Naphthenic acids, cobalt salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-10-09 to 2014-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

yes

Remarks:

, the study was performed with the strain TA 100 only.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspection dates: 25 April, 23/25 and 26 July 2012

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

cobalt sulfate

IUPAC Name:

cobalt sulfate

Constituent 2

Reference substance name:

Cobalt sulphate

EC Number:

233-334-2

EC Name:

Cobalt sulphate

Cas Number:

10124-43-3

Molecular formula:

Co.H2O4S

IUPAC Name:

cobalt(2+) sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Cobalt sulfate heptahydrate- Molecular formula: CoSO4 · 7H2O- Storage condition of test material: at room temperature

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9: phenobarbital/β-naphthoflavone induced rat liver S9 will be used as the metabolic activation system (prepared from 8 – 12 weeks old male Wistar rats weight approx. 220 – 320 g).

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)Experiment Ia: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)Experiment II: 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)Experiment IIa: 31.25, 62.5, 125, 250, 375, 500, 750, and 1000 μg/plate (without metabolic activation)Experiment IIb: 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al., 1981)*.*Reference:- Maron D.M., J. Katzenellenbogen and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test Mutation Res. 88, 343-350.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: concentration: 10 µg/plate; vehicle: deionised water

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control with metabolic activation: concentration 2.5 µg/plate; vehicle: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; Pre- experiment/Experiment I & Ia) & preincubation (Experiment II, IIa, and IIb)Pre-Experiment/Experiment ITo evaluate the toxicity of the test item a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were as described below (plate incorporation test).Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.The pre-experiment is reported as main experiment I, since the following criteria are met: evaluable plates (>0 colonies) at five concentrations or more in all strains used.Experimental Performance (Pre-experiment/Experiment I, Ia, Ib, II, IIa, and IIb):For each strain and dose level, including the controls three plates were used.The following materials were mixed in a test tube and poured onto the selective agar plates:- 100 μL test solution at each dose level (solvent or reference mutagen solution (positive control)),- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),- 100 μL bacteria suspension (cf. test system, pre-culture of the strain),- 2000 μL overlay agarIn the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark (de Serres & Shelby, 1979)*.The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. ACCEPTABILITY OF THE ASSAY:The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:- regular background growth in the negative and solvent control- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data- the positive control substances should produce a significant increase in mutant colony frequencies- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.*Reference:- de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay Mutation Res. 64, 159-165

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed (Hollstein et al., 1979)*.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (de Serres & Shelby, 1979)*.An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.*Reference:- de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay Mutation Res. 64, 159-165- Hollstein,M., J. McCann, F.A. Angelosanto and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens Mutation Res. 65, 133-226

Statistics:

A statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: the test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in the absence and presence of metabolic activation in experiment I. The undissolved particles had no influence on the data recording.DOSE SELECTION:The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate was chosen as maximal concentration.Two further tests and confirmatory experiment was performed.RESULTS:- the plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all experiments.- no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.- no increase in revertant colony numbers of strain TA 100 was observed following treatment with cobalt sulfate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) in experiment I. A weak increase in revertant colony numbers was observed following treatment with cobalt sulfate in strain TA 100 in the absence of metabolic activation in experiment II. The threshold of twice the number of the corresponding solvent control was just exceeded (induction factor 2.1) at 100 and 333 μg/plate, no increase were observed at higher concentrations, those effects were not based on overlapping toxic effects. In the confirmatory experiment the threshold of two was reached at 125 and 1000 μg/plate. Since there is no dose dependency and reproducible increase the test item is considered non-mutagenic. Based on the equivocal results obtained confirmatory experiments were performed. The plate incorporation and pre incubation assay was repeated under identical conditions. In experiment Ia a minor and dose dependent increase was observed in the absence of metabolic activation at 5000 μg/plate, but the threshold of two was not reached and the values are within the historical control range of the solvent and untreated control. In experiment IIb a minor increase was observed in the absence of metabolic activation at 2500 μg/plate, but the threshold of two was not reached and the values are also within the historical control range of the solvent and untreated control. Therefore the test item is judged as non-mutagenic.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeCobalt sulfate did not induce gene mutations by base pair changes in the genome of the strain TA 100 in the absence or presence of metabolic activation.