Mutation Research 417 Ž1998.

19–30

Recommendations for statistical designs of in vivo mutagenicity

tests with regard to subsequent statistical analysis
Ilse-Dore Adler a,) , James Bootman b, John Favor a , Graham Hook c ,
Gerlinde Schriever-Schwemmer a , Gerhard Welzl d , Elbert Whorton e,
Isao Yoshimura f , Makoto Hayashi g
a
¨ Saugetiergenetik,
GSF-Institut fur ¨ D-85758 Neuherberg, Germany
b
Pharmaco-LSR, Eye, Suffolk IP23 7PX, UK
c
National Institute of EnÕironmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, USA
d
¨ Medizinische Informatik und Sytemforschung, D-85758 Neuherberg, Germany
GSF-Institut fur
e
DiÕision of Epidemiology and Biostatistics, UniÕersity of Texas Medical Branch J47, GalÕeston, TX 7555-1047, USA
f
Faculty of Engeneering, Science UniÕersity of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan
g
DiÕision of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1, Kamiyoga, Satagaya-ku, Tokyo 158, Japan
Received 12 February 1998; revised 15 June 1998; accepted 25 June 1998

Abstract

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherberg, Germany, to start a discussion of
experimental design and statistical analysis issues for three in vivo mutagenicity test systems, the micronucleus test in mouse
bone marrowrperipheral blood, the chromosomal aberration tests in mouse bone marrowrdifferentiating spermatogonia, and
the mouse dominant lethal test. The discussion has now come to conclusions which we would like to make generally known.
Rather than dwell upon specific statistical tests which could be used for data analysis, serious consideration was given to test
design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are
interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system.
Concerning the statistical sensitivity parameters, a type I error of 0.05 Žone tailed., a type II error of 0.20 and a dose related
increase of twice the background Žnegative control. frequencies were generally adopted. It was recommended that sufficient
observations Žcells, implants. be planned for each analysis unit Žanimal. so that at least one adverse outcome Žmicronucleus,
aberrant cell, dead implant. would likely be observed. The treated animal was the smallest unit of analysis allowed. On the
basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000
immature erythrocytesranimal should be scored for micronuclei from each of at least 4 animals in each comparison group in
the micronucleus assays. A minimum of 200 cells should be scored for chromosomal aberrations from each of at least 5
animals in each comparison group in the aberration assays. In the dominant lethal test, a minimum of 400 implants Ž40–50
pregnant females. are required per dose group for each mating period. The analysis unit for the dominant lethal test would be
the treated male unless the background frequency of dead implants ŽDI. is so low that multiple males would need to be

)
Corresponding author. Tel.: q49-89-3187-2302; Fax: q49-89-3187-3099; E-mail: adler@gsf.de

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 9 1 - 6
20 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30

integrated to meet the minimum observation of one adverse outcome ŽDI. per analysis unit. A three-step strategy of data
analysis was proposed for the cytogenetic assays. Use of negative historical controls was allowed in certain circumstances
for interpretation of results from micronucleus tests and chromosomal aberration tests. q 1998 Elsevier Science B.V. All
rights reserved.

Keywords: Experimental design; Statistical analysis; Micronucleus test in mouse bone marrowrperipheral blood; Chromosomal aberration
tests in mouse bone marrowrdifferentiating spermatogonia; Mouse dominant lethal test

1. Introduction response w7x that ‘statistical techniques are an essen-

During the Melbourne Workshop on Harmoniza-
tion of Test Guidelines it was decided to organize a
separate meeting in which the statistical considera-
tions of experimental design and the statistical treat-
ment of data could be discussed and harmonized.
The test systems considered during the statistics
workshop held at the GSF in Neuherberg, Germany,
on September 13 and 14, 1993, were in vivo mi-
cronucleus tests for bone marrow and peripheral
blood, tests for chromosomal aberrations in bone
marrow cells and differentiating spermatogonia, and
the rodent dominant lethal assay. Even though, some
years have passed between the statistics workshop
and the present publication, we are convinced that
these considerations are still of general value w1x.
A number of guidelines and reviews on the statis-
tics of the tests under consideration already exist
w2–5x. Therefore, the workshop focused on general
concepts for the design of experiments rather than
specific statistical methods for data analysis of each
test system. To approach this, we analyzed data
bases which were provided for this workshop and
characterized the nature of the data for each test.
2. General comments
The following statements represent a description
of the framework within which any statistical design
can be applied.
First, statistics can provide guidance for objective
decision making, however, final conclusions have to
be based on biological judgements. Even though
Ashby and Tinwell w6x suggested that there was no
need for statistics in their sequential approach of the
micronucleus test, we agree with Festing and Lovell’s
tial tool in the interpretation of results’ w8x. We go
even one step further in emphasizing that experimen-
tal design should be based on statistical considera-
tions w9x.
Second, the smallest unit for statistical compar-
isons is the treated animal. Limited animal resources
and animal protection criteria indicate that it is im-
portant to design the experiment with the minimal
but optimal animal number using statistical consider-
ations.
Third, the design of the experiment has to be
based on the a-level Žtype I error. for recognizing a
predetermined increment over the spontaneous level
with an acceptable statistical power Ž1-type II error..
Fourth, all experiments must have a concurrent
negative control and all concurrent negative controls
must be added to the negative historical control data
base.
Concurrent negative controls are essential for
) controlling the technical conduct of the ex-
periment and
) appropriate statistical treatment of the data.
Historical negative controls when available are the
basis for
) designing the experiment and
) accepting a test Žby comparing concurrent
negative control values to the historical negative
control, Fig. 1, step 1..
Lastly, the increment in mutational events over
the spontaneous level to be recognized is determined
by biological criteria rather than by purely statisti-
cally achievable precision.
With the tests under study, the ability to detect a
doubling of the observed endpoint at an a-level of
0.05 and a power of 80% was considered to be an
easily achievable minimum standard. The sample
size should be large enough to assure that at least
 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30 21

Fig. 1. Strategy of statistical evaluation of test data for micronucleated PCE and aberrant bone-marrow cells. The strategy applies for data
which contain several doses and a negative control. Historical negative controls serve two purposes; first to accept or reject a set of data, and
second, to evaluate individual data points by pair-wise comparison.

one adverse event Žmicronucleus, aberrant cell, dead
implant. will be recorded for each analysis unit. It
should be generally required that a clear description
of the design of the experiment including the power
of the test, the statistical methods used to analyse the
data and the level of significance actually achieved is
required in every data report.
3. Micronucleus test
The analysed variables are firstly, the proportion
of immature erythrocytes with micronuclei ŽMN. and
secondly, for bone marrow toxicity, the proportion of
immature erythrocytes among total erythrocytes.
3.1. Historical control data analyzed
The historical control data of the micronucleus
test have been provided by 12 laboratories Ž7 from
Japan, 2 from UK, 2 from USA, and 1 from Ger-
many. and are comprised of 1299 experiments with a
total of 6883 mice ŽTable 1.. In most laboratories,
the Poisson index of dispersion was approximately
1.0 as expected with two exceptional laboratories, A
and K ŽTable 1.. The data of laboratory K could
deviate from the theoretical distribution because of
the rather small number of groups. The reason, how-
ever, for the small Poisson index for the data of
laboratory A is not clear. Although there were these
two exceptions, it can be concluded that the frequen-
cies of spontaneous micronucleated immature ery-
throcytes in negative Žno treatment or vehicle. con-
trols follow a Poisson distribution Ža binomial distri-
bution, as well. thus the differences among animals
are minimal.
The results of the F-test on the micronucleus data
excluding the two groups with exceptional factors
are summarized in Table 2. Data for the F-test were
not transformed because some data were only pro-
vided as percent micronucleated cells. A preliminary
trial with selected data showed no difference be-
tween the results of F-tests with square-root trans-
formed and non-transformed data. When F-ratios are
relatively small then these factors do not seriously
affect the background level. However, there are sev-
eral factors affecting the frequencies of micronucle-
ated immature erythrocytes, i.e., years when data
were obtained Žpossibly by different investigators.,
strains of mice, and vehiclersolvents.
22 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30

Table 1
Characteristics of the historical negative control data provided from micronucleus assays
X
Laboratory Total no of Total no of No. of animals per group Frequencies of MNPCE Mean T
animals groupsa Mean Max Min Mean S.D. Max Min
A 555 119 47 8 2 0.187 0.071 0.500 0.067 0.585
B 357 62 5.8 7 2 0.127 0.076 0.317 0.000 1.083
C 483 84 5.8 6 3 0.214 0.076 0.440 0.083 0.945
D 122 20 6.1 15 2 0.216 0.054 0.233 0.050 0.980
E 470 84 5.6 6 4 0.122 0.049 0.240 0.025 1.090
F 1026 193 6.3 12 1 0.278 0.087 0.600 0.000 0.903
G 395 69 5.7 6 4 0.208 0.067 0.400 0.075 1.021
b
H 601 121 5.0 5 3 0.185 0.074 0.380 0.020
I 599 120 5.0 5 4 0.090 0.053 0.244 0.000 1.041
J 1182 234 5.1 7 4 0.073 0.039 0.218 0.010 0.918
K 222 34 6.5 9 5 0.133 0.033 0.200 0.071 0.743
L 871 159 5.5 10 3 0.175 0.067 0.350 0.030 1.093
Total 6883 1299
a
Males and females in an experiment were considered as different groups.
b X
T could not be calculated because data were provided in percent with a significant figure of 2.
X
T : ŽPoisson index of dispersion.rŽgroup size minus 1..

On that basis, historical negative controls should the criteria necessary for quality of the historical
be compiled separately for strains and vehicles within control data are met, these data may be combined
each laboratory and it should comprise at least 10 over vehicles and time. To assure the quality of the
separate experiments. It was agreed that as long as historical control data there must exist no systematic

Table 2
Summary of F-tests of the historical negative control data provided from micronucleus assays
Laboratory Year Strain Vehicle Route No. of Sample time Cell type Sex No. of cells
treatments analysed
per animal
A ? q " y q q y " 1000
B ? BDF1 q q y y B " 1000
C ? BDF1 y y y q B y 1000
D " CD-1 y y y 24 B M 1000
E ? q y y y q B M y
F ? q nt nt $ $ P q 1000
G " ddY q " y y B M 1000
H q Swiss " po " y q y D
I ? ICR q po q q B y 1000
J ? CD-1 " ? q y B q q
K ? 102 = C3H y ip 1 24 B y 2000
L q B6C3F1 " ip 3 " B M 2000

q: Significant; ": Ambiguous; y: Not significant; ?: No data available; D: Probably identical numbers of cells were evaluated; nt: No
treatment, therefore ‘Number of treatments’ and ‘Sample time’ do not apply Ž$..
When name of strain, treatment Žpo or ip., Number of treatments Ž1 or 3., sample time Ž24., cell type ŽB s bone marrow or P s peripheral
blood., sex ŽM s males., and number of cells analysed per animal Ž1000. appear in the table, these factors were used exclusively in the
respective laboratory.
 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30
differences between the various control groups, cur-
rent and historical w10x. Factors in achieving the
above include:
1. The method of scoring the response must be
unchanged during the relevant period.
2. The experimental units must be comparable
throughout the period.
3. The experimental protocol must have remained
fixed throughout the period covered by the histor-
ical data and the current experiment.
3.2. Size of the experiment
On the basis of the historical control data Žaround
1–2 MNr1000 immature erythrocytes. a per animal
sample size of 1000 immature erythrocytes is insuffi-
cient to detect at least one micronucleus per animal.
To avoid too many zero counts a minimum of 2000
immature erythrocytes per animal should be scored.
The minimum number of animals is 4 per group if
the historical control is 1.5 MNr1000 ŽTable 3 and
Appendix A.. The protocol described above is suffi-
cient to detect at least 80% of chemicals which
induce a 2-fold increase in micronucleated immature
erythrocytes over the historical control level at the
significance level of 0.05 ŽTable 3, Fig. 3.. If a
laboratory does not follow this sample size recom-
mendation it should justify their own experimental
design and report the power of detection and the
assayed increment over the negative control.
3.3. Statistical methods to be proposed
An experiment typically comprises a negative
Žsolvent. control group, three dose groups and a
positive control group w11x. Three consecutive steps
Table 3
Power Ž%. for the micronucleus assay based on the mean frequencies of micronucleated cells in the negative controls under the given
sample size to detect a doubling of the spontaneous frequency with a significance of 5%
Percent of MN cells Number of animals per group Ž2000 cells per animal.
in negative 2 3
historical controls
0.10 42.9
0.15 63.6
0.20 73.1
0.25 82.1
0.30 87.1
23
of statistical evaluation are proposed w12,13x. The
steps are illustrated in Fig. 1.
Ž1. Acceptance of the test: A test is acceptable
only if the mean of the concurrent negative control is
within the mean " 3SD of the historical control,
where SD relates to the interanimal variability. Non-
acceptance requires a new experiment Žtest..
Ž2. Dose–response analysis: When accepted, the
data are evaluated by an appropriate trend analysis
using the concurrent negative control data w22x. A
significant positive trend test signifies an effect of
the treatment.
Ž3. Evaluation of individual group responses: Each
treated group mean is then evaluated by pair-wise
comparison to the historical negative control. If none
of the individual treated groups shows a significant
difference but significance was shown in the trend
test the interpretation requires biological judgement.
The same applies to data which show no trend but a
significant difference between an individual treat-
ment group and the historical negative control pro-
vided the pair-wise evaluation was corrected for
multiplicity of comparisons w14x. The test may then
be considered equivocal.
The importance of the historical control mean and
variance makes it necessary that both should be
reported. We agree with Margolin and Risko w5x that
historical control can be easily misused without
proper safeguard Žsee Section 3.1..
This strategy is an illustration of an approach
rather than a recommendation and laboratories should
feel free to choose their own approaches as long as
they justify them adequately.
The procedure described here is applicable only to
the data of the frequencies of micronucleated imma-
4 5 6
65.2 73.4 82.8 86.4
78.4 86.3 91.8 96.2
86.7 93.2 96.6 98.6
91.7 96.6 98.7 99.4
95.8 98.3 99.5 99.8
24 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30

ture erythrocytes and not to the ratio of the immature
erythrocytes to total erythrocytes. The latter can be
approximated by a Gaussian distribution and stan-
dard methods of statistical analysis are acceptable
w4x.
4. Chromosomal aberration tests
full potential, i.e., the variables analysed should in-
clude both the
) percentage of damaged cells, and
) the types of aberrations observed, i.e., gaps,
breaks and exchanges.
The more laborious and skill-demanding cytoge-
netic test is best used for validationrcharacterization
purposes.

The following considerations pertain to the analy-
sis of structural chromosome aberrations in bone
marrow cells as well as in differentiating spermato-
gonia. To make the effort of scoring all types of
aberrations meaningful the test should be used to its
4.1. Historical control data analyzed
The historical control data of the chromosomal
aberration test have been provided by 2 laboratories
from the USA and are comprised of 135 experiments

Fig. 2. Comparison of negative control data from the bone marrow chromosomal aberration tests in two laboratories over time
Žmeans" SEM..
 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30 25

with 1073 mice. Vehicles, means and variances are

summarized in Table 4. The analysis of the historical
control data indicated that laboratory, vehicle, and
year contribute to total variability ŽFig. 2 and Table
4.. Thus, historical negative controls have to be
compiled separately for strains and vehicles within
each laboratory and it should comprise at least 10
separate experiments. The same criteria for historical
data quality apply as in the micronucleus test.

4.2. Size of the experiment

As in the micronucleus test, an experiment typi-

cally comprises a negative Žsolvent. control group,
three dose groups and a positive control group.
On the basis of the historical control data Žaverag-
ing 1.0% damaged cells., a per animal sample size of
100 cells is insufficient to detect at least one aberrant
cell per animal. To avoid too many zero counts, a
minimum of 200 cells per animal should be scored.
The minimum number of animals is 5 per compari-
son group. This sample size is sufficient to detect at
least 80% of chemicals which induce a 2-fold in-
crease in aberrant cells over the historical control
level of 1.0% and above at the significance level of
0.05 ŽTable 5, Fig. 4.. If a laboratory does not follow
this recommendation they should justify their own
experimental design and report the power of detec-
tion and the assayed increment over the negative
control.
Since the background frequency for exchanges is
below 1r1000 cells, statistical evaluation of ex-
change frequencies in treated groups has to rely on
historical negative controls. However, until the ex-
change rate in the treatment group is very high the
assay will not show anything with its presently rec-
ommended size. It would require 25 000 cells per
Table 4
The historical negative control data for the bone marrow chromo-
somal aberration test Žpercent of aberrant bone marrow cells.
Laboratory Vehicle No. of Mean Variance
animals
1 corn oil 366 0.69 0.76
buffered saline 120 0.77 0.74
2 corn oil 428 1.19 1.60
buffered saline 159 0.89 1.37
Fig. 3. The type I error and the power of the proposed three-step
procedure for the micronucleus assay. For the simulation, dose
levels were set as Ž d 0 , d1 , d 2 , d 3 . s Ž0, 1, 2, 4. and the assumed
population proportions of micronucleated cells of historical con-
trols as p s 0.1%Ž`., p s 0.15% Žv ., p s 0.2% ŽI., and
p s 0.25% ŽB.. The dose responses were assumed as Žp 0 , p 1 ,
p 2 , p 3 ,. s Žp , p , p , p . for the type I error and as Ž1=p ,
1.25=p , 1.5=p , 2.0=p . for the power. A total of 5000 quasi
experiments were performed in the present simulation study. To
keep the family-wise type I error at 5%, the nominal significance
levels for step 2 ŽCochran–Armitage trend test. and step 3 Žcom-
parison with a historical control. were set as 20% and 5% Ž15r3.,
respectively.
group to detect a doubling over a control rate of 1
exchange in 1000 cells. Using 200 cells from each of
5 animals per group would only have a power of
15–20% to detect a doubling over the background
rate.
4.3. Statistical methods for data eÕaluation
The same three consecutive steps of statistical
evaluation as for the micronucleus data are recom-
mended. However, if a different experimental design
is used and justified, i.e., employing only one or two
dose groups, the statistical evaluation may be re-
stricted necessarily to pair-wise comparisons be-
tween treated and concurrent negative controls w4x.
5. Dominant lethal test
The performance of dominant lethal experiments
can serve either to identify germ cell mutagens or to
26 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30

Table 5
Power Ž%. for the chromosomal aberration assay based on the mean frequencies of aberrant cells in the negative controls under the given
sample size to detect a doubling of the spontaneous frequency with a significance of 5%
Percent of aberrant Number of cells per group
cells in negative 400 600 800 1000 1200 1400 1600 1800
historical controls
0.5 23.8 38.9 43.9 56.0 66.0 65.8 73.8 78.7
1.0 44.7 66.7 73.0 83.5 86.6 91.3 93.6 96.2
1.5 65.8 78.9 87.4 92.4 95.6 97.2 98.7 99.1
2.0 75.3 94.2 95.0 96.5 98.6 99.3 99.4 99.9

characterize mutagenic effects in germ cells. De-
pending on the purpose, continuous treatment of all
developmental stages from meiosis to mature ga-
metes followed by a short mating period or single
acute treatment followed by sequential matings are
commonly used. The statistical treatment of domi-
nant lethal data has been described by various au-
thors w2,15–20x. The present recommendations of the
statistical test design are directed towards the classi-
Fig. 4. The type I error and the power of the proposed three-step
procedure for the bone marrow chromosomal aberration test. For
the simulation, dose levels were set as Ž d 0 , d1 , d 2 , d 3 . s Ž0, 1, 2,
4. and the assumed population proportions of micronucleated cells
of historical controls as p s 0.5% Ž`., p s1.0% Žv ., p s1.5%
ŽI., and p s 2.0% ŽB.. The dose–responses were assumed as
Žp 0 , p 1 , p 2 , p 3 ,. s Žp , p , p , p . for the type I error and Ž1=p ,
1.25=p , 1.5=p , 2.0=p . for the power. A total of 5000 quasi
experiments were performed in the present simulation study. To
keep the family-wise type I error at 5%, the nominal significance
levels for step 2 ŽCochran–Armitage trend test. and step 3 Žcom-
parison with a historical control. were set as 20% and 5% Ž15r3.,
respectively.
cal dominant lethal test with treated males. They are
applicable to experiments for identification of andror
for characterization of germ cell mutagens.
5.1. Definition of Õariables
Many authors have defined the possible variables
to be included in the evaluation of dominant lethal
data w15,21–24x. The main parameters of dominant
lethality are )Postimplantation loss: the proportion
of dead implants ŽDI.. )Preimplantation loss: aver-
age number of total implants per pregnant female in
the control minus average number of total implants
per pregnant female in the treated group. If corpora
lutea are counted, then preimplantation loss is calcu-
lated as corpora lutea minus total implants.
Other variables to be evaluated are:
) pregnancy rate,
) live implants per pregnant female,
) dead implants per pregnant female,
) contribution of early death and late death to
total death.
The analysis of all variables should be male ori-
ented. Although the smallest unit for analysis may be
the individual male, an analysis unit may involve
multiple males, that is, it may be necessary to com-
bine the results for two or more males to achieve a
sufficient number of implants per analysis unit to
analyse the DI. The results for each variable can be
analyzed using analysis of variance methods after
transformation to achieve reasonable normality and
similar intra-group variances w15,25x. It is particu-
larly useful to present the data not only in tabular
form containing all these parameters but to illustrate
the data graphically w26,27x.
 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30 27

5.2. Size of the experiment Table 7

It is known that DI-frequencies in controls are
different for different laboratories and different
strains of mice. Binomial distribution was assumed
with different control rates of DI-frequencies of 5%,
10% and 15% in order to determine the number of
total implants needed in each comparison group to
achieve desired power with a one sided type I error
of 0.05 ŽTables 6 and 7.. Extra-binomial variability,
as cautioned to exist by Generoso and Piegorsch
w28x, would call for increases in the implant require-
ments Žsample sizes. in each comparison group or
would result in slight decreases in the assay’s power.
The actual variance, whatever its size may be, will
be aptly considered when appropriately employing
the analysis of variance. The test assesses a no effect
hypothesis against a one-sided increase above con-
trols.
In Table 6 it is seen that using at least 333
implants per group would allow detection of a 10%
DI-frequency in the treated group above a 5% con-
trol frequency with 80% power; fewer implants would
be needed to detect a doubling over a 10% and 15%
control frequency. However, in excess of 500 im-
plants are needed to detect an absolute increase of
5% DI over a 10% and 15% background.
Table 7 shows that using 400 implants per group
achieves 86% power to detect an absolute increase of
5% DI over a 5% control frequency and somewhat
less power to detect an absolute increase of 5% DI
over control frequencies of 10% and 15%. The power
is excellent for detecting doublings of control rates
in all instances when controls are greater than 5%
DI. Even when a control rate is as low as 3% DI the
Approximate power achievable to detect actual increases above
negative control frequencies of dead implants ŽDI. when 400
implants are available per group
DI-frequencies DI-frequencies in exposure groups Ž%.
in control 10% 15% 20% 25%
groups
5% 86.0 99.9 99.9 99.9
10% na 70.0 99.0 99.9
15% na na 60.0 97.0
na s not applicable.
power to detect a doubling is 70%, and to detect an
absolute increase of 5% DI the power is 95%. Thus,
it is recommended that at least 400 total implants
Žrequiring about 40 pregnant females. be attained for
each comparison group and that each analysis unit
contains sufficient numbers of females and implants
so that at least one DI is expected w20x.
Table 8 gives the approximate number of implants
Žand females. needed in each analysis unit to achieve
at least one expected DI according to the size of the
background rate and assuming 10 total implants per
pregnant female. It is seen that with mating ratios of
1 male to 3 females the male is acceptable as an
analysis unit when a control rate is 3% DI or more.
Using a mating ratio of 1:2, the male is acceptable as
the analysis unit when the control rate is 5% DI or
more. Results from two males may be randomly
combined to form an analysis unit Žclustering. which
meets the recommendation. Males should be ran-
domly integrated into analysis units and the identities
preserved before the experiment is initiated to pre-
serve data analysis uniqueness w20x. Using a 1:1

Table 8
Approximate number of implants and number of pregnant females
Table 6
in each analysis unit needed to yield at least one dead implant
Approximate number of implants Žpregnant females. needed in
ŽDI. in negative controls with a range of DI-frequencies
each group to detect actual increases in the proportion of dead
implants ŽDI. when a s 0.05 and power is 80% DI-frequencies in negative controls
DI frequencies DI-frequencies in exposure groups Ž%. 3% 5% 7% 10% 12% 15%
in control 10% 15% 20% 25% Implants 30 20 15 10 8 7
groups Females 3 2 2 1 1 1
5% 333 Ž33. 105 Ž11. 55 Ž6. 35 Ž4. Males Ž1:3. a 1 1 1 1 1 1
10% na 535 Ž54. 155 Ž16. 76 Ž8. Males Ž1:2. a 2 1 1 1 1 1
15% na na 710 Ž71. 195 Ž20. Males Ž1:1. a 3 2 2 1 1 1
a
na s not applicable. Mating ratio.
28 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30
mating ratio will require male integrations when
control rates are lower than 7% DI and the individual
male is sufficient when control rates exceed 7% DI.
5.3. Statistical methods to be proposed
Each outcome variable which is analysed should
first be computed from the data for each analysis
unit. All statistical analyses should be based on the
analysis-unit results and any appropriate statistical
method may be used, so long as test-procedure re-
quirements are reasonably met. However, using the
analysis of variance on transformed results Žto
achieve reasonable normality and equal variance. or
non-parametric tests are to be preferred. Although
data may be transformed for analysis to determine
the levels of significance, a summary of the original
results should be presented in tabular form. It should
contain original counts of pregnant females, live and
dead implants as well as the unit of the original
variables according to dose and mating period. This
should be accomplished by back-transforming the
relevant means and standard errors. A description of
all the statistical procedures which are used is re-
quired together with a reasonable assurance that they
met the conditions necessary for their use. Doses and
mating periods should be evaluated statistically for
trend andror differences and the observed levels of
significance Žp-values. should always be stated for
each statistical test conducted.
6. Conclusion
Our work group has prepared recommendations
for test designs and statistical analyses by reviewing
negative control data collected from different labora-
tories. For all assay systems considered, we found it
possible to recommend practical sizes of experiments
which give a high Ž80%. probability of detecting a
doubling of the spontaneous rate. This was consid-
ered relevant for hazard identification. It must be
understood by all scientists performing these assays
that statistical analysis is an important aid to data
interpretation. In particular, the power of the selected
test design and the a probability threshold selected
will determine the utility of a negative result for
hazard identification.
Acknowledgements
We wish to thank all those who generously pro-
vided raw data of in vivo cytogenetic tests to us:
Biosafety Research Center, Foods, Drugs and Pesti-
cides, Japan; Food and Drug Safety Center, Hatano
¨ Saugtier-
Research Institute, Japan; GSF Institut fur ¨
genetik, Germany; Hazleton, WA, USA; Hazleton
Microtest, UK; Itoham Food, Central Research Insti-
tute, Japan; Japan Tobacco, Toxicology Research
Laboratories, Japan; Japan Bioassay Laboratory,
Japan Industrial Safety and Health Association,
Japan; Life Science Research, Industrial Toxicology,
UK; Merck Research Laboratories, Genetic and Cel-
lular Toxicology, USA; Mitsubishi-Kasei Institute of
Toxicological and Environmental Sciences, Japan;
National Institute of Health Sciences, Japan; Na-
tional Institute of Environmental Health Sciences,
NIEHSrNTP, USA; SRI International, Toxicology
Laboratory, USA. We also wish to thank C. Hamada,
University of Tokyo, for performing the analysis of
the historical micronucleus data.
Appendix A
The historical control data provided for this task
showed that the mean frequencies of micronucleated
immature erythrocytes ranged from 0.073 to 0.278%
ŽTable 1 in the text.. Also published negative control
data ranged from 0.0 to 0.3 w29x. We performed a
small simulation study to assess the power for the
micronucleus assay and the chromosomal aberration
test according to the statistical strategy agreed to by
the working group. The assumptions of the simula-
tion study were as follows: Ž1. The experiment con-
sists of three treatment groups and a concurrent
negative control. The dose levels were set as Ž d 0 , d1 ,
d 2 , d 3 . s Ž0, 1, 2, 4.. Ž2. The assumed population
proportions of micronucleated cells of historical con-
trols were set as p s 0.1%, 0.15%, 0.2%, 0.25%,
and 0.3% for the micronucleus assay and p s 0.5%,
1.0%, 1.5%, and 2.0% for the chromosomal aberra-
tion test. Ž3. The dose–responses were assumed as
Žp 0 , p 1 , p 2 , p 3 ,. s Žp , p , p , p . for the type I
error and Ž1 = p , 1.25 = p , 1.5 = p , 2.0 = p . for
the power for both tests. Ž4. The statistical procedure
was described in the text and Fig. 1. In the first step,
 I.-D. Adler et al.r Mutation Research 417 (1998) 19–30
when the value of the negative control in the simula-
tion was out of the 3)SD range, new series of
random numbers were generated. In the second step,
the Cochran–Armitage trend test using logŽdose.
was applied and in the third step w10,30,31x, each
value of the treatment group was evaluated by com-
paring it with the historical control approximated by
the binomial distribution BŽ2000, p . and BŽ200, p .
for the micronucleus assay and the chromosomal
aberration test, respectively w12x. Ž5. To keep the
family-wise type I error at 5%, after trial and error
trials the nominal significance levels for step 2 and
step 3 were set as 20% and 15%, respectively.
Actually, in the third step, the significance level for
each group was 5% Ž15r3. because multiplicity of
comparisons was considered. Ž6. A total of 5000
quasi experiments were performed and the probabil-
ity of a type I error or the power of the procedure
were estimated with the coefficient of variation of
about 0.06.
The results of the simulation study are shown in
Figs. 3 and 4, for the micronucleus assay and the
chromosomal aberration test, respectively. The simu-
lation results show that with the use of four or more
animals per group and the analysis of 2000 cells per
animal the power to detect a doubling of the histori-
cal control level is more than 80% when the mean
percent of the micronucleated cells in the historical
control is 0.15% or higher. Even for the historical
control value as low as 0.1%, analysing 2000 cells
from each of five animals gives more than 80%
power. In the chromosomal aberration test, with 5
animals per dose and 200 cells scored per animal the
power to detect a doubling of the historical control
level is more than 80% when the mean percent of
cells with chromosomal aberrations in the historical
control is greater or equal to 1.0%.
Here, we did not consider the downturn phe-
nomenon in the present simulation. Sometimes, in
the micronucleus assay downturns have been ob-
served. Several procedures have been reported to
overcome this phenomenon w32–37x. The procedure
proposed by Simpson and Margolin w36,37x is recom-
mended for the micronucleus assay. Their original
method was nonparametric testing but the strategy
can be applied to parametric procedures as well. The
simple trend test for monotone dose–response data
as the second step of the proposed method can be
29
replaced by such a procedure for a downturn in the
dose–response data.
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