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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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 473) and according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide
EC Number:
229-245-3
EC Name:
3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide
Cas Number:
6448-95-9
Molecular formula:
C24H18N4O4
IUPAC Name:
3-hydroxy-4-[(2-methyl-5-nitrophenyl)diazenyl]-N-phenyl-2-naphthamide
Test material form:
not specified
Details on test material:
Name of test material (as cited in study report): Chromosomal Abberation Test with Cultured Mammalian Cells: C.I. Pigment Red 22
Analytical purity: 99.8 %

Method

Species / strain
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital and 5,6-benzoflavone induced rats
Test concentrations with justification for top dose:
cell growth inhibition test: 0, 8.4, 16.8, 33.6, 67.2, 134.4, 268.8, 537.5, 1075, 2150, 4300 µg/mL
chromosomal abberation test: 0, 37.5, 75, 150, 300, 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance is insolube in water, DMSO and acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for continuous treatment and short treatment in absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
for short treatment method in the presence of metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration:
continuous treatment: 24 or 48 h
short treatment: 6 h
- Expression time (cells in growth medium):
short treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
continuous treatment: 24 or 48 h
short treatment: 24h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2 h before completion of incubation)
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: Doubling time: 16.2 h

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cell survival ratio
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The final judgement on the outcome of the test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5, the result was assessed as negative; from 5-10% (exclusive of 10%), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural abberations with gaps and of cells without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrarions.
Statistics:
No significance tests were performed, since the incidence of cells with chromosomal aberrations was assessed according to the evaluation criteria described above.

Results and discussion

Test results
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation occurred at concentrations of 268.8 µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at concentrations of 268.8 µg/mL and higher

ADDITIONAL INFORMATION ON CYTOTOXICITY:
in the cell growth inhibition test no cytotoxicity was noted.
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

The test item is not clastogenic in Chinese hamster lung fibroblast (CHL/IU) cells under the experimental conditions described in the study.
Executive summary:

In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.

Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 4300 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 268.8 µg/mL and higher.

In the chromosomal aberration test, concentrations of 0, 37.5, 75, 150, 300 and 600 µg/mL were tested. A continuous exposure (24 or 48 h) and a short time exposure (6 h with 18 h expression time) in the presence and absence of S9-mix were performed.

At least 200 metaphases per culture were evaluated for structural chromosome aberrations.

Appropriate mutagens were used as positive controls. They produced markedly positive results.

It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.

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