Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 02, 2010 - March 23, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
perfomed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Steam Activated Carbon (High Skeletal Density activated carbon)
- Physical state: solid
- Analytical purity: confidential
- Lot/batch No.: confidential
- Expiration date of the lot/batch: confidential
- Stability under test conditions: Stable in water
- Storage condition of test material: At room temperature, protected from moisture

Method

Target gene:
His-gene: Amino acid histidine
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: TA 1537: his C 3076; rfa-; uvrB-; TA 98: his D 3052; rfa-; uvrB-; R-factor; TA 1535: his G 46; rfa-; uvrB-; TA 102: his G 428; rfa-; uvrB+; R-factor; TA 100: his G 46; rfa-; uvrB-; R-factor.
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar rat liver S9 induced by Phenobarbital/B-Naphthoflavone
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Strain TA 100 and TA 1535: sodium-azide; Strain TA 1537 and TA 98: 4-nitro-o-phenylene-diamine; Strain TA 102: methyl methane sulfonate; With metabolic activation: All strains: 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes
- Selection time (if incubation with a selection agent): at least 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
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 (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
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.
Statistics:
Mean and Standard Deviation

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tube from 1000 µg/plate up to 5000 µg/plate in both experiments. Precipitation of the test item on the incubated agar plates was observed from 333 µg/plate up to 5000 µg/plate in experiment I and from 1000 µg/plate up to 5000 µg/plate in experiment II. The undissolved particles of the test item had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1537 at 5000 µg/plate in the presence of metabolic activation.
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):
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Steam Activated Carbon is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Steam Activated Carbon to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 ug/plate.

The plates incubated with the test item showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used.

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 substantial increase in revertant colony numbers of any of the five test strains was observed following treatment with Steam Activated Carbon at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Steam Activated Carbon is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.