Reaction product of soluble nickel salt, cobalt salt, manganese salt with alkalines

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

initiated on 2010-02-08 and completed on 2010-03-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Hypothesis for read-across: Cobalt dihydroxide oxide and nickel dihydroxide are the main components of the target UVCB-substance, and considered the major drivers for adverse effects based on its properties and relative quantity in the substance. For read-across purpose, data from the constituents (cobalt hydroxide oxide and nickel dihydroxide) of the target UVCB-substance is used in the REACH registration. A GLP-following and guideline study, performed with cobalt oxide hydroxide.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt hydroxide oxide

EC Number:

234-614-7

EC Name:

Cobalt hydroxide oxide

Cas Number:

12016-80-7

Molecular formula:

CoHO2

IUPAC Name:

cobalt hydroxide oxide

Details on test material:

- Name of test material (as cited in study report): cobalt oxide hydroxide
- Physical state: solid, black powder
- Storage condition of test material: stored at 15-25ºC in the dark

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media, containing 100 units/mL Penicillin, 100 µg/mL Streptomycin, 2.5 µg/mL Amphotericin B, 0.5 mg/mL Pluronic (except for RPMI 20) and heat inactivated horse serum (0% v/v for RPMI A; 10% v/v for RPMI 10 and 20% v/v for RPMI 20).

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range-Finder (with and without S9-mix): 1.796, 3.591, 7.183, 14.37, 28.73, 57.46, 114.9, 229.9, 459.7 and 919.4 µg/mL.
Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.
Experiment I (with and without S9-mix): 0.25, 0.50, 1, 2, 4, 6, 8, 15, 30, 50 and 100 µg/mL;
Experiment II (with and without S9-mix): 1, 2, 4, 6, 8, 10, 12.5, 15, 25 and 50 µg/mL.

Cultures selected for mutation assessment:
Experiment I (with and without S9-mix): 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 15 µg/mL;
Experiment II (with and without S9-mix): 0, 2, 4, 6, 8, 10 and 12.5 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: methyl cellulose
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that cobalt oxide hydroxide was not soluble in 1M hydrochloric acid, tetrahydrofuran (THF) and dimethyl formamide (DMF). The test article was formulated as a suspension (at 10 mg/mL) in 0.5% w/v methyl cellulose (0.5% MC) which was considered acceptable. The nominal solubility limit in culture medium was less than 500 μg/mL, as indicated by the appearance of persistent precipitate at this concentration 3 hours after test article addition.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% MC diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline 1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% MC diluted 10-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed.
- Selection time (if incubation with a selection agent): At the end of the expression period, cell were plated for 6TG resistance. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 days) and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6 -thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was performed in duplicate cultures (single cultures only used for positive control treatments).

DETERMINATION OF CYTOTOXICITY
- Method: relative survival
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described for the Mutation Experiments. Single cultures only were used and positive controls were not included. Following treatment, cells were washed with tissue culture medium and resuspended in 20 mL of RPMI 10. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for
determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 8 days.

OTHER:
Analysis of results: All calculations were performed either manually or by computer using validated software.
- Plating efficiency (PE) in any given culture is therefore: PE = P/No of cells plated per well;
- Percentage relative survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [PE (test)/PE (control)] x 100;
- Mutant frequency (MF) is usually expressed as "mutants per 10^6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated: MF = [PE (mutant)/PE (viable)] x 10^6

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05).
2. there was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05).
3. the effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared
with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with cobalt oxide hydroxide at any concentration tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment and Experiment I.
- Effects of pH and osmolality: No marked changes in osmolality or pH, compared to the concurrent vehicle controls were observed in the Range-Finder Experiment at 7.183 μg/mL in the absence of S9 and at 3.591 μg/mL in the presence of S9, the highest concentration tested (individual data not reported). No marked changes in osmolality or pH, compared to the concurrent vehicle controls were observed in Experiment I at 15 μg/mL, the highest concentration tested in the absence and presence of S9 (individual data not reported).
- Precipitation: Highest tested concentrations were limited by the appearance of post-treatment precipitate.

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, ten concentrations were tested in the absence and presence of S9, ranging from 1.796 to 919.4 μg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentrations plated for survival were 7.183 μg/mL in the absence of S-9 and 3.591 μg/mL in the presence of S9 (both limited by the appearance of post-treatment precipitate), which gave 81% and 112% relative survival (RS), respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison of controls with historical means.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest concentrations analysed were 7.183 μg/mL in the absence of S9 and 3.591 μg/mL in the presence of S9, which gave 81% and 112% RS, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cobalt Oxide Hydroxide (CoOOH): summary of results

 

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		%RS	MF§					%RS	MF§
0		100	2.61			0		100	1.90
0.25		119	2.38		NS	0.25		93	2.49		NS
0.5		132	2.97		NS	0.5		87	2.11		NS
1		129	2.99		NS	1		93	1.99		NS
2		135	3.75		NS	2		105	2.35		NS
4		109	0.50		NS	4		95	1.71		NS
6		108	2.30		NS	6		89	1.57		NS
8		129	1.57		NS	8		97	0.19		NS
15	PP	118	2.50		NS	15	PP	82	0.80		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		85	7.52			2		53	8.86
0.15		99	14.63			3		23	8.57

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		%RS	MF§					%RS	MF§
0		100	2.77			0		100	1.29
2		90	1.03		NS	2		89	1.72		NS
4		92	2.24		NS	4		89	0.31		NS
6		106	1.51		NS	6		88	0.48		NS
8		98	1.28		NS	8		82	1.72		NS
10		95	1.91		NS	10		92	1.59		NS
12.5	PP	83	1.54		NS	12.5	PP	91	1.38		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		73	10.26			2		58	21.34
0.15		61	19.23			3		51	29.81

 

§                            6‑TG resistant mutants/10⁶viable cells 7 days after treatment

%RS                      Percent relative survival adjusted by post treatment cell counts

NS                         Not significant

PP                          Precipitation observed following treatment incubation period

Justification for read-across: Cobalt oxide hydroxide and nickel dihydroxide are the main components of the target UVCB-substance, and considered the major drivers for adverse effects based on its properties and relative quantity in the substance. For read-across purpose, data from the constituents (cobalt oxide hydroxide and nickel dihydroxide) of the target UVCB-substance is used. According to section 1.5, annex XI of regulation (EC) 1907/2006, such studies do not need to be conducted, since adequate data from structurally analogous substances are available. Therefore read-across from these compounds is justified.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that cobalt oxide hydroxide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

Cobalt oxide hydroxide was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6 -thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). The test article was formulated in 0.5% w/v methyl cellulose. In the cytotoxicity Range-Finder Experiment, ten concentrations were tested in the absence and presence of S9, ranging from 1.796 to 919.4 μg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentrations plated for survival were 7.183 μg/mL in the absence of S9 and 3.591 μg/mL in the presence of S9 (both limited by the appearance of post-treatment precipitate), which gave 81% and 112% relative survival (RS), respectively.

Accordingly, for Experiment I eleven concentrations, ranging from 0.25 to 100 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 15 μg/mL (limited by the appearance of post-treatment precipitate), which gave 118% and 82% RS in the absence and presence of S9, respectively. In Experiment II ten concentrations, ranging from 1 to 50 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 12.5 μg/mL (limited by the appearance of post-treatment precipitate), which gave 83% and 91% RS in the absence and presence of S9, respectively.

Vehicle and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals.

No statistically significant increases in mutant frequency were observed following treatment with cobalt oxide hydroxide at any concentration tested in the absence and presence of S9 in Experiments I and II and there were no statistically significant linear trends.