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Standard for Investigation by Mutagenicity Studies using Microorganisms Notification for prescribing the standard set forth by Minister of Health, Labour and Welfare pursuant to the provisions of Paragraph 1 of Article 57-3 of Industrial Safety and Health Law In Japanese
Standard for Investigation by Mutagenicity Studies using Microorganisms
Sept. 1, 1988 ML Notification No. 77 Amended on June 2, 1997 ML Notification No. 67 Amended on December 25, 2000 ML Notification No. 120 Pursuant to the provisions of Paragraph 1 of Article 57-3 of Industrial Safety and Health Law (Law No. 57 of 1972), the standard prescribed by Minister of Health, Labour and Welfare shall be set forth as follows and applied on and after October 1, 1988.
(Scope of Application)
Article 1 The present notification shall apply to the investigation by mutagenicity studies (limited to those using microorganisms. The same applied to hereinafter.) of the toxicity investigations as provided in Paragraph 1 of Article 57-3 of the Industrial Safety and Health Law (Law No. 57 of 1972. Referred to as the "Law" in the following paragraph).
2 Of the toxicity investigations as provided in Paragraph 1 of Article 57-3 of the Law, the standard for the investigation other than mutagenicity studies, etc. shall be prescribed by Director-General of Labour Standards Bureau of MHLW.
(Kinds of Mutagenicity Studies)
Article 1-2 The mutagenicity studies shall be carried out by the dose-range finding study and the main study.
2 The dose-range finding study shall be a study for determining the highest dosage of the test substance (which indicates chemical substance used in the study; the same applies hereinafter.) in the main study.
3 The main study shall be a study to investigate whether the test substance is mutagenic or not.
(Method of the Mutagenicity Studies)
Article 2 The dose-range finding study and the main study shall be carried out by the pre-incubation method, the plate method, or an equivalent method that makes it possible to obtain findings equal to or better than the two former methods.
2 The dose-range finding study and the main study shall be carried out with and without the metabolic activation system (which means a system in which an auxiliary factor such as coenzyme is added to supernatant fraction of homogenate of animal liver treated to induce a drug metabolizing enzyme system).
(Strains used for the Mutagenicity Studies)
Article 3 The bacterial strains used for the dose-range finding study and the main study shall be those listed in each following Item. However, if it is assumed necessary to carry out mutagenicity studies using bacterial stains other than these in view of the characters of the test substance, such strains must be added.
	1.	Salmonella typhimurium TA98
	2.	Salmonella typhimurium TA100
	3.	Salmonella typhimurium TA1535
	4.	Salmonella typhimurium TA1537, TA97 or TA97a
	5.	Escherichia coli WP2uvrA, Escherichia coli WP2uvrA (pKM101) or Salmonella typhimurium TA102
(Dose level of Test substance)
Article 4 The dose level of the test substance shall conform to those prescribed below:
	1.		The highest dose level for the dose-range finding study shall be 5 mg per plate.
	2.		The highest dose level per plate for the main study shall be the dose specified according to any of the following cases.
		a.	The dose level that indicates growth inhibition of the bacterial strain by test substance, if any growth inhibition of the bacterial strain by the test substance in the dose-range finding study was observed.
		b.	The dose level at which precipitation of the test substance is observed, if no growth inhibition is observed against the strain by the test substance in the dose-range finding study and precipitation of the test substance is observed.
		c.	5 mg, if no growth inhibition is observed against the strain by the test substance in the dose-range finding study and precipitation of the test substance is not observed.
	3.		Dose levels shall be set for at least five stages or more at proper intervals.
(Control substance)
Article 5 The control substance in the dose-range finding study and the main study shall be the solvent used for dissolving the test substance for the negative control and a proper known mutagenic substance for the positive control.
(Number of Plates Used)
Article 6 The number of plates used for the dose-range finding study and the main study shall be two or more for each dose level of the test substance, and for the negative control and the positive control, respectively.
(Observation)
Article 7 In the dose-range finding study and the main study, the state of growth inhibition of the strain by the test substance and the state of the precipitation of the test substance must be ascertained.
2 The ascertainment of the preceding paragraph shall be carried out during counting the number of colonies resulted from reverse mutation.
(Reproducibility)
Article 8 The results of mutagenicity studies must be reproducible.
(Particulars)
Article 9 In addition to what have been provided from Article 2 to the preceding Article, necessary particulars for performing the mutagenicity studies shall be prescribed by Director General, Labour Standards Bureau, MHLW.

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