Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: Chromosome aberration study

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Chinese hamster lung.
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
1242 ug/mL without S-9 mix (6 hr short-term treatment)
1005 ug/mL with S-9 mix (6 hr short-term treatment)
775 ug/mL without S-9 mix (24 hr continuous treatment)
Vehicle / solvent:
- Vehicle(s) used:CMC (carboxymethyl cellulose);
- Justification for choice of solvent/vehicle:
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
Two plates were used for each concentration. The cells were treated continuously with the test substance for 24 hr in the absence of metabolic activation (continuous treatment) or shortly for 6 hr (short-term treatment) in the presence or absence of metabolic activation. In the short-term treatment, cells were incubated for additional 18 hr in a fresh culture medium without the test substance. The co-factor-supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavon. Mytomycin C or Benzo[a]pyrene was used as a positive control for the assay.
Colcemid (0.1 ug/ml) was added to the culture medium 2 hr before cell harvesting and then chromosome preparations were made. A hundred well-spread metaphase plates were observed under a microscope for each plate. Incidence of polyploid cells and that of cells with structural aberrations such as chromatid breaks and exchanges, chromosome breaks, exchanges and gaps, and others were recorded.
The results were considered negative when the incidence was less than 5%, and considered positive when the incidence was more than 10%.
The results were considered equivocal when the incidence was 5-10%.
Evaluation criteria:
The results were considered negative when the incidence was less than 5%, and considered positive when the incidence was more than 10%.
The results were considered equivocal when the incidence was 5-10%.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In this study, an increase in structural chromosomal aberrations, attributed to a decrease of culture media pH, was observed. Therefore, the confirmation tests were conducted with or without a buffered medium, which was prepared with a double amount of sodium hydrogen carbonate.

In the main and confirmation tests conducted with buffered medium, the number of cells with structural chromosomal aberrations except gaps increased at dose of1000 ug/mL and higher after continuous treatment without metabolic activation (frequency: 5-12 %).
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Colcemid (0.1 ug/ml) was added to the culture medium 2 hr before cell harvesting and then chromosome preparations were made. A hundred well-spread metaphase plates were observed under a microscope for each plate. Incidence of polyploid cells and that of cells with structural aberrations such as chromatid breaks and exchanges, chromosome breaks, exchanges and gaps, and others were recorded.
The results were considered negative when the incidence was less than 5%, and considered positive when the incidence was more than 10%.
The results were considered equivocal when the incidence was 5-10%.

Applicant's summary and conclusion

Conclusions:
This chromosome aberration study found p-toluic acid to increase structural chromosomal aberrations.