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Genetic toxicity: in vitro

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in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, near guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference Type:
Chromosome aberration and sister chromatid exchange test results with 42 chemicals
Anderson BE, Zeiger E, Shelby MD, Resnick MA, Gulati DK, Ivett JL and Loveday KS
Bibliographic source:
Environmental and molecular mutagenesis vol. 16, suppl. 18: 55-137

Materials and methods

Test guideline
equivalent or similar to guideline
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
mixed xylenes
EC Number:
mixed xylenes
Details on test material:
no further details


Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cells were cloned at Litton Bionetics Inc. Cells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% foetal calf serum at 37°C using 5% CO2. Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
supernatant from the 9,000g fraction of the livers from Aroclor 1254-induced male Sprague Dawley rats with NADP and isocitrate in serum-free medium
Test concentrations with justification for top dose:
without S9: 0, 20.1, 50.3, 100.5 µg/mL
with S9: 0, 15.1, 20.1, 50.3 µg/mL
Vehicle / solvent:
Controlsopen allclose all
Negative solvent / vehicle controls:
Positive controls:
without S9
Positive control substance:
mitomycin C
Migrated to IUCLID6: 0.25 & 0.75 µg/mL
Positive controls:
with S9
Positive control substance:
Migrated to IUCLID6: 7.5 & 37.5 µg/mL
Details on test system and experimental conditions:
c.a. 24 hr before treatment, cells were initiated at a density of 1.2-1.75 x 10^6 cells/75 cm2 flask.
Trials without S9: cells incubated with appropriate control or chemical for 8 hr. Cells then washed and Colcemid added for a 2-2.5 hr exposure.
Trials with S9: cells treated with S9 and test chemical in serum-free medium for 2 hr, washed, resuspended in medium containing serum, and incubated for an additional 8-10 hr, with Colcemid present for the final 2 hr.
Cells were harvested by mitotic shake-off and stained with Giemsa.
Evaluation criteria:
Cells with good morphology and with a chromosome number of 21 ± 2 selected for analysis. 200 cells/dose scored
Scoed for "simple" (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), "complex" (interstitial deletions, triradials, quadriradials, rings, and dicentrics), and `other' (pulverized, polyploids, and endoreduplications) aberrations. These categories were combined to form the category "total".
Statistical analysis was conducted only on "total" aberrations. The percent of cells with aberrations, rather than aberrations per cell, were analyzed to avoid distorting the data for cases where a small number of cells had a large number of aberrations. Gaps and endoreduplications were scored but not tabulated in the totals or included in the statistical analyses. The data were evaluated for both trend and dose point increase over the solvent control. A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett's method were considered significant if <0.05, whereas a trend of P < 0.003 was significant.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
at higher doses than those tested
Vehicle controls validity:
Positive controls validity:
Additional information on results:
Harvest time was 12 and 10 hours with and without S9 activation respectively.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Mixed xylenes did not induce cytogenetic change in Chinese hamster ovary cells using an assay to detect chromosome aberration.
Executive summary:

Mixed xylenes did not induce cytogenetic change in Chinese hamster ovary cells using an assay to detect chromosome aberration at a concentration of 50.5 µg/mL with activation and 100.5 µg/mL without activation.