Nickel(2+) propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

read across to nickel acetate (CAS 373-02-4): negative in the S. typhimurium reversion test

read across to nickel acetate (CAS 373-02-4): negative in in vitro gene mutation study in mammalian cells

read across to nickel acetate (CAS 373-02-4): negative in in vitro chromosome aberration study

read across to nickel sulphate hexahydrate (CAS 10101-97-0): positive in in vitro chromosome aberration study

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the read across justification (IUCLID6 section 13).

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mammalian cell line, other: Mouse mammary carcinoma cells (FM3A)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the read-across justification (IUCLID6, section 13).

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97, and E. Coli

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Reference 3

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:

weight of evidence

Justification for type of information:

Please refer to the read across justification (IUCLID6 section 13).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mammalian cell line, 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

Key result

Species / strain:

mammalian cell line, other: C3H mouse mammary carcinoma cells (FM3A)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

other: Maron and Ames 1983

Principles of method if other than guideline:

Study conducted according to Maron and Ames 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research. 113:173-215.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Nickel acetate
- Substance type: Pure product
- Physical state: Liquid
- Analytical purity: Reagent grade

Species / strain / cell type:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97 and E. Coli

Metabolic activation:

with and without

Metabolic activation system:

S9, The S9 mix was prepared from Aroclor-treated Sprague-Dawley rats, according to the procedures of Ames et al (1975).

Test concentrations with justification for top dose:

not reported

Vehicle / solvent:

- Vehicle/solvent 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:

METHOD OF APPLICATION: According to Ames et al. (1975)
DURATION: Details not reported, According to Ames et al. (1975)
SELECTION AGENT: Details not reported, According to Ames et al. (1975)
NUMBER OF REPLICATIONS: The assay was conducted with all strains, both in the presence and absence of S9, in duplicate or triplicate plates.
NUMBER OF CELLS EVALUATED: Details not reported, According to Ames et al. (1975)

DETERMINATION OF CYTOTOXICITY
- Method: Details not reported, According to Ames et al. (1975)
- Various dilutions of the substance were tested, starting at the solubility or cytotoxicity limit.

Evaluation criteria:

According to Ames et al. (1975)

Statistics:

not reported

Key result

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA97, and E. Coli

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In both the presence and absence of the metabolic activation system, nickel acetate was found not to be mutagenic under the conditions tested.

Conclusions:

The test substance nickel acetate was non-mutagenic both with and without metabolic activation in the bacterial reverse mutation assay.

Executive summary:

 

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The publication lacks information on cell and slide preparation, the purity of test material, and source of cells used. Culture conditions are not reported. Criteria for scoring chromosomal aberrations not identified. Gaps were recorded separately. Percentage of cells with chromosomal aberrations not reported. No positive control included. Low number of metaphases scored.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

FM3A cell lines used are not specified in OECD 473.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Reported as Nickel acetate;
-Purchased from Wako Pure Chem. Indust. Ltd.

Species / strain / cell type:

mammalian cell line, other: Mouse mammary carcinoma cells

Cytokinesis block (if used):

colcemid

Metabolic activation:

without

Test concentrations with justification for top dose:

0.20, 0.32, 0.64, and 1 mM

Vehicle / solvent:

Eagle's minimal essential medium with 10 % fetal bovine serum, penicillin (100U/mL) and streptomycin (100 µg/mL)

Details on test system and experimental conditions:

C3H mouse mammary carcinoma cells (FM3A) (1 x 10e5 cells/mL) were exposed to various concentrations of the test substance for 24 or 48 hours. Two hours prior to the end of the treatment period, colcemid was added to the culture (final concentration = 10e-7 M).
Slides were prepared using the flame-drying method. 100 metaphases were scored. Experiments were repeated.

Key result

Species / strain:

mammalian cell line, other: C3H mouse mammary carcinoma cells (FM3A)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

Nickel acetate induced few chromosomal aberrations and showed no dose-response.

Conclusions:

The test substance was negative in the chromosomal aberration study.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

In the publication there are no information about replications and the purity of the test substance given.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Qualifier:

according to guideline

Guideline:

other: Nakamura et al.

Version / remarks:

Year: 1983 (unclear if "Year of test guideline" or "Year of study completion".)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Reported as Ni(CH3COO)2-4H2O
- Source: Wako Pure Chemical Industries

Target gene:

HPRT, XPRT

Species / strain / cell type:

mammalian cell line, other: Mouse mammary carcinoma cells (FM3A)

Metabolic activation:

with and without

Metabolic activation system:

2.5 % S15

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 exposed to hypoxanthine, aminopterin, and thymidine for 1 week prior to use in the mutation assay, then cultured in Eagle's minimum essential medium supplemented with 3% fetal bovine serum, 1% non-essential amino acids, hypoxanthine, and thymidine. Cultured FM3A cells were then exposed to various concentrations of NiCl2 for 3-48 hours, washed in Hank's balanced salt solution, and then cultured for an additional 7 days. Then, 2 x 10e6 cells were inoculated into 40 mL of selective agar medium containing 10 µg/mL 6-thioguanine (6-TG) and cultured for 14 days. The number of 6TG colonies were counted and mutation frequency (per 10e6 surviving cells) was determined.

Statistics:

Statistical analysis was performed using the Welch test.

Key result

Species / strain:

mammalian cell line, 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:

There was a concentration-dependent decrease in percent cell survival following treatment. At 0 M treatment, survival was 76%, with 100% relative plating efficiencies. This decreased to 13% survival with relative plating efficiencies of 18% after treatment at 4.0 x 10e-4 M. The number of mutants per 10e6 surviving cells increased in with Ni exposure, although results were not significant (P<0.05).

Conclusions:

The test substance was not mutagenic in the in vitro mammalian gene mutation test.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

the publication lacks information on replication. No positive control group was included. No information is given on a vehicle (acetone) control. Only a single dose level was used for the chromosome aberration tests; no dose-response can be determined. No statistical evaluation of the data was reported. A non-standard cell line was used (embryo cells).

Qualifier:

no guideline followed

Principles of method if other than guideline:

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

GLP compliance:

not specified

Type of assay:

other: Sister chromatid exchange assay and Chromosomal aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Nickel Sulfate Hexahydrate, 10101-97-0
- Purity: 97 %

Species / strain / cell type:

mammalian cell line, other: Syrian hamster embryo cells and human lymphocyte cultures

Details on mammalian cell type (if applicable):

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.

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 1.0, 2.5, 5.0 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: Acetone

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:

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 °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 µg BrdUrd/mL medium and incubated for 24 hours. Four hours prior to harvest, cells were treated with Colcemide (0.13 µg/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)

Key result

Species / strain:

mammalian cell line, 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 µg/mL: 15.95 (0.92)
2.5 µg/mL: 17.25 (1.44)
5.0 µg/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 µg/mL: 16.50%, 0.16 (0.03) (Chromosomal aberrations noted in embryo cells included gaps, breaks, exchanges, and minutes)

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

read across to nickel sulphate hexahydrate (CAS 10101-97-0): positive in in vivo insect germ cell cytogenetic assay

read across to nickel sulphate hexahydrate (CAS 10101-97-0): negative in in vivo mammalian germ cell study: cytogenicity/chromosome aberration

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the read-across justification (IUCLID6 section 13).

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no cytotoxicity effects on bone marrow even at maximum tolerated dose established based on mortality and clinical observations

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo insect germ cell cytogenetic assay

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to the read across justification (IUCLID6 section 13).

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

nickel sulfate hexahydrate

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: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 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/solvent used: Cell culture control water
- Lot No: 017156 and 01100526

Details on exposure:

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

Duration of treatment / exposure:

3 days

Frequency of treatment:

Once daily

Post exposure period:

24 hours after 3rd dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males

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 bw.

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 slides, 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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no cytotoxicity effects on bone marrow even at maximum tolerated dose established based on mortality and clinical observations

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 bw/day
- No cytotoxicity observed at 750 or 1000 mg/kg bw/day. Mean PCE:NCE ratios were 0.32 and 0.64 compared to 0.81 (control)
The maximum tolerated dose was estimated at 500 mg/kg bw/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 substance nickel sulfate hexahydrate was evaluated as negative in the rat bone marrow micronucleaus assay under the conditions of this assay.

Reference 4

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo insect germ cell cytogenetic assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A standard Test Guideline was not specified in this study.

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

- Name of test material: Nickel sulphate hexahydrate

Species:

Drosophila melanogaster

Strain:

other: For SLRL, white males were treated and crossed to BASC females. For SCL, ring X males of the genotype XC2 y B/sc8Y were treated and crossed to y 2 w a females.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS/ENVIRONMENTAL CONDITIONS
- Stocks maintained in mass cultures at 25 +/- 1 °C.
- Food medium was 9.2 % agar-agar, 15.4 % D-glucose, 21.5 % sugar, 38.5 % corn meal, 9.2 % yeast, 3.1 % propionic acid, 3.1 % nipagin and 1000 mL distilled water.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: 5% sucrose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Adult males 0-48 h old were injected i.p. with NiSO4-6H20. Treated and control males were crossed with BASC virgin females 1-3 days old for two days at a ratio of one male x two females. The progeny of each treated male was kept separately to identify possible clusters of lethals.

Brood males (A,B,C,D) were transferred to fresh virgin females at intervals of 2, 2, 3, and 3 days. F1 females fertilzied by F1 males were intercrossed in vials and the F2 scored for sex-linked recessive lethals. F3 was retested.

SCL Test: 0-2 day old ring X males, treated or untreated, were mass mated in bottles with 1-3 day old virgin females for 2 days followed by 1 or 2 successive broods, at a ratio of 3F per 1M. The F1 offspring were scored for exceptional XO males and XXY females.

Duration of treatment / exposure:

one injection

Frequency of treatment:

one injection

Post exposure period:

2 days

Dose / conc.:

0 ppm

Remarks:

nominal

Dose / conc.:

200 ppm

Remarks:

nominal

Dose / conc.:

300 ppm

Remarks:

nominal

Dose / conc.:

400 ppm

Remarks:

nominal

No. of animals per sex per dose:

100 males treated/concentration and control

Control animals:

yes, concurrent vehicle

Positive control(s):

none reported

Tissues and cell types examined:

not reported

Details of tissue and slide preparation:

not reported

Statistics:

SLRL were calculated by the Kastenbaum-Bowman (1970) tables. SCL chi square was applied to test significance.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Induction of sex-linked recessive lethals occurred at all concentrations tested. Total sex-chromosome loss was only detectable at significant numbers at the highest concentration (400 ppm). NiSO4 is mutagenic in the SLRL assay.

Table 1: Induction of sex-linked recessive lethals in Drosophila males injected with the test substance

	A		B		C		D		A+B+C+D^a
Conc. [ppm]	0-2 days	%	2-4 days	%	4-7 days	%	7-10 days	%	0-10 days	%
Control	4/1204	0.33	4/1436	0.28	2/926	0.22	2/1230	0.16	12/4796	0.25
200	8/1102	0.73^b	2/1024	0.19	2/1100	0.18	12/1582	0.76^b	24/4818	0.50^b
300	6/708	0.85^b	2/1078	0.19	2/1028	0.19	20/1376	1.45^b	30/4190	0.72^bb
400	10/1226	0.82^b	3/1006	0.30	2/1140	0.17	28/1700	1.65^b	43/5072	0.85^b

 

a: Sum of 2 Experiments

b: Significant at the 5% level (Kastenbaum-Bowman test).

 

Table 2: Frequencies of offspring resulting from sex-chromosome loss in Drosophila males injected with the test substance

			Exceptional
Conc [ppm]	brood	Total offspring	Males X0	%	Females XXY	%
Control	A	3560	3	0.08	2	0.06
	B	3137	1	0.03	1	0.03
	C	3230	3	0.09	1	0.03
Total	A+B+C	9927	7	0.07	4	0.04
200	A	3269	8	0.24	2	0.06
	B	3557	4	0.11	4	0.11
	C	3421	7	0.20
Total	A+B+C	10247	19	0.19	6	0.06
300	A	3415	8	0.23	2	0.06
	B	3156	3	0.10	6	0.19
	C	3260	8	0.24	2	0.06
Total	A+B+C	9831	19	0.21	10	0.10
400	A	3337	8	0.24	2	0.06
	B	3138	10	0.32	7	0.22
	C	3212	8	0.25	2	0.11
Total	A+B+C	9687	26	0.27^b	11	0.11

 

Conclusions:

NiSO4 is mutagenic in the SLRL assay and was shown to have a potential to cause total chromosome loss.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

In vitro studies

In vitro gene mutation study in bacteria

No data on the substance itself is available. Therefore, read across to nickel acetate (CAS 373-02-4) was applied.

De Flora et al. (1984) evaluated the genotoxicity of multiple chemicals and chemical classes of compounds, including nickel acetate. The compounds were assayed comparatively using 1) the Ames reversion test (with his- S. typhimurium strains TAl538, TA1537, TA1535, TA98, TA100 and, in part, TA97), and a DNA-repair test.  The test substance was tested with and without metabolic activation system (S9-mix). Nickel acetate was negative in the S. typhimurium reversion test for all six strains.

In vitro gene mutation study in mammalian cells

No data on the substance itself is available. Therefore, read across to nickel acetate (CAS 373-02-4) was applied (Morita et al., 1991).

FM3A cells were exposed to hypoxanthine, aminopterin, and thymidine for 1 week prior to use in the mutation assay, then cultured in Eagle's minimum essential medium supplemented with 3% fetal bovine serum, 1% non-essential amino acids, hypoxanthine, and thymidine. Cultured FM3A cells were then exposed to various concentrations of NiCl2 for 3-48 hours, washed in Hank's balanced salt solution, and then cultured for an additional 7 days. Then, 2 x 10e^6 cells were inoculated into 40 mL of selective agar medium containing 10 µg/mL 6-thioguanine (6-TG) and cultured for 14 days. The number of 6TG colonies were counted and mutation frequency (per 10e^6 surviving cells) was determined. Statistical analysis was performed using the Welch test. The positive control substance was N-methyl-N'-nitro-N-nitrosoguanidine. Cells were also tested in the presence of a metabolic activation system (2.5% S15). There was a concentration-dependent decrease in percent cell survival following treatment. At 0 M treatment, survival was 76%, with 100% relative plating efficiencies. This decreased to 7% survival with relative plating efficiencies of 9% after treatment at 4 x 10e^-4 M. The number of mutants per 10e^6 surviving cells increased in a concentration-dependent manner, with a significant increase seen at 4 x 10e^-4 M. The test substance was not mutagenic in this in vitro mammalian gene mutation test.

In vitro cytogenicity/ chromosome aberration study in mammalian cells

No data on the substance itself is available. Two studies with nickel compounds are available. Therefore, read across to nickel acetate (CAS 373-02-4) and to nickel sulphate hexahydrate (CAS 10101-97-0) was applied.

In the first study (Umeda et al., 1979), C3H mouse mammary carcinoma cells (FM3A) (1 x 10e^5 cells/mL) were exposed to various concentrations of the test substance nickel acetate for 24 or 48 hours. The study was conducted without metabolic activation. Two hours prior to the end of the treatment period, colcemid was added to the culture (final concentration = 10e^-7 M). Slides were prepared using the flame-drying method. 100 metaphases were scored. The experiments were repeated. As only few chromosomal aberrations were induced by the test substance, it was considered not to cause chromosomal aberrations.

In the second study (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 10e^7 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 (10e^6 cells/100 mm dish) were treated with nickel sulphate (prepared in acetone) and 10 µg  BrdUrd/mL medium and incubated for 24 hours. Four hours prior to harvest, cells were treated with Colcemide (0.13 µg/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 10e^6 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 sulphate (prepared in acetone) and 30 µg BrdUrd/mL medium and incubated for 48 hours. Two hours prior to harvest, cells were treated with Colcemide (0.13 µg/mL), then collected, centrifµged, 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 µg/mL: 17.20 (0.90); 5.0 µg/mL: 18.95 (1.52)

Hamster embryo cells [frequency of SCEs/metaphase (standard error)]: BrdUrd control: 11.55 (0.84); 1.0 µg/mL: 15.95 (0.92); 2.5 µg/mL: 17.25 (1.44); 5.0 µg/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 µg/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 µg/mL: 16.50%, 0.16 (0.03), (Chromosomal aberrations noted in embryo cells included gaps, breaks, exchanges, and minutes).

This summary was obtained from the ECHA disseminated dossier of nickel sulphate hexahydrate (CAS 10101-97-0).

In vivo studies

In vivo insect germ cell cytogenetic assay

No data on the substance itself is available. Therefore, read across to nickel sulphate hexahydrate (CAS 10101-97-0) was applied.

In this assay (Rodriguez-Arnaiz et Ramos, 1986), the test substance nickel sulphate hexahydrate was injected into Drosophila melanogaster males at different concentrations in order to test the chemical for the induction of SLRL (sex linked recessive lethals) and SCL (sex chromosome loss) in germ cells. The test substance was dissolved in 5% sucrose solution at 200, 300 and 400 ppm. In a preliminary test the LD50 was found to be at 400 ppm. Adult males (0-48 h old) were injected intraperitoneally with the salt. Control males were injected with 5% sucrose solution. For the SLRL test, white males (0-2 days old, treated and control) were crossed with BASC virgin females (1-3 days old), for two days at a ratio of one male × two females. The progeny of each treated male was kept separately to identify possible clusters of lethals. To test the sensitivity of different germ cell stages, a brood pattern analysis was performed (Broods A, B, C, D) by transferring the males to fresh virgin females at intervals of 2-2-3-3 days. F1 females autofertilized by F1 males were intercrossed in vials and the F2 scored for sex-linked recessive lethals. F3 was retested. For the SCL test, 0-2 day old ring X males (genotype X^c2yB/sc⁸Y), treated or kept untreated as controls, were mass-mated in bottles with 1-3 day old virgin y²w^a females for 2 days (A) followed by 1 or 2 successive broods (B, C), at a ratio of 3 females per male. The F1 offspring were scored for exceptional XO males and XXY females. As a result, the test substance induced SLRL at all concentrations, with the peak of activity at premeiotic and postmeiotic stages. It failed to produce SCL except at the highest concentration tested, where induction of XO males was significant for the pooled data. The test substance was considered genotoxic in this assay. 

In vivo mammalian germ cell study: cytogenicity/chromosome aberration

No data on the substance itself is available. Therefore, read across to nickel sulphate hexahydrate (CAS10101-97-0) was applied. The study data were obtained from the ECHA disseminated Dossier of Nickel sulphate (CAS 7786-81-4).

The study according to OECD Guideline 474 was conducted with nickel sulphate hexahydrate to evaluate its potential to induce chromosome aberrations in vivo. The study was conducted with male Sprague-Dawley rats. Each day the test article was prepared freshly by adding water. Formulations were held at room temperature. The animals were dosed by oral gavage once daily for three consecutive days. The dose levels were 125, 250, or 500 mg/kg bw/day. Cyclophosphamide was used as the positive control administered by oral gavage dissolved in deionized water at a dose of 60 mg/kg bw. The 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. Following centrifugation to pellet the marrow, the supernatant was spread on slides, fixed with methanol and stained in acridine orange, dried, and analyzed under fluorescent microscopy. 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. As a result, no genotoxicity was observed, and no cytotoxicity effects on bone marrow even at maximum tolerated dose established based on mortality and clinical observations.

Overall conclusion on genotoxicity

The source substance nickel sulphate hexahydrate (CAS 10101-97-0) was positive in one in vitro chromosomal aberration study. It was furthermore positive in the in vivo insect germ cell cytogenetic assay. In line with the classification of the source substances nickel acetate (CAS 373-02-4) and nickel sulphate hexahydrate (CAS 10101-97-0), the target nickel substance is classified as mutagenic into category 2. For further details, please refer to the read across justification.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The studies were considered reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the results, the target and source substances are to be classified for mutagenicity into category 2 and labelled with H341 (suspected of causing genetic defects).