6.4.2.Chromosomal Aberration Test

Test Description

This assay evaluates potential mutagenic properties which cause structural changes to the chromosome in a mammalian cell line from the Chinese hamster ovary (CHO). The chromosomes are examined for aberrations during metaphase of the cell cycle, with or without mammalian chromosome activation (rat liver S-9 fraction) following the methodology of Galloway et al. (1985). Sample mutagenicity is confirmed by determining a dose-response curve for any presumptive positive response.

The test article was extracted in McCoy’s 5A Medium. A monolayer of CHO cells was exposed to the test article extract in triplicate cultures and in the presence and absence of S9 metabolic activation. Parallel testing was also conducted with a negative and positive control. Culture medium was used as the negative control and the positive control was mitomycin C (MMC) in the absence of S9 and cyclophosphamide (CP) in the presence of S9.

Test Results

Under the conditions of this assay, the extract was not considered genotoxic to Chinese Hamster Ovary cells in the presence or absence of S9 metabolic activation. As expected, positive controls showed significant evidence of genotoxic activity.

6.4.3. Sister Chromatid Exchange

Test Description

This assay evaluates potential mutagenic properties in device extracts that could cause primary DNA damage in the cell. Differential staining of labeled sister chromatids is used to identify damage. Tests are run with and without mammalian microsome activation (rat liver S-9 fraction) according to the methodology of Galloway et al. (1985). Potential sample mutagenicity is confirmed by constructing a dose-response curve on any positive response.

A monolayer of CHO cells was exposed to the test article extract in triplicate cultures in the presence and absence of S9 metabolic activation. Parallel testing was also conducted with negative and positive controls. Culture medium was used as the negative control and the positive control was mitomycin C (MMC) in the absence of S9 and cyclophosphamide (CP) in the presence of S9.

Test Results

Under the conditions of this assay, the test extract was not considered genotoxic to Chinese Hamster Ovary cells in the presence or absence of S9 metabolic activation. The negative and positive controls performed as expected.

2.2.2.1.4. Cytotoxicity Testing in the l-929 Mouse Fibroblast Cell Line

The purpose of cytotoxicity testing is to assess and measure cell damage, changes in cell growth and changes in cellular metabolism. Two ways have been identified to conduct these evaluations in cell cultures: tests using extracts from the material and tests using direct contact of the material with the cultured cells. The importance of using materials in a form as close to the final product has been emphasized by testing guidelines. The laboratory with whom we have worked (NAmSA) recommended using a minimum essential medium (MEM) elution method using a serum-based extraction medium to maintain the culture. Direct contact methods were not considered to be as sensitive or reliable as MEM methods.

Test Description

An extract of leachables from the test article was prepared using Minimum Essential Medium (MEM). This test extract was placed onto a confluent monolayer of L-929 mouse fibroblast cells. Separate flasks were prepared for duplicate negative controls and for a positive control (end-point titration procedure). The monolayers in both test and negative control flasks were examined microscopically at 24, 48, and 72 hours to determine any change in cell morphology. The monolayer in the positive control flasks was examined at 24 hours and the result was compared to the NAmSA historical value.

Test Results

Under the conditions of this study, the MEM test extract showed no evidence of causing cell lysis or toxicity. Negative and positive controls performed as expected.