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Genetic toxicity: in vitro

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in vitro DNA damage and/or repair study
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
EU Method B.19 (Sister Chromatid Exchange Assay In Vitro)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells

Test material

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:
with and without S9: 0, 5.0, 16.7, 50.0 µg/mL
Vehicle / solvent:
Controlsopen allclose all
Negative solvent / vehicle controls:
Positive controls:
without S9
Positive control substance:
mitomycin C
Migrated to IUCLID6: 0.0010 & 0.0100 µg/mL
Positive controls:
with S9
Positive control substance:
Migrated to IUCLID6: 0.40 & 2.00 µg/mL
Details on test system and experimental conditions:
Cultures at a density of 1-1.25 x 10^6 cells/75 cm2 flask were initiated 24 hr prior to treatment.
For tests without S9, the test and positive control chemicals were added to flasks which were incubated at 37°C for 2 hr prior to the addition of bromodeoxyuridine (BrdUrd) (10-5M) and then for a further 24 hr. After this time, the cells were washed and medium containing BrdUrd and Colcemid added and incubated for an additional period of 2-2.5 hr.
For tests with S9, cells in serum-free medium were treated with S9 and the test chemical or appropriate control and then incubated at 37°C for 2 hr. After this time, the cells were washed and medium containing serum and BrdUrd was added to the flasks. Colcemid was added to the flask after 24 hours of incubation. A further period of incubation for 2-2.5 hr followed.
For both tests, a visual estimate of the confluency of each flask was made for signs of toxicity. Mitotic cells were harvested and the medium returned to the original flasks which were reincubated in case a delayed harvest was required. The harvested mitotic cells were treated with hypotonic buffer and suspended in fixative. Slides were made, air-dried, stained using Hoechst 33258, rinsed and mounted. The ratio of first-division metaphase (Ml) to M2 cells was determined. Where cell cycle delay was observed, the cells from the original flask were harvested 4-5 hr following the original harvest. Where cell cycle delay was anticipated, the cells were harvested approximately 30-34 hr following the addition of BrdUrd, with Colcemid present for the final 2 hr.
Evaluation criteria:
Cells with good morphology and with a chromosome number of 21 ± 2 were selected for analysis. 50 cells per dose were scored.
The data were evaluated for both trend and dose point increase over the solvent control. In the SCE assay a trend of P < 0.005 or an individual dose with a 20% increase over the solvent control were considered significant. 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 25-29 hours after addition of BrdUrd.
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 sister chromatid exchange.
Executive summary:

Mixed xylenes did not induce cytogenetic change in Chinese hamster ovary cells using an assay to detect sister chromatid exchange at a concentration of 50.0 µg/mL both with and without metabolic activation.