M. Aida et al., PROTOCOL RECOMMENDED BY THE CSGMT JEMS.MMS FOR THE SHORT-TERM MOUSE PERIPHERAL-BLOOD MICRONUCLEUS TEST/, Mutagenesis, 10(3), 1995, pp. 153-159
Although the target cells for the bone marrow (BM) and peripheral bloo
d (PB) micronucleus tests are the same, erythroblasts, the PB method o
ffers important advantages over the BM method. We propose a protocol f
or the shortterm peripheral blood micronucleus test. This assay is int
ended primarily for the identification of a wide variety of chemical c
lastogens and spindle poisons, and secondarily for risk assessment. Th
e recommended experiment size, seemingly small, has adequate detection
power. Experimental results obtained from Collaborative Study Group f
or the Micronucleus Test (CSGMT) studies and data collected from a sur
vey of the literature provided the basis of the proposed protocol. Our
protocol, designed for mice, includes the following features. (i) The
maximum tolerated dose (MTD) is determined experimentally with a smal
l number of animals treated i.p. or per os (or by other routes, if cal
led for) in a dose-finding test, which can be conducted simultaneously
with tests for finding both number of treatments and optimal sampling
time. (ii) At least three groups of five mice (at least four effectiv
e animals per group), males or females, are given i.p, or per os doses
of, for example, the MTD, 1/2 MTD and 1/4 MTD once, twice or more, 24
h apart. (iii) Peripheral blood samples are taken before treatment (t
he 0 time control) and twice at 48 and 72 h for a single treatment, on
ce between 24 and 36 h after the second treatment for double treatment
s, or once 24 h after the final treatment for multiple dosing. Or, if
an optimal sample time is established in a preliminary test, samples a
re taken at that time, (iv) Samples are stained with acridine orange,
and 2000 immature erythrocytes per animal are examined. (v) The combin
ed data of 0 time samples are the negative control. When statistical a
nalysis shows that the negative control is out of the historical contr
ol range or experimental results are equivocal, a repeat test is neede
d.