Cobalt dichloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-07-08 to 2013-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Principles of method if other than guideline:

This study design was chosen to re-assess the findings in the study report by Pagano and Zeiger (1992)(please refer to the enpoint study record w_Pagano_Zeiger_1992_bacterial reverse mutation assay (CoCl2)). Due to genetic instability the Salmonella strain TA97 used in the Pagano and Zeiger study, the strain TA97a was used in this experiment.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt dichloride

EC Number:

231-589-4

EC Name:

Cobalt dichloride

Cas Number:

7646-79-9

Molecular formula:

Cl2Co

IUPAC Name:

cobalt(2+) dichloride

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Cobalt dichloride- Material: Cobalt dichloride hexahydrate- Molecular formula: CoCl2 6H2O- Molecular weight: 238 g/mol- Physical state: solid (crystals)- Storage condition of test material: in tightly closed containers protected from direct sunlight in a cool, dry, well-ventilated place. Containers that have been opened must be carefully released and kept upright.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 0.316, 1.0, 3.16. 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate (with and without metabolic activation)Main study: 10.0, 31.6, 100, 316, 1000, and 3160 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Batch no. 1345/12; LAB-SCAN Analytical Sciences, 44-101 Gliwice, Poland)Cobalt dichloride was completely dissolved in dimethylsulfoxide (DMSO). A correction factor of 1.8 was used to account for the water content of the test item.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and preincubationPlate incorporation method (Experiment 1)Sterile top agar containing 0.6% agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45°C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 10^8 viable cells in the late exponential or early stationary phase), 0.1 mL of test item solution (or 0.1 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer.The test components were mixed and then poured onto a minimal glucose agar plate (Minimal Glucose Agar medium E) and finally allowed to solidify.Immediately after solidification, the plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter (Biocount 5000 γ, Biosys), and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate was evidence of bacterial toxicity.Preincubation method (Experiment 2)The test item/test solution was preincubated with the test strain (containing approximately 10^8 viable cells in the late exponential or early stationary phase) and sterile buffer or the metabolic activation system (0.5 mL) for 30 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of the test item/test solution (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.NUMBER OF REPLICATIONS: three (main study)DETERMINATION OF CYTOTOXICITYPrior to the main test two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA97a.Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Evaluation criteria:

A test item is considered to show a positive response if- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY, Colquhoun, 1997)* compared with the solvent control to at least 2-fold of the solvent control in both independent experiments;- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient, Colquhoun, 1997)* isobserved;- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.*Reference:- Colquhoun, D.: Lectures on Biostatistics, Clarendon Press, Oxford (1971)

Statistics:

Please refer to the field "Evaluation criteria" above.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TEST:Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 μg/plate in both experiments. Hence, 3160 μg cobalt dichloride/plate was chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.MAIN STUDY:Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at the top concentration of 3160 μg/plate in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.No increase in revertant colony numbers as compared with control counts was observed for cobalt dichloride, tested up to the cytotoxic concentration of 3160 μg/plate, in test strain TA97a in two independent experiments without and with metabolic activation (plate incorporation and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Cobalt dichloride tested up to the cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strain TA97a neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.