97%
- Other details not reported or not applicable

Method

Target gene:

Not applicable

Species / strain

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

Controls

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

Results and discussion

Test results

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

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

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)