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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Performed to GLP and current guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Chromium (III) hydroxide
EC Number:
215-158-8
EC Name:
Chromium (III) hydroxide
Cas Number:
1308-14-1
Molecular formula:
Cr (OH)3
IUPAC Name:
chromium (III) hydroxide
Test material form:
solid
Specific details on test material used for the study:
Identification: Chromium (III) HydroxidePhysical state/Appearance: Green powderBatch: 001Purity: >99.6%Expiry Date: Not suppliedStorage Conditions: Room temperature in the dark

Method

Target gene:
Salmonella typhimurium Strains Genotype Type of mutations indicatedTA1537 his C 3076; rfa-; uvrB-: frame shiftTA98 his D 3052; rfa-; uvrB-;R-factorTA1535 his G 46; rfa-; uvrB-: base-pair substitutionTA100 his G 46; rfa-; uvrB-;R-factorEscherichia coliStrain Genotype Type of mutations indicatedWP2uvrA trp-; uvrA-: base-pair substitution
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction induced with Phenobarbitone/-Naphthoflavone
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate Experiment 150 to 5000 μg/plate Experiment 2Top dose maximum for guideline
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
In solubility checks performed in–house the test item was noted as insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).2. A reproducible increase at one or more concentrations. 3. Biological relevance against in-house historical control ranges.4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight thinning without S-9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains (except TA1537 dosed in the absence of S9-mix) at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Weakened bacterial background lawns were noted for TA1537 dosed in the absence of S9-mix at 5000 μg/plate following the change in test methodology from plate incorporation to pre-incubation. A test item precipitate (green and powdery in appearance) was noted under an inverted microscope at 1500 μg/plate and by eye at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.

Applicant's summary and conclusion

Conclusions:
Chromium (III) Hydroxide was considered to be non-mutagenic under the conditions of this test.
Executive summary:

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5, 500 and 5000 μg/plate (TA100 dosed in the absence of S9-mix only). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle control.

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