Report from the in vitro micronucleus assay working group.
At the Washington "2nd International Workshop on Genotoxicity Testing" (25-26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth "3rd International Workshop on Genotoxicity Testing" (28-29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7.


METHODS
statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.
Kirsch-Volders
Micheline
M
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium. mkirschv@vub.ac.be
Sofuni
Toshio
T
Aardema
Marilyn
M
Albertini
Silvio
S
Eastmond
David
D
Fenech
Michael
M
Ishidate
Motoi
M
Kirchner
Stephan
S
Lorge
Elisabeth
E
Morita
Takeshi
T
Norppa
Hannu
H
Surrallés
Jordi
J
Vanhauwaert
Annelies
A
Wakata
Akihiro
A
D000818:Animals; D001681:Biological Assay / Q000592:standards*; D002455:Cell Division / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D048629:Micronuclei, Chromosome-Defective; D009152:Mutagenicity Tests / Q000592:standards