Data
(f) Data and reporting(1) Treatment of results. (i) Data shall include cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. In the case of a positive response in the L5178Y TK =/ test, colonies are scored using the criteria of small and large colonies on at least one concentration of the test substance (highest positive concentration) and on the negative and positive control. The molecular and cytogenetic nature of both large and small colony mutants has been explored in detail and is discussed in the references under paragraphs (g)(22) and (g)(23) of this section. In the TK =/ test, colonies are scored using the criteria of normal growth (large) and slow growth (small) colonies (a scoring system similar to the one described in the reference under paragraph (g)(24) of this section may be used). Mutant cells that have suffered the most extensive genetic damage have prolonged doubling times and thus form small colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible chromosome aberrations. The induction of small colony mutants has been associated with chemicals that induce gross chromosome aberrations. Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies. (ii) Survival (relative cloning efficiencies) or relative total growth shall be given. Mutant frequency shall be expressed as number of mutant cells per number of surviving cells. (iii) Individual culture data shall be provided. Additionally, all data shall be summarized in tabular form. (iv) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing, and the metabolic activation conditions. (2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. (ii) A test substance, for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this system. (iii) Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. (iv) Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration-response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used. (3) Test report. The test report shall include the following information: (i) Test substance: (A) Identification data and CAS no., if known. (B) Physical nature and purity. (C) Physicochemical properties relevant to the conduct of the study. (D) Stability of the test substance. (ii) Solvent/vehicle: (A) Justification for choice of vehicle/solvent. (B) Solubility and stability of the test substance in solvent/vehicle, if known. (iii) Cells: (A) Type and source of cells. (B) Number of cell cultures. (C) Number of cell passages, if applicable. (D) Methods for maintenance of cell cultures, if applicable. (E) Absence of mycoplasma. (iv) Test conditions: (A) Rationale for selection of concentrations and number of cell cultures including e.g., cytotoxicity data and solubility limitations, if available. (B) Composition of media, CO2 concentration. (C) Concentration of test substance. (D) Volume of vehicle and test substance added. (E) Incubation temperature. (F) Incubation time. (G) Duration of treatment. (H) Cell density during treatment. (I) Type and composition of metabolic activation system including acceptability criteria. (J) Positive and negative controls. (K) Length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate). (L) Selective agent(s). (M) Criteria for considering tests as positive, negative or equivocal. (N) Methods used to enumerate numbers of viable and mutant cells. (O) Definition of colonies of which size and type are considered (including criteria for small and large colonies, as appropriate). (v) Results: (A) Signs of toxicity. (B) Signs of precipitation. (C) Data on pH and osmolality during the exposure to the test substance, if determined. (D) Colony size if scored for at least negative and positive controls. (E) Laboratory's adequacy to detect small colony mutants with the L5178Y TK =/ system, where appropriate. (F) Dose-response relationship, where possible. (G) Statistical analyses, if any. (H) Concurrent negative (solvent/vehicle) and positive control data. (I) Historical negative (solvent/vehicle) and positive control data with ranges, means, and standard deviations. (J) Mutant frequency. (vi) Discussion of the results. (vii) Conclusion.