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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
O-isopropyl ethylthiocarbamate
EC Number:
205-517-7
EC Name:
O-isopropyl ethylthiocarbamate
Cas Number:
141-98-0
Molecular formula:
C6H13NOS
IUPAC Name:
O-isopropyl ethylthiocarbamate
Constituent 2
Reference substance name:
Carbamothioic acid, ethyl, O-(1-methyl-ethyl) ester
IUPAC Name:
Carbamothioic acid, ethyl, O-(1-methyl-ethyl) ester
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): IPETC
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: ambient

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
Human peripheral blood was obtained by venipuncture from healthy donors known to be without any medication and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to MARON and AMES (1983). S9 was collected from 20 - 30 rats. The pooled fraction was tested for:
Test concentrations with justification for top dose:
10, 26, 103, 257, 1029 and 2571 µg IPETC per mL medium
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
was used as the positive control for the study in the absence of metabolic activation.
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
was used as the positive control for the study in the presence of metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours in experiment 1 and 24 hours in experiment 2

SPINDLE INHIBITOR (cytogenetic assays): colcemid® to accumulate cells in a metaphase-like stage of mitosis (c-metaphase).

Evaluation criteria:
The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o the number of chromosomal aberrations is significantly (at p  0.05) increased compared with the solvent control
o the increase observed is concentration-dependent
o both duplicate cultures lead to similar results
o the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:
The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p  0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was noted in the experiment without and with metabolic activation (24-h or 4-h exposure, respectively) starting at 1029 µg IPETC /mL. Haemolysis was noted at concentrations of 2571 and 5143 µg/mL in both experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: human peripheral lymphocytes
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 present test conditions, IPETC, tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.