Cobalt(2+) hydrogen citrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Cobalt hydrogen citrate Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA97a, TA98, TA100, and E.coli: negative with and without metabolic activation (according to OECD 471) Cobalt citrate Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA97a, TA98, TA100, and TA102: negative with and without metabolic activation (according to OECD 471): Cytogenicity in mammalian cells ( chromosome aberration test): human lymphocytes: ambiguous with and without metabolic activation (according to OECD 473) Read-across with cobalt chloride hexahydrate Cytogenicity in mammalian cells (micronucleus test): mouse bone marrow cells: negative with and without metabolic activation Cytogenicity in-vivo (micronucleus test): male mice: positive

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

CoCl2*6H2O was administered once by intraperitoneal injection to male mice. The test item was investigated for its ability to induce micronuclei in bone marrow cells.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: BALB/c AnNCrj

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 22-27 g
- Diet (e.g. ad libitum): CRF-1 diet supplied by Charles River Japan, ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

one injection

Frequency of treatment:

one injection

Post exposure period:

30 hours

Remarks:

Doses / Concentrations:
0, 25, 50, 90 mg/kg bw
Basis:
analytical conc.

No. of animals per sex per dose:

5 male animals per dose

Control animals:

yes

Positive control(s):

none

Tissues and cell types examined:

bone marrow cells

Statistics:

The data for micronucleus induction were analysed statistically by the Kastenbaum-Bowman method [Kastenbaum and Bowman, 1970]. Data for the P/N ratio were analysed statistically using student´s t-test and one-way layout analysis of variance.

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Treatment with the test item induced a dose-dependent increase in MPCE frequency. The P/N ratio was the lowest at 90 mg/kg bw of CoCl2*6H2O in the in vivo micronucleus test.

Dose of CoCl2*6H2O [mg/kg bw]	MPCE frequency [%]	P/N ratio
90	0.75±0.43**	0.87±0.19**
50	0.46±0.09**	1.44±0.44
25	0.12±0.13	2.47±0.17
0	0.18±0.15	1.98±0.32

Results show mean ± SD

** P < 0.05 statistically different from the solvent control

Doses of CoC6H6O7:

26.2, 52.3 and 94.2 mg/kg bw CoC6H6O7 (recalculated values; equivalent to 25, 50 and 90 mg/kg bw CoCl2*6H2O)

Conclusions:

Interpretation of results (migrated information): positive

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

Cobalt hydrogen citrate was investigated in a bacterial reverse mutation assay following OECD 471 (Andres, 2013). The S. typhimurium strains TA 1535, TA 97a, TA 98 and TA 100 and E.coli were used as tester strains. The test item was applied in concentrations up to 5000 µg/plate in 2 independent experiments (standard plate test and preincubation test). No increase in the number of revertants compared to the vehicle control (water) was observed in any strain at any concentration with or without metabolic activation. No signs of toxicity were observed. The background lawn was visible and the number of revertant colonies was not reduced. However, as no complete dissolution of the test item in water was possible, undissolved particles were visible on the plates.

In addition, cobalt citrate was investigated in a bacterial reverse mutation assay following OECD 471 (Andres, 2012). The S. typhimurium strains TA 1535, TA 97a, TA 98, TA 100 and TA 102 were used as tester strains. Cobalt citrate was applied in concentrations up to 5000 µg/plate in 2 independent experiments (standard plate test and preincubation test). No increase in the number of revertants compared to the vehicle control (water) was observed in any strain at any concentration with and without metabolic activation. No signs of toxicity were observed. The background lawn was visible and the number of revertant colonies was not reduced. However, no complete dissolution was possible and undissolved particles were visible on the plates.

The genotoxic potential of cobalt citrate was assessed in a chromosome aberration test in human blood lymphocytes according to OECD 473 (Markx, 2012). The test item was suspended in RPMI 1640 medium without fetal calf serum. The exposure duration was 4 h with and without S9 mix. For the preliminary cytotoxicity test, concentrations up to 5000 µg/mL were tested. At concentrations ≥ 627.5 µg/mL, no viable cells were found both in the presence and in the absence of metabolic activation. At concentrations of 39.2 to 313.8 µg/mL with and without metabolic activation, the mitotic index was reduced to 12 to 70% compared to the negative control. Based on the result of the cytotoxicity test, the final test concentrations selected for the evaluation of chromosomal damages were 39.2, 78.4, 230.9 and 313.8 µg/mL with and without metabolic activation. The chromosome aberration test yielded a positive result at the 313.8 µg/mL both in the presence and in the absence of metabolic activation. However, at this concentration cytotoxicity had reduced the mitotic index to 70% of the vehicle control value. Furthermore, the intervals between the concentrations evaluated for chromosome aberration differed by factor 2 or 3. The actual guideline requires a factor of max. 2. A confirming experiment should have been carried out with adjusted concentration levels. However, the authors assumed the test substance to be positive in this chromosome aberration test. Taking into account the above mentioned methodical deficits, the result of this study is considered "ambiguous".

Read-across with cobalt chloride hexahydrate

In mouse bone marrow cells, micronucleus formation was not significantly altered by treatment with cobalt chloride hexahydrate in the presence or absence of S9 mix (Suzuki et al., 1993). However, no positive control substance was used to validate this assay.

In an in-vivo micronucleus test, mice were injected intraperitoneally with 25, 50 and 90 mg/kg bw cobalt chloride hexahydrate.

30 hours following the single injection, a dose-dependent increase in micronucleus formation was seen at 90 and 50 mg/kg bw, but not at 25 mg/kg bw cobalt hydrogencitrate (recalculated values, equivalent to 26.2, 52.3 and 94.2 mg/kg bw cobalt hydrogen citrate) (Suzuki et al., 1993).

In summary, an Ames test with cobalt hydrogen citrate showed a negative result. Also an Ames test with cobalt citrate produced a negative response. An in-vitro chromosome aberration assay tested with cobalt citrate was ambiguous. The read-across substance cobalt chloride hexahydrate was also negative in an in-vitro micronucleus assay. There is only one positive result in a micronucleus assay with mice. In this study, mice were intraperitoneally injected with cobalt chloride hexahydrate. Based on the positive result, cobalt hydrogen citrate was considered to be mutagenic.

Justification for selection of genetic toxicity endpoint
The read-across substance cobalt chloride hexahydrate led to a positive result in an in-vivo micronucleus assay. Based on the positive response, cobalt hydrogen citrate was considered to be mutagenic.

Justification for classification or non-classification

Based on read-across, the available data on genetic toxicity meet the criteria for classification as Category 2 (H341) according to Regulation (EC) 1272/2008 and as R68, Category 2 according to Directive 67/548/EEC.