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Genetic toxicity: in vitro

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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:
2013-07-04 to 2013-07-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 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 NTP (NTP, 1998)(please refer to the endpoint study record w_NTP_1998_bacterial reverse mutation assay (CoSO4)). Since the results of the previous test should only be verified a test with only one strain was conducted with and without metabolic activation instead of a full test according to OECD 471 (1997).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cobalt sulphate heptahydrate- Molecular weight (if other than submission substance): 281.1 g/mole- Physical state: rose coloured powder- Storage condition of test material: 2 to 8°C, protected from moisture and light, in a tightly closed container.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Mutation experiment 1: 5.0, 15.81, 50.0, 158.1, 500.0, 1581, and 5000 µg/plate (with or without metabolic activation)Mutation experiment 2: 156.3, 312.5, 625.0, 1250, 2500, and 5000 µg/plate (with or without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: preliminary solubility data indicated that cobalt sulphate heptahydrate was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations equivalent to at least 100 mg/mL.Test article stock solutions were prepared by formulating cobalt sulphate heptahydrate under subdued lighting in DMSO with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4.5 hours of initial formulation.A correction factor of 1.8 was used to account for the water content of the test item.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
purified water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: 2 µg/plate (stock solutions were formulated in purified water)
Untreated negative controls:
yes
Remarks:
purified water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: 5 µg/plate (stock solutions were formulated in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and preincubationExperiment 1 (Plate incorporation) anad Experiment 2 (Preincubation)Cobalt sulphate heptahydrate was tested for mutation (and toxicity) in one strain of Salmonella typhimurium (TA100), in two separate experiments, using triplicate plates without and with S-9. Negative (vehicle) controls were included in quintuplicate, and untreated controls and positive controls were included in triplicate in both assays without and with S-9. These platings were achieved by the following sequence of additions to 2.0 mL molten agar at 46±1°C:• 0.1 mL bacterial culture• 0.1 mL test article solution or control• 0.5 mL 10% S-9 mix or buffer solutionfollowed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S-9 mix, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2.0 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments). The background lawn was inspected for signs of toxicity.DETERMINATION OF CYTOTOXICITYThe background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made.ACCEPTANCE CRITERIAThe assay was to be considered valid if the following criteria were met:1. The vehicle control counts fell within the laboratory’s historical control ranges.2. The positive control chemicals induced increases in revertant numbers of ≥2-fold the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:1. A concentration related increase in revertant numbers was ≥2-fold the concurrent vehicle control values.2. The positive trend/effects described above were reproducible.The test article was considered positive in this assay if all of the above criteria were met.The test article was considered negative in this assay if none of the above criteria were met.Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).However, adequate interpretation of biological relevance was of critical importance (OECD, 1997)*.*Reference- OECD (1997). “Bacterial Reverse Mutation Test”, in: OECD Guideline for the Testing of Chemicals, Test Guideline 471

Results and discussion

Test results
Species / strain:
S. typhimurium TA 100
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
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY AND SOLUBILITY:Experiment 1: evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 μg/plate in the absence and presence of S-9.Experiment 2: evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 μg/plate in the absence and presence of S-9.The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.MUTATION:Following cobalt sulphate heptahydrate treatments of the test strain in the absence and presence of S-9, no increases in revertant numbers were observed that were greater than 2-fold the concurrent vehicle control. This study was considered therefore to have provided no evidence of any cobalt sulphate heptahydrate mutagenic activity in this assay system.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negativeCobalt sulphate heptahydrate did not induce mutation in one histidine-requiring strain (TA100) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).