Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-08-11 to 2010-09-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The present study was performed according to the respective OECD guideline and EU method

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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
Principles of method if other than guideline:
no aplica
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Charcoal
EC Number:
240-383-3
EC Name:
Charcoal
Cas Number:
16291-96-6
Molecular formula:
C
IUPAC Name:
Charcoal
Details on test material:
CAS no.: 16291-96-6
Batch no.: 29004
Purity: C-Fix: 80.5 %; dose calculation not adjusted to purity
Storage conditions: At room temperature, moisture protected
Expiry date: December 2030

An extract of the test item was prepared using the following procedure: 250 g of the test item was extracted with 1 L acetone/n-hexane 50:50 (v:v) at 50°C in the ultrasonic bath for 1 h. After cooling to room temperature the extract was filtered. This procedure was repeated; thereafter the sample was mixed mechanically with 250 mL of the abovementioned solvent mixture. After a filtration step, the volume of the extract was reduced in several steps to approximately 150 mL using a rotary evaporator at 50°C. The extract was quantitatively filled up to 250 mL in a volumetric flask. Accordingly, the nominal concentration of the extract was 1,000 mg/mL.
The extract, a pale yellowish-brown liquid, was then stored in the refridgerator at 2-8°C.

Method

Target gene:
In this study in vitro mouse lymphoma assay (MLA), employing the in L5178Y cells carrying a single gene mutation in the tk (thymidine kinase) are employed. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-), producing the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products. In the presence of a toxic pyrimidine analogue 5-trifluorothymidine (TFT), the enzyme will incorporate this analogue into the cells.
Cells deficient in thymidine kinase (TK) due to the mutation tk +/- → tk -/- are resistant to the cytotoxic effects of TFT. Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT (the mutant cells have non-functional pyrimidine salvage pathway), whereas normal cells, which contain thymidine kinase, are not.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver homogenate obtained from rats induced with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
128; 320; 800; 2,000 and 5,000 µg/mL
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
concentration: of 0.2 µg/mL for the 3-h treatment and 0.1 µg/mL for the 24-h treatment; for tests without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
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:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Assay 1
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

Under the conditions of this study, the extract of the test item charcoal (Probe 2: C-Fix=80.5%) did not induce gene mutations in the cultured mammalian cells (L5178Y TK+/- cells, neither in the presence nor in the absence of metabolic activation by rat S9 mix.
Accordingly, the test item was concluded to be non-mutagenic under the conditions of the present study.

Categories Display