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Guideline

Standard for Investigation by Chromosome Aberration Test
using Cultured Mammalian Cells

Attached paper 1

In Japanese
Attached paper 1
Standard for Investigation by Chromosome Aberration Test
using Cultured Mammalian Cells
1. Kind of Chromosome Aberration Test
(1)   Chromosome aberration test using cultured mammalian cells shall be tests by short-term treatment process and by continuous treatment process.
(2)   Tests by short-term treatment process and by continuous treatment process shall be carried out for identifying substances that cause chromosome aberration.
(3)   Cell growth inhibition test shall be a test for determining the highest dose level of the test substance, for the test by short-term treatment process and by continuous treatment process.
2. Method of Chromosome Aberration Test
(1)   Cell growth inhibition test and test by short-term treatment process shall be carried out with and without metabolic activation system (which means a system in which an auxiliary factor such as a coenzyme is added to supernatant fraction of homogenate of animal liver treated to induce a drug metabolizing enzyme system).
(2)   If it is judged as negative in a test by short-term treatment process, a test by continuous treatment process shall be carried out.
(3)   The test by continuous treatment process shall be carried out without the metabolic activation system.
3. Cells used for Chromosome Aberration Test
  Cells used for tests by short-term treatment process and by continuous treatment process shall be the primary, or stock cultures or cell-line of Chinese hamster fibroblasts, human peripheral lymphocytes or other cultured mammalian cells.
4. Dose Levels of Test Substance
  The highest dose level of the test substance must conform to the provisions as set forth below.
(1)   The highest dose level of the cell growth inhibition test shall be 5 mg/ml or 10 mM, whichever is lower.
(2)   The highest dose level of the test by short-term treatment process and by continuous treatment process shall be as follows.
a.   The dose level at which 50 or more inhibition of cell growth is clearly observed, when 50% or more inhibition of cell growth is clearly observed in the cell growth inhibition test
b.   The dose level of 5 mg/ml or 10 mM, whichever is lower, when 50% or more inhibition of cell growth is not observed in the cell growth inhibition test (excluding the case mentioned in Item c)
c.   The dose level at which precipitation is observed, if clear inhibition of cell growth is not observed in the cell growth inhibition test and precipitation of the test substance is observed when treatment is completed.
(3)   Dose levels shall be set that enable chromosome analysis in 3 or more stages at appropriate intervals
5. Control Substance
  Control substances in tests by short-term treatment process and by continuous treatment process shall be a solvent used for dissolving the test substance for negative control and a proper known substance which induces chromosome aberration for positive control.
6. Number of Plates Used
  The number of plates used for tests by short-term treatment process and by continuous treatment process shall, in principle, be 2 or more, for each dose level of test substance as well as negative control and positive control.
7. Observation and Records
(1)   In tests by short-term treatment process and by continuous treatment process, slide samples must be coded and observed under the condition that treatment conditions cannot be identified.
(2)   With respect to chromosome structural aberration, 200 or more well-spread metaphase cells in which the number of chromosomes is equal to mode ± 2 shall be observed for each dose level, and the number of cells with chromosome structural aberration and the number of cells in accord with the kind of aberration must be recorded.
(3)   With respect to the chromosome numerical aberration, 200 or more well-spread metaphase cells must be observed and incidence of polyploids must be recorded.
8. Judgment of the Results
(1)   Positive determination is given, when the appearance frequency of cells with chromosome structural aberration and numerical aberration apparently increases as compared to the negative control and in its action the dose dependency or reproducibility is observed.
(2)   When it is impossible to give a clear positive or negative determination, a confirmation test must be carried out under proper experimental conditions.
9. Application
  This standard shall be applied on and after April 1, 1998.


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