National Toxicology Program

Two in vitro assays for chromosomal damage were conducted in cloned Chinese hamster ovary cells (CHO-W-B1) to identify chemicals capable of inducing chromosomal aberrations (CA) or sister chromatid exchanges (SCE) in mammalian cells. Procedures for both assays are described in detail by Galloway et al. (1985, 1987). The CA assay only detects structural chromosomal damage; it does not detect numerical chromosome changes (aneuploidy) induced by chemical exposure.

Test chemicals were supplied to the testing laboratory as coded aliquots by Radian Corporation (Austin, TX). They were tested in cultured CHO cells for induction of SCE and CA, both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least 3 doses of test chemical; the high dose was limited by toxicity or solubility, or in the absence of either of these factors, the high dose was limited to 5 mg/mL . A single culture flask per dose was used. Tests yielding equivocal or positive results generally were repeated.

The SCE Test

Sister chromatid exchanges are a measure of DNA damage and increased levels of DNA damage are associated with mutation induction and cancer. Assaying for SCE requires examining cells that have entered their second mitotic division after the initiation of chemical exposure. Therefore, in the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. 5-Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium with test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, without test chemical. Incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test chemical. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were scored blind and slides from a single test were read by the same person. Fifty second-division metaphase cells were scored to determine the frequency of SCE/cell for each dose level. If significant chemical-induced cell cycle delay was seen in treated cultures, the incubation time was lengthened to ensure the accumulation of a sufficient number of scorable (second-division metaphase) cells.

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response (Galloway et al., 1985). The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive); increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial. Positive and weak positive trials were repeated. The overall assay result was based on an evaluation of the responses in all trials within an activation condition.

The CA Test

Because many birth defects and most cancers are associated with abnormal chromosome complements, it is important to identify chemicals that can induce chromosome damage. To assess induction of CA, cells were harvested in their first mitotic division after the initiation of chemical exposure. Therefore, in the CA test without S9, cells were incubated for 8-12 hours with the test chemical in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics; Colcemid was added and incubation continued for 2 hours. The incubation time and the dose levels selected were determined from the information on cell cycling and toxicity obtained from the SCE test; if cell cycle delay was anticipated in the CA test, the incubation period was extended to permit accumulation of sufficient cells in first metaphase for analysis. The cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. For the CA test with S9, cells were treated with the test chemical and S9 for 2 hrs, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hrs. Cells were harvested in the same manner as for the treatment without S9.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred or two hundred first-division metaphase cells were scored at each dose level. The classes of aberrations that were recorded included "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations), and "other" (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Chromosomal aberration data are presented as the percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987). For a single trial, a statistically significant (P<0.05) difference for one dose point and a significant trend (P<0.015) was considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A strong trend (P < 0.003) with a single significant dose level was designated weak positive *, to indicate a high level of induced aberrations. A strongly positive trend (P < 0.003), in the absence of a statistically-significant increase at any one dose point, led to an equivocal call. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers. Trials that gave a weak positive or positive result were repeated. The overall result for the CA assay was based on an evaluation of the responses in all trials within an activation condition.

Galloway SM, Armstrong MJ, Reuben C. et al. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Mol. Mutagen. 10(Supplement 10): 1 - 175.

Galloway SM, Bloom AD, Resnick M et al. (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ. Mutagen. 7: 1-51.