Modified Ames Test

How it works: In developing the assay, the standard Ames mutagenicity assay was modified to provide maximum sensitivity to the class of compounds chiefly responsible for the mutagenicity and carcinogenicity of petroleum oils, polycyclic aromatic compounds (PAC). A series of oils already evaluated in the mouse skin-painting bioassay was tested using the modified mutagenicity test, and their mutagenic responses quantified by regression analysis of the dose responses. The slopes resulting from these analyses, termed Mutagenicity Indices, or MIs, were then correlated with analogous Carcinogenicity Indices (CIs) calculated from the tumor responses seen in the skin-painting bioassays. The standard curve thus derived was used to establish an MI threshold above which an oil would be expected to demonstrate statistically significant carcinogenic activity in a skin-painting assay. Since the correlation between MI and CI holds because both the mutagenicity and carcinogenicity of oils are mediated largely, if not exclusively, by PACs, the Modified Ames Test's applicability is limited to test materials containing these compounds. This category includes petroleum refinery streams, as well as used or re-refined oils, coal-derived tars and oils, and formulated products comprised in part of such materials, provided there are no components present in the formulation that interfere with the performance or interpretation of the test.

The endpoint: The endpoint of the Modified Ames Test is Mutagenicity Index or MI. It is the slope of the dose-response curve for mutagenicity, as determined using linear regression analysis. MIs can range from zero for highly refined oils to greater than 1,200 for certain catalytically cracked stocks.

Interpretation of MI: Correlation of MI with dermal carcinogenic potential in the mouse skin-painting bioassay has shown that most oils with MIs greater than 1.0 are carcinogenic, while most with MIs less than 1.0 are not. The exception to the latter specification is the class of oils known as bright stock extracts or residual aromatic extracts which, to be certified non-carcinogenic in the European Union, must have MIs less than 0.40.

Logistics: We prefer that samples submitted for Modified Ames testing be no larger than 25 ml. Minimum sample size for a definitive MI determination is 0.2 ml, but limited data can be obtained on samples as small as 0.05 ml, if a known volume of the oil is premeasured into a container suitable for extraction using a five-fold volume of DMSO relative to sample size. Assay turnaround time depends on workload, but when sponsors require results on a rush basis, we can usually accommodate by offering seven-day-a-week testing and reporting services at no additional charge. Results, in the form of MIs, are provided to the sponsor by e-mail upon completion of testing. Detailed written reports (also sent by e-mail) usually follow within a month or two, but can also be supplied on an expedited basis as needed. Paper copies of reports are available upon request. We welcome inquiries regarding customized report formats or data analyses, and are happy to provide tutorials on the use and interpretation of the Modified Ames Test or our other tests as part of our consulting services.

Work Product: PetroLabs conducts Modified Ames Tests in strict accordance with ASTM Method E 1687 10. Consistent with that protocol, we test a full dose range of the reference oil (positive control) with each assay to ensure that the metabolic performance of the S-9 preparation used is within the acceptable historical range. We pride ourselves on providing only unequivocal MI data to our sponsors. If any of the control parameters for a given test are out of range or the dose-response curve for mutagenicity cannot be well fit (e.g. R^2 < 0.90 for active materials), we repeat the test at no additional cost. We conduct the Modified Ames Test by the most rigorous scientific standards and laboratory practices, but we do not adhere to all the documentation and quality assurance provisions of the Good Laboratory Practice (GLP) guidelines. Therefore, at present, our studies are intended for sponsor information only, and are not suitable for regulatory submissions, if the agency in question requires full GLP adherence.