INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 51
GUIDE TO SHORT-TERM TESTS FOR DETECTING MUTAGENIC
AND CARCINOGENIC CHEMICALS
Prepared for the IPCS by the International Commission
for Protection Against Environmental Mutagens and Carcinogens
This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organisation, or the World Health Organization.
Published under the joint sponsorship of
the United Nations Environment Programme,
the International Labour Organisation,
and the World Health Organization
World Health Orgnization
The International Programme on Chemical Safety (IPCS) is a
joint venture of the United Nations Environment Programme, the
International Labour Organisation, and the World Health
Organization. The main objective of the IPCS is to carry out and
disseminate evaluations of the effects of chemicals on human health
and the quality of the environment. Supporting activities include
the development of epidemiological, experimental laboratory, and
risk-assessment methods that could produce internationally
comparable results, and the development of manpower in the field of
toxicology. Other activities carried out by the IPCS include the
development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and
promotion of research on the mechanisms of the biological action of
ISBN 92 4 154191 1
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GUIDE TO SHORT-TERM TESTS FOR DETECTING MUTAGENIC AND CARCINOGENIC
2. DESCRIPTION OF WIDELY-ADOPTED PROCEDURES
2.1. Bacterial mutation assays
2.1.1. Principles and scientific basis of the assay
2.1.2. Relevance and limitations of the assay
2.1.3. The procedure
188.8.131.52 Outline of the basic procedure
184.108.40.206 Critical factors in the procedure
2.1.4. Presentation and interpretation of data
220.127.116.11 Data-processing and presentation
18.104.22.168 Interpretation of data in terms of
positive and negative
22.214.171.124 Dealing with ambiguous results
126.96.36.199 How the most critical factors identified
above can influence the validity of the
188.8.131.52 Interpretation of the results in terms of
the intrinsic mutagenic activity of the
2.2. Genotoxicity studies using yeast cultures
2.2.2. Genetic end-points
184.108.40.206 Point mutation
2.2.3. Information required
220.127.116.11 Significance of positive results in yeast
18.104.22.168 Negative results in yeast assays
2.3. Unscheduled DNA synthesis in cultured mammalian cells
2.3.2. Chemical exposure and UDS
22.214.171.124 The choice of a suitable cell line
2.3.4. The elimination of semiconservative replication
2.3.5. Chemical exposure
2.3.6. Radiolabelling procedures
2.3.7. Detection of UDS
2.3.8. Data processing and presentation
126.96.36.199 Choice of cell line
188.8.131.52 Choice of protocol
184.108.40.206 Method of activating proximate carcinogens
2.4. In vitro cytogenetics and sister-chromatid exchange
2.4.2. Procedure: chromosomal aberrations
220.127.116.11 Cell types
18.104.22.168 Culture methods
22.214.171.124 Chromosome assay
2.4.3. Procedure: sister chromatid exchange
2.4.4. Procedure: scoring
2.4.5. Extent of testing
2.4.6. Data processing and presentation
126.96.36.199 Critical factors
188.8.131.52 Experimental design and analysis
2.5. In vitro cell-mutation assays
2.5.1. Principles and scientific basis of the assay
2.5.2. Relevance and limitations
184.108.40.206 Outline of the basic technique
220.127.116.11 Cell types and selective systems
18.104.22.168 Culture conditions
22.214.171.124 Expression time
126.96.36.199 Choice and concentration of selective
188.8.131.52 Stability of the spontaneous mutant
184.108.40.206 Provision for metabolic conversion
220.127.116.11 Controls and internal monitoring
18.104.22.168 Population size, replicates, and
2.5.4. Data processing and presentation
22.214.171.124 Treatment of results
126.96.36.199 Evaluation of results
188.8.131.52 Ambiguous results
184.108.40.206 Mutant selection
220.127.116.11 Expression time
18.104.22.168 Cell numbers
22.214.171.124 Metabolic conversion
2.6. The use of higher plants to detect mutagenic chemicals
2.6.2. Test systems
126.96.36.199 Detection of mitotic chromosome damage
188.8.131.52 Detection of aberrations in meiotic
184.108.40.206 Detection of gene mutations at specific or
2.7. The Drosophila sex-linked recessive lethal assay (SLRL)
220.127.116.11 Test organism life cycle
18.104.22.168 Stock cultures
22.214.171.124 List of nomenclature
126.96.36.199 Equipment and laboratory techniques
2.7.3. Principle of the recessive lethal assay
2.7.4. Metabolic activation
2.7.5. Test performance
188.8.131.52 Treatment procedures
184.108.40.206 Toxicity tests
220.127.116.11 Control and replicate experiments: sample
2.7.6. Data processing and presentation
18.104.22.168 Disadvantages of the recessive lethal test
22.214.171.124 Weak mutagens and non-mutagens
126.96.36.199 Data base
188.8.131.52 Correlation with mammalian carcinogenicity
184.108.40.206 Recent developments
2.8. In vivo cytogenetics: bone marrow metaphase analysis and
220.127.116.11 Current understanding of the formation of
18.104.22.168 Classification of chromosomal aberrations
22.214.171.124 The basis for micronucleus formation
126.96.36.199 Experimental animals
188.8.131.52 Treatment and sampling
184.108.40.206 Dose levels
220.127.116.11 Number of cells scored per animal
18.104.22.168 Positive and negative controls
22.214.171.124 Preparation procedure for bone-marrow
126.96.36.199 Preparation procedure for micronuclei
188.8.131.52 Microscopic analysis
2.8.3. Data processing and presentation
184.108.40.206 Chromosomal aberrations
220.127.116.11 Statistical evaluation
18.104.22.168 Possible errors in microscopic evaluation
22.214.171.124 Comparison of test sensitivity
126.96.36.199 Application of the method to other tissues
2.9. Dominant lethal assay
2.9.2. Procedure for male mice
188.8.131.52 Standard and test conditions
184.108.40.206 Test conditions to be established by each
2.9.3. Dominant lethals in female germ cells
2.9.4. Data processing and presentation
3. LABORATORY FACILITIES AND GOOD LABORATORY PRACTICE
3.2. Laboratory facilities and equipment
3.2.1. Microbial laboratories
3.2.2. Tissue culture laboratories
3.2.3. Facilities for other procedures
3.3. Good laboratory practice in genetic toxicology
3.3.1. Origins and nature of GLP
3.3.2. GLP requirements
3.3.3. Summary of resources and records needed
4. SELECTION, APPLICATION, AND INTERPRETATION OF SHORT-TERM TESTS
4.2. Selection of assays
4.2.1. Detection of the major types of genetic damage
220.127.116.11 Gene mutations
18.104.22.168 Chromosomal damage
22.214.171.124 DNA damage
4.2.2. Scientific validity
126.96.36.199 Genetic basis
188.8.131.52 Metabolic capability
4.2.3. Predictive value
184.108.40.206 Mutagenic activity
220.127.116.11 Carcinogenic activity
18.104.22.168 Relevance to chemical class
4.2.4. Available expertise and facilities
4.3. Application of assays
4.3.1. The phased approach
22.214.171.124 Phase 1 - the basic screen
126.96.36.199 Supplementary tests
4.3.2. Nature and extent of potential human exposure
188.8.131.52 Limited or negligible distribution
184.108.40.206 Medium distribution, limited exposure
220.127.116.11 Extensive distribution, intentional or
4.3.3. Regulatory requirements
4.4. Acceptablility and reliability of data
4.5. Interpretation of results and significance for human
4.5.1. General principles
18.104.22.168 Results of individual assays
22.214.171.124 Results from combinations of assays
4.5.2. Influence of the extent of exposure and
126.96.36.199 Pharmaceutical compounds
188.8.131.52 Chemical compounds in food
184.108.40.206 Domestic chemical compounds
220.127.116.11 Chemical compounds used in industry
NOTE TO READERS OF THE CRITERIA DOCUMENTS
Every effort has been made to present information in the
criteria documents as accurately as possible without unduly
delaying their publication. In the interest of all users of the
environmental health criteria documents, readers are kindly
requested to communicate any errors that may have occurred to the
Manager of the International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland, in order that they may be
included in corrigenda, which will appear in subsequent volumes.
* * *
A detailed data profile and a legal file can be obtained from
the International Register of Potentially Toxic Chemicals, Palais
des Nations, 1211 Geneva 10, Switzerland (Telephone no. 988400 -
The following experts participated in the preparation of this
Dr I.-D. Adler, Institute for Genetics, GSF, Neuherberg, Federal
Republic of Germany
Dr J. Cole, MRC Cell Mutation Unit, University of Sussex, Brighton,
Sussex, United Kingdom
Dr N. Danford, University College of Swansea, Swansea, United
Dr U. Ehling, Institute for Genetics, GSF, Neuherberg, Federal
Republic of Germany
Dr M. Parry, Genetics Department, University College of Swansea,
Swansea, United Kingdom
Dr R. Roderick, Sittingbourne Research Centre, Sittingbourne, Kent,
Dr S. Venitt, Chester Beatty Research Institute, Institute for
Cancer Research, Royal Cancer Hospital, Pollards Wood Research
Station, Chalfont St. Giles, Buckinghamshire, United Kingdom
Dr W. Vogel, Department of Radiation Genetics and Chemical
Mutagenesis, Sylvius Laboratories, Leiden, The Netherlands
Dr R. Waters, Genetics Department, University College of Swansea,
Swansea, United Kingdom
During the past few years, great advances have been made in
understanding the processes leading to malignant disease. It is
clear that alterations in genetic material are involved in these
processes and that a great many carcinogens are capable of inducing
such alterations under appropriate conditions. Heritable
alterations in germ cells may also be induced by certain chemicals
and may constitute a genetic risk. Numerous short-term tests have
been developed to detect the ability of chemicals to cause such
changes and are being used routinely and successfully, on a large
scale. There is a widespread desire to evaluate the data obtained
from short-term tests and to generate such data in areas of the
world where the necessary combinations of expertise may not yet be
The International Commission for Protection Against
Environmental Mutagens and Carcinogens (ICPEMC), an assembly of
scientists with expertise in the fields of environmental
mutagenesis, carcinogenesis, genetic toxicology, and epidemiology,
was therefore pleased to respond to the request of the
International Programme on Chemical Safety of the World Health
Organization, to prepare a document containing guidance in the
field of short-term testing for mutagens and carcinogens with
genetic activity. This document represents the views of ICPEMC and
is published by IPCS in an attempt to stimulate scientific
discussion, as well as to provide guidance on the use of
genotoxicity tests in chemical safety programmes. Although short-
term tests to screen for mutagenicity and carcinogenicity are
useful, they have their difficulties and limitations. For example,
while the majority of chemical initiators of carcinogenesis give
positive results in tests for genetic change, it is not necessarily
true that all chemicals with genetic activity are carcinogens.
Moreover, there are carcinogens and cocarcinogens that are not
readily detected by mutagenicity tests and that may act by
mechanisms of quite a different nature. Such substances are
necessarily excluded from consideration here, but that does not
mean that they may not be of the same importance. There are
differences of approach in genetic toxicology, as in most other
branches of science. The present document reflects a widely-used
approach that may be regarded as good contemporary practice. It
should be emphasized that it does not claim to be definitive or to
contain recommendations for regulatory action either in connection
with the kind or number of tests that should be carried out, or the
regulatory decisions that may be taken on the basis of the results
of such tests. It is designed to explain the types of test that
are commonly employed and the meaning that the results of such
tests may have in the assessment of possible human hazard, as far
as is possible with current scientific knowledge.
It is obvious that any assessment of test results in terms of
mutagenic or genotoxic hazard can be properly made only in the
context of the whole toxicological profile of a substance and its
use. ICPEMC is currently working towards a position with regard to
the selection of short-term tests and its recommendations should be
available in the near future.
Current practice is still rapidly evolving and should not be
considered as fixed. Moreover, what might be considered feasible
in scientifically advanced countries with large resources of
expertise might be quite inappropriate in developing countries.
The latter, however, provide the raison d'être of the present
document, which is offered in a spirit of helpfulness in the hope
that it may enable short-term genotoxicity tests to be used in a
It has been known for many years that some chemicals can cause
cancer in man. More recently, there has been a growing awareness
of the possibility that chemicals may also produce mutations in
human germ cells thus influencing the frequency of genetic or
heritable diseases. Many thousands of chemicals, including
pharmaceutical products, domestic and food chemicals, pesticides,
and petroleum products are present in the environment and new
chemicals are being introduced each year. In addition, there are
many compounds that occur naturally, which are known to be
mutagenic and/or carcinogenic (e.g., mycotoxins in foods). It is
important, therefore, that chemicals to which people are exposed,
either intentionally (e.g., therapeutically), in the course of
their daily life (e.g., in domestic products, cosmetics, etc.), or
inadvertently (e.g., in pesticides) are tested for their potential
to produce cancer and genetic damage (mutations).
A few chemicals have been identified as carcinogenic because of
their known association with cancer in man. However, carcinogenic
activity is usually determined by the ability of a chemical to
produce tumours during the life-time exposure of laboratory
animals. Studies of this kind may last for two or three years and
require the use of scarce resources and expertise. This has led to
the search for alternative ways of detecting chemicals with cancer-
producing potential and a number of relatively inexpensive assays
have been developed, many using biological systems rather than
whole mammals. Because such assay systems need far less time to
complete than classical long-term studies in rodents they are
referred to as "short-term tests".
Although the results of epidemiological studies have confirmed
that exposure to a number of chemicals, such as vinyl chloride and
beta-naphthylamine, can cause cancer in man, convincing
epidemiological evidence that chemicals constitute a mutagenic
hazard for man is not yet available. It is known that genetic
defects cause a significant proportion of human diseases, but the
contribution of environmental chemicals to genetic disease is
unknown. This is not surprising as the possibility of such a
danger to health has only been apparent for about one generation.
The information that determines the characteristics of a cell
or organism is contained in the genetic material of the cell, which
is composed of deoxyribonucleic acid (DNA). DNA is composed of
sub-units of deoxyribonucleotides, which themselves consist of a
pentose sugar (2-deoxyribose), a phosphate ester, and a purine or
pyrimidine nitrogenous base. These sub-units form a 3-dimensional
helical double-stranded structure (Watson & Crick, 1953). Each of
the two strands consists of a linear array of the deoxyribose sugar
molecules linked together in a chain by phosphate molecules. The
strands are joined side-by-side by hydrogen bonds between
complementary pairs of the purine and pyrimidine bases. The
complementary pairs of bases are guanine (a purine) paired with
cytosine (a pyrimidine) and adenine (a purine) paired with thymine
(a pyrimidine). The unique sequence of bases, taken in groups of
three, or triplets, forms the genetic code, each triplet coding for
a particular amino acid. Sequences of triplets provide uniquely
the information necessary for the synthesis of a functional protein
or enzyme. Such a functional sequence of bases is known as a gene.
Genetic information is passed from one cell generation to the next
by precise duplication of the strands and equal distribution of the
DNA, prior to cell division (i.e., the mitosis of somatic cells or
the meiosis of germ cells) and is responsible for the faithful
handing on of all the characteristics of one generation to the next
generation. This fundamental genetic process is common to all
organisms ranging from a simple bacterial cell to a complex mammal
or plant. In higher organisms, the long strands of DNA are bound
to proteins (histones) and are organised into a number of complex
structures called chromosomes, located in the nucleus of the cell.
With the exception of the germ cells, which carry a single set
of chromosomes and are termed haploid, the cells of higher
organisms contain duplicate sets of chromosomes, one set derived
from each parent, i.e., they are diploid.
Diploid cells, therefore, carry a pair of each of the
functional genes, which occupy a precise position or locus along
the length of the DNA of a particular chromosome. The paired genes
may be identical (homozygous) or functionally different
(heterozygous); heterozygous forms of the same gene are called
alleles. In many cases, one of the two alleles has a dominant
function over its partner. Such partners are called dominant (or
partially dominant) and recessive (or partially recessive).
Occasionally, a pair of alleles are expressed independently of each
other and are regarded as co-dominant.
Genes carried by the X-chromosome behave differently. As males
have only one X-chromosome, the genes are not carried in pairs and
a gene on this chromosome is expressed as a dominant or recessive
X-linked characteristic. Although females have two X-chromosomes,
a similar, though more complex situation exists, as only one of the
two X-chromosomes expresses its genes in a particular cell type.
The fact that the basic helical structure of DNA and the genetic
coding is common to all living organisms, whether they are
bacteria, plants, or mammals, means that data obtained from studies
of the effects of a chemical on one species can be used to predict
the possible genetic response of another species to the same
Alterations to the information carried by the DNA occur as a
result of small changes in the structure of the DNA molecule,
whereby the base sequence transmitted to the next generation is
changed, and this may result in descendants with different
characteristics to the parent. The alterations are, in effect,
mutations and, though many of them are detrimental, some mutations
are compatible with a normal, healthy existence, being responsible
for the subtle differences between members of a species and
constituting the driving force of evolution.
Mutagenic chemicals interact with DNA causing changes in its
structure. This may result in the loss, addition, or replacement
of bases, thus altering their sequence in the DNA and affecting the
fidelity of the genetic message.
The effect of these mutational changes may be to prevent the
synthesis of functional proteins completely (i.e., inhibition of
gene expression) or may lead to the synthesis of proteins with
modified structure and enzymes with altered activity and
specificity. Where mutations lead to changes in the genetic
information carried by male or female germ cells, the progeny of
such affected parents may express the mutation as some form of
heritable abnormality or disease. When mutations occur in the
somatic cells of a complex organism, they may produce irreversible
changes in the cell that may ultimately be involved in producing a
Mutation of one of a pair of genes may lead to a change in gene
expression that can override the function of the normal partner and
is called a dominant mutation. Recessive mutations are expressed
only when both genes carry the same recessive mutation. For
example, a recessive mutation inherited from the male parent will
only be expressed in the progeny if the same recessive gene is also
inherited from the female parent.
Cells can survive potentially lethal damage to the DNA, because
of the activity of a series of enzyme-mediated processes that are
generally termed DNA repair. The simplest form of DNA repair
involves removal (excision) of the chemically-altered portion and
the repair of the gap left in the DNA strand by the synthesis of
new DNA using the undamaged sister strand as a template. Damage to
DNA that is not repaired by this mechanism interferes with normal
DNA synthesis (i.e., DNA replication). This stimulates another
kind of DNA repair (post-replication repair) which, because it is
not always accurate (i.e., it is error-prone), may lead to
mutagenic changes in the DNA. The detection of unusual excision
DNA repair activity, so called unscheduled DNA synthesis, in
mammalian cells (as a response to damage to the DNA) forms the
basis of an important assay for identifying chemicals that cause
damage to DNA (section 2.3).
Damage to DNA may be expressed as mutations at the chromosome
level (i.e., chromosomal aberrations or chromosomal mutations) or
at the level of the gene (i.e., gene or point mutations).
Chromosomal mutations may be observed as changes in the structure
of the chromosome (structural aberrations) or in the number of
chromosomes in a cell (numerical aberrations or aneuploidy).
Structural aberrations are a consequence of DNA damage while
numerical changes are usually caused by defects in the accurate
distribution of chromosomes during cell division, i.e., DNA damage
may not be involved.
Mutations contribute significantly to human diseases and
congenital malformations, though the extent of this contribution is
not precisely known. Some diseases such as Down's syndrome
(Trisomy 21) are associated with structural or numerical
chromosomal abnormalities. Others are a result of mutations of
single genes and there are other diseases and congenital
abnormalities for which a genetic alteration may be partly
responsible. Sickle cell anaemia is a disease caused by the
inheritance of a single mutant gene that is responsible for the
synthesis of an abnormal haemoglobin molecule. In the homozygous
state, i.e., where the mutant gene is inherited from both parents,
the resulting disease is severe. Heterozygous individuals carry
only a single mutant gene and suffer from the relatively mild
sickle cell trait. Because of the resistance of sickle cell red
blood cells to the malarial parasite, carriers of the trait have a
selective advantage over unaffected individuals and the mutant gene
is maintained at a high frequency in certain populations.
Although there is no definitive evidence that exposure to
chemicals is responsible for any of the known human genetic
disorders, experimental evidence from other mammals have shown that
chemicals can produce both chromosomal and gene mutations of the
type that are associated with human genetic diseases. There is
little direct evidence to suggest that man is any less susceptible
than other mammals to the effects of exposure to mutagenic
Alterations in the structure and function of DNA are believed
to play a crucial role in the production of cancer by chemicals.
Carcinogenesis is a multistage process that may take years to
evolve and a number of different factors influence the progression
from a normally functioning cell to an invasive neoplastic tumour.
(A carcinogen is defined as an agent that significantly increases
the frequency of malignant neoplasms in a population; carcinogens
may be physical, chemical, or biological agents). The complex
mechanisms by which chemicals induce malignancy are not fully
understood, but there is evidence that suggests the occurrence of
four major stages following an adequate exposure of a mammal
(including man) to a chemical carcinogen:
(a) transport of the chemical from the site of entry into
the body and, in many cases, metabolic modification
of the chemical (principally in the liver) to a more
(b) interaction of the molecule or its reactive metabolite
with the molecular target in the cell (the most
important of which is DNA);
(c) expression of the DNA damage as a potentially
carcinogenic lesion; and
(d) progression, influenced by modifying factor(s), and
proliferation to form a malignant tumour.
Some carcinogenic chemicals appear to be responsible for only one
part of the process and are not regarded as complete carcinogens.
For example, many chemicals that interact with DNA and are thus
mutagenic appear to initiate the process by inducing the primary
DNA lesion. These are called initiators and the damage they cause
is generally irreversible. Other compounds have been shown to
influence the expression and progression of the initial DNA change
and are called tumour enhancers. Some of these do not interact
with DNA, they are not mutagenic and include the so-called tumour
promoters. A third group includes chemicals known as complete
carcinogens in that they are probably capable of both initiating
and promoting activity. All chemicals that produce DNA damage
leading to mutations or cancer, including initiators and complete
carcinogens, are described as genotoxic.
The animal bioassay for detecting carcinogenic chemicals is a
large, complex and very expensive scientific study using some
hundreds of rodents to which the suspect chemical is administered
for most of their life span. Similarly, the specific locus test in
mice (Searle, 1975), which is one of the few currently available
assays for detecting heritable gene mutations in mammals, requires
the examination of many thousands of offspring and is equally
expensive and time-consuming. Thus, of the multitude of chemicals
introduced into the environment this century, and the hundreds of
new compounds being synthesized each year, only a small fraction
can be tested in conventional animal studies. For this reason, the
last decade has seen the introduction of a number of relatively
rapid tests for detecting mutagenic and carcinogenic chemicals.
Such tests are economical in resources and produce results in a
matter of weeks. Almost all of these short-term procedures are
based on the demonstration of chromosomal damage, gene mutations,
or DNA damage, and many of them are in vitro assays (i.e.,
conducted in experimental biological systems without the use of
live animals). As will be described in section 2, the test
organisms range from bacteria and yeasts to insects, plants, and
cultured animal cells and there are also short-term tests in which
laboratory animals are exposed to test chemicals for periods of a
few hours to, at most, a few weeks. In practice, a suspect
chemical is first tested using in vitro procedures, to study its
ability to react with DNA and thus induce mutations. It may then
be necessary to determine its genotoxicity for intact animals by
testing in short-term mammalian ( in vivo) assays.
The concept that carcinogenic chemicals cause cancer by a
mutagenic mechanism is the basis of the somatic mutation theory of
cancer induction. Between 1955 and 1970, there were many attempts
to demonstrate the mutagenicity of carcinogens using simple
bacterial assays but these experiments failed to show a direct
relationship between mutation and cancer. Following the pioneering
studies of Miller & Miller (1966), it was realized that the
majority of chemical carcinogens were biologically inactive until
they were enzymatically converted into reactive molecules. Such
chemicals are referred to as pro-carcinogens. Intermediate
metabolites that are the precursors of the ultimate reactive
molecule (i.e., the molecule capable of reacting with DNA) are
known as proximate carcinogens. Cancer-inducing chemicals are
often poorly soluble in water and, like most foreign compounds
entering an organism, undergo a sequence of metabolic reactions
intended to detoxify them and if necessary convert them into more
water-soluble products, which can then be excreted by the kidneys.
In some cases, these metabolic reactions produce carcinogenic
chemicals, converting them into proximate and ultimate carcinogenic
metabolites. Most ultimate carcinogenic molecules are
electrophilic reactants capable of binding with nucleophilic sites
on DNA and other macromolecules (Miller & Miller, 1971).
The major classes of chemical carcinogens are activated by an
oxidative reaction catalysed by microsomal mixed-function oxidases,
though other enzyme systems influence this activation. Appropriate
enzymes are present in most mammalian tissues, the highest activity
occurring in the liver. In bacteria and most cultures of mammalian
cells, mixed-function oxidase activity is either absent or very low
and they are therefore not capable of activating the majority of
carcinogens at a significant rate. Insects have a complex
metabolic capability and appear to be capable of activating a wide
range of pro-carcinogens. Yeast cells are also capable of limited
foreign-compound metabolism. In the early 1970s, Garner et al.
(1971) and Malling (1971), recognizing the significance of the
metabolic activation of chemicals, devised experiments in which
mammalian enzymes and bacterial cells were combined in a single
assay. This led to the introduction of the first useful screening
assay for carcinogenic chemicals, i.e., the Salmonella typhimurium
reversion test described by Professor Bruce Ames and his colleagues
(Ames et al., 1973). Essential aspects of mammalian metabolism are
now introduced into many short-term in vitro assays by the
incorporation of an enzyme-rich, cell-free extract of mammalian
tissues. The most commonly used preparation is the post-
mitochondrial supernatent (referred to as the "S9" fraction),
obtained after high speed centrifugation of a homogenate of rat
Although more than a hundred "test systems" for investigating
genotoxicity have been described in the literature, ranging through
the biological phyla from bacteriophage to mammals, less than 20
are in regular use and some of these are only available in
specialised laboratories. The most widely-accepted systems are
summarized below and described in detail in section 2.
Assays that involve the use of bacteria for detecting mutagenic
chemicals are the most-extensively used and, in general, the most
thoroughly validated. Unlike higher organisms, in which the DNA is
organized into complex chromosomal structures, bacteria contain a
single circular molecule of DNA that is readily accessible to
chemicals that can penetrate the cell wall. Bacterial tests also
have the advantage that a population of many millions of cells with
a relatively short generation time can be tested in a single assay.
In the classical techniques, strains of bacteria are used that
already contain mutations of specific genes. For example, a
mutation at the histidine locus in S. typhimurium removes the
ability of the bacterium to synthesize histidine and such mutants
cannot survive in culture medium lacking this nutrient. Reversion
at this locus enables the cell to synthesize histidine again and
thus proliferate in medium lacking the amino acid. Mutations
induced by test-chemicals, i.e., "reverse" mutations, are detected
by the growth of the "revertant" bacteria to form colonies in
appropriate selective culture media. Reverse mutation refers to
reversion of an existing mutation, while forward mutation refers to
the formation of a new mutation (section 2.1). Bacterial assays
can be adapted for detecting mutagenic metabolites in body fluids
(e.g., urine, blood, plasma) from exposed animals or human beings.
Yeast and fungi occupy a position between bacteria and animal
cells in terms of genetic complexity. The structure of fungal DNA
and its organisation into chromosomes is similar in many ways to
that of mammals. Both haploid and diploid forms can be used in
genetic assays. Tests using yeasts, such as Saccharomyces
cerevisiae, are available for detecting both forward and reverse
mutations and a variety of other genetic changes (section 2.2).
Certain strains of yeast can be used to detect chemicals that
induce aneuploidy (i.e., unequal distribution of chromosomes during
cell division) and there is some evidence that non-genotoxic
carcinogens can be identified using these strains.
The demonstration of DNA repair activity in mammalian cells is
indirect evidence of DNA damage. DNA repair can be detected in a
simple mammalian cell culture assay that involves the measurement
of "repair" or "unscheduled" DNA synthesis (UDS) (section 2.3).
The assay is based on the fact that thymidine is incorporated into
DNA during both normal and repair synthesis. Cells treated with
the suspect chemical are exposed to radioactive isotope-labelled
(i.e., tritiated) thymidine at a stage when normal DNA synthesis is
dormant or suppressed. The amount of radiolabelled thymidine
detected in the DNA is a measure of DNA repair synthesis and, thus,
an indication of primary DNA damage.
Chemicals can be tested for their ability to induce chromosomal
damage either in mammals, insects, cultured mammalian cells, or in
plants. Mammalian cell cultures provide a convenient test system
and either established cell lines or human blood lymphocytes can be
used (section 2.4). Analysis of metaphase chromosomes in cells
from the bone marrow of rats, mice, or hamsters is a well-
established technique for studying chromosome damage in vivo
(section 2.8). Alternatively, chromosome fragments can be
identified as micronuclei in certain bone marrow cells and in other
tissues and the "micronucleus test" has proved to be a relatively
simple assay for detecting chemicals capable of damaging
Cultures of mammalian cells can also be used to investigate the
induction of gene mutations by chemicals (section 2.5). The
principles involved are similar to those of microbial assays, i.e.,
cells are cultured in medium containing the suspect chemical and
are then sub-cultured into a selective medium in which only mutant
cells can survive. The number of cells that proliferate to form
colonies is a measure of the number of cells that have undergone a
forward mutation at the specific gene locus.
As described earlier in this section, bacteria, yeasts, and
cultured mammalian cells may lack the enzymes necessary for the
conversion of many carcinogens and mutagens to a molecular form
that will react with DNA. Assays using these systems must,
therefore, be supplemented with a suitable mammalian microsomal
enzyme preparation. Many insects are able to activate a wide range
of genotoxic chemicals and the demonstration of genetic changes in
Drosophila melanogaster (the fruit fly) forms a useful assay for
investigating carcinogenic and mutagenic chemicals (section 2.7).
Because of the tremendous advances made in the use of microbial
and mammalian cell procedures in genetic toxicology, plant material
is less often used for studying mutagenic chemicals than
previously. However, the use of plants such as the bean (Vicia
faba), the onion (Allium cepa), the spiderwort (Tradescantia
paludosa), maize (Zea mays), barley (Hordeum vulgare), and the
soybean (Glycine max) may have significant advantages over other
systems and their value in screening chemicals for mutagenic
activity has still to be fully explored (section 2.6).
Investigation of genetic changes at both the gene and chromosomal
level can be conducted in plants without the complicated laboratory
facilities required for other types of assay and this may be a
great advantage under certain circumstances. A possible
disadvantage is that the metabolic pathways in plants differ in
many respects from those in mammals. Thus, meaningful
extrapolation to man of data obtained in plant studies is uncertain
Data obtained from non-mammalian organisms and cultured
mammalian cells determine whether a chemical or its metabolite(s)
is capable of interacting with DNA and producing genetic damage.
Two procedures are described in section 2 that are used to
investigate the mutagenic activity of chemicals in the intact
animal. The first (section 2.8) is designed to detect chromosome
damage in the somatic cells of rodents. The second (section 2.9)
is the dominant lethal assay that can identify chemicals capable of
inducing genetic damage in the reproductive or germ cells of
animals. In this test, male rats or mice are dosed with the
suspect chemical and mated with untreated females. Certain types
of chromosomal damage induced in the male germ cells are lethal to
the fertilised ova and this can be detected by examination of the
Before short-term assays can be used to screen chemicals for
potential carcinogenic activity with any degree of confidence,
their sensitivity and accuracy for this purpose must be thoroughly
validated. The first comprehensive validation of bacterial tests
for detecting carcinogens was conducted by Ames and his colleagues
(McCann et al., 1975) using a combination of bacteria (Salmonella)
and mammalian microsomal enzymes. In this study, in which 300
chemicals were tested, approximately 90% of carcinogens were
bacterial mutagens and 90% of non-carcinogens failed to show
mutagenic activity. Following this, Purchase et al. (1978)
investigated 120 carefully-selected chemicals in a series of six
short-term tests. Again, the Ames bacterial mutation test gave a
predictive value for carcinogenic potential of about 90%. Analyses
of these and later studies showed that the success rate of
mutagenicity tests for detecting carcinogens was influenced by the
type or class of chemical selected for testing and the criteria on
which the carcinogenic activity in animals was judged. Rinkus &
Legator (1979) reviewed data from bacterial tests on 465 known or
suspected carcinogens. The compounds were divided into a number of
separate categories, depending on their chemical structure. The
chemicals that showed the best correlation (94%) between mutagenic
and carcinogenic activity were those that either could react
directly with DNA (i.e., ultimate electrophiles) or could be
activated by metabolic enzymes to DNA reactants. Chemicals that,
from their structure, appeared unlikely to react with DNA, showed a
very poor correlation between mutagenic and carcinogenic activity.
These chemicals appear to cause cancer by a different, possibly
The most ambitious validation exercise to date was the
International Program for the Evaluation of Short-term Tests for
Carcinogenicity (de Serres & Ashby, 1981) in which some thirty in
vitro and in vivo assays involving more than fifty laboratories
were evaluated for their ability to discriminate between
carcinogenic and non-carcinogenic compounds. Twenty-five
carcinogens and 17 non-carcinogens, including 14 pairs of
carcinogenic/non-carcinogenic analogues, were tested in most of the
assays. Animal cancer bioassay data from the 42 chemicals were
critically evaluated by an expert committee. Bacterial mutation
assays gave the best overall performance producing reliable results
in a large number of laboratories, and were confirmed as the first
choice as an initial screening test. However, it was also noted
that some known rodent carcinogens were not detected or were only
detected with difficulty by the standard Ames procedure. Other
assays that discriminated well between carcinogens and non-
carcinogens included in vitro tests for chromosome damage and
unscheduled DNA synthesis and assays using yeasts, and, although
the database was smaller, results from Drosophila tests and in
vitro gene mutation assays suggested that they were useful
components of a testing battery. In vivo tests for chromosome
damage showed their ability to differentiate between a number of
carcinogen/non-carcinogen pairs and thus confirmed their value for
investigating the in vivo behaviour of chemicals found to be
mutagenic in in vitro tests.
A further major collaborative study, which was designed to
complement the International Program referred to above, was
conducted under the auspices of the International Programme on
Chemical Safety. The object of this project was to identify the
best in vitro test or tests to complement the bacterial reversion
assay (Ashby et al., 1985).
Although bacterial mutation assays have a high predictive value
for carcinogenicity, in most validation studies at least 10% of
compounds give results that conflict with the animal cancer data.
For this reason, it is generally accepted that bacterial assays
should not be used in isolation for the testing of chemicals and it
is common practice to use a "battery" or "package" of short-term
assays as a preliminary screen.
Carcinogenic and/or mutagenic chemicals may induce one or more
of a number of genetic changes and an assessment of the possible
genotoxic hazard posed by a chemical should normally contain assays
capable of detecting changes at both the gene and chromosomal
level, and in some cases, tests for DNA damage. Some authorities
such as the Organisation for Economic Cooperation and Development
(OECD), the European Economic Community (EEC) and the US
Environmental Protection Agency (US EPA), require specific tests to
be carried out on certain types of chemicals. The application and
interpretation of short-term tests are discussed in detail in
section 4 and additional information is presented by Dean et al.
Section 2 contains descriptions in phylogenetic order of the
most commonly used and most widely-accepted assays, some of which,
however, are used more often than others. In each procedure,
specific minimal scientific and technical criteria can be
identified that are critical factors in obtaining data of
acceptable quality and reliability. These factors are emphasized
in section 2 and should be carefully considered by scientists
contemplating setting up a testing facility and by those who are
responsible for judging the validity of submitted data and
assessing the genotoxic hazard associated with the use of
chemicals. Additional criteria that relate to good laboratory
practice in genetic toxicology and to the type and quality of
laboratory facilities are discussed in section 3.
2. DESCRIPTION OF WIDELY-ADOPTED PROCEDURES
2.1. Bacterial Mutation Assays
2.1.1. Principles and scientific basis of the assay
Bacteria have proved to be most suitable for the study of
mutations, which are rare events, occurring naturally at a specific
locus in less than one in a million bacteria at each cell division.
However, as bacteria are single-celled organisms that divide
rapidly and can be grown in large numbers in a few hours, it has
proved to be relatively easy to grow tens of millions of organisms
under circumstances where the one in a million event can be
detected. Furthermore, a great deal is known about the
biochemistry and genetics of bacteria, and it has, therefore, been
possible to develop special strains that are sensitive to a wide
range of mutagens.
Bacterial test systems fall into 3 main classes, namely, those
that detect backward mutations, those that detect forward
mutations, and those that rely on a DNA repair deficiency. By far
the most widely-exploited method is the induction of backward or
reverse mutations in Salmonella typhimurium or, less frequently,
Escherichia coli. The important point of this type of test is
that, from the very beginning, each strain of bacterium already
possesses a selected mutation that prevents it performing an
essential biochemical function, such as the synthesis of one out of
the twenty or so amino acids necessary for the synthesis of
proteins, unlike a non-mutant, "wild-type", strain of the same
species. A wild-type (prototrophic) strain can synthesize all its
amino acids from inorganic nitrogen (e.g., ammonium phosphate) when
provided with a suitable source of carbon (e.g., glucose). The
strains of S. typhimurium used in reverse mutation tests cannot
synthesize the amino acid histidine and are therefore designated as
"his-". Similarly, E. coli strains that cannot synthesize the
amino acid tryptophan have been used and are referred to as "trp-".
Such strains are said to be auxotrophic. The reverse mutation test
is so named because it can show whether a test material can reverse
the effect of the pre-existing mutation (e.g., his-) by causing a
second mutation which allows the bacterium to synthesize histidine
from inorganic nitrogen. This process is often abbreviated to
"his-" to "his+" and is referred to a reversion from auxotrophy
to prototrophy. The resulting mutants are also called revertants.
In order to make the test more sensitive, the tester strains
have been made more susceptible to mutagens by genetically changing
the structure of their cell walls so that they become more
permeable to large fat-soluble molecules. Other genetic
manipulations have reduced their ability to repair regions of DNA
that have been damaged by chemicals and various types of radiation.
Further sensitivity in the tester strains has been attained by
introducing plasmids (small DNA molecules) that carry genes that
interfere further with DNA repair, making the host bacteria even
more vulnerable to the mutagenic effects of chemicals.
Because there are many different types of DNA damage, and
because the use of the reverse mutation test requires the test
chemical to hit a very small target in order to overcome the effect
of the pre-existing mutation, several different targets are
presented to the test chemical. This is achieved by using several
strains of the same species of bacterium, each one carrying a
different pre-existing mutation in the same amino-acid gene. Two
types of mutation are employed: base-pair substitution and
frameshift. For example, there are several different mutations in
the histidine gene of S. typhimurium, each different mutation
being carried in a different strain, but all strains sharing the
other traits (e.g., DNA-repair defects and cell-wall defects that
make them very sensitive).
As mentioned in section 1, E. coli and S. typhimurium lack
most of the enzymes that can perform the type of metabolic
activation characteristic of mammalian biotransformation. The
enzymes are therefore added in the form of a liver extract prepared
from laboratory animals, usually rats. The rats are given
chemicals that increase the amount of metabolic-activation enzymes
in the liver and are then left for a few days before they are
killed, to allow the enzymes to build up. This is known as
induction, and the chemicals that are used are called "inducers".
The most widely used inducer is Aroclor(R) 1254, a mixture of
polychlorinated biphenyls. Phenobarbital plus 5,6-benzoflavone is
also used for induction. The liver is ground up and centrifuged at
high speed; the supernatant liquid contains the metabolic enzymes
(some of which are bound to membranes (microsomes)) and is called
S9 (short for "9000 g supernatant").
In a bacterial mutation assay, his- bacteria are mixed with S9
and several doses of the test chemical and are allowed to divide by
providing a small amount of histidine. If the test chemical is
itself mutagenic, or if the enzymes in the S9 act on the test
chemical to produce substances (metabolites) that are mutagenic,
this will be shown by the appearance of a small proportion of
bacteria, which will continue to grow and divide, even when the
supply of histidine has been used up. These revertants can be
detected easily, since their DNA has been permanently changed so
that they can make histidine from inorganic nitrogen, and can grow
indefinitely without added histidine. Thus, each mutant eventually
grows into a microscopic colony and it is the count of these that
is the end-point of the assay.
2.1.2. Relevance and limitations of the assay
Bacterial mutation assays are used in a large number of
laboratories throughout the world. Several large-scale trials,
carried out to test the usefulness of these assays in detecting
potential carcinogens and mutagens (Purchase et al., 1978; McMahon
et al., 1979; Bartsch et al., 1980; de Serres & Ashby, 1981), have
shown that bacterial mutation assays are very good at picking out
chemicals known to cause cancer. Moreover, relatively few
chemicals that do not cause cancer have given positive results in
these tests. In general, therefore, chemicals that are mutagenic
in bacteria are more likely to cause cancer than chemicals that are
not mutagenic, i.e., mutagenicity is a characteristic property of a
large number of carcinogens. It is important to understand,
however, that there does not seem to be any useful quantitative
relationship between the ability of a chemical to cause mutations
in bacteria and cancer in animals or people. In other words, a
chemical that is a strong mutagen in bacteria is not necessarily a
strong carcinogen in animals, nor is it always the case that a weak
bacterial mutagen will be a weak carcinogen.
A second limitation of bacterial mutation tests is their
inability to detect chemicals that are thought to induce cancer,
not by causing DNA damage, but by other means, as yet poorly
understood. Such substances include asbestos, nickel, arsenic, and
hormone-like chemicals such as diethylstilboestrol. There are
other substances, e.g., phorbol esters, which are extracts of
certain species of the plant genus Euphorbia, and certain
secondary bile acids, which usually do not cause cancer when given
alone to animals, but which increase the effects of other cancer-
causing chemicals. This so-called tumour promotion does not come
about because of the production of DNA damage. Thus, it will not
be detected by mutagenicity assays which detect only substances
that can initiate cancer. Nevertheless, promoters may well play a
significant part in human cancer and it is important to recognise
that bacterial mutagenicity tests cannot detect promoting activity.
Substances are known that cause genetic defects (and possibly
cancer) in higher organisms by interfering with the machinery that
controls the exact distribution of chromosome sets from one
generation of cells to the next, and from parents to children,
causing mistakes in the number and structure of chromosomes
delivered to cells during cell division. Such substances do not
always cause DNA damage, and will not be detected in bacterial
mutagenicity tests, since bacteria do not have chromosomes.
The use of cell-free extracts (S9) from rats to represent the
metabolism of chemicals in mammals is another limitation that must
be borne in mind, when interpreting the results of bacterial
mutation tests. Studies have shown that breaking up liver cells
can, in some cases, distort the pattern of metabolism, resulting in
levels and proportions of metabolites that would not be produced in
the intact liver. Moreover, because the test is carried out in a
test-tube rather than in an animal, it is impossible to allow for
several other factors, which can in some cases give a misleading
impression of the mutagenic or carcinogenic effects of a chemical.
The way the chemical enters the body and is distributed to the
various organs, how each organ metabolises it, and how the chemical
or its metabolites leave the body can all play a part in
determining if, and to what extent, the chemical is mutagenic or
carcinogenic for the animal. None of these factors can be
reproduced in a test-tube containing bacteria, S9, and a chemical.
Despite these limitations, bacterial mutation tests have been
found by trial and error to be extremely valuable as the first in a
series of tests for screening chemicals for potential mutagenic and
carcinogenic activity. Moreover, bacterial tests have been
validated in far greater detail than any other tests currently used
in genetic toxicology.
2.1.3. The procedure
The bacterial mutation test that forms the basis of all
screening programmes was devised by B.N. Ames and his co-workers
and is usually referred to as the " Salmonella/microsome test". It
is essential that workers who intend to use this test, and those
who review the results of such tests read the following papers:
Ames et al. (1975), McCann et al. (1975), McCann & Ames (1976),
Maron et al. (1981), Levin et al. (1982), and Maron & Ames (1984).
The following technical details are not intended as a defined
recommended protocol, but represent good current practice and good
criteria for successful bacterial tests.
18.104.22.168. Outline of the basic procedure
In the Salmonella/microsome test, several his- strains of
S. typhimurium are used in order to detect several different types
of DNA damage. A set of sterile test-tubes is held at 45 °C.
Molten soft agar ("top agar") (2 ml) containing a low concentration
of histidine is added to each tube followed by 0.1 ml of a culture
of the required bacterial strain, which has been grown over the
previous night in a very rich nutrient liquid ("nutrient broth").
This "overnight culture" contains about 1 x 109 bacteria per ml, so
that each tube contains about 1 x 108 bacteria. A range of doses
of the test chemical (dissolved in a suitable solvent) is then
added, each dose to a separate tube. Dimethylsulfoxide (DMSO) is
the most widely used solvent. It dissolves numerous different
kinds of chemicals, is miscible with water and, at the amount used
in the test (0.1 ml or less), is not toxic to bacteria.
Several tubes are set aside to act as "controls", i.e., tubes
that will receive the solvent but not the test chemical and will
therefore indicate the background (spontaneous) level of mutation.
It is essential to know the level of background mutation for each
bacterial strain in each experiment in order to tell whether the
test chemical has had any mutagenic effect. Finally, 0.5 ml of
S9-mix is added to each tube and the contents mixed thoroughly by
rapid shaking. S9-mix consists of S9 (usually between 4 and 30% by
volume) to which has been added nicotinamide-adenine dinucleotide
phosphate (NADP) and glucose-6-phosphate (which together provide
energy for metabolism), phosphate buffer to maintain pH, and salts
of magnesium and potassium. A set of tubes is also prepared
without S9. This is to check whether the test chemical can cause
mutation without the need for metabolic activation. Chemicals of
this type are directly-acting mutagens: certain directly-acting
mutagens can be made non-mutagenic by S9; thus, it is important to
include this check.
The additions of bacteria, test chemical, and S9-mix are made
in rapid succession, in order to avoid the potentially harmful
effects of the rather high temperature (45 °C) necessary to keep
the soft agar molten. As soon as possible after mixing, the
contents of each tube are poured on to the surface of 30 ml of
solid 1.5% agar ("bottom agar") which contains glucose, ammonium
and other salts, and phosphate buffer in a 9-cm petri dish
("plate"). The plate is shaken to distribute the top agar in a
thin, even layer over the bottom agar. The lid of the plate is
replaced and each plate is placed on a level surface: the top agar
then cools and solidifies. When all the tubes have been poured,
and the plates have cooled, they are inverted and placed in an
incubator at 37 °C for 48 h.
This is called the plate incorporation technique, since all the
ingredients of the test are incorporated into a thin layer of soft
agar on the surface of harder agar in a plate. During the first
few hours of incubation, all the his- bacteria will grow, since
there is a trace of histidine present. At the same time that the
bacteria are dividing, the enzymes in the S9-mix, supported by the
energy provided by the NADP and G-6-P, may act on the test chemical
to form metabolites that can enter the rapidly dividing bacteria.
Some of these metabolites, or the test chemical itself, may react
with the bacterial DNA, causing DNA damage, some of which will lead
to mutation in a very small fraction of the progeny of the 100
million bacteria present at the start of incubation. When all the
histidine has been used up, the bulk of bacteria will stop
dividing, and a thin, visible confluent lawn of bacteria will have
formed in the soft agar. However, bacteria that have sustained DNA
damage leading to a mutation with the effect of reverting the his-
gene to his+ will continue to divide, since they can now
synthesize their own histidine from the ammonium salts in the
bottom agar. Each single revertant his+ (mutant) bacterium can
produce enough daughter bacteria in 48 h to form a single colony of
bacteria, easily visible to the naked eye. Therefore, the number
of such colonies on the plate is an accurate reflection of the
number of his+ revertants that have arisen spontaneously or by the
action of the test chemical. If there are significantly more
revertant colonies on treated plates than on control (solvent only)
plates, and if the numbers of revertants rise with increasing dose,
the result of the test is positive, and the chemical is a bacterial
22.214.171.124. Critical factors in the procedure
There are several conditions that must be met in order to
ensure an adequate test: these are briefly discussed below. More
extensive discussion can be found in IARC (1980a) and Venitt et al.
It is essential that a base-line protocol should be written
before starting a screening programme. Methods for the preparation
and storage of S9 and bacterial strains, and other procedures
should be thoroughly checked by performing assays with reference
mutagens and authenticated bacterial strains, under conditions
prescribed by the chosen protocol. Advice should be sought from
Choice, checking, storage, and culture of bacterial strains
The following strains of S. typhimurium are most-commonly used
for routine screening (Ames et al., 1975): TA 1535, TA 1538, TA 98,
and TA 100. Strains TA 97 and TA 102 are also considered useful,
under some circumstances (Maron & Ames, 1983). In addition,
E. coli WP2uvrApKM101 is often included. This trp- strain is very
sensitive to a wide range of mutagens (McMahon et al., 1979; de
Serres & Ashby, 1981; Matsushima et al., 1981; Venitt & Crofton-
Bacterial strains should be regularly checked for their
characteristic genetic traits, including: amino acid requirement;
background mutation; induced mutation with reference mutagens;
presence of plasmids where appropriate; presence of cell-wall and
Authenticated "master cultures" should be stored at a
temperature below -70 °C. Overnight cultures for routine assays
should be prepared by inoculation from master cultures or from
plates made from a master culture - never from a previously-used
overnight culture. The overnight culture should contain at least
109 viable bacteria per ml, and should be freshly prepared for each
Negative and positive controls
Each assay should include negative controls (addition of the
solvent but no test chemical) in order to check the background
mutation and positive controls (addition of reference mutagens to
check that the assay is performing correctly). A list of
appropriate positive control mutagens is given by Maron & Ames
(1984). Where possible, the compounds selected as positive
controls should be structurally related to the compound under test.
Test material and solvents
All data available on the substance to be tested should be
provided and recorded, including its lot or batch number, physical
appearance, chemical structure, purity, solubility, reactivity in
aqueous and non-aqueous solvents, temperature- and pH-stability,
and sensitivity to light. A sample of each substance to be assayed
for mutagenicity should be retained for reference purposes.
Solutions of test substances should be freshly prepared for
each experiment, and unused portions should be discarded. The
nature and percentage of impurities should be given: if a known
impurity is present in the test substance, it too should be assayed
for mutagenicity at doses equivalent to those that would be present
in the chosen doses of the major constituent. If a mixture is to
be tested, this should be stated.
The proposed uses of the test substance should be known, since
antibiotics, surfactants, preservatives, and biocides pose special
problems in bacterial mutation assays.
It is essential to devise operating procedures that minimize
the hazards from storage, handling, weighing, pipetting, and
disposing of mutagens and carcinogens, and that deal with
accidental contamination (Montesano et al., 1979; IARC, 1980b;
University of Birmingham, 1980; MRC, 1981). Laboratories should
follow the guidelines laid down for Good Laboratory Practice (GLP)
(PMAA, 1976; Federal Register, 1978). These important matters are
discussed further in section 3.
In most cases, DMSO is the best solvent, but, in cases where it
is unsuitable, other solvents may be used (Maron et al., 1981).
Preparation and use of S9
The animals should be free of disease and infection, kept clean
and at a reasonable temperature and should not be stressed by
careless handling. Dosing with inducing agents should be
consistent from one batch of animals to the next. Animals should
be killed humanely and the livers removed and chilled as soon as
possible. S9 should be stored at, or below, -70 °C.
Optimum mutagenesis with a particular test compound depends on
the amount of S9 added per plate. Too much as well as too little
S9 can drastically lower the sensitivity of the test. The optimum
S9 level for a given compound should therefore be checked. The
amount of S9 per plate is best expressed as mg liver protein per
plate calculated from the protein concentration of the S9.
There are two widely-accepted methods of using S9-mix:
(a) S9-mix is mixed with the top agar, bacteria, and test
substance, and the whole mixture is immediately
poured on to the surface of the bottom agar (Ames et
al., 1975); and
(b) in the pre-incubation method, the test substance,
bacteria, and S9-mix are mixed and incubated for 30
min; top agar is then added, and the mixture is
poured on to the bottom agar. This modification is
often more efficient in detecting certain classes of
mutagens, for example, aliphatic N-nitroso compounds
(Bartsch et al., 1976; Yahagi et al., 1977).
Design of experiments
A minimum of three plates per dose should be used in all
experiments. Doses of test chemicals should be spaced at intervals
differing by factors of less than 5. Narrow spacing of doses
avoids missing mutagens that are very toxic and that produce very
steep dose-response curves with sharp cut-offs. Combining the
requirement for narrow spacing of doses with the need to encompass
a very wide range of doses, two strategies emerge:
(a) a large experiment with closely-spaced doses ranging
from sub-microgram to milligram levels, using 7 or 8
doses together with positive and solvent controls; and
(b) two experiments, the first using sub-microgram to tens
or hundreds of micrograms. If the results are
positive, this should be confirmed in the strains and
the dose range in which the positive effect was
observed. If negative results are obtained, the
second experiment should be carried out at a higher
dose range, using the highest dose from the first
experiment as the lowest dose in the second
experiment, and extending the dose-range well into
the milligram range.
All experiments should be repeated at least once. If the first
experiment produces a weak or equivocal result (e.g., a dose-
related but less than 2-fold increase in revertants per plate), the
experiment should be repeated until a consistent picture emerges.
Incubation and examination of plates
Plates should be incubated at 37 °C for at least 48 h before
being scored. It is important to ensure that volatile test
compounds and gases are incubated in closed systems. After
incubation, it is essential to inspect the background lawn of both
treated and control plates with a dissecting microscope in order to
check for toxic effects (thinning of the lawn) or excess growth,
which may indicate the presence of amino acids in the test
2.1.4. Presentation and interpretation of data
126.96.36.199. Data-processing and presentation
The description of the protocol should be detailed enough to
allow independent replication of the assay. If a published
protocol has been used, this should be referred to, and any
deviations from it should be indicated. The following information
should be included in reports: source of the S9 (strain and
species of animal); details of inducers; percentage of S9 in the
S9-mix; mg liver protein per plate; concentration of buffer and
cofactors; items bought in from proprietary sources (e.g., S9,
ready-poured plates) should be noted. "Raw" data should be
provided: individual values of numbers of mutant colonies per
plate should be tabulated in ascending order of dose, starting
with the solvent controls. Data from positive controls should be
clearly identified and separated from the results obtained for the
test substance. The doses of test compound should be expressed by
weight per plate and not by volume. If the test substance is a
formulation or mixture, results should also be expressed per weight
of active ingredient(s). Providing that a complete raw data set is
provided, it is also useful to present graphs showing dose-response
188.8.131.52. Interpretation of data in terms of positive and negative
For a substance to be considered positive in a plate-
incorporation test it should have induced a dose-related and
statistically significant increase in mutations compared with
appropriate concurrent controls, in one or more strains of
bacteria, in the presence and/or absence of S9, in at least two
separate experiments. Experience has shown that a doubling or more
of the background mutation, combined with a dose-response curve,
indicates a positive response.
A test substance is considered negative if it does not produce
any increase in mutation at any dose, in at least 2 separate
experiments that complied with the base-line protocol submitted
with the test report. This protocol should include the following
requirements: the strains used, e.g., S. typhimurium strains TA
1535, TA 1538, TA 98, TA 100; E. coli uvrA(pKM101); testing at
doses spaced at 4-fold intervals or less and extending to the
limits imposed by toxicity or solubility, or, where the substance
is very soluble, into the milligram range; adequate concurrent
negative and positive controls, including positive controls to test
the efficiency of the S9-mix; tests in the presence and absence of
S9-mix; and finally, evidence of the identity of the bacterial
strains used in each experiment.
184.108.40.206. Dealing with ambiguous results
An ambiguous result arises when, at one or more doses, there
are more revertants per plate than are seen on concurrent control
plates, but there is not a clear dose-response relationship. This
increase may be consistent in two or more experiments. The effect
might occur in just one tester strain and at one particular level
of S9 in the S9-mix. Such a result cannot be classified as
negative, neither is it positive. The use of historical control
values to interpret ambiguous results is not recommended.
Ambiguous results may be caused by a technical problem, such as
the presence of nutrients in the test substance or the
bacteriostatic effect of the test substance; or it might be an
indication that a change in experimental procedure is required. In
addition, in the course of several replicate experiments, one or
two assays might be positive, and some might be negative. Results
of this type may be classified as "irreproducible". Under these
circumstances, the use of alternative protocols may resolve the
problem. See Venitt et al. (1983) for further discussion.
220.127.116.11. How the most critical factors identified above can influence
the validity of the data
The conduct of bacterial mutation tests requires close
attention to every aspect of the experimental procedure. Success
in running large numbers of such tests in routine screening
programmes depends on the establishment of consistent methods for
every phase of the experiment. A deficiency in just one area will
jeopardize the whole enterprise.
18.104.22.168. Interpretation of the results in terms of the intrinsic
mutagenic activity of the test material
A bacterial mutagenicity assay simply determines whether the
substance under investigation is or is not a bacterial mutagen in
the presence and/or absence of an exogenous metabolizing system
derived from a mammal (S9). Such a test cannot determine whether
the test substance is mutagenic and/or carcinogenic in any other
species. However, it may be concluded that a substance found to be
mutagenic in properly-conducted bacterial mutation assays should be
regarded as potentially mutagenic or carcinogenic for mammals
(including man) until further evidence indicates otherwise.
2.2. Genotoxicity Studies Using Yeast Cultures
The budding and fission yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe, respectively, are among the most
extensively studied of the eukaryotes and provide convenient tools
for use in genetic toxicology studies of environmental chemicals.
The internal structure of the yeast cells shows strong similarities
to that of the cells of higher organisms, in that they possess a
differentiated nucleus containing a nucleolus. The accurate
functioning of cell division depends on the synthesis of a spindle
apparatus; however, unlike mammalian cells, yeasts and other fungi
maintain their nuclear membrane during cell division.
The budding and fission yeasts are distantly related and differ
significantly in the persistance of the diploid phase of the life
cycle. S. cerevisiae haploid strains of the a and mating type, and
diploid cultures heterozygous and homozygous for mating-type may be
cultivated in the vegetative phase. In contrast, in S. pombe, the
vegetative haploid cells of mating type h+ and h- fuse to produce
a zygote, which undergoes immediate reduction division (meiosis) to
produce 4-spored haploid asci. Thus, S. cerevisiae strains are
suitable for routine use as both vegetative haploids and diploids,
whereas S. pombe strains are suitable for use only for the
measurement of genetic end-points detectable in haploids. Diploid
vegetative cultures of S. pombe have been produced by special
treatments but have not been used in genotoxicity studies so far.
Yeasts are physiologically-robust organisms, tolerating pH
values between 3 and 9; they survive at temperatures from freezing
to above 40 °C, and growth can occur over a range of approximately
18 °C - 40 °C. Growth is optimal at 28 °C and 32 °C for
S. cerevisiae and S. pombe, respectively, using a carbon source
such as glucose.
Diploid cultures of S. cerevisiae undergo meiosis under a
variety of conditions, such as those found in exhausted medium and
in the presence of 1% potassium acetate. Thus, by varying the
medium, it is possible to study S. cerevisiae during both mitotic
and meiotic cell division. The uncontrolled induction of meiosis
and spore formation in exhausted vegetative growth medium can lead
to problems during long periods of treatment. Such problems are
eliminated by the use of diploid strains such as JDI (described
later), which are unable to undergo meiosis and spore formation.
For both fission and budding yeast, there is an extensive data
base of experiments involving their use in studies on the
genotoxicity of chemicals. This data base has been reviewed by the
US Environmental Protection Agency Gene-Tox Program (Loprieno et
al., 1983; Zimmermann et al., 1984). However, readers should be
aware that significant numbers of chemicals in the yeast data base
were screened prior to the introduction of techniques involving in
vitro mammalian activation mixes. Thus, many of the apparently
negative results in the literature may stem from the use of
The primary advantages of yeasts in genotoxicity studies can be
summarized as follows:
(a) eukaryotic chromosome organization;
(b) variety of genetic end-points can be assayed;
(c) cost-effective assays requiring limited technical and
laboratory facilities using a "robust" organism.
2.2.2. Genetic end-points
When used in genotoxicity studies, yeast cultures have
significant advantages over other test systems in terms of the
comprehensive range of genetic end-points that can be assayed.
These end-points include:
(a) point mutation in chromosomal and mitochondrial genes;
(b) recombination, both between and within genes; and
(c) chromosome aneuploidy during both mitosis and meiosis.
A number of other variables including membrane damage,
differential killing in repair-deficient strains, and selective
effects have also been studied. However, the data base for such
events is still limited, and this section will be confined to the
events classified into groups (a), (b), and (c).
22.214.171.124. Point mutation
A variety of forward mutation systems has been used with yeast
cultures. However, the most extensively studied system has been
the one based on the induction of defective alleles of the genes
for adenine synthesis. The system involves the use of cultures
carrying defective mutations of the genes Adenine-1 and Adenine-2
of S. cerevisiae and Adenine-6 and Adenine-7 of S. pombe, the
presence of which results in the production of red-pigmented
colonies in S. cerevisiae and red/purple colonies in S. pombe,
owing to the presence of an intracellular pigment (aminoimidazole
carboxylic acid ribonucleotide in the case of adenine-2 mutations).
Forward mutations are detected in such strains by the induction
of further mutations at 5 genes that precede the production of the
red/purple pigment in the adenine synthetic pathway. Such
mutations result in the production of doubly-defective colonies,
which can be visually observed as white colonies or sectors. The
system can be illustrated as shown below:
Strain P1 of S. pombe genotype: ade6-60, rad10-198, h-
ade6-60 ----------------> ade6-60 ade x <- new mutation
red/purple forward mutation white
colonies produced by both colonies
The assay involves the treatment of red/purple cultures with
the test agent and the visual screening of colonies produced on
low-adenine medium for the production of whole white or sectored
colonies (Loprieno, 1981).
The most widely-used yeast strain for the detection of reverse
mutation is the haploid strain of S. cerevisiae XV185-14C,
developed by von Borstel and his colleagues (Mehta & von Borstel,
1981), which has the following genotype:
a ade2-1, arg4-17, lys1-1, trp5-48, his1-7, hom3-10
The markers ade2-1, arg4-17, lys1-1, try5-48 are ochre
"nonsense" mutations, which are revertible by base-substitution
mutagens that induce site-specific mutations or ochre-suppressor
mutations in t-RNA loci. The marker his1-7 is a missense mutation
that is reverted mainly by second site mutations; hom3-10 is
believed to be a frameshift defect because of its response to a
range of diagnostic mutagens. As with most, if not all, of the
frameshift mutations identified in yeast, the hom3-10 allele
reverts at a relatively low frequency and its use in testing
protocols requires the screening of large populations of cells.
In diploid strains of S. cerevisiae, the only mutation marker
that has been extensively used is the ilvl-92 mutation that is
present in the homoallelic condition in strain D7. Unfortunately,
the marker responds to only a limited range of mutagens and it
would be inappropriate to regard it as a comprehensive point
mutation screening system for environmental chemicals.
The induction of mutations that lead to defects in
mitochondrial function in yeast may be detected by the assay of the
frequency of respiration-deficient "petite" colonies, which are
incapable of aerobic respiration. Such colonies are characterized
by their small size and their inability to grow on non-fermentable
carbon sources such as glycerol. Petite colonies may be produced
by the induction of both chromosomal and extrachromosomal events
but, in diploid cells, those detected are predominantly of
extrachromosomal origin. In yeasts, extrachromosomal mutations are
induced at high levels by a wide range of chemical mutagens.
However, at present, the significance of such events is far from
clear (Wilkie & Gooneskera, 1980).
In eukaryotic cells, genetic exchanges between homologous
chromosomes are generally confined to a specialized stage of
meiotic cell division which, in yeasts, occurs during the process
of sporulation. Recombinational events in yeasts may also be
detected during mitotic or vegetative division, though the
spontaneous frequency is generally at least 1000 times less than
that observed during meiosis. Sporulating yeast cultures can be
used to study the rates of both spontaneous and induced meiotic
recombination, but it is generally the mitotic events that are of
practical value in genotoxicity studies.
Mitotic recombination can be detected in yeasts both between
genes and within genes. The former event is called mitotic
crossing-over and generates reciprocal products whereas the latter
is most frequently non-reciprocal and is called gene conversion.
Crossing-over is generally assayed by the production of recessive
homozygous colonies or sectors produced in a heterogenous strain.
Gene conversion is assayed by the production of prototrophic
revertants produced in a heteroallelic strain carrying two
different defective alleles of the same gene. Mitotic gene
conversion can be distinguished functionally from point mutation by
the elevated levels of prototrophy produced in heteroallelic
strains compared with levels in homoallelic strains (carrying two
copies of the same mutation).
Genetic loci in this paper are labelled as follows:
abbreviation for gene
ade 6 - 60
Capital letters indicate the wild-type form of the gene and lower
case the mutant form. The suffixes r and s indicate resistance and
sensitivity to antimicrobial agents, respectively. rad loci -
indicate genes involved in DNA repair. ‚------- represents a
chromosome with its centromere.
The value of assaying mitotic recombination in yeast in
genotoxicity studies stems from the observation that both events
are elevated by exposure to genotoxic chemicals. These increases
are produced in a non-specific manner, i.e., levels are increased
by all types of mutagens, irrespective of their mode of action.
Thus, the primary advantage of assaying for the induction of
mitotic recombination is that the events involved are reflective of
the cell's response to a wide spectrum of genetic damage. A number
of suitable strains of S. cerevisiae have been constructed for use
in genotoxicity testing. However, for the purposes of this
document it will be confined to those most frequently used and
convenient for use.
Mitotic gene conversion can be assayed using selective medium
in the diploid S. cerevisiae strain D4 (Zimmermann, 1975). The
genotype of D4 is as follows:
Chromosome III ‚--------------- Chromosome XII ‚---------------
ade2-2 leu1 trp5-12
Chromosome XV ‚--------------- Chromosome VII ‚---------------
ade2-1 LEU1 trp5-27
ade2-2, ade2-1, trp5-12, and trp5-27 are heteroalleles at
the ADE2 and TRP5 loci, respectively. These alleles undergo
mitotic gene conversion to produce prototrophic colonies
carrying one wild-type allele which makes growth possible on
selective medium lacking either tryptophan or adenine, e.g.,
cell division ‚-----------------
with chromosome ‚-----------------
ade2-2 replication / ade2-2
‚----------------- \ or
The D4 strain has been extensively used in the study of
genotoxic chemicals (Zimmermann et al., 1984) and has proved to be
a valuable tool. However, the use of the strain is limited by the
relatively high spontaneous reversion frequencies of the ADE-2
marker which means that, if this loci is to be used, cultures with
low spontaneous prototroph frequency must be selected prior to
Mitotic gene conversion can also be assayed in the strain JDI
(Sharp & Parry, 1981), which is capable of simultaneously assaying
mitotic crossing-over on chromosome XV. The genotype of JDI is as
Chromosome III --------------‚-------------
ade2 ser1 his8
Chromosome XV ‚-------------------------------------
ADE2 SER1 HIS8
Chromosome VII ‚-------------------
his4C, his4ABC, trp-U9, and trp5-U6 are heteroalleles at the
HIS4 and TRP5 loci, respectively. These alleles undergo mitotic
gene conversion to produce proto colonies carrying one wild-type
allele which makes growth possible on selective medium lacking
either tryptophan or histidine. Mitotic crossing-over can be
assayed by the production of red colonies or sectors homozygous for
ade-2 and the markers distal on chromosome XV, e.g.,
ade2 ser1 his8
ade2 ser1 his8
ade2 ser1 his8 \ / ade2 ser1 his8
‚------------------- \/ ‚------------------
O------------------- --> /\ ----> O------------------
ade2 ser1 his8 / \ ade2 ser1 his8
ADE2 SER1 HIS8
ADE2 SER1 HIS8
white colonies mitotic crossing-over red colonies or
for adenine serine
Thus, using the strain JDI, it is possible to assay mitotic
gene conversion at two separate loci and also to detect one of the
possible homozygous chromosome combinations produced by mitotic
crossing-over (but not both reciprocal products). The strain has
been selected for its inability to undergo sporulation and is thus
suitable for long periods of treatment. Protocols are available
for the use of this strain under conditions of optimal cytochrome
P-450 concentrations (Kelly & Parry, 1983a).
A particularly convenient multipurpose strain of S. cerevisiae
is D7 (Zimmermann, 1975) which carries a set of genetic markers
that allow the simultaneous assay of mitotic crossing-over, gene
conversion, and point mutation.
The genotype of D7 is as follows:
a trp5-12 cyhr 2
Chromosome III ‚------- Chromosome VII ‚----------------------
alpha trp5-27 CYHS 2
Chromosome XV ‚-------- Chromosome V ‚----------------
The heteroalleles of the TRP-5 locus, tryp5-12 and trp5-27,
undergo mitotic gene conversion to produce prototrophic colonies
carrying one wild-type allele which makes growth possible on
selective medium lacking tryptophan. While ade2-40 is a
completely inactive allele of ADE-2 that produces deep red
colonies, ade2-119 is a leaky allele (only partially defective)
causing accumulation of only a small amount of pigment and thus
producing pink colonies. In heteroallelic diploids, the ade2-40
and ade-2-119 alleles complement to give rise to white adenine-
independent colonies. Mitotic crossing-over in D7 may give rise
to the production of cells homoallelic for the ade2 mutations and
thus lead to the observation of both red and pink reciprocal
‚-------------- ‚--------------- red colonies
ade2-40 ade2-40 O---------------
‚--------- ‚-------------- / ade2-40
O--------- --> \/ /
ade2-119 /\ ade2-119 /
O-------------- \ ade2-119
ade2-119 ‚--------------- pink colonies
The frequency of induced reciprocal mitotic crossing-over can
be unambiguously confirmed in D7 by the visual observation of
treated colonies. Mitotic crossing-over can also be assayed in D7
by the use of the recessive cycloheximide resistant cyhr 2 allele
on chromosome VII. Crossing over between CYH2 and the centromere
of chromosome VII results in the production of colonies that are
capable of growth on medium containing cycloheximide (Kunz et al.,
cyhr 2 cyhr 2 cyhr 2
‚---------- ‚----------------- ‚-------------
O---------- ---> ---> O-------------
CYHS 2 cyhr 2
cycloheximide- mitotic crossing-over cycloheximide-
sensitive colonies resistant
The final genetic event that can be assayed in strain D7 is the
induction of base-substitution mutation at the homallelic- ilvl-92
markers by the production of prototrophs that grow on selective
minimal media that lack isoleucine.
Abnormal chromosome segregations leading to the production of
numerical chromosome aberrations can be detected in yeasts by
genetic means using appropriate yeast strains. Suitable strains of
S. cerevisiae are available that are capable of detecting and
quantifying the reduction of monosomy (chromosome loss) during
mitotic cell division from the 2n to the 2n-1 condition and the
production of disomy (chromosome gain) and diploidisation in spores
produced during meiotic cell division (sporulation) (Fig. 1).
Although a number of strains have been developed for the
detection by genetic means of chromosome aneuploidy, only two have
been extensively used in the screening of environmental chemicals.
These are D6 described by Parry & Zimmermann (1976) and DIS13
described by Sora et al. (1982), which have been developed for the
assay of induced aneuploidy during mitotic and meiotic cell
The genotype of S. cerevisiae diploid strain D6 is as follows:
ade3 leu1 trp5 cyhr 2 met13
ADE3 LEU1 TRP5 CYHS 2 MET13
Chromosome XV ‚-------------- Chromosome III ‚------------
This strain forms red colonies because of the presence of ade2
in a homozygous condition and is sensitive to the presence of
cycloheximide in the medium because the cyhr 2 resistance allele is
recessive. The loss of the chromosome VII homologue carrying the
dominant wild-type allele of this group of linked markers results
in cells that form white (due to the expression of ade3) and
cycloheximide-resistant (due to the expression of the selective
cyhr 2 marker) colonies that also express the markers leu1, trp5
and met13 (defined as Go). Treatment protocols may involve the
treatment of stationary phase cells in an appropriate buffer,
exponential phase cells in buffer for a short period before they
enter Go, or growing cells in nutrient medium or on overlay plates.
The vast majority of the experimental studies on chemical
mutagens using yeasts have involved liquid-suspension assays.
Plate assays are nevertheless also possible and have been used
for the assay of mitotic crossing-over, point mutation, and gene
conversion (Fink & Lowenstein, 1971; Parry et al., 1976; Kunz et
al., 1980). The advantages of liquid-suspension assays with regard
to their ability to quantify cellular toxicity has, however, led to
a preponderance of the studies in the literature. Cells of both
yeast species have also been used extensively in host-mediated
assays (Fahrig 1975; Loprieno et al., 1976) where they appear to
tolerate incubation in mammals for long periods without eliciting
Liquid-suspension assays involve treatment of cells with test
chemicals for periods of preferably less than 24 h, removal of the
test chemical, followed by plating on nutrient and selective medium
for quantitation of both cell viability and the genetic end-point.
Appropriate treatment media for the strains described here can be
found in the publications of Loprieno, (1981), Mehta & von Borstel
(1981), Parry & Sharp (1981), Sharp & Parry (1981), and Zimmermann
& Scheel (1981) for both S. pombe and S. cerevisiae. Specific
stages of mitotic cell division such as G1, S, and G2, can be
investigated using synchronized cultures or, more conveniently, the
separation of exponential phase cells by means of a zonal rotor
(Davies et al., l978).
Exposure of yeast cells to test chemicals is generally
performed at the optimal growth temperature for the two species,
i.e., 28 °C and 30 °C for S. cerevisiae and S. pombe,
respectively. When mammalian metabolic activation preparations are
used (see later) it may be appropriate to incubate cultures at
37 °C for a proportion of the total treatment time. In all such
treatments, it is essential that media are adequately buffered at
pH 7.0, as yeast cultures rapidly acidify their media. However,
advantage can be taken of the pH tolerance of the organisms for the
testing of chemicals that are biologically active at acid pHs.
When direct comparisons have been made between liquid yeast
suspension assays and bacterial plate assays, there has been a
close similarity in the sensitivity of the two assays (Parry &
The assessment of the genotoxicity of chemicals in yeasts
during meiosis involves the treatment of cells during the process
of sporulation. Sporulation can be induced by the transfer of
vegetative cultures to a medium containing only potassium acetate,
but maximum levels of sporulation are obtained if the culture is
pre-grown in a pre-sporulation medium containing both acetate and
nutrients. Chemicals can be assayed by exposure of cells
throughout the sporulation period or by treatment at specific
stages during meiosis. Suitable protocols for the assay of the
effects of chemicals, during meiosis, on mutation and chromosome
aneuploidy have been described in detail by Kelly & Parry (1983b)
and Parry & Parry (1983), respectively. During sporulation, the
treatment medium undergoes an alkaline pH change which may result
in the detoxification of some test compounds.
Mammalian metabolic activation preparations have been employed
in the assay of genotoxic chemicals using both fission and budding
yeasts, and suitable formulae for such mixes have been described by
Loprieno (1981) and Sharp & Parry (1981), for use with
Schizosaccharomyces and Saccharomyces, respectively. Most
preparations are based on those used in bacterial assays, which
have been described earlier in this report. Relatively few studies
have been performed with yeasts using various enzyme-inducing
agents, mammalian species, and liver fractions, though there is
considerable scope for such studies (Wilcox et al., 1982). There
is now evidence that, unlike Salmonella, yeast cells have a
significant endogenous metabolic capacity of their own (Callen &
Philpot, 1977) and protocols have been developed that produce
relatively high levels of cytochrome P-450 for periods of up to
18 h of chemical treatment (Kelly & Parry, 1983a).
In yeast liquid-suspension assays, the time of exposure to the
test chemical depends on the nature of the protocols used, the
specific chemical being tested, and the yeast strain and genetic
end-point being studied. Thus, no specific recommendations can be
made with regard to the optimal time of exposure required to
adequately test chemicals. There are a number of factors that
should nevertheless be borne in mind when designing an experiment:
(a) In studies with vegetative cells, care must be taken
that, when diploid cells are used, the exposure times
are not such as to lead to the induction of
sporulation. If long exposure times are necessary,
cells should be checked for spore formation or use
made of non-sporulating strains such as JDI.
(b) Exposure times should be sufficient to allow for
entry of the test chemical into the cell and the
production of the damage that provides the substrate
for the induction of the specific end-point being
(c) Provision must be made to allow for the expression of
the end-point, e.g., in the case of the assay of
induced chromosome aneuploidy a period of
post-treatment cell division must take place before
exposure to a selective agent.
Dose selection is another parameter that is highly dependent on
a number of experimental variables such as: the culture used, the
end-point measured and the nature of the chemical being tested. In
general, dose ranges should be selected on the basis of
cytotoxicity and solubility to include concentrations that range
from the "no-effect" dose level up to 90% cell lethality with
approximately 1/3 log spacing. Dose selection is more difficult
with assays such as that for chromosome loss where "humped" dose-
response curves are a common feature (Parry et al., l980) and
maximum induction of the end-point may occur at non-toxic doses.
Similar problems of dose selection have also been encountered with
specific chemicals such as the dinitropyrenes, whereas in yeasts,
the induction of mitotic gene conversion is detectable only at non-
toxic doses and is reduced at higher concentrations (Wilcox &
Parry, 1981). In such cases, there is probably no alternative but
to test a chemical down to arbitrary concentrations of at least
0.1 µg/ml or to relate the minimum test concentrations to potential
After exposure to the test chemical, yeast cells are washed and
plated after dilution on nutrient medium and the appropriate
screening medium for the end-point under test. In the case of cell
viability and genetic end-points that provide a large number of
scorable events per plate, 3 replicate plates are appropriate.
However, in the case of relatively rare events, such as mutation in
frameshift marker strains, and non-selectable events, such as
forward mutation at the adenine loci, the plate numbers must be
increased to ensure the statistical significance of the data, as
with small numbers the standard deviation of the plate counts will
be large. There is no generally agreed method of analysing the
data generated by yeast genotoxicity tests and a number of suitable
methods have been described, e.g., Loprieno et al. (1976), Sharp &
Parry, (1981), and Kelly & Parry (1983a).
It is essential that all experiments using yeast cells should
be independently repeated and ambiguous results may require further
experimentation with careful selection of sample size, treatment
concentrations, culture stage, or metabolic activation system. The
aims of such repeated experiments should be to increase the
statistical validity of the results.
2.2.3. Information required
Data are best presented in tabular form supplemented with the
appropriate graphical treatment. A test report should include the
(a) strain of yeast used and genotype;
(b) description of the test conditions, including growth
phase of cells used, whether growing or non-growing;
details should be provided of length of treatment,
dose levels, toxicity, medium, and treatment
procedure; the negative and positive controls used
should be clearly specified;
(c) raw data should be provided to include plate counts
of viability and colony type selected, calculations
of survival and frequency of genetic end-point under
study, and dose-response relationships if applicable;
(d) the results should be evaluated using an appropriate
statistical procedure and interpretation provided.
126.96.36.199. Significance of positive results in yeast assays
1. A positive response in mutation assays is indicative
of the ability of a chemical to induce point
mutations in eukaryotic DNA.
2. A positive response in assays for mitotic
recombination indicates the potential of a chemical
to produce DNA interactions in a eukaryotic cell.
The majority of such chemicals will be capable of
producing either point mutations or chromosome
aberrations in mammalian cells.
3. A positive response in assays for chromosome
aneuploidy indicates the potential of a chemical to
produce changes in chromosome number in eukaryotic
cells. However, in at least a proportion of such
chemicals, the effect may be specific for yeasts and
requires confirmation in a mammalian system.
Such a response should be reproducible in independent
experiments and should be significant when evaluated by an
appropriate statistical test. However, care should be taken to
ensure that the cultures used showed the "normal" levels of
spontaneous frequency for the event scored and that treatment
conditions were not such as to induce sporulation in vegetative
cultures. Considerably more weight can be placed on results if an
unambiguous dose response is observed, though deviations from
linearity are common for many of the genetic end-points of yeast.
There are no published data suggesting that yeast assays produce
false-positive results for any consistent reason.
188.8.131.52. Negative results in yeast assays
Negative results may be obtained in yeast assays of chemicals
for genetic activity for a number of reasons:
(a) the test compound is inactive in eukaryotic cells;
(b) the compound has not been exposed to the appropriate
metabolic activation system;
(c) the relevant genetic end-point is not detectable in
yeast cultures, e.g., the induction of chromosome
(d) the compound has not been tested over an appropriate
dose range, e.g., a fungicide may lead to cellular
toxicity before the genetically active cellular
concentrations are achieved. Another such example
may be found in assays such as those for chromosome
aneuploidy which frequently generate "humped"
dose-response curves where the genetically active
range requires the use of extensive concentration
The validity of a negative result with a test chemical will be
of more general relevance if data are accompanied by appropriate
responses from positive control chemicals.
2.3. Unscheduled DNA Synthesis in Cultured Mammalian Cells
The ability of living cells to remove damage induced in DNA was
first reported in 1964 (Boyce & Howard-Flanders, 1964; Setlow &
Carrier, 1964). It is now clear that cells can cut out portions of
DNA damage in one strand of the double helix, replace the excised
portion with undamaged DNA nucleotides by using the opposite strand
as a template and rejoin the newly synthesized section to the pre-
existing DNA strand (Hanawalt et al., 1979) (Fig. 2). This process
is called excision repair and restores the original integrity of
the DNA molecules.
Unscheduled DNA synthesis (UDS) is the term used to describe
the synthesis of DNA during the excision repair of DNA damage and
as such is distinct from the semiconservative replication that is
confined to the "S" phase of the eukaryotic cell cycle. Rasmussen
& Painter (1964) first reported the incorporation of 3H thymidine
into the DNA of cultured mammalian cells during the repair of
damage induced by ultraviolet irradiation. These authors used
autoradiography to detect UDS. This method involves culturing
cells on glass slides, exposing them to a DNA-damaging agent in the
presence of a medium containing high specific activity 3H
thymidine, and observing the radiolabel incorporated during UDS
into cells that are not semiconservatively replicating DNA. This
is done by way of an emulsion or film that detects the beta
emission from the tritium. The ability of substances to induce UDS
in cultured cells is now widely used routinely to assess the
genotoxic activity of compounds in mammalian systems. The assay is
therefore a measure of the amount of repair produced and monitors
neither the original lesion nor the consequences of repair. The
amount of DNA replication associated with UDS is relatively low
compared with the amount associated with semiconservative
replication. If autoradiography is used to monitor this process,
"S" phase cells that are undergoing semiconservative replication
are readily eliminated from the analysis because of their heavy
labelling indices. In this section, the measurement of
radiolabelled thymidine incorporated during UDS by either
autoradiography (Cleaver & Thomas, 1981) or liquid scintillation
counting (LSC) will be considered (San & Stich, 1975; Martin et
al., 1978). Unfortunately, the second method cannot distinguish
between semiconservative and repair replication. It measures the
total amount of DNA replication by monitoring the incorporation of
3H thymidine into the total DNA, including cells that are actively
replicating DNA semiconservatively. The elimination of the
semiconservative replicative process is therefore an essential
prerequisite for this approach and can be achieved by various
methods to be discussed later.
UDS has been detected in cells cultured from many mammalian
species, in various cell types, and with different inducing agents.
Human fibroblasts (San & Stich 1975) or human transformed cell
lines such as HeLa (Martin et al., 1978) are often used. One
disadvantage of these cell lines is that they do not possess the
ability to activate proximate carcinogens as does the liver in
vivo, and thus additional metabolic activation in the form of a
liver microsomal extract is required during chemical exposure.
Alternative approaches to metabolic activation have been developed
using epithelial cells derived from liver. These have been shown
to retain some of the ability to activate proximate carcinogens
(Williams, 1977; Dean & Hodson-Walker, 1979).
2.3.2. Chemical exposure and UDS
A large number of mutagens/carcinogens capable of inducing
many types of DNA damage are known to induce UDS. The exact
amount of this repair synthesis depends on (a) the particular
mutagen/carcinogen in question, (b) the type of DNA repair process
that operates on the damage induced, and (c) the size of the repair
patch that is cut out to remove the damage prior to subsequent
resynthesis. For example, it is known that some types of DNA
damage, such as those induced by gamma or X irradiation, are
repaired relatively quickly in mammalian cells and involve the
excision of only one to three bases per lesion (Regan & Setlow,
1974). Other types of damage such as UV radiation-induced
pyrimidine dimers, are repaired more slowly and involve the
replacement of from twenty to seventy bases per lesion (Regan &
Setlow, 1974). The ability to detect UDS is further influenced by
more obvious factors such as whether the cells used take up and
incorporate (3H)thymidine readily, the concentration and specific
activity of the (3H)thymidine, and the efficiency of the
scintillation counter or film used to detect the radiolabel.
184.108.40.206. The choice of a suitable cell line
Transformed cell lines, i.e., those possessing an infinite life
("immortal" cells) are preferred by some workers, but others have
used primary cell lines with a finite life of some 30 - 40
generations. Generally, transformed cells (e.g., Hela) are easier
to culture and grow more rapidly than primary cells. Rapid growth
is an advantage for general cell culture, but it also means that
the number of cells actively undertaking semiconservative DNA
replication in the population at a given time is higher, and the
sensitivity of the assay is thus slightly reduced. This fact is
less significant if autoradiography is used as the means of
detecting UDS, since replicating "S" phase cells are readily
excluded from cells at other stages of the cell cycle. However, if
scintillation counting is used as the means of detecting UDS,
precautions must be taken to greatly reduce the amount of
semiconservative replication in the cell population. So far, it
has not been possible to eliminate background levels of residual
semiconservative replication entirely. In this respect,
untransformed cell lines have a distinct advantage in that they
stop dividing when they reach confluence because of contact
inhibition when the amount of residual semiconservative DNA
replication is considerably less than that seen in an actively
A second important factor influencing the choice of cell line
involves the activation of proximate carcinogens. Activation can
be undertaken by the cell itself or by a microsomal liver extract
added to the cell culture. The advantages of adding microsomal
extract are that the same extract can be used in a range of other
tests on different organisms, hence facilitating legitimate
comparisons between various end-points. Furthermore, the source of
extract can be easily varied if there is concern about the effects
of a compound in a particular species, organ, or tissue. The major
disadvantage is that certain concentrations of the extract iself
can be toxic to some cell lines. The use of hepatocytes, because
of their ability to activate proximate carcinogens without
microsomal extract, would seem to offer a considerable advantage.
However, a careful analysis of the hepatocyte line selected is
essential prior to its use for the routine monitoring of DNA
damaging agents, because it is also known that that such cells can
exhibit a reduction in activation ability after a number of
passages. Consequently, many research workers prefer to use
freshly isolated primary hepatocytes for each experiment (Williams,
1977). Two further problems have been identified in the use of
primary rat hepatocytes to screen chemicals for genotoxic activity
(Lonati-Galligani et al., 1983). First, hepatocytes show a high
cytoplasmic background labelling because of the incorporation of
radioactive thymidine into mitochondrial DNA. Second, a large
variation in the functional state of isolated hepatocytes affects
the reproducibility of the system. Whatever cell type is chosen,
it is imperative that a control chemical, known to require
metabolic activation in order to induce DNA damage, is included in
each experiment in order to monitor the activity of the endogenous
metabolizing enzymes in that particular cell population.
For the autoradiography method, cells are cultured on cover
slips or glass slides, and, for scintillation counting, the cells
are cultured in disposable petri dishes.
2.3.4. The elimination of semiconservative replication
The minimizing of semiconservative DNA replication is an
absolute prerequisite for the measurement of UDS by liquid
scintillation counting. Although this is less important for
autoradiography, some research workers prefer to suppress the
semiconservative process in actively-dividing cells, especially if
the percentage of such cells is high, and if long repair times are
to be studied. In this last instance, the proportion of cells
entering new rounds of DNA replication could be substantial.
Hydroxyurea, is commonly used at 10-3M in UDS studies to
suppress semiconservative replication, and, at this dose, it has
little effect on the amounts of UDS observed. It should be noted
that this drug may react with the microsomal activation mixture
to produce DNA damage (Andrae & Greim, 1979) and that, at high
concentrations, inhibitory effects on DNA repair have been reported
(Collins et al., 1977). Thus, unless its inclusion is essential,
this drug is best avoided. When hydroxyurea is used, the
appropriate controls to monitor possible effects such as reaction
with microsomal extract to produce DNA damage should be undertaken.
Additional treatments, such as incubation with medium containing
low serum, or, as mentioned, growing cells to a monolayer are
also used, as both approaches result in a cell population that
contains considerably fewer replicating cells. Generally, when
scintillation counting is the method selected for the detection of
UDS, one or other of these two approaches is tried, before addition
2.3.5. Chemical exposure
Prepared cultures are exposed to a range of doses of the
chemical to be tested with, if required, the addition of microsomal
extract and appropriate cofactors. The choice of a suitable dose
range is governed by the toxicity of the compound. Studies should
usually be undertaken at doses that induce 50% or less cytotoxicity
as measured by, for example, Trypan Blue exclusion. In each
experiment, it is imperative to include the appropriate control
cultures to ensure that the system is functioning correctly. Thus,
background UDS in untreated cells in the presence or absence of
activation systems and solvents should be included as well as an
appropriate positive control known to be activated by the
microsomal activation system used (e.g., n-acetylamino-fluorene).
Cell cultures are usually exposed to five dose levels of the test
compound and, ideally, each dose is duplicated. Exposure times are
usually of the order of one to a few hours, but longer periods have
2.3.6. Radiolabelling procedures
A typical procedure involves adding to the culture medium
(3H)thymidine of a specific activitya greater than 20 Ci/mmol at
10 µCi/ml. This is usually carried out immediately after the test
compound is added so that both the radiolabel and compound are
present simultaneously for the duration selected for the
2.3.7. Detection of UDS
The autoradiographic method involves the removal of the medium
containing the test compounds, followed by rinsing and fixation of
the cells, coating the slides with autoradiographic emulsion and
then drying them prior to developing. Procedures will vary with
the particular process used and are described in detail by Cleaver
& Thomas (1981). After developing, the cells are stained and the
grains in the emulsion over the cell nuclei of control and treated
samples are either observed microscopically and counted visually or
with an electronic counter. The data are expressed as grains per
In the liquid scintillation method, culturing and exposure
procedures are similar to those for autoradiography, except that
more cells and more replicate samples are usually analysed. The
data are expressed as disintegrations per min (dpm) of incorporated
(3H) thymidine per µg of DNA. Hence, not only the amount of
radioactivity per sample needs to be determined, but also the
amount of DNA. This can be carried out by DNA extraction with
perchloracetic acid hydrolysis (Schmidt & Thannhauser, 1945),
using one aliquot for reaction with diphenylamine to measure DNA
concentration (Burton, 1956) and a second aliquot for scintillation
counting to measure (3H) thymidine incorporation. Alternative
methods for estimating DNA concentrations are available (San &
2.3.8. Data processing and presentation
For a compound to be accepted as positive with the UDS assay,
there should be: (a) a dose-related increase in UDS, and (b) a
statistically-significant increase in UDS above that of a negative
control. Data are usually presented as grain counts per nucleus
(often as histograms), or (3H) incorporation as dpm per µg DNA, as
determined by scintillation counting. The data should include the
results from all treatments and controls. At high concentrations
of test agents, the amounts of UDS may plateau because of the
saturation of repair mechanisms, or they may even decrease due to
cytotoxicity. This again emphasises the importance of undertaking
experiments over a wide dose range, and then selecting a narrow
range from the initial data to verify a potentially positive
a The specific activity denotes how much of the thymidine is
Various criteria have been used for the definition of a
positive result. Investigators have considered a compound positive
when it induced at least 150% of the control levels of UDS as
measured by liquid scintillation counting (San & Stich, 1975), or
when it induced at least 6 grains per nucleus in excess of
background levels with autoradiography (Williams, 1977). In
addition to such basic criteria, the data should also be subjected
to statistical analysis to determine whether or not the increases
are significant. Cleaver & Thomas (1981) recommend that, when 40 -
100 cells per slide are counted for several slides per treatment,
the average grain number for each slide can be used as a measure of
UDS, and the average and standard error of these averages would be
the more suitable parameter for the amount and accuracy of the
data. When liquid scintillation counting is employed, the standard
deviation or standard error of the mean should be included to
describe the distribution of the data. Additional analyses such as
analyses of variance, non parametric comparisons of grain
distribution, and estimates of the correlation between UDS and dose
can be undertaken, and the selection of the most appropriate method
will depend on the design of the experiment. The t-test, used by
some, increases the chances of obtaining false positives, whereas
though the analysis of variance does not introduce this problem it
does reflect cytotoxic effects. Ideally, the analysis should be
complemented with a contrast analysis that can distinguish between
treatments giving negative, positive, or cytotoxic effects. For a
more complete review of the statistical analysis of UDS data, the
Gene-tox report on UDS tests by Mitchell et al. (1983) can be
UDS is a relatively straightforward approach for measuring DNA
repair and as such is extremely useful for examining compounds that
are potentially genotoxic for mammalian cells. Nevertheless, it is
usually undertaken as part of a battery of screening tests. In
exceptional cases, it may be the only assay to provide a positive
result and, in such cases, in vivo tests should be undertaken to
clarify this result. It should also be noted that this assay
detects the repair of DNA damage. The assay would not detect a
compound that induced an unrepairable lesion in DNA, though the
same compound would be expected to induce genetic damage, e.g.,
mutations, in other test systems. The usefulness of DNA repair
assays in screening is more fully reviewed by Cleaver (1982).
It is obvious from the procedural discussion in this section
that a number of different systems are currently used to measure
UDS. Thus, it would seem appropriate to list and briefly evaluate
critical factors that can influence each type of assay, and which
have been mentioned at various stages in the text.
220.127.116.11. Choice of cell line
If possible, untransformed human fibroblasts or primary rat
hepatocytes should be used, because semiconservative DNA
replication is more readily suppressed in the former, whereas the
latter are essentially non-dividing and can themselves activate
proximate carcinogens. Furthermore, a relatively large pre-
existing data base is available for both cell types, which enhances
the possibility of making comparisons with other compounds tested.
However, it should be remembered that primary rat hepatocytes
exhibit high levels of incorporation of radioactivity into the
cytoplasm. It has been suggested that for autoradiographic
estimates, instead of subtracting cytoplasmic grains from nuclear
grains, as is usually done to account for non-nuclear
incorporation, grains overlying the nucleus and a cytoplasmic area
should be scored and plotted separately in these cells (Lonati-
Galliganai et al., 1983). The variation in the functional state of
freshly isolated hepatocytes can be a problem and it is imperative
to undertake adequate controls to verify their ability to activate
18.104.22.168. Choice of protocol
If rat hepatocytes are used, autoradiography is the method of
choice, as liquid scintillation counting (LSC) requires such a
large number of cells. Human fibroblasts can be analysed either by
autoradiography or by LSC, but it should be borne in mind that the
latter approach requires more cells per sample, more duplicate
samples, and the addition of hydroxyurea to supress residual
semiconservative replication. At concentrations above 10-3 M,
hydroxyurea can inhibit DNA repair and can also induce DNA damage
by interacting with microsomal extract. These facts have to be
considered when interpreting data. Where costs have to be kept to
a minimum, it should be noted that a liquid scintillation counter
is expensive compared with the cost of a microscope for
autoradiography. However, a counter can process many samples
automatically, whereas microscopic analysis is more time consuming.
22.214.171.124. Method of activating proximate carcinogens
The use of rat hepatocytes removes the necessity for the
addition of microsomal extract. Whatever kind of microsomal
activation is used, it is important to include appropriate controls
to verify that the activation system is functional.
Finally, it should be emphasized that, regardless of the system
used, the most important features in undertaking UDS studies are a
full understanding of, and extensive experience with, the test
2.4. In Vitro Cytogenetics and Sister-Chromatid Exchange
In vitro cytogenetic tests are designed to demonstrate the
induction of chromosome damage (aberrations), visible under the
light microscope, in cultured cells (Fig. 3). This usually
involves examination at the metaphase stage of the cell cycle
(Evans & O'Riordan, 1975; Savage, 1976). Though other methods such
as anaphase analysis and enumeration of micronuclei have been used,
they are not generally considered suitable for routine testing in
cultured cells. A physical or chemical agent is classified as a
clastogen if it produces an increase in the number of breaks in
chromosomes over that found in control samples. Cytogenetic tests
therefore assess gross damage to the DNA involving at least one
double-strand break. A detailed discussion of the theoretical
aspects of the development of chromosome aberrations is given in
Many agents only induce visible chromosome damage after the
cells have undergone a round of DNA replication, and the test must
be designed to allow enough time after treatment for aberrations to
develop. However, damaged cells may not survive for more than one
or two cell cycles after aberrations have been induced, and it is
essential that cells should be examined in their first metaphase
after treatment (Evans, 1976).
Induction of chromosome aberrations involves major damage to
chromosome structure, and thus to the DNA, and so clastogenic
agents must be viewed as potentially harmful. Although cells with
visible chromosome aberrations are unlikely to have the potential
to survive, repair of DNA damage may have occurred in apparently
undamaged cells, and if this is error-prone, mutations could
result. Certain types of chromosome damage, such as some deletions
and rearrangements (translocations, inversions), may not be lethal.
The comparatively high level of chromosomal disorders in man
emphasizes the importance of chromosome changes in human
populations (DHSS, 1982).
The preparation of material for examination of the chromosomes
is technically simple, and this has undoubtedly contributed, in
part, to the widespread use of short-term cytogenic tests.
However, accurate and reliable scoring of metaphase chromosomes for
aberrations does require a high level of expertise.
As its name implies, sister chromatid exchange (SCE) involves
an apparently symmetrical change between chromatids within a
chromosome (i.e., between identical sequences of DNA). SCEs are
only visible under the microscope if sister chromatids can be
distinguished (Fig. 4); this requires different culture methods
from those used in the preparation of metaphases for the scoring of
chromosome aberrations. Because of the ease of preparation and
scoring, it is a very widely-used test in the study of mutagens.
SCE induction alone is not generally accepted as sufficient
evidence to classify an agent as mutagenic. The mechanism of SCE
induction is not fully understood, though a number of models have
been proposed (Wolff, 1982). Some clastogens induce only a small,
or no, increase in SCEs, X-irradiation being a particularly
striking example. There is a high background level of SCE compared
with chromosome aberrations; it is rare to find many cells without
SCEs in untreated samples. This may be partly due to the
5-bromodeoxyuridine (BrdUrd) that is added to the culture medium in
order to visualise SCEs, since BrdUrd itself is known to induce
SCEs (Latt et al., 1981). This basal level also implies that cells
with SCEs are capable of subsequent growth. SCE evaluation may
thus be a more valid indicator than chromosome breakage of events
compatible with cell survival, hence its widespread use in
mutagenicity screening programmes.
2.4.2. Procedure: chromosomal aberrations
The techniques used in the study of chromosomal aberration have
been described in detail by Evans (1976) and in the Gene-Tox
reports of Latt et al. (1981) and Preston et al. (1981).
126.96.36.199. Cell types
Two types of cells are used most widely for both tests. These
are CHO, an established fibroblast cell line, derived from Chinese
hamster ovary, and human peripheral blood lymphocytes (mononuclear
white blood cells). The small number of chromosomes in CHO cells
(modal number 22) makes scoring relatively straight forward. It is
an easy cell line to maintain, using standard tissue culture
techniques, and, with a cell cycle time of 12 - 14 h, grows very
rapidly (Latt et al., 1981; Preston et al., 1981; Dean & Danford,
Human peripheral lymphocytes do not divide spontaneously in
culture, but can be stimulated to divide by treatment with a
mitogen such as phytohaemagglutinin (PHA). Cultures are initiated
from fresh blood samples and are not maintained for more than a few
cell divisions. The first metaphase after the addition of the
mitogen is not reached for about 36 - 40 h, after which the cells
divide about every 18 h, with considerable variation, both within
and between cultures. Culture methods are described in detail by
Dean & Danford (1975) and Evans & O'Riordan (1975).
Other cell lines have been used in cytogenetic assays, for
example, lines with endogenous metabolizing capacity, such as rat
liver epithelial cells (Dean & Hodson-Walker, 1979). It is
important for a cell line to be fully validated with a range of
suitable chemicals before being used for routine testing.
Initially, information on the toxicity of a test agent is
required, and subsequently, concentrations up to a level where some
toxicity is observed are used in the cytogenetic assay. Toxicity
can be assessed, for example, by measurement of the mitotic index
or by cell counts. To obtain sufficient data from the chromosomal
aberration assay, a minimum of 3 replicate cultures of each of 3
doses, or 2 replicates of 4 doses is advisable, in addition to a
negative control (solvent only) and a positive control. With
lymphocyte cultures, it is recommended to use blood from at least
two different donors in each experiment. Under rare circumstances,
it may be possible to use a positive control structurally related
to the test material; more commonly, the positive control will be a
known clastogen. A direct-acting clastogen or one requiring
metabolic activation is used, as appropriate (see below). The
doses of the test agent should range from a concentration showing
some toxicity down to 1/4 or 1/8 of this, or equivalent log doses.
Since many clastogens only show effects close to the toxic dose,
there is rarely any advantage in selecting lower concentrations for
188.8.131.52. Culture methods
Cell lines are grown either in small tissue-culture flasks or
directly on sterile microscopic slides or cover slips until the
cells are proliferating. The agent under study is then introduced,
preferably by replacing the culture medium with medium plus agent.
Lymphocytes do not attach to culture flasks or slides, and are
usually grown in small bottles as suspension cultures. The test
agent can be added to lymphocyte cultures when they are set up. It
is preferable, however, to allow time for the cells to leave the GO
stage before adding the test agent, because toxic concentrations of
the test agent may prevent the cells from entering the cell cycle.
Thus, addition of the test agent after 24 - 36 h of culture is more
likely to be effective.
Modifications to either system may be required; for example,
volatile or gaseous compounds should be tested in a closed system.
In some instances, components in the serum used in the culture
medium may bind to the test agent, in which case it is desirable to
treat the cells in serum-free medium for a few hours, and then
continue culturing in normal medium. Erythrocytes (red blood
cells), present in lymphocyte cultures set up from whole blood, can
also bind the test agent. Various methods of separating out the
lymphocytes are available (Boyum, 1968), though whole blood
cultures are more widely used. Unless the cell line has been shown
to have intrinsic metabolizing capabilities, or there is strong
evidence that the agent under test is direct-acting (requiring no
metabolism), a metabolizing system, such as an S9 microsome
fraction obtained from rat liver, must be included (section 2.1).
When testing chemicals of unknown mutagenic activity, assays both
with and without a metabolizing system are required. If there is
compelling evidence that the agent is either direct-acting or
requires activation, then the first test can be carried out either
without or with a metabolizing system. If equivocal or negative
results are obtained, further tests will be necessary.
The times of exposure differ between the two test systems, and
are detailed below, but it is important to bear in mind that S9 can
be toxic to mammalian cells. Thus the time of exposure to the test
agent may be limited to only 1/2 - 3 h in assays using S9. In
these cases, the medium containing the test material and S9 is
replaced by normal medium, until the cells are harvested.
Treatment at toxic doses may extend the cell cycle time, and the
period between treatment with the test chemical and harvesting
should be extended accordingly. It may be necessary to use more
than one sampling time; this is discussed in more detail in section
184.108.40.206. Chromosome assay
Following the introduction of the test agent, the cells are
incubated for between one and two cell cycles so that the majority
of the mitotic cells are in the first metaphase after treatment,
when harvested. Before harvesting, a spindle poison such as
colchicine is added to arrest cells at metaphase. The cells are
treated with a hypotonic solution, such as 0.56% (0.075 M)
potassium chloride, and then fixed, usually with a freshly prepared
3:1 mixture of methanol and glacial acetic acid. Subsequently, the
cells are stained with Giemsa, orcein, or other chromosome stain,
and are then examined under the microscope. The slides should be
coded randomly and independently, and at least 100 metaphases
scored from each replicate. However, if the mitotic index is
greatly reduced at the highest dose, it is not always possible to
score 100 cells. Aberration types are usually classified into
chromatid (involving only one chromatid) and chromosomal or
isochromatid (affecting both chromatids) and are clearly described
by Evans & O'Riordan (1975), Savage (1976), ISCN (1978), and Scott
et al. (1983). Other classification systems have been devised,
such as that of Buckton et al. (1962), in which chromosome
aberrations are classified as stable (Cs), such as translocations,
or unstable (Cu), such as dicentrics, depending on whether or not
they can be maintained through successive cell cycles. Banding
techniques (Evans, 1976), commonly used for detailed chromosome
analysis, are rarely used for routine screening.
2.4.3. Procedure: sister chromatid exchange
Cultures to demonstrate SCEs are set up as for chromosome
assays, but in addition to the test agent, BrdUrd is present, at
concentrations of 10 - 25 µM, throughout the period from treatment
to harvesting. The cells must be allowed to pass through two
rounds of DNA replication (S-phases) before harvesting. BrdUrd is
incorporated into the newly synthesized DNA in place of thymidine,
and thus, at the first metaphase, all chromatids possess one DNA
strand containing BUdR and one containing thymidine. The
chromatids separate at anaphase, and at the second S-phase, the DNA
synthesized again contains BUdR. Since one DNA template is
unsubstituted and the other substituted with BUdR, the chromosomes
now contain DNA with one chromatid completely (bifiliarly)
substituted and the other, half (unifiliarly) substituted with
BrdUrd. These chromatids stain differentially following the
treatment described below.
The cultures are harvested as usual but, thereafter, stained
with Hoechst 33258 solution, exposed to a light source emitting
long-wave UV radiation, and then stained with Giemsa. The
bifiliarly substituted chromatids stain to a much lesser degree
than the unifiliarly substituted chromatids. Where SCE has
occurred, a change in the staining intensity can be seen on one
chromatid, with a reciprocal change on the other (Perry & Wolff,
1974). These second metaphase chromosomes are referred to as
harlequin chromosomes. First metaphase cells have uniformly darkly
stained chromatids, and third and subsequent metaphases have a
mixture of pale-staining and harlequin chromosomes. Since second
division metaphases can be recognized by having entirely harlequin
chromosomes, the problem of mitotic delay is not so great with SCE
analysis as with assessment of chromosome aberrations. Though
there may be a reduction in the proportion of second-division
metaphases at the highest doses, these can be recognized and
scored. A number of reviews of SCEs are available; Latt et al.
(1977), Perry (1980), and Wolff (1982) include photographs of
differentially stained chromosomes.
The slides should also be coded, and at least 30 (preferably 50
or more) metaphases examined from each culture.
2.4.4. Procedure: scoring
Accurate identification of chromosome aberrations and scoring
of SCEs requires a high degree of skill, and should only be
undertaken by suitably trained and experienced personnel.
Descriptions of aberrations do not usually include details of
potential artefacts, and it is essential to appreciate that there
are a number of normal chromosome orientations which the
inexperienced may score as aberrations. In addition, the quality
of the material must be sufficiently high for accurate assessment,
and analysis of "fuzzy", overlapping, or highly-scattered
chromosomes should not be attempted.
Results, including types of aberrations observed, should be
recorded on a suitable score-sheet. For chromosome aberrations,
some record, either the vernier reading on the microscope stage or
a photograph of each aberrant cells, is usually taken. It is
obviously important to ensure that the same metaphases are not
scored twice. For further details, see section 2.8.
2.4.5. Extent of testing
The decision as to whether a chemical has been tested
sufficiently to classify it as positive or negative in these test
systems is not always clear cut. If a clear positive response is
seen, or if there is no increase above negative control levels at
any dose, provided there is evidence of toxicity at the highest
dose, no further testing is necessary. If, however, the negative
control gives unusually high values, or the positive control fails
to induce the expected number of aberrations, this would suggest
that the experiment should be repeated. A weakly positive response
will often need to be confirmed in an additional experiment,
though, under some circumstances, adequate data may be obtained
from simply scoring additional metaphases on slides already
examined. Otherwise, it may be necessary to repeat the experiment,
either exactly as before or using different dose levels or exposure
times. A particularly important aspect of cell kinetics under the
influence of toxic doses is the delay in the cell cycle time.
Thus, an apparently negative dose-response can be obtained if cells
at higher doses have not undergone a round of DNA replication
between treatment and harvesting, when examining an S-dependent
agent. In these cases, a later sampling time is required in a
2.4.6. Data processing and presentation
When a clear dose-related increase in chromosomal aberrations
is obtained, or when there is clearly no increase above control
levels, the result may be obvious without the need for statistical
analysis. It is, however, always advisable that results should be
subjected to an appropriate statistical analysis. Metaphase
analysis, particularly if gaps are excluded, can yield such small
numbers of aberrations that objective interpretation is only
feasible after such statistical tests as the Chi2 and Fisher's
Exact test (Sokal & Rohlf, 1969).
The data are best presented in tabular form showing the
results for each dose, and the positive and negative controls,
and including details of the cell line, culture conditions, and
slide codes. The minimum data required are: the doses, number
of metaphases observed from each culture, and either the
percentage of aberrant metaphases, including and excluding gaps,
and the total number of aberrations, or the average number of SCEs
per cell. Further breakdown of chromosomal aberrations into
chromatid/chromosomal-type aberrations, and classes within these
groups (gaps, breaks, exchanges, etc.) is also essential. Graphic
representation of a dose-response can also be helpful.
Test agents are regarded as unequivocably positive if a dose-
related increase is observed over 3 or 4 doses (including the
negative control), and/or 3 or 4 doses give aberration or SCE
levels significantly higher than the negative control level.
If no dose-related increase is observed, and no dose gives a
significantly higher frequency of chromosome aberrations or SCEs
than the control, then the data are interpreted as negative. Weak
or marginal findings usually require additional data or testing,
but a reproducible dose response rising just above control levels,
provided this is statistically significant, is the criterion for
the designation of a weakly positive result.
Compounds should normally be tested up to concentrations that
induce detectable toxicity or reduction in mitotic index. Agents
that show no evidence of toxicity in preliminary studies should
be tested to the limit of solubility. However, very high
concentrations of some non-toxic chemicals may interfere with
culture conditions, and the maximum dose level should be decided on
a case by case basis.
Occasionally, a single high value is found at one dose or
replicate culture. This requires a repeat experiment, using a
narrower dose range above and below that dose. If the increase in
aberrations or SCEs at the dose is not reproduced, then the
isolated result can be discounted. Another potential problem of
interpretation is an increase in chromatid gaps at the highest
dose. The significance of gaps is discussed in section 2.8. In
the absence of an increase in other types of aberrations, it is
possible that the increase in gaps is associated with the
cytotoxicity of the test material and is not necessarily of
genotoxic significance. In such instances, it is particularly
important to consider data from other test systems before
evaluating the mutagenicity of the chemical.
220.127.116.11. Critical factors
The assessment of cytogenetic damage and SCEs depends crucially
on accurate scoring. It must be emphasized that this, in turn,
depends on the training and experience of the scorer as well as the
quality of the material. Both under- and over-estimation can
result from failure to meet these criteria.
It is also necessary for the cell line used to be suitably
validated with known clastogens before it is used routinely. Even
where an apparently strongly positive response is obtained, the
results may be viewed with question if, for example, a highly
unstable cell line is used. This emphasizes the need to use either
the standard cell types (CHO or lymphocytes) or to establish an
adequate data base for a new cell line, particularly with regard to
its metabolizing capacity and the level of aberrations in untreated
18.104.22.168. Experimental design and analysis
If information is available on the chemical structure and
metabolizing requirements of the test agent, the most suitable
medium for treating the cells (complete or serum-free) can be
assessed. The value of using toxic doses is emphasised in cases
where serum components are suspected to react with the test agent,
as it serves to confirm that the agent has entered the cells.
A particularly important aspect is the influence of the test
agent on the cell cycle time. Mitotic delay is frequently found at
toxic doses, and this may result in the examination of metaphases
in which visible aberrations have not had time to develop.
However, the next lowest dose should indicate whether or not this
is the case; this can be further checked by testing additional
doses and sampling times. With SCEs, the problem still arises, but
because second division metaphases can be recognised and are
scored, it is possible to assess whether or not significant mitotic
delay has occurred.
The system used to ensure that metabolic activation has
occurred can lead to a number of problems. The toxicity of S9 has
already been mentioned, and cells retaining metabolic activation
may vary in the extent and range of activation, and, furthermore,
some may lose their activating ability following repeated
subculturing. A positive control structurally related to the test
compound, in addition to a standard positive control, is ideal, but
in practice this is rarely available.
An assessment of clastogenicity can only be accepted if the
results from negative and positive controls are within the expected
range. The occurrence of a high value in the negative control
cultures is by no means unusual, but, in a well-characterized line,
its validity can be tested statistically. Any indication that a
positive control agent (especially one requiring activation) has
not been detected would also require a repeat of the experiment.
Further studies are necessary to clarify unexpected or
ambiguous results, in which case the use of additional sampling
times will often provide confirmatory data. If a clear negative or
positive result is obtained, there is usually no need for
verification in a second experiment. If toxic doses cannot be
achieved, a different solvent or a variation in the exposure time
should be considered. Unfortunately, the low tolerance of cultured
mammalian cells to pH changes and organic solvents precludes more
than slight variations in the culture conditions.
The ability of a chemical to produce undoubted double-strand
breaks, i.e., a strong clastogenic action, is an important finding
as is strong evidence that there is no such activity. Thus, it is
important to try to resolve the ambiguous and weakly positive
results. These can usually be clarified by further testing. An
increase in gaps alone only implies a discontinuity in staining,
and therefore may not be due to double-strand breaks. Agents that
induce gaps may cause disturbances in normal chromosome structure,
but not necessarily chromosome breakage (section 2.8). Since the
precise mechanism of SCE induction is not fully understood, the
genetic significance of SCEs cannot be determined, at present.
However, they reflect a direct interaction of chemicals with DNA
and represent a useful system for the detecion of genotoxic
chemicals. With certain exceptions, chromosome-aberration and SCE
assays correlate well with other tests.
2.5. In Vitro Cell-Mutation Assays
2.5.1. Principles and scientific basis of the assay
The use of cultured mammalian cells, including human cells, for
mutation studies can give a measure of the intrinsic response of
the mammalian genome and its maintenance processes to mutagens,
while offering rapidity of assay and ease of treatment compared
with the use of whole animals.
Several forward and reverse mutation selection systems are
available for use with cultured cells (Abbondandolo, 1977). The
basis of the majority is that the cells are cultured in a
"selective medium" containing a toxic compound or anti-metabolite
(called the "selective agent") which is toxic to all normal, non-
mutant cells, but in which rare, mutant cells can continue to grow
to form colonies. A major requirement for such assays is that
evidence should be provided that the end-point of the measurement
is a mutational event that occurs at a specific gene locus, that
is, it should be consistent with the induction of a heritable
alteration in the DNA sequence. In general, detailed biochemical
analysis of the gene product and cytogenetic study of the
chromosomes points to the mutational origin of the selected
colonies, though it is possible that some of the phenotypic changes
observed may be the result of the kinds of non-mutational changes
in gene expression that occur during normal development
("epigenetic events") (Siminovitch, 1976).
One problem associated with the selection of mutants of
mammalian cells is that two copies of each gene are present in a
normal diploid cell, and, in many cases, the mutant gene product
acts recessively: that is, adequate gene product is transcribed
from one (non-mutant) gene copy to fulfil the cell's needs. In
this case, mutation of both genes in a diploid cell (a very rare
event) is necessary to detect the mutant phenotype. Therefore,
mutation of such genes in cultured mammalian cells is studied in
the hemizygous or heterozygous state, using either X-chromosome
genes (where only one X is present in male cells, or only one
active X in female cells) or autosomal genes that have either been
found, or deliberately selected to be hemizygous in some cells
An example of an X-chromosome-located gene that is the basis of
a mutation selection system is the gene coding for the enzyme
hypoxanthine-guanine phosphoribosyltransferase (HPRT). HPRT is one
of a number of "salvage" enzymes in which the function is to
salvage the degradation products of nucleic acid synthesis (purines
and pyrimidines), but which are not essential for the survival of
the cultured cells, since these bases can be synthesized de novo.
HPRT catalyses the conversion of guanine and hypoxanthine to the
corresponding nucleoside-5'-monophosphates. In cells containing
HPRT, toxic purine analogues, for example 6-thioguanine or
8-azaguanine, are also incorporated, and this forms the basis of
the selection of mutants. Thus, cells with normal, non-mutant HPRT
are killed when they are cultured in the presence of these
selective agents, but mutants, with altered, or non-functional HPRT
(or its complete absence) are able to survive and form colonies,
because the toxic analogues are not incorporated into DNA or RNA.
Purines continue to be made in the mutant cells by the de novo
pathway. It is interesting to note that human beings with a rare
sex-linked recessive disease, the Lesch-Nyhan syndrome, are mutant
at the HPRT locus. All cells from males with this disorder are
HPRT deficient and are resistant to the toxic effects of
6-thioguanine and 8-azaguanine.
Another gene coding for a "salvage" enzyme, this time on an
autosomal chromosome, is the thymidine kinase (TK) gene. This
enzyme incorporates exogenously supplied thymidine, and its toxic
analogues, into the cell. In this case, both homozygous (TK+/+)
and heterozygous (TK+/1) diploid cells contain sufficient
thymidine kinase for the cells to be killed when they are cultured
in the presence of toxic pyrimidine base analogues such as
5-bromodeoxyuridine or trifluorothymidine. Mutants that do not
contain any functional TK (TK-/-) do not incorporate the analogues,
and are therefore able to survive and form colonies in the presence
of these selective agents. Normal diploid cells contain two copies
of the TK gene, and as simultaneous mutation of both genes is a
very rare event, a heterozygous(TK+/-) cell line must first be
constructed for mutation assays based on this enzyme to be
Since complete loss of the "salvage" enzymes HPRT and TK is
not deleterious to cultured mammalian cells, all types of
mutations, including base-pair substitution (which may result in
altered gene product), frame-shifts, and deletions (which result in
complete lack of enzyme) should be detected. Evidence to support
this has been presented in the considerable literature available on
the HPRT locus (Caskey & Krush, 1979) and the TK locus (Hozier et
In both the above cases, the mutant gene product acts
recessively. A few mutation systems rely on the semi-dominant
action of the mutant gene product, and in these cases mutation in
only one of the two genes present in a diploid cell is necessary to
detect the mutant phenotype. For example, mutation to the semi-
dominant phenotype of ouabain-resistance involves an essential
enzyme, the membrane-bound Na+/K+-dependent ATPase (Baker et al.,
1974). Ouabain kills cells by binding to this enzyme and causing
an imbalance in ion flow, but rare mutants can be found that fail
to bind ouabain while retaining functional ATPase activity. The
range of mutations detected by ouabain resistance would be expected
to be much more restricted than for the non-essential salvage
enzymes, because, if a large mutagenic change, e.g., a deletion,
occurred in the gene coding for the Na+/K+ ATPase, the essential
enzyme function would be lost together with ouabain binding, and
the mutant cell would die. Some evidence has been presented to
support this conclusion (Baker, 1979).
2.5.2. Relevance and limitations
In the testing of potential chemical mutagens, a major
limitation of cultured mammalian cell systems is the difficulty of
simulating in vitro the type and quantity of metabolic activation
that may occur in different tissues in vivo. This is because the
cultured cells lack the full range of enzymes required to activate
the diverse range of potential mutagens and carcinogens encountered
in the environment. Thus, the experiments must be conducted in the
presence of an "exogenous" metabolic activation system. This may
be supplied by the use of rat liver homogenates ('S9') or by co-
cultivating the tester cells with metabolically competent cells,
such as freshly isolated rat hepatocytes. The choice of the
metabolizing system(s) and the way that it is applied in the assay
has great importance for the efficiency of the test in predicting
the mutagenic or carcinogenic potential of chemicals that require
activation. Considerable further work is required to determine the
optimal conditions for the in vitro activation of many chemicals.
22.214.171.124. Outline of the basic technique
A large population of cells is exposed to the test substance,
with and without an exogenous metabolic activation system, for a
defined period of time. After removal of the test substance, the
cytotoxicity is determined by measuring the colony-forming ability
and/or the growth rate of the cultures after treatment. Bulk
cultures of the treated cells are maintained in a growth medium for
a sufficient period of time to allow the newly induced mutations to
be detected. During this period, known as the expression time,
the growth rate can be monitored and the cells sub-cultured if
necessary. The mutant frequency is then determined by seeding
known numbers of cells at high density in a medium containing a
selective agent to detect the number of mutant colonies, and at
a lower density in a medium without selection to determine the
cloning efficiency. After a suitable incubation time, colonies
are counted. The mutant frequency per viable cell is derived by
adjusting the number of mutant colonies in the selective medium by
the estimate of viable, colony-forming cells obtained from the
number of colonies in the non-selective medium.
Cloning efficiency (CE) Mean colonies per plate in non-
Total cells seeded per plate in
Mutant frequency (ME) Mean mutant colonies per plate
in selective medium
Total cells seeded per plate
in selective medium
Mutant frequency per survivor = MF
Each experiment contains "control" (untreated) cultures so that
the background (spontaneous) mutant frequency can be determined.
126.96.36.199. Cell types and selective systems
Many different cell types, including cells of human, rat,
mouse, and hamster origin, and a wide variety of selective systems
are available for potential gene mutation assay (Abbondandolo,
1977; Holstein et al., 1979). Many of these fulfil the criteria
suggested for the use of mammalian cells in such assays, for
example, a sound genetic basis for the system, high cloning
efficiency, low spontaneous mutation frequency, and a demonstrated
sensitivity to a variety of chemical mutagens. However, in
practice, only three cell lines, the V79 and CHO Chinese hamster-
derived cell lines, and L4178Y mouse lymphoma cells have been
widely used for large-scale mammalian cell in vitro assays. In
all three cases, HPRT and the Na+/K+ ATPase genes have been used as
the genetic systems for the basis of mutant selection. In
addition, an L5178Y cell line, heterozygous at the TK locus (L5178Y
TK+/-), has been developed by Clive and co-workers (Clive et al.,
1972), and has been extensively used for the selection of TK
mutants in mutation assays. More recently, a CHO cell line,
heterozygous at the TK locus, has also been developed (Adair et
al., 1980). Several reviews are available which discuss the
general principles of mammalian cell assays (Hsie et al., 1979;
Fox, 1981) and the V79 (Bradley et al., 1981), CHO (Hsie et al.,
1981), and L5178Y (Clive et al., 1983) cell lines, in particular.
Many of the critical factors involved are discussed in these papers
and the extensive available literature is cited. Considerable
variations exist in the protocols for mutation assays using
different mammalian cell lines and selective systems. These should
be carefully studied, preferably in close consultation with
experienced investigators in this field.
Some of the problems associated with this assay have been
highlighted and discussed in the recently conducted collaborative
study on short-term in vitro tests, by Ashby et al. (1985), and
some recommendations for future improvements have been made.
188.8.131.52. Culture conditions
Culture conditions should be well-defined, and the cells should
be maintained under optimal growth conditions throughout the
experiment. Cultured cells require the presence of serum to
maintain growth, and the serum batch may affect growth rate,
cloning efficiency, and mutant frequency. Batches of serum should
therefore be carefully pretested and a large volume of a suitable
batch stored frozen. Medium, pH, temperature, humidity, and cell
dispersion techniques are among the critical factors in mammalian
cell culture techniques and should be carefully controlled if
reproducible data are to be obtained.
To ensure that all stages of the cell cycle are exposed to the
test substance, exponentially growing cells in tissue culture
medium should normally be used. Great care should be taken to
standardize the treatment conditions. Medium, pH, serum content,
incubation conditions, and cell density during treatment should all
be carefully controlled. The cultures should be protected from
light during treatment, and suspension cultures should be shaken.
The test substance should be dissolved just before use, preferably
in tissue culture medium. Other vehicles may be used, for example,
dimethyl sulfoxide, but each should be tested to be certain that
its presence has no effect on cell viability or growth rate. For
initial toxicity studies, a wide range of molarity of the test
substance should be used. When the toxic response has been
determined, the mutation experiment should cover several
concentrations (usually a minimum of four) ranging from non-toxic
(90 - 100% survival of the treated cells) to toxic (1 - 10%
survival). Greater levels of kill are not recommended (Bradley et
al., 1981). Treatment time is generally for 1 - 5 h, although 16 h
(Bradley et al., 1981) or longer may be appropriate (Cole et al.,
184.108.40.206. Expression time
After the cells have been exposed to a mutagen, they must be
cultured in a non-selective medium for a period of time so that (a)
the mutagen-induced damage can be "fixed" in the DNA, and (b) the
constitutive level of the non-mutant enzyme, and its mRNA, can
decrease to a negligible level. The time required for a new
mutation to be phenotypically expressed as a mutant enzyme (the
"phenotypic expression period") will depend on the initial number
of non-mutant enzyme (and mRNA) molecules, their half life under
physiological conditions, and the rate of cell division. The
expression time varies with the cell line, the selective system,
and possibly also the mutagen treatment. After the maximum induced
mutant frequency has been observed, there may be a plateau in the
frequency of mutants, while in other cases, there may be a peak in
the number of mutants followed by a fall in the frequency. For
example, it has been found that induced ouabain-resistant mutants
are first observed within 24 h of mutagen treatment, reach a
maximum 48 - 72 h after treatment, and later remain at an
approximately constant value. The thioguanine-resistant phenotype,
however, requires a minimum of 6 - 7 days after treatment before
new mutations are fully expressed, after which there is again a
plateau in the mutant frequency. In contrast to these
observations, mutations at the TK locus reach a peak value 48 -
72 h after treatment, and then there is a marked decline in
frequency with time. For quantitative mutation studies, it is very
important that near-optimal phenotypic expression of induced
mutation should be observed, and the shape of the expression time
curve for newly-induced mutants must be carefully determined by
experiment by each laboratory, under well-defined conditions, using
a number of different mutagens.
220.127.116.11. Choice and concentration of selective agent
The concentration of the selective agent is one of the most
critical factors (Thompson & Baker, 1973). The dose should be
high enough for complete kill of non-mutant cells and the mutant
frequency at the chosen concentration should not be affected by
small variations that may occur in day-to-day culture conditions.
6-Thioguanine is generally considered to be a more stringent
selective agent for the selection of HPRT mutants than
8-azaguanine, and is the agent of choice for mouse cells, as
azaguanine is non-toxic to these cells. Trifluorothymidine, which
is recommended for the selection of TK mutants, is both heat and
light unstable and must be handled with great care.
18.104.22.168. Stability of the spontaneous mutant frequency
A high and variable spontaneous mutant frequency can cause
considerable problems with data interpretation. Several methods
are available for maintaining a low, stable frequency:
(a) The cell line can be re-cloned to establish a suitable
sub-line. A large frozen stock can then be stored in
liquid nitrogen and one vial used for each experiment.
(b) Cells regularly sub-cultured to maintain stocks should be
diluted to low density to remove pre-existing mutants.
(c) For the TK and HPRT systems, pre-existing mutants lacking
these enzymes can be removed from the population by
growing the cells in medium containing aminopterin. This
anti-metabolite blocks the de novo purine and pyrimidine
synthesis pathways. If thymidine and hypoxanthine are also
added to the medium (called "HAT" medium), non-mutant cells
containing TK and HPRT continue to grow using the "salvage"
pathway. Mutant TK- or HPRT- cells die in HAT medium,
because they are unable to use either the de novo or the
"salvage" pathways for nucleotide synthesis. After "HAT"
treatment, again, a large stock of cells can be stored in
liquid nitrogen for future use.
22.214.171.124. Provision for metabolic conversion
Three methods of supplying exogenous mammalian activation
systems are available (Bartsch et al., 1982).
(a) Rodent liver preparations ("S9") (see also section above)
These can be prepared from untreated animals (usually rats)
or from animals pre-treated with "inducing agent" (e.g.,
phenobarbital, 3-methylcholanthrene, or Aroclor(R) 1254) to induce
high levels of the mixed-function oxidases that catalyse the
metabolic activation steps. Such preparations have been widely
used with mammalian cells (Kuroki et al., 1977, 1979; Bartsch et
al., 1979; Clive et al., 1979; Amacher & Turner, 1981, 1982a,b).
These papers contain detailed methods for the preparation of both
S9 and the NADPH energy generating systems "co-factor mix" for use
with mammalian cell cultures.
A batch of S9 should be prepared, tested for sterility, and
stored for up to 3 months at -70 °C, or in liquid nitrogen. Both
S9 and the co-factor mix should only be thawed immediately before
(b) Cell-mediated metabolism
In this case, the indicator cells (e.g., L5178Y or V79) are co-
cultivated with metabolically-competent cells, e.g., freshly-
isolated rat hepatocytes (Amacher & Paillet, 1983) or hamster cell
lines such as BHK or SHE (Langenbach et al., 1981; Bartsch et al.,
1982). The pro-mutagen or -carcinogen is metabolized to the active
product by the competent cells and diffuses into the indicator
cells, where it reacts with the DNA.
(c) The host-mediated assay
Finally, the cultured cells may be placed inside the body of an
animal (usually a mouse) which is treated with the test substance.
After a suitable period, the cells are withdrawn, and the mutant
frequency determined. For example, L5178Y cells can be grown in
the peritoneal cavity of compatible mice (Fischer et al., 1974) or
V79 cells in diffusion chambers in mice (Sirianni et al., 1979).
126.96.36.199. Controls and internal monitoring
For each experiment, positive and negative controls are
required. A negative control is necessary to check the background
mutant frequency. It should consist of no treatment and/or the
solvent as used to dissolve the test substance. Two separate
positive controls (to check that the assay is performing correctly)
are necessary, one of which should require metabolic activation.
It is an advantage if a positive control with a known dose-response
is used, so that the sensitivity of the assay can be assessed in
Cell cultures should be periodically checked for mycoplasma
contamination (Russel et al., 1975) and can be periodically
karyotyped to check chromosome stability.
188.8.131.52. Population size, replicates, and reproducibility
(a) Population size
The power and sensitivity of the test should be pre-determined,
taking the toxic effect of the test substance and the mutant
frequency in the untreated population into account. The number of
cells to be treated, sub-cultured, and exposed to selection should
be sufficient for a particular increase over the control mean to be
detected. The precise numbers depend on the cell line and
selective system, but as a general guide it has been suggested that
ten times the inverse of the spontaneous mutant frequency should be
used. This means, for example, that if the spontaneous mutant
frequency is 1 x 10-6, then 107 visible cells should be used for
each treatment level. If there is substantial initial toxicity,
this number should be increased correspondingly. Similar care
should be taken over the numbers of cells sub-cultured during the
expression period, to avoid sampling error. The number of cells
exposed to selection should be such that the numbers of mutant
colonies observed on both control and test plates are sufficient
for statistical analysis.
One protocol recommends that duplicate samples should be
treated, sub-cultured, and plated in every experiment (Clive et
al., 1979). Alternatively, single very large populations can be
used for each treatment level.
The determinations should be quantitative and reproducible.
The whole experiment should be carried out at least twice using
freshly prepared test substance, though not necessarily over
precisely the same dose range. If both experiments give a positive
or negative result, this could be considered acceptable. However,
for low or equivocal responses, further experimentation may be
2.5.4. Data processing and presentation
184.108.40.206. Treatment of results
The test report should include precise details of all methods
used in the test procedure. All validation data should be provided
and retained for further reference.
Data should be presented in tabular form. All original data,
including toxicity data, absolute cloning efficiency of the control
cultures, and individual colony counts for the treated and control
groups should be presented for both mutation induction and survival
plates. Survival and cloning efficiencies should be presented as a
percentage of the controls. Mutant frequency should be presented
as per 106 clonable cells. Possible toxicity of the vehicle should
220.127.116.11. Evaluation of results
Several criteria have been suggested for determining a positive
result, one of which is a statistically significant, concentration-
related increase in the mutant frequency. An alternative is based
on the detection of a reproducible and statistically significant
positive response for at least one concentration of the test
substance. The problem with such an approach is that, although
several methods of statistical analysis have been published (Clive
et al., 1979; Amacher & Turner, 1981; Snee & Irr, 1981; Tan & Hsie,
1981), there is, at present, no general concensus as to the most
appropriate method. Further work is required on the optimum
experimental design and statistical analysis of mammalian cell
A substance that does not produce either a reproducible
concentration-related increase in mutant frequency or a
reproducible significant positive result at any one test point is
considered non-mutagenic in this test.
18.104.22.168. Ambiguous results
Ideally, experimental design should be such that ambiguous
results do not occur. Examples of ambiguous results might be very
early expression (day 0 or 1) of induced TK or HPRT mutants, marked
variations in colony numbers at different expression times or an
inverse concentration-related effect. Repeat experiments, paying
particular attention to growth conditions, stringent mutant
selection, and all the critical culture conditions may be necessary
to resolve ambiguous results.
Mammalian cell gene mutation assays have a sound genetic and
biochemical basis. Defined protocols have been developed for the
three most commonly-used cell lines and reproducible results have
been produced using a number of chemical mutagens. A limited
amount of testing has been done using carcinogens (mainly using the
L5178Y TK system) and the role in predicting carcinogenicity
requires further study. Some systems are capable of determining
multiple genetic end-points (Cole et al., 1982; Gupta & Singh,
1982), and these are potentially advantageous as mutagen-screening
One of the most important factors influencing the validity of
the data is that the investigator should have a thorough
understanding of the particular cell system in use. This includes
the culture conditions that will support good cell growth and an
awareness of the many possible causes of sub-optimal growth. Slow
growth rate may result in reduced incorporation of analogues and
incomplete kill of wild-type cells. Other factors that deserve
particular emphasis are described below.
22.214.171.124. Mutant selection
It is very important to ensure stringency of selection for each
particular cell line. Pool sizes differ between cell lines and
under different growth conditions, and the relative affinity of
salvage enzyme for analogue and natural substrate may differ
markedly. The kill curve of the selective agents used must be
carefully checked, and the concentration chosen should not be
within or close to the range in which exponential fall in survival
occurs. This is especially important if a high and variable
spontaneous mutant frequency is found, as this makes data
interpretation particularly difficult.
126.96.36.199. Expression time
It is essential that a near-optimal expression time for the
induction of mutants should be used if accurate data are to be
obtained for the analysis of concentration-related effects. The
expression time should be carefully defined for each selective
system using a number of mutagens. The use of a single "standard"
expression time may give misleading results and, ideally, at least
two expression times should always be used so that it is clear that
the peak has been observed. This is particularly important if an
unusual dose-response relationship is obtained, for example few
mutants being induced with increased dose.
188.8.131.52. Cell numbers
Experiments should be designed to maximize the possibility of
statistical analysis of the data. If small effects are to be
detected, it is most important that the spontaneous mutant
frequency should be borne in mind, and that sufficient cells should
be exposed to treatment and cloned in selective medium to provide
reasonable numbers of mutants as a basis for analysis.
184.108.40.206. Metabolic conversion
This is a major area in mammalian cell assays requiring further
research. At present, no single experimental design is ideal for
detecting all compounds that require metabolic conversion. The
factors requiring consideration are species and inducer used for
tissue homogenate (S9) preparation, the correct final concentration
of the homogenate, and the use of intact cells rather than
homogenate. These factors may make a considerable difference to
the apparent mutagenicity of the test compound, and the laboratory
conducting the test should be able to provide evidence that, using
a clearly defined protocol for metabolic conversion, mutagens from
different classes of chemicals requiring metabolic activation
(e.g., benzo( a)pyrene, N-nitrosodimethylamine, and N-acetyl-2-
aminofluorene) induced mutations in a dose-dependent fashion.
Flexibility is important as a single compromise protocol may not be
appropriate in every case.
Mammalian cell lines have been used in the study of chemically
and physically induced specific locus mutations since 1968.
Clearly defined methods for mutagenesis assays using cultured
mammalian cells have been developed and a detailed examination of
the genetic basis of the markers used has been made. Although a
number of areas requiring further study remain (Ashby et al.,
1985), criteria have been established for an evaluation to be made
of the induction of specific locus mutations in mammalian cells,
and of the role of such assays in predicting carcinogens.
2.6. The Use of Higher Plants to Detect Mutagenic Chemicals
Many of the fundamental concepts of modern genetics were
established in higher plants and the term "mutation" was introduced
by the Dutch botanist, Hugo de Vries, in 1909, to describe a sudden
hereditary change in Oenothera lamarckiana. Plant systems played a
major part in early investigations of the genetic changes caused by
radiation (Read, 1959; Revell, 1959) and a variety of plants have
been used to study the mutagenic effects of chemicals at the gene
and chromosome levels. With the increasing concern over the
genotoxicity of sophisticated techniques for studying mutations in
bacteria, lower plants, insects, and mammalian cells, there has
been a loss of interest in the testing of potentially mutagenic
chemicals in higher plant systems. This is surprising as plants
appear to offer significant advantages over other organisms in
certain circumstances, though they have, of course, important
Techniques for studying mutagenic chemicals have been developed
in about 10 species of higher plants and a whole range of specific
genetic end-points are available. Mitotic chromosome alterations
can be studied in the somatic cells from root tips, or pollen tubes
in, for example, barley, the broad bean, or the onion. Pollen
mother cells from a number of species are suitable for detecting
chemically-induced chromosomal aberrations in meiotic cells. Gene
mutations at specific loci can be investigated in maize or soybean
plants and multilocus mutation systems are available in barley and
maize. The chromosome systems allow the observation of structural
chromosome damage and effects on chromosome segregation and general
mitotic function. The chromosomes are morphologically similar, and
appear to respond to treatment with mutagens in a similar way to
those of mammals and other eukaryotes.
A survey of the literature prepared under the US Environmental
Protection Agency Gene-Tox Program (Constantin & Owens, 1982)
revealed that about 350 compounds, covering a wide range of
chemical classes, had been tested for mutagenic activity in plants.
The same authors also compared the results of testing eight model
mutagens in plants with the results obtained in other systems.
They claimed that the correlation between plant data and results
from cultured mammalian cells was at least as good as that with
data derived from bacteria and Drosophila. A comparison of the
results of testing a series of pesticides in plant root tips and
mammalian cells for chromosomal aberrations showed a remarkable
qualitative similarity between the two sets of results. However,
the data on chromosome damage in mammalian cells for some of the
pesticides was not truly representative of the literature on these
chemicals. Although a database representing more than 350
compounds tested in plant systems has been assembled, a large
proportion of the chemicals tested were shown to be mutagenic in
one plant system or another, and there is a significant lack of
information on non-mutagenic chemicals.
In spite of the above comments, it is apparent that plant
assays possess some advantages over other systems that remain to be
fully exploited in the area of genetic toxicology. Chromosome
assays on plants are rapid and inexpensive and do not require
elaborate laboratory facilities, and a wide range of genetic end-
points is available. However, before the full potential of plant
systems can be exploited, some serious limitations have to be
overcome. There is a lack of knowledge concerning many of the
critical molecular processes in plants, particularly those
influencing the metabolism of foreign compounds; thus, it is
difficult to assess the significance for mammals, including man, of
data derived from plant experiments. There are also fundamental
differences in structure between plant and mammalian cells. The
rigid cellulose wall of plant cells almost certainly affects the
penetration of certain chemicals and there may be selective
differences between plant and mammalian cells in the kinds of
molecules that can be absorbed. However, the DNA of plants and
animals appears to be similar in structure and function and the
mechanism of protein synthesis seems to be the same. Higher plants
have more cytoplasmic (mitochondrial) DNA than animal cells and, in
addition, the chloroplasts contain DNA.
Mitotic chromosome division in plants follows a similar course
to that in mammalian cells, though meiosis and gametogenesis are
very different. In plants, cell division is accompanied by the
formation of a plate that separates the daughter cells while in
mammals, the cells divide by constriction of the cytoplasm.
2.6.2. Test systems
Although about 25 different test-systems have been described in
10 plant species, the following have been established as practical
and useful for testing chemicals for mutagenic activity:
(a) mitotic chromosomal damage;
(b) aberrations in meiotic chromosomes; and
(c) gene mutations at specific or miltiple loci.
220.127.116.11. Detection of mitotic chromosome damage
Growing root tips of the broad bean, Vicia faba (Ma, 1982b),
the onion, Allium cepa (Grant, 1982), the spiderwort, Tradescantia
paludosa (Ma, 1982a), and of barley, Hordeum vulgare (Constantin &
Nilan, 1982) provide a readily available source of material for
studying the damaging effects of chemicals on chromosomes.
(a) Vicia faba
The six pairs of chromosomes can be clearly observed at the
metaphase stage of mitosis, and it is possible to identify all
types of chromatid and chromosome aberrations. In addition to
conventional metaphase analysis, methods are also available for
detecting chromosome damage by counting micronuclei and for
recording sister chromatid exchanges. The technique is most
suitable for studying water-soluble chemicals, but by using organic
solvents, e.g., dimethyl sulfoxide, other compounds can also be
tested. Normally, stock solutions of the test compound are added
to the growth solution; appropriate buffers should be used to
correct extremes of pH. Freshly prepared solution should always be
used. The technique (Kihlman, 1971) is relatively simple, and
requires only a minimum of laboratory equipment. Seeds are
softened by soaking in water for 6 - 12 h, then allowed to
germinate in moist vermiculite or similar medium at a temperature
of about 19 °C. After germination (4 days), the growing shoot is
removed and the seedlings transferred to a tank of water, which
should be fully aerated. After 24 h in the tank, primary root
growth is sufficiently active for study. It is important to
control the pH and temperature of the water as both may affect the
frequency of chromosome aberrations induced by a given chemical.
Treatment times may vary between 1 and 24 h, though short
treatment times are preferable for the identification of the most
sensitive mitotic stages. However, it is conventional to
incorporate two or three different treatment times, when testing
chemicals of unknown mutagenic activity. The mitotic cycle of
Vicia is between 18 and 22 h and, as the interphase stage is the
most sensitive to the majority of chemicals, it is necessary to
allow a recovery period of about 8 h in the absence of the test
chemical. This ensures that roots are fixed and processed at a
stage where chromosomes damaged by the chemical will be in the
first metaphase after treatment. In some cases, an additional
recovery period of 30 - 40 h may be used, so that chromosomes can
be examined at the second metaphase. Both treatment and recovery
should take place in the dark. Before the roots are fixed, they
are transferred to a solution of 0.02 - 0.05% colchicine and
agitated in this solution for 2 - 4 h. This treatment blocks the
cell cycle at the metaphase stage and leads to an accumulation of
metaphase chromosomes that are suitable for analysis.
For most purposes, fixation in ethanol: acetic acid (3:1)
gives satisfactory results. This is best carried out at 4 °C
(refrigerator temperature) for a minimum of 20 min; fixation from
2 - 24 h is more effective for permanent preparations. For
preliminary analysis or when permanent preparations are not
required, chromosomes can be stained using the aceto-orcein method.
The Feulgen squash technique of Darlington & Lacour (1969) is
preferable for permanent slides followed by rapid freezing,
dehydration in alcohol, and mounting.
The various kinds of chromosomal aberrations can be scored in
metaphase preparations and, for many chemicals, the scoring of
chromatid-type aberrations in the first metaphase after treatment
gives the most reliable measure of mutagenic activity. However,
some compounds, e.g., maleic hydrazide, produce a peak of activity
during the second metaphase and this should be determined before a
chemical is regarded as inactive. Examination of anaphase
chromosomes for fragments and bridges is a useful technique for
rapid screening and for obtaining preliminary information on
clastogenic (i.e., chromosome-breaking) activity, mitotic delay,
and the absence of cell division. Such information is useful for
deciding treatment concentrations and times and recovery periods
for subsequent metaphase studies. For a detailed description of
metaphase and anaphase aberrations, see Kihlman (1971).
Micronuclei resulting from chromosome fragments or lagging
chromosomes can be scored at the interphase following treatment
(Ma, 1982a), and a technique has been described for investigating
sister-chromatid exchanges (SCE) in root tips (Kihlman & Andersson,
A sufficient number of root tips should be used for each of a
wide range of concentrations of the test compound to give an
adequate number of data for subsequent interpretation and, if
necessary, statistical evaluation. A minimum of 100 metaphase
cells should be analysed from at least 10 roots for each
experimental group. Doses should be selected within half-log
intervals and compounds should be tested up to obviously cytotoxic
or inhibitory (i.e., reduction in mitotic index) concentrations.
It is usual to conduct preliminary experiments to identify a
suitable range of concentrations. More than one exposure period
and two or three recovery periods may be necessary to obtain the
maximum incidence of chromosome damage and a major objective is to
determine a dose-response relationship for chemicals that appear to
be mutagenic. Control experiments are needed for each assay and
should include a negative control, consisting of roots cultured in
the growth solution including any solvent used, and a positive
control, consisting of roots treated with a known mutagen such as
Results are usually expressed as the number of aberrations per
100 cells, per group and the number in each experimental group is
compared with the values from the negative control group. In most
cases, positive results are so obvious that statistical analysis is
unnecessary. Where the number of aberrations is low, a simple
t-test or Chi-squared test, using a significant level of 1% to
determine positive results, is usually adequate.
(b) Allium cepa
Although a number of species of Allium have been used for
genetic studies, the common onion, Allium cepa, has proved to be
the species of choice for root-tip chromosome studies (Grant,
1982). Mitotic cells of Allium contain 8 pairs of large
chromosomes. The technique for root-tip chromosome preparations is
very similar to that described for Vicia. The outer scales are
removed from young bulbs to expose the root primordia and they are
then supported in a rack over a suitable tank containing water at
20 °C. Adequate root growth should be obtained in 2 - 4 days. The
roots are then ready for treatment with the test chemicals followed
by processing and mounting as described above. In an even simpler
technique, Allium cepa seeds are germinated on layers of paper
towelling soaked with the test solution in a culture dish. Primary
roots are usually 0.5 - 1.0 cm long after 3 days, and they can then
be processed for analysis.
(c) Tradescantia paludosa
Compared to Allium and Vicia, only a few chemicals have been
tested for mitotic chromosomal aberrations in Tradescantia, but it
has the advantage that both meiotic and mitotic chromosomal damage
and gene mutations can be tested in the same species. Dividing
cells in the root tip of Tradescantia contain 12 large metacentric
chromosomes. A large number of roots can be obtained from cuttings
from mature plants in about a week. These rooted cuttings can then
be used for chromosome studies in much the same way as those of
Allium or Vicia (Ahmed & Grant, 1972).
(d) Hordeum vulgare
Both root-tip and shoot-tip cells can be used to investigate
mitotic chromosome changes in barley. The chromosomes are large,
12 in number, and very suitable for the rapid scoring of
aberrations. The procedure is similar to that described above.
Barley seeds are allowed to germinate while in contact with the
test solution. Five to seven primary roots develop from each seed
and the roots are usually fixed between 24 and 48 h after
germination and then processed for metaphase chromosome analysis.
Squash preparations can be made from a number of growing points on
the developing shoot and numerous cells are usually available for
metaphase analysis. Frequency of chromosome damage may vary
between root tips and shoot preparations because of differences in
the effectiveness of transport of different chemical molecules
(Constantin & Nilan, 1982).
In general, the root tip procedures are relatively simple and
sensitive assays for clastogenic chemicals. The species described
have small numbers of large chromosomes, which simplifies analysis,
and aberrations can be scored at either metaphase or anaphase.
They are more suitable for testing water-soluble compounds than
those that are not easily soluble. It should be emphasized that
the metabolic pathways required for the activation of many
chemicals have not been fully characterized in these plant systems.
Thus, the relevance of these results for mammalian cells cannot be
properly assessed, at present.
18.104.22.168. Detection of aberrations in meiotic chromosomes
Although the processes of sexual reproduction in plants are
greatly different from those in mammals, there are some
similarities in meiotic cell division and chromosome behaviour.
The induction of anomalies in the chromosomes of, for example,
pollen mother cells, may be analagous to meiotic chromosome damage
in mammalian reproductive cells, though convincing evidence for
this is lacking. A number of plants including Vicia and Hordeum
offer relatively easy means of studying meiotic events including
numerical (e.g., non-disjunction) as well as structural chromosome
changes. A method is also described for counting micronuclei in
4-cell stages as a measure of chromosome breakage (Ma, 1982a). The
techniques are simple, involving fixing the flower buds in ethyl
alcohol/acetic acid, staining the anthers using a squash technique,
and then analysing the chromosomes in the pollen mother cells.
(a) Vicia faba
For the examination of meiotic chromosomes in Vicia, it is
necessary to raise the plants to maturity in either growth chambers
or glasshouses. This is fairly time-consuming and requires much
more space than the root-tip assay. Chemicals can be applied
either by spraying in solution on the young flower buds or by
exposing the buds to the chemical in the form of a gas or vapour in
an appropriate chamber (Tomkins & Grant, 1976). Suitable
concentrations of the chemical and exposure times are determined
from preliminary experiments and it is usual to allow a recovery
period before processing the pollen tubes for the analysis of
anaphase cells for bridges and fragments.
(b) Tradescantia paludosa
Strains of T. paludosa that proliferate and propagate easily
and quickly under local environmental conditions should be used. A
suitable clone should grow to maturity from cuttings in 40 - 60
days. Since the chromosomes of pollen mother cells are not of
adequate quality for the detailed analysis of metaphase
aberrations, a technique has been developed for detecting
chromosome breakage on the basis of micronuclei at the tetrad
stage. In practice, the inflorescences are removed from the plant
and the stems placed in solutions of the test chemical.
Alternatively, the buds can be exposed to gaseous materials in a
suitable chamber. The optimum length of treatment is determined
experimentally and a recovery period of 24 - 30 h is necessary to
allow chromosome damage in early prophase 1 to reach the tetrad
stage where micronuclei can be scored. Micronuclei are assumed to
be a result of either chromosome fragmentation or of whole
chromosomes lost during meiosis and are therefore a measure of both
structural damage and aneuploidy (or non-disjunction). It is usual
to score between 1000 and 1500 tetrads from each experimental group
including both negative and positive controls.
(c) Hordeum vulgare
Chromatid and chromosomal aberrations can be investigated in
pollen mother cells (microsporocytes), which are present in large
numbers in the developing barley spike. The spike is produced when
the shoot apex undergoes a transition from a new leaf promordium to
an inflorescence primordium. The spike is collected for
cytogenetic analysis at approximately the same time as the last
leaf (i.e., the flag leaf) emerges. As meiosis in the pollen
mother cells is not synchronized, spikes can be used for testing
over a period of up to 40 h during development. Chemicals can be
applied by spraying the spike or adjoining areas at selected times,
before removing the spikes. The entire spike is fixed in
ethanol/acetic acid and processed in the normal way (Constantin &
22.214.171.124. Detection of gene mutations at specific or multiple loci
A specific locus is a region of a chromosome that controls the
development of a phenotypic characteristic. It is equivalent to
the classical Mendelian gene and can mutate to a new allele with an
associated change in phenotype. Although there are a number of
specific loci that are potentially useful for studying chemical
mutagens, only a few systems are sufficiently well characterized to
be used in practice. An example of these is the waxy mutation as
expressed in pollen grains of maize.
(a) Waxy locus mutations in Zea mays
Maize has a long history of use in genetic studies and hundreds
of genotypically defined strains are available. The pistillate
flowers containing the female spores (megaspores) develop on a
separate part of the plant to the characteristic tassels containing
the male spores (microspores). Tetrads of haploid microspores
develop in the anthers through a process of meiosis and then, by
mitotic division, the male gametophyte or pollen grain is formed.
The haploid, female megaspore develops from the megasporocyte by
meiotic division and, after a complex process of mitosis, the
female gametophyte is produced.
The waxy locus assay is based on dominance or recessiveness in
a gene that determines the presence of amylose in the kernel. In
the recessive genotype (wx), the kernels have a waxy appearance
and the starch of the endosperm contains only amylopectin. The
starch in the dominant (Wx) form consists of a mixture of
amylopectin and amylose. Kernels carrying the Wx allele stain a
dark blue-black when stained with iodine while wx/wx kernels,
which have no amylose, stain a red colour. The waxy phenotype can
also be detected in pollen grains using the iodine reaction and
this forms the basis of the assay.
The assay can be conducted by the direct treatment of the
tassels, which are harvested at an appropriate time and stored in
70% ethanol. Homozygous Wx plants are exposed to the test
chemical and forward mutations are detected by a lack of amylose in
the iodine-treated pollen. A reverse mutation assay using plants
of the Wx/wx genotype can be used in a similar technique.
It is usual to analyse some 250 000 pollen grains per tassel in
5 - 10 plants. The frequency of mutants in pollen from treated
plants is compared with that from the untreated controls.
Further details of this and other mutation assays in
maize, and information on the application and interpretation
of these procedures are given in the review by Plewa (1982).
(b) Chlorophyll-deficient mutations in Hordium vulgare
Chlorophyll synthesis and its control is governed by a large
number of genes and a variety of recessive mutations can be
detected after treatment of barley seed with mutagens or by
exposure of the plant during its complete life cycle. The
procedure for detecting chlorophyll-deficient mutations is
relatively time-consuming as they are observed in the second (M2)
generation after treatment of the seed. The system is reviewed by
Constantin (1976). The waxy pollen test can also be applied in
barley (Sulovska et al., 1969).
(c) Somatic mosaicism in Glysine max
The induction of spots of contrasting colour in the leaves of
soybean seedlings appears to have many attributes as a useful
short-term test for mutagenic chemicals. The spots result from a
variety of genetic changes in either meiotic or mitotic cells, the
assay can be completed in 4 - 5 weeks, and its requires a minimum
of laboratory facilities. The test is based on the Y11 locus and
its mutation to y11. The homozygous Y11 Y11 has dark green leaves
that may show light green or very dark green spots, the
heterozygous Y11 y11 has light green leaves showing dark green,
yellow, or twin (dark green/yellow) spots. Although cytological
evidence of the genetic basis of the mosaicism is limited, it has
been inferred from the phenotypic expression that the spots may be
a result of somatic crossing over, non-disjunction, chromosome
deletion, gene mutation, or somatic gene conversion.
In studies on the induction of leaf mosaics, seeds are treated
with the test chemical during germination. They are then planted
in a non-nutritive medium and grown under controlled conditions in
a glasshouse until the second compound leaf unfurls (4 - 5 weeks).
The number and type of spots per leaf on each plant is recorded and
the numbers of spots on treated plants compared with the untreated
control values. An appropriate positive control group (i.e.,
mitomycin C, N-methyl- N-nitrosourea) is included in each assay.
For a detailed review of the assay see Vig (1982).
(d) Somatic gene mutations in Tradescantia
The Tradescantia assay, which involves a change in flower
colour from blue to pink, is particularly suitable detecting
mutagens in the atmosphere (Schairer et al., 1978). The hybrid
clone 4430 is heterozygous for a specific flower colour locus. The
dominant blue allele produces the phenotypically blue colour in the
petals. The recessive pink phenotype is only expressed by mutation
or deletion at the blue allele. The pink colour is detected as
pink cells in the stamen or as sectors in the petals. For
laboratory studies, cuttings bearing a young inflorescence are
treated with liquid or gaseous compounds for periods of a few hours
to a number of days. The cuttings are then transferred to growth
chambers under standard conditions, until the necessary
observations have been carried out. Mutations are expressed as
single pink cells or as strings of pink cells in the stamen hairs.
Some 40 - 75 hairs can be obtained from each bud. Details of the
technique and its application for detecting gaseous mutagens in the
environment are given by Van't Hof & Schairer (1982).
There are about 10 test systems in plants that can be used to
investigate the mutagenic effects of chemicals and they cover a
full spectrum of genetic end-points. They range from the rapid and
simple root-tip assays for structural chromosome damage to
relatively complex tests for specific locus mutations. Plant
assays have been used extensively to test chemicals in solution and
some of the systems are uniquely fitted for detecting low
concentrations of atmospheric mutagens. A test using homosporus
ferns (Klewoski, 1978) is being developed for detecting water-borne
mutagens in natural waters and effluents.
Although the literature on plant mutagenesis is extensive,
there are few data comparing the results observed in plants with
those in mammals, and extrapolation between the two remains
somewhat tenuous. Some mammalian carcinogens that are known to
require metabolic conversion to reactive molecules are detected as
mutagens in plant systems (e.g., some nitrosamines). In the
limited comparisons available, there is a positive correlation
between mutagenicity in plants and mammalian cells. However, there
appear to be two serious limitations in the interpretations of the
results of plant assays in terms of human hazard. First, though
there are data on up to a hundred chemicals in some systems, the
majority of the chemicals tested have been mutagens (in some assays
as many as 95% of chemicals tested). Thus, many more data on the
response of plants to chemicals shown to be non-mutagenic in other
systems are required. The second limitation is related to the
fundamental differences in the metabolism of foreign compounds
between plants and mammals, and information is lacking on the
mutagenicity and metabolic mechanisms in plants for many of the
major classes of mammalian carcinogens.
In spite of these reservations, it must be recognised that
plant systems have many attributes in terms of cost and technical
simplicity that recommend their use in specific circumstances for
the initial screening of chemicals for mutagenic activity.
2.7. The Drosophila Sex-Linked Recessive Lethal Assay (SLRL)
The fruitfly Drosophila melanogaster is a test organism in
which it is possible to analyse in vivo heritable mutations and
chromosomal aberrations in the same population of treated germ
cells. Special strains (stocks) are available or can be
constructed to study gonadal or somatic tissue for gene mutations,
deletions, and for almost all possible types of chromosomal
rearrangements. In addition, special test protocols have been
devised to detect aneuploidy resulting from nondisjunctional
events. Comparative investigations on the reliability of these
different genetic end-points have clearly revealed that the
X-linked recessive lethal test is by far the most sensitive and
reliable assay in Drosophila to screen compounds for heritable
genetic damage. One of the major reasons is that the phenomenon of
"recessive lethality" can have different origins: recessive
lethals comprise point mutations (intragenic changes), deletions
affecting more than one gene, and both small and large
rearrangements (Auerbach, 1962a). Thus, a mutagen that only
produced gene mutations would not be detected in a test for
translocations, but would still be picked up in the recessive
lethal assay. In this section, a brief outline of the performance
and the most essential points of the recessive lethal method will
126.96.36.199. Test organism life cycle
Drosophila melanogaster undergoes complete metamorphosis. The
egg produces a larva that undergoes two molts, so that the larval
period consists of three stages (instars). The third instar larva
becomes a pupa which, in turn, develops into an imago, or adult.
Depending on the temperature, this fly requires 9 - 20 days to
complete one generation. At 25 °C, the culture temperature
preferred in most laboratories, the major stages in the life cycle
are: embryonic development, 1 day; first larval instar, 1 day;
second larval instar, 1 day; third larval instar, 2 days; prepupa,
4 h; pupa, 4.5 days. Thus, at 25 °C, one generation lasts only
9 - 10 days.
188.8.131.52. Stock cultures
Glass milk bottles of about 200 ml volume are used for stock
cultures. For smaller cultures, e.g., pair matings in the
recessive lethal test, vials of about 40 ml are used. The culture
media most widely used are banana medium and cornmeal medium, i.e.,
74.3 g water, 1.5 g agar, 13.5 g molasses, 10.0 g cornmeal, and
0.7 g methyl- p-hydroxybenzoate (to reduce growth of moulds).
184.108.40.206. List of nomenclature
The book of Lindsley & Grell (1968) entitled "Genetic
Variations of Drosophila melanogaster" represents the exhaustive
compilation of the mutants of Drosophila. This book gives the
nomenclature used by Drosophila geneticists, together with a
detailed description of mutants, chromosomal aberrations, special
balancer chromosomes, cytological markers, and wild-type stocks.
This guide is indispensable when working with Drosophila.
220.127.116.11. Equipment and laboratory techniques
There are several detailed descriptions of mutation work on
Drosophila, including culture medium, equipment, stock culturing,
and handling of flies (Abrahamson & Lewis, 1971; Demerec & Kaufman,
1973; Würgler et al., 1977).
2.7.3. Principle of the recessive lethal assay
Individual chromosomes of Drosophila melanogaster have been
labelled X, Y, 2, 3, and 4. The female chromosomes consist of
three pairs of autosomes (2, 3, 4) and one pair of rod-shaped X
chromosomes. The chromosome complex (2n) of the male has three
pairs of autosomes, one X and one J-shaped Y chromosome. The X and
Y chromosomes, therefore, are called the sex chromosomes.
The recessive lethal test can be readily designed to detect the
induction of heritable genetic lesions in a large part of the
Drosophila genome. Two generations are required for the detection
of recessive lethals on the X-chromosome, which represents about
20% of the entire genome. It is estimated that about 700 - 800 of
the 1000 loci on the X-chromosome can mutate to give rise to
recessive lethal mutations.
The most relevant features of the X-chromosomal recessive
lethal test (also referred to as sex-linked recessive lethal assay)
are illustrated in Fig. 5. Males from a wild-type laboratory
strain are treated (or kept untreated as controls) and are then
mated (P1) with virgin females that are homozygous for the X-linked
markers B (Bar, semi-dominant; eye restricted to a narrow vertical
bar in male and in homozygous female. Heterozygous female has a
number of facets intermediate between homozygous female and wild-
types) and " wa" (white-apricot, recessive; eye colour yellowish
pink), affecting the shape and colour of the eyes (Lindsley &
Grell, 1968). This "Basc" balancer X-chromosome also carries an
inversion to prevent crossing-over of a lethal from the treated
(paternal) X-chromosome to its homologue in the heterozygotes (F2-
P2). Thus, the two "marker genes" B and " wa" serve to distinguish
"treated" (paternal) from "untreated" (maternal) chromosomes. The
F1-P2 generation is intercrossed. In the F2, which splits into
four genotypes that can easily be identified by their different
phenotypes, it is possible to distinguish the two classes of flies
carrying copies from a treated chromosome (left side) from those
that do not (right side). If a complete recessive lethal mutation
is induced in an X-bearing germ cell of the treated P1 male, all
the somatic cells of the resulting F1 female will be heterozygous
for this mutation, and also 50% of its eggs will carry it. Half of
the F2 males will be hemizygous carriers for it and will therefore
die. But this can be seen only when single-mating is conducted in
the F1, which is an absolute prerequisite for the proper
performance of the assay.
Female treatment is not recommended in routine testing
procedures, because the females may contain pre-existing lethals
that have to be crossed out before starting an experiment. The
major advantages of the recessive lethal test are:
(a) The criterion used to decide whether a mutation is
present or not is very objective. The decision is
based on whether, in the F2-generation, one entire
class of males is absent or not (Fig. 6). Therefore,
personal bias is reduced to a minimum.
(b) Lethals are much more frequent than other types of
genetic lesions, i.e., viable visible mutations or
large structural aberrations.
(c) A representative part of the Drosophila genome is
covered by this multi-locus procedure.
2.7.4. Metabolic activation
The organism itself has a complex metabolic system (Vogel et
al., 1980). The presence in Drosophila of cytochrome P-450-
dependent oxygenase, cytochrome b5, aryl hydrocarbon hydroxylase,
and other components of the xenobiotic-metabolizing enzymes has
been demonstrated. There is substantial experimental evidence
supporting the conclusion that Drosophila has the enzymic
potential for converting a wide array of pro-mutagens/pro-
carcinogens (about 80 pro-carcinogens to date) into genetically-
2.7.5. Test performance
18.104.22.168. Treatment procedures
Chemicals are most commonly administered to Drosophila, either
by injection into the body cavity or by feeding, at the adult or
larval stage. Other methods of treating flies include treatment
through inhalation or using aerosols. Adult males are recommended
for testing purposes, since females are more readily sterilized by
chemicals and have, so far, proved more refractory to the induction
of heritable genetic changes.
Experience with several classes and types of mutagens indicates
the importance of a flexible protocol, when using the recessive
lethal assay. There are several examples in which the route of
administration has been shown to have a profound effect on the
mutagenicity detected. Injection seems to be more reliable for the
detection of highly reactive mutagens such as the unstable beta-
propionylactone and chloroethylene oxide. Adult feeding is more
effective in cases where a single injection (pulse treatment) is
highly toxic, as has been demonstrated with the carcinogen
N-nitrosodiethylamide (DEN). Several solvents (ethanol, Tween 60,
Tween 80, special fat emulsions) can be used to dissolve or
emulsify chemicals of low water solubility. The use of DMSO and
DMF should be avoided. These solvents have recently been shown to
inhibit chemical mutagenesis in some pro-carcinogens by blocking
their metabolic activation (Zijlstra et al., 1984).
22.214.171.124. Toxicity tests
Pilot studies should give concentration-mortality relationships
to express the general biological reactivity of the chemical under
test. The availability of such toxicity data aids the adequate
design of the genetic studies and, if the compound under test is a
mutagen, provides the condition that will produce the maximum yield
of mutations without killing the animal as a result of lethal
overdose. Thus, pilot studies should give an approximate idea of
the possible toxicity (LD50) of the test compound. Technical
aspects of toxicity tests are described by Würgler et al. (1977).
Actual results of toxicity tests with a series of monofunctional
alkylating agents were reported by Vogel & Natarajan (1979).
Regarding the general testing strategy, the highest possible
concentrations that can be used for testing for recessive lethal
mutations should be used first. Acute toxicity, reduced fertility,
and solubility problems may then be the limiting factors. Use of
two different dose levels is recommended, i.e., the MTD (maximum
tolerated dose) and 1/4 or 1/5 of it.
It is well known that chemical mutagens often exhibit stage
specificity, i.e., show more or less pronounced mutagenic effects
at different stages in germ-cell development. It is, therefore,
essential to analyse the progeny from treated spermatozoa, late and
early spermatids, and spermatocytes. Analysis of offspring from
treated spermatid stages is of particular importance because there
is considerable evidence, derived from experiments with
alkaryltriazenes, nitrosamines, and other pro-mutagens, that
release of ultimate mutagenic metabolites from the parent pro-
mutagen takes place directly in metabolically-active spermatid
stages, whereas spermatocytes are highly susceptible to killing.
On the other hand, there seems no need to include in the test the
analysis of spermatogonia, because there are only few cases of
mutagens that affect spermatogonia, but are not active in meiotic
or postmeiotic cells (Auerbach, 1962b).
With the brooding technique, the spatial pattern of
spermatogenesis is translated into a temporal pattern of successive
broods. Treated wild-type males are therefore re-mated at regular
intervals of 2 - 3 days with fresh virgin Basc females. An excess
of 3 - 5 females per male serves to sample all germ cells that are
in the mature stage. A total sampling period of 7 - 9 days (3 to 4
broods) is considered sufficient for mutagen testing.
126.96.36.199. Control and replicate experiments: sample size
Würgler et al. (1975) prepared sample-size tables that are very
helpful for adequately planning recessive lethal tests. The most
significant points in this respect are:
(a) the dependence of the outcome of the genetic test on
the number of chromosomes tested;
(b) the dependence of the result on the frequency of
spontaneous mutations; and
(c) on statistical grounds, the optimal number of tests
to be performed.
It is essential that, before starting a study, particular
attention should be paid to these statistical questions. To give
an example, with a spontaneous mutation frequency of 0.2% lethals
(10 lethals in 5000 progeny) and a sample size of 4500 in the
treated group, 0.47% was the lowest value to prove statistically
that a mutagenic effect was observed (Fig. 6). It is also obvious
from Fig. 6 that an increase in the number of tested chromosomes
above 5000 does not really help to improve the resolving power of
the assay. The most effective way of planning a study in order to
achieve results of statistical significance is to test about equal
numbers of chromosomes in the control and the treated groups.
Two studies, consisting of three successive broods each, can be
carried out easily within one week, with the aid of one technician.
If 600 - 800 cultures are set up for each brood, there is a testing
capacity of 1800 - 2400 chromosomes per study. One to two
replicate studies should be sufficient to classify a given test
substance. Complicated cases with mutation frequencies slightly
higher than the spontaneous background, will need further studies.
On the average, 80 man-hours are involved in testing an unknown
Experience with several tester stocks has shown that the
spontaneous mutability (about 0.1 - 0.2%) remains fairly
constant over the years. Initially, control studies should
be run concurrently; after more experience, concurrent control
studies are not mandatory, if the recessive lethal test is
either clearly positive (> 1 - 2% lethals) or negative (lethal
frequency < historical controls from the same laboratory). If the
percentage of lethals falls between the historical control and 1%,
concurrent control runs are obligatory. At least one replicate
study should be conducted in all cases.
Performance of, and possible pitfalls in, the recessive lethal
test have been extensively described by Auerbach (1962a),
Abrahamson & Lewis (1971), and Würgler et al. (1977).
2.7.6. Data processing and presentation
The experimental data to be reported should include the strains
and mating schedule used, the number of chromosomes tested, and
both the number and percentage of lethals. The aim of a study is
to find out whether the mutation frequency obtained from the
treated group is significantly higher than the spontaneous
background. After subtracting from the total number of F2 cultures
those that are sterile, the experimental data consist of the
following numerical values:
Nc = number of tested X-chromosomes (number of progeny) in the
Mc = number of recessive lethals in the control group;
Nt = number of tested X-chromosomes (number of progeny) in the
treated group; and
Mt = number of recessive lethals found in the treated group.
The basic question to be answered is: is the mutation frequency
pt (%) = Mt/Nt x 100, determined for the treated group,
significantly higher than pc = Mc/Nc x 100 for the control? For
statistical consideration of the data, the simple significance test
developed by Kastenbaum & Bowman (1970) should be applied.
Statistical analysis of mutagenicity data from the recessive lethal
assay is further described by Würgler et al. (1975, 1977).
188.8.131.52. Disadvantages of the recessive lethal test
The scoring of induced recessive lethal mutations is a highly
objective method of exploring the mutagenic potential of a
chemical. Nevertheless, there are a few cases of misclassification
through incorrect performance of the test. It may, therefore, be
profitable to summarize some of the obvious problems that can arise
in the design of such studies:
(a) As a standard rule, single-mating (one male and 3 - 5
females) should be applied to identify the very rare
cases of spontaneous clusters, i.e., mutants of common
origin. It is then possible to keep track of the F1
family of cultures derived from each P1 culture.
Clusters will tend to appear in families. If, in
postmeiotic broods, large clusters of lethals are
observed among F2 progeny derived from the same P1
male, it is recommended that these should be eliminated
from the final score, because they may reflect
spontaneous mutations that arose in dividing
spermatogonia during the development of that particular
(b) Great care must be exercised in the scheme to ensure
the use of virgin females in the P-generation. Thus,
all F1 females must be heterozygous for the treated
X-chromosome and the Basc balancer chromosome, and at
least three of the four different phenotypes must be
present in the F2 generation.
184.108.40.206. Weak mutagens and non-mutagens
Relatively large sample sizes are needed to discriminate
between weak mutagens and non-mutagens. It is possible to use
either concurrent negative controls, or historical controls. In
the latter case, at least 10 000 control tests (chromosomes) should
exist for a particular tester strain and each particular solvent
(e.g., Tween 80/ethanol). Gocke et al. (1982) reported a very
extensive set of historical controls, collected over many years.
It has to be stressed that results obtained with a large number of
tests, but with only one type of exposure or application, are only
informative with regard to that particular set of experimental
conditions. Recent instances of weak mutagenic activity in the
recessive lethal test are provided in an extensive study on some
carcinogenic polycyclic hydrocarbons and aromatic amines (Vogel et
al., 1983). No single technique (injection; feeding) was found to
be suitable for all the carcinogens investigated; hence, very
extensive experiments had to be carried out, using a flexible test
220.127.116.11. Data base
The recessive lethal assay has been well developed and
calibrated against a wide array of direct-acting agents and pro-
mutagens. Interlaboratory variability has not been a problem with
this assay. According to a report of the US EPA Gene-Tox Program,
421 compounds have been tested in the recessive lethal assay (Lee
et al., 1983). Of these, 198 compounds were found to be positive
and 46 negative, at the highest concentration tested. A third
group containing as many as 177 compounds, was not classified as
either positive or negative, because the very rigid criterion used
was the test of at least 7000 chromosomes in both the control and
the treated groups (per dose level), with a spontaneous frequency
of 0.2%. With the fulfillment of this criterion, it would be
possible to detect a doubling of the recessive lethal frequency
(Lee et al., 1983). The problem with this approach is that
flexibility is diminished, and that too much weight is put on one
experimental condition. The alternative procedure, which seems
more realistic in view of the fact that Drosophila constitutes a
very complex metabolic system, would be to use a flexible test
protocol in studies with weak mutagens. Reliance should not be
placed on a large number of chromosomes tested at only one
concentration. A variety of experimental conditions (e.g.,
injections versus feeding) can be used to identify optimal
experimental conditions for a given genotoxic agent. A good
example of the latter approach is the demonstration by Zijlstra &
Vogel (1984) that 7,12-dimethyl-benz( a)anthracene, methyltosylate,
and nor-nitrogen mustard are strongly mutagenic, weakly mutagenic,
or even ineffective in the recessive lethal assay, depending on the
route of administration used.
18.104.22.168. Correlation with mammalian carcinogenicity data
In the Gene-Tox report by Lee et al. (1983), there were 62
compounds that could be classified as positive or negative for both
carcinogenesis in mammals and mutagenesis in the recessive lethal
assay. Of the 62 compounds, there was agreement between
carcinogenic activity and mutagenesis classification in 56 cases
(50 positive and 6 negative), i.e., 90% would have been correctly
classified as to carcinogenicity using only the SLRL test. The
data were derived from a list in which 198 National Cancer
Institute (NCI) bioassays were evaluated for carcinogenicity
(Griesemer & Cueto, 1980).
In another comparative analysis (Vogel et al., 1980), 85 out of
107 carcinogens (79%) were found to be mutagenic in Drosophila. Of
the remaining compounds, 17 were negative and another 5 were not
sufficiently tested to reach meaningful conclusions. The
documentation by Vogel et al. (1980) is based predominantly on the
142 chemicals considered in the IARC Monographs volumes 1 - 20 for
which there is "sufficient evidence of carcinogenicity" in
experimental animals, according to evaluations by expert committees
(IARC, 1979). A second source for the documentation of
carcinogenicity data was a list prepared by the US EPA (1976).
22.214.171.124. Recent developments
The recessive lethal assay is a relatively time-consuming
method compared with systems using bacteria or lower eukaryotes.
This disadvantage may, however, be offset in the future when, in
addition to overall metabolic considerations, attention is directed
to differences in metabolism existing between somatic and gonadal
tissue, as was recently demonstrated for the inducibility of AHH
(aryl hydrocarbon hydroxylase) activity. Thus, somatic assay
systems might be particularly valuable as a complement to recessive
lethal tests on the germ line. One system is based on eye-colour
markers (Becker, 1966), and another on wing-hair markers (Garcia-
Bellido et al., 1976). Both systems are currently under validation
in several laboratories (Graf et al., 1983; Vogel et al., 1983).
With the white/white-coral system (Becker, 1966), which has been
calibrated against 35 reference mutagens, it is possible to test
about 4 - 6 chemicals in 2 weeks. Moreover, tests based on the
detection of genetic changes in somatic cells have the advantage
that they can be performed within one generation.
2.8. In Vivo Cytogenetics: Bone Marrow Metaphase Analysis and
In vivo bone marrow tests, which include metaphase chromosome
analysis, and the micronucleus assay are used to identify
clastogenic compounds, that is, those that are capable of inducing
structural changes in chromosomes. Chromosomal aberrations are
analysed in mitotic metaphases from proliferating tissue, such as
bone marrow samples from laboratory animals. In the micronucleus
test, clastogenic effects can be measured indirectly by counting
small nuclei in interphase cells formed from acentric chromosome
fragments or whole chromosomes.
Both tests are widely used, and they are regarded as of
particular importance by many regulatory authorities, because, in
the whole animal, the obvious deficiencies in artificial metabolic
activation systems used in in vitro systems are avoided.
126.96.36.199. Current understanding of the formation of chromosomal
Chromosomal aberrations occur because of lesions in the DNA
that lead to discontinuities in the DNA double helix. The primary
lesions, which include single- and double-strand breaks, base
damage, DNA-DNA and DNA-protein crosslinks, alkylations at base or
phosphate groups, intercalations, thymine dimers, apurinic and
apyrimidinic sites, are recognized by DNA-repair processes.
Therefore, the lesions may be corrected or transformed, to
restitute the original base sequence or produce chromosomal
aberrations and/or gene mutations.
The breakage-reunion hypothesis (Sax, 1938) implies that a
discontinuity in the DNA may be stabilized to appear as a break at
metaphase. Alternatively, the discontinuity may be restituted by
repair processes to the original state, whereby the chromosome does
not exhibit visible structural changes. Two DNA discontinuities in
temporal and spatial proximity may interact in the reunion of the
broken ends, thus forming exchange configurations. The exchange
hypothesis (Revell, 1959) postulates that all aberrations are the
result of exchange processes that involve interaction between two
local instabilities in close proximity. Experimental data have
been provided in support of both hypotheses.
Recent experimental results support the breakage-first
hypothesis. The evidence for double-strand breaks being the
ultimate DNA lesion for chromosomal aberrations has been summarized
by Obe et al. (1982). Double-strand breaks lead directly to
chromosomal aberrations; all other primary lesions require
transformation to double-strand breaks by DNA replication and/or
Double-strand breaks can be induced directly by ionizing
radiation and S-independent chemicals, e.g., bleomycin. Double-
strand breaks may lead to an immediate fixation of the aberration
by misrepair. Depending on the time of induction within the cell
cycle, the types of aberrations observed at the succeeding
metaphase are of a chromosome (from G1) or chromatid (from G2)
nature, i.e., involve both or only one chromatid (Evans, 1962).
Most chemical mutagens do not cause double-strand breaks directly.
The preliminary lesions are transformed in S-phase, and the
aberrations observed at metaphase are of the chromatid type (Evans
& Scott, 1964). A classification of chemicals according to their
mode of action during the different phases of the cell cycle was
given by Bender et al. (1974) and is still valid (Brewen & Stetka,
188.8.131.52. Classification of chromosomal aberrations
Aberrations are divided into chromatid-type and chromosome-
type, the first involving only one chromatid, the latter, both
chromatids at identical sites. Furthermore, breaks can be
distinguished from exchange configurations by their physical
appearance at metaphase rather than by their mode of formation.
Breaks are true discontinuities with clearly dislocated fragments
and also include fragments without obvious origin. They should not
be confused with achromatic lesions (gaps), which do not represent
true discontinuity in the DNA. It is generally assumed that gaps
are sites of despiralization in the metaphase chromosome that
render the DNA non-visible under light microscopy. It has been
proposed that an achromatic lesion may actually be a single-strand
break in the DNA double helix as a result of incomplete excision
repair and, thus, may represent a point of possible instability
(Bender et al., 1974). Therefore, gaps are always noted but
reported separately from true chromosomal aberrations.
Exchange configurations can be subdivided into intrachanges,
i.e., exchange within one chromosome, and interchanges, i.e.,
exchange between two chromosomes. The classification into intra-
and interchanges applies to chromatid- as well as to chromosome-
type aberrations. Depending on the location of the original
discontinuities and the ways of reunion, further classification of
exchanges is possible such as symmetrical or asymmetrical, complete
or incomplete. The terminology is complex but has been clearly
reviewed by Savage (1976) and Scott et al. (1983).
The majority of aberrations observed at first metaphase after
exposure are lethal to the cell that carries them or to the
daughter cells. Whenever acentric fragments are formed, genetic
imbalance will result. In the case of chromosome-type aberrations,
both daughter cells will die, since both chromatids are affected.
In some cases of chromatid-type aberrations, only one chromatid is
affected and only one of the daughter cells may die. Only
symmetrical forms of chromosome or chromatid exchanges, with no
loss of genetic material, will survive cell division and be
transmitted to future cell generations. Chromatid-type aberrations
that survive the first division are converted to derived
chromosome-type aberrations. The balanced "stable" types of
aberrations are reciprocal translocations and inversions. Since
there is no rule without exceptions, the occasional balance of
genetic material will allow cell survival. Chromosomal syndromes
in human diseases such as Cri du Chat (deletion of chromosome 5) or
Down's syndrome (trisomy 21) are due to structural or numerical
chromosomes changes, i.e., genetic imbalance.
Some more unspecific chromosomal changes should be mentioned
for the sake of completeness. So-called sub-chromatid aberrations
have been primarily observed at anaphase, most typically as "side
arm bridges", after exposure of cells to ionizing radiation during
prophase of cell division. A model of sub-chromatid aberrations
has been discussed by Klasterska et al. (1976). However, another
phenomenon, namely chromosome stickiness, cannot be discriminated
from subchromatid aberrations, when cells are scored at metaphase
rather than anaphase.
Chromosome shattering can be seen at metaphase, similarly
chromosome pulverisation has been described. There is no clear
distinction between these two phenomena, which simply represent
different degrees of damage inflicted on the chromosomes. In the
case of shattering, chromosomes appear to have been broken up into
many small pieces of various lengths. Sometimes just a few,
sometimes all, chromosomes are shattered but usually intact
chromosomes or conventional chromatid-type aberrations remain
recognizable. In cells with pulverisation, the chromosomes can be
reduced to masses of fragments. The phenomenon of premature
chromosome condensation (PCC) is sometimes confused with shattering
or pulverisation. However, its appearance is quite different. Thin
chromosomal fragments of various lengths lie among the debris. The
PCC phenomenon was shown to arise from the virus-mediated fusion of
a cell in division to a cell in S-phase, which brings about
visualization (condensation) of chromosomes in the process of
duplication (Johnson & Rao, 1970). PCC was also described in
Chinese hamster bone marrow after chemical treatment (Kürten & Obe,
1975). Here, it was explained as condensation of chromatin in
micronuclei, induced by mitotic condensation of the chromosomes in
the main nuclei while the micronuclei were still in S-phase.
Polyploidy and endoreduplication are frequently described in
cultured cells, though they are less often seen in bone-marrow
material. Although it is important to assess aneuploidy, the
routine scoring of numerical aberrations in bone-marrow metaphases
is not recommended, because deviations in chromosome number often
arise as preparational artifacts.
184.108.40.206. The basis for micronucleus formation
Micronuclei originate from chromosomal material that has lagged
in anaphase. In the course of mitosis, this material is
distributed to only one of the daughter cells. It may be included
in the main nucleus or form one or more separate small nuclei,
i.e., micronuclei. The micronuclei mainly consist of acentric
fragments as demonstrated by DNA content measurements (Heddle &
Carrano, 1977). They may also consist of entire chromosomes and
may result from non-disjunction due to malfunction of the spindle
apparatus. These larger micronuclei are formed by spindle poisons
(Yamamoto & Kikuchi, 1980). Micronuclei can be observed in any
cell type of proliferating tissue. They are, however, most easily
recognized in cells without the main nucleus, namely erythrocytes.
The scoring of micronuclei in bone-marrow cells was proposed as
a screening-test by Boller & Schmid (1970) and Heddle (1973). The
frequency of micronuclei can be evaluated most readily in young
erythrocytes, shortly after the main nucleus is expelled. The
young ones are termed polychromatic erythrocytes (PCEs), the mature
ones normochromatic erythrocytes (NCEs). With conventional
staining techniques, PCEs stain bluish to purple because of the
high content of ribonucleic acid in the cytoplasm. NCEs stain
reddish to yellow. The PCEs are also slightly larger than the
In mouse-bone marrow, the maturing erythroblasts go through six
or seven cell divisions with a cell-cycle length of about 10 h
(Cole et al., 1979). About 10 h after the last mitotic division,
the expulsion of the main nucleus is completed and the resulting
PCE remains in the bone marrow for another 10 h. Treatment-induced
micronuclei derived from chromosomal fragements produced during the
preceeding cell cycle will thus appear in PCEs not earlier than
10 h after injection of the animal with the test chemical.
Experience with known chemical mutagens has shown that, in fact,
micronuclei appear much later than this. Jenssen & Ramel (1978)
demonstrated by simultaneous treatment of mice with methyl
methanesulfonate (MMS) and 3H-thymidine labelling that nuclear
expulsion was delayed by about 9 h. Even though an increase in
micronuclei levels compared with controls was already seen after
12 h, the curve rose steeply between 18 and 24 h, corresponding to
the MMC-induced delay of the last cell cycle up to nuclear
Detailed experimental procedures are described by Adler (1985).
220.127.116.11. Experimental animals
Bone-marrow studies can be carried out with most laboratory
mammals. Chinese hamsters may be preferred for metaphase analysis
because of their low chromosome number (2n = 22) and their readily-
distinguishable chromosomes. Mice (2n = 40) or rats (2n = 42) are
also frequently used. For the micronucleus test, the use of rats
is less convenient. Rat tissue is rich in mast cells. In the
course of bone marrow preparation, these cells shed granules
containing heparin, which stain in a similar manner to micronuclei,
and thus, make scoring of true micronuclei rather difficult.
The animals used in bone-marrow studies should be young adults.
The high proliferative activity and the low fat content of bone
marrow in young animals favour the quality of the preparations.
Each group of test animals should consist of equal numbers of males
and females to allow for sex differences in response to the
18.104.22.168. Treatment and sampling
The treatment should generally comprise a single application
of the test compound, followed by multiple sampling of groups
of animals at different times (Preston et al., 1981). The most
commonly used routes of application are intraperitoneal
injection and oral intubation. Other routes of application, e.g.,
inhalation, are possible. The time of maximum response may vary
from chemical to chemical, depending on the sensitive cell-cycle
stage and the influence of the chemical on cell-cycle length.
Moreover, absorption, distribution, and metabolism may influence
the optimum interval between treatment and sampling. Therefore, a
single, generally applicable sampling time cannot be recommended.
The central sampling interval after dosing is usually 24 h for
chromosome analysis and 30 h for the micronucleus test. In
addition, one earlier and one later sampling interval should be
used, e.g., between 12 - 18 and 36 - 48 h for metaphase analysis
and 12 - 18 and 60 - 72 h for the micronucleus test.
Earlier publications on bone-marrow cytogenetics and the
micronucleus test (Matter & Schmid, 1971; Schmid et al., 1971)
recommended two treatments with a 24-h interval between the two.
Other authors have used 5 daily applications of the test compound.
The single treatment schedule is preferable for the following
(a) Cell killing
Chromosomal aberrations do not accumulate over successive cell-
cycles, because, in most cases, they are cell-lethal. Thus, true
clastogens also kill cells. In repeated treatment schedules, the
first dose kills off the most sensitive cells leaving a changed
cell population of more resistant cells for the following
(b) Analysis of first post-treatment mitosis
Chromosomal aberrations can only be assessed quantitatively if
scored at the first post-treatment mitosis. If multiple treatments
are applied at 24-h intervals, the cells damaged by the first
application will have gone through one or more cell divisions.
Aberrations that are cell-lethal will have been lost by death of
the cells. Scorable chromatid-type aberrations, if the cells have
not been rendered non-viable, will have transformed into derived
chromosome-type aberrations. These can only be recognized with
banding techniques and karyotyping of each cell, a procedure that
is too laborious for screening purposes. Thus, the chromatid-type
aberrations scored after long-term treatments will represent only
the effect of treatment on the penultimate cell cycle.
In theory, micronuclei may accumulate after treatment of two or
more cell cycles, since they are scored in a cell type that does
not undergo further cell division (Salamone et al., 1980).
However, as with metaphase analysis, the cell-killing effect will
adversely influence the micronucleus yields after repetitive
treatment of the proliferating precursor cells. In order to
measure the accumulation of damage under conditions of low cell-
toxicity, the spacing of treatment should be governed by the length
of the cell-cycle. Cole et al. (1981) recommend that for studies
using the bone marrow of adult mice, 10-h intervals should be used
and erythrocytes should be sampled 25 h after the last treatment.
But, even if cell killing does not occur at low doses, cell-cycle
delay caused by the test chemical, as described in the previous
section for MMS, may defeat the purpose of the study.
Repeated treatment schedules can occasionally be justified for
pharmacological reasons. For example, if a compound requires
metabolic activation by an enzyme that is induced by the chemical
itself, it can be argued that, to establish the necessary enzyme
level, the compound should be administered several times. However,
7,12-dimenthylbenz( a)anthrazene and benzo( a)pyrene, which are
metabolized by self-induced enzyme systems, readily induced
micronuclei after a single application and required relatively late
sampling (36 h or 48/72 h) (Salamone et al., 1980; Kliesch et al.,
1982). An increase in micronuclei was not observed after benzo
( a)pyrene was administered in a 5-day treatment schedule (Bruce &
Heddle, 1979). Thus, for the micronucleus test, the advantage of
repeated treatments is questionable.
Because of cell-killing effects and the necessity to analyse
first post-treatment mitoses, long-term treatments are not suitable
for chromosome studies with proliferating tissues. If, for
whatever reason, prolonged treatment is required, e.g., in a
feeding study, non-proliferating cells such as peripheral
lymphocytes should be sampled from the treated animals. These
cells can be stimulated to cycle in vitro so that it is possible
to score first mitoses after treatment or to count micronuclei in
second interphase cells.
22.214.171.124. Dose levels
The choice of test dose levels is based on the maximum
tolerated dose (MTD) of the compound in the species used for the
test. The MTD is defined by the cellularity of the bone marrow and
the yield of analysable metaphases or PCEs. A rule of thumb is to
use the MTD as the highest dose. To accept a positive result, it
is usually necessary to demonstrate an increase in effect with
increasing dose. If the test results are negative, the conclusion
is only acceptable when two or three dose levels have been tested
or the test has been repeated. Testing with additional dose levels
can be restricted to the interval of maximum effect with the
highest dose level or to the central sampling interval in case of
negative results with the highest dose, bearing in mind the fact
that cell-cycle delay is related, not only to the nature of the
chemical, but also to the dose level.
126.96.36.199. Number of cells scored per animal
The number of metaphases scored or PCEs counted per animal is
governed by the number of animals in each group and the statistical
procedure used for planning and evaluating the study. At least 500
metaphases or 4000 PCEs should be scored for a single-dose group.
188.8.131.52. Positive and negative controls
A negative vehicle-control (solvent) is an essential part of
each study. A positive control is generally required. The
positive control is only meaningful if it demonstrates the
test sensitivity with the lowest positive dose of a known
clastogen, e.g., 0.16 mg/kg of mitomycin C or 3.1 mg/kg of
procarbazine (Kliesch et al., 1982). When the chemical under test
is given in repeated treatments, the positive control has to
demonstrate that treatment with a known clastogen at low dose
levels produces an effect.
184.108.40.206. Preparation procedure for bone-marrow metaphases
The preparation procedure has been described in detail by Dean
(1969). Animals are injected with colchicine or Colcemid solutions
prior to bone marrow sampling, in order to accumulate metaphases.
Other spindle poisons can also be used. Dose levels and timing
depend on the animal species. For mice, 4 mg colchicine/kg body
weight is usually given, 1 - 1.5 h prior to sacrifice. Chinese
hamsters require a longer period of colchicine treatment. In vitro
colchicine treatment is also possible, after collection of bone
marrow (Tjio & Wang, 1965).
Bone marrow is flushed from the femur into a neutral medium
such as 2.2% sodium citrate, Hank's balanced salt solution (HBSS).
After the sampling of bone marrow from all animals into individual
centrifuge tubes is completed, the cells are centrifuged for 5 min
at 100 x 6. The supernatant is discarded completely and a
hypotonic solution is slowly added while agitating the tube to
disperse the pellet. The hypotonic medium can be 1% sodium
citrate, 0.56% potassium chloride, or the medium diluted with
distilled water (1:1). The duration of the hypotonic treatment
depends on the animal species and ranges from 15 to 30 min at room
temperature. Hypotonic effects may be intensified at 37 °C;
however, clumping of cells due to collagens and fat in the bone
marrow is also increased. After centrifugation, at the end of
hypotonic treatment, the cells are fixed by the addition drop-wise
of freshly prepared cold methanol/acetic acid mixture (3:1) to the
resuspended pellet. The fixative is changed 3 times. In between,
the cell suspensions should be stored in the refrigerator and can
remain there overnight before slide making. It is essential that
fresh fixative be prepared just prior to fixation; it cannot be
kept overnight because ester formation will weaken the fixation
For slide making, the most crucial factor is that the glass
slides are absolutely clean and grease-free. They can be kept in
70% alcohol (overnight), used wet, and then flame-dried which
facilitates chromosome spreading. Other methods of slide making
include cooling the slides in an ice-box before use or storing them
in cold distilled water and using them wet. Shortly after the cell
suspension has been applied (2 - 3 drops per slide), they can be
dried on a warm plate.
Slides are usually stained for 10 min in a 5% Giemsa solution
(pH 6.8). The staining solutions have to be filtered, immediately
before use. The stained slides are washed in distilled water, air-
dried, cleared in xylene, and cover-slipped using a suitable
mounting medium. Staining with 2% acetic orcein for 30 min is also
220.127.116.11. Preparation procedure for micronuclei
The method, which has been described in detail by Matter &
Schmid (1971), Heddle (1973), and Schmid (1976), includes the
following basic steps. Bone marrow is flushed from the femur into
fetal calf serum, the cells are centrifuged for 5 min at 100 x g,
and the supernatant is discarded as completely as possible. The
pellet is resuspended and care should be taken to prevent the loss
of any material into the wide part of the pasteur pipette. One
drop of the bone-marrow suspension is placed on one end of a clean,
grease-free slide, and pulled behind a glass cover slip to produce
a cone of bone-marrow smear. The slides are air-dried before
staining, possibly overnight, and double-stained with May-Grünwald
and Giemsa as described by Schmid (1976). The only change adapted
by various laboratories is the replacement of distilled water by
phosphate buffer (pH 6.8) for the Giemsa solution.
18.104.22.168. Microscopic analysis
Slides are coded before scoring and only decoded after scoring
of the entire study is completed.
(a) Chromosomal aberrations at metaphase
Slides are screened for analysable metaphases under low-power
magnification (16 or 25 x objective). High magnification (oil
immersion objective) is used for examination of each individual
metaphase. Only cells with the complete number of centromeres are
included. Each aberration is noted separately on a scoring sheet.
Vernier readings can be taken for all cells or only for those that
carry an aberration.
Selection of analysable metaphases may pose a certain bias.
Criteria for rejecting a metaphase include: incomplete number of
centromeres; loss of chromatid alignement and/or centromere
splitting due to extended colchicine treatment; extensive overlap
of chromosomes; and poor fixation of the chromosomes.
The mitotic index, which is the fraction of cells in a given
population that undergo mitosis at a given time, indicates cell
proliferation activity. The number of mitoses should be determined
for each animal by counting 500 nuclei. Changes in the mitotic
index reflect the cytotoxic effect of the treatment.
(b) Micronuclei in polychromatic erythrocytes (PCEs)
Polychromatic erythrocytes are counted in each field of high-
power magnification (oil immersion objective), and the number of
those with micronuclei is determined. The ratio of PCEs to NCEs is
established for each animal by counting a total of 1000
erythrocytes. Changes in the ratio of PCEs to NCEs reflect the
cytotoxic effect of the treatment. The number of NCEs with
micronuclei is also noted.
2.8.3. Data processing and presentation
22.214.171.124. Chromosomal aberrations
From the raw data on the scoring sheets, two ways of tabulating
the individual animal data should be used, i.e., number of
aberrations per cell, and number of cells with aberrations. These
express the severity of damage to the affected cell, and to the
cell population, respectively. In the individual animal data
sheets, results from each animal within one experimental group are
listed, and the various types of aberrations are recorded. In the
summary reporting sheets, mean values and standard deviations over
all animals are given for the various experimental groups. While
gaps and breaks are kept separate in the summary reports, the
various forms of exchange can be summarized, but should be
separated according to chromatid- or chromosome-type. Two columns
should give the mean of all aberrations per cell (including and
excluding gaps) and the average percent of cells with aberrations
(including and excluding gaps). Changes in the mitotic index
should be reported separately.
Individual animal-report sheets are compiled from the raw data.
They contain the total number of PCEs counted, the ratio of PCEs to
NCEs, and the number of PCEs with micronuclei. From the individual
animal reporting sheets, the summary report is compiled by giving
the average frequency of micronucleated PCEs (for each experimental
group), the average ratio of PCEs/NCEs, and the average
126.96.36.199. Statistical evaluation
Katz (1978) stresses the point that the best designed studies
are those with approximately equal numbers of individuals in the
experimental and control groups. He also points out that the
minimum number of animals in the study is governed by the
spontaneous incidence and the required sensitivity.
A statistical design for the micronucleus test was described by
Mackey & MacGregor (1979). They used a sequential sampling
strategy and based the statistical analysis on the negative
binomial distribution or the binomial distribution. The number of
animals in their design was not fixed and the number of PCEs per
animal was arbitrarily chosen to be either 500 or 1000. Decision
limits were given on the basis of the spontaneous micronucleus
incidence of 2/1000, a required 3-fold increase for a positive
result and an 0.01 probability of error to both sides. Sampling
and treatment of animals in this design have to be continued as
long as the cumulative micronucleus counts fall between the given
Equally as important as the number of animals, is the number of
cells scored. This again depends on the spontaneous frequency of
the parameter under test and the required increase for a positive
result. Grafe & Vollmar (1977) published a table that related
these two factors to the minimum number of cells required in the
micronucleus test. The table was based on the assumption of
binomial distribution and a probability of error of 0.05.
According to the table, a sample of 15 700 cells would be necessary
to recognize an increase by a factor of 2 over the spontaneous
micronucleus frequency of 2/1000. This approach is useful when the
total number of cells is scored with no regard for interanimal
variation. However, because there is usually a lack of homogeneity
between treated animals, it is the number of animals in the
experimental group that determines the precision of the statistical
procedure rather than the total number of cells scored.
The inadequacy of the currently available statistical
approaches lies in the fact that the number of cells per animal or
the number of animals per group is chosen arbitrarily. So far,
none of the published recommendations for the statistical planning
and evaluation of cytogenetic in vivo tests (including the
micronucleus test) has dealt with the problem of interanimal and
within-animal variability in treated groups. The distribution of
cytogenetic variables remains debatable and difficult to determine.
Until more satisfactory statistical models become available, it
may be prudent to use a non-parametric statistical procedure to
determine whether or not two samples, one drawn from the control
group and one from the group of treated animals, belong to the same
population (null-hypothesis). The rank tests by Mann & Whitney
(1947), based on the so-called Wilcoxon Test, seem to be the
methods of choice. Correction for tied ranks is possible (Walter,
1951). If more than two independent samples are to be compared,
the test described by Kruskal & Wallis (1952) can be used. These
tests require that the per animal sample size of cells is constant.
From the tables of critical values of the test statistic U, it can
be deduced that at least 4 animals in both groups are required
before a test ( P = 0.05) can be applied.
Data from control animals should be tested for inhomogeneity by
means of the binomial dispersion test (Snedecor & Cochran, 1967)
and should only be accepted if homogeneity is obtained. In
experimental groups, individual animal response to the treatment
may vary such that inhomogeneity is obtained. Therefore, standard
deviations should be calculated with the sample size given
according to the number of animals and not the total number of
For micronuclei in mouse bone marrow, the spontaneous rate in
the controls is 0.2% and fairly constant. Scoring 2000
polychromatic erythrocytes per animal, 1000 per slide, has proved
practical, but is still an arbitrary value. Likewise, the
spontaneous frequency of breaks in mouse bone-marrow metaphases is
0.5 - 1.0%, and the arbitrarily chosen number of cells to be scored
per animal is 125. Practical experience with known clastogens has
shown that, using these samples with 4 animals per group,
significant differences can be recognized by the rank test if the
experimental value is twice as high as the control value, i.e., the
spontaneous rate is doubled.
188.8.131.52. Possible errors in microscopic evaluation
Microscopic evaluation of metaphase chromosomes for chromosomal
aberrations is somewhat subjective. The criteria for the
discrimination between gaps and breaks, for instance, has been
discussed frequently, and two general opinions exist:
(a) A gap is an unstained region in the chromatid that is
smaller than the width of the chromatid. If the
unstained region is larger, it is termed a break.
(b) An unstained region in the chromatid is termed a gap
if no dislocation of the fragment is recognizable.
The length of the unstained region is not important.
The dislocation characterizes the break.
As discussed in the introduction, it is generally believed
that, unlike breaks, gaps do not represent true discontinuities
with DNA. This view suggests that the second criterion should be
chosen. Opinions differ, however; for example, Scott et al. (1983)
recommend the first criterion.
Another subjective element is the choice of analysable
metaphases, particularly as the loss of chromatid alignment can
contribute to failures in the recognition of aberrations of the
Mouse chromosomes carry a heterochromatic region near to the
centromere. Often, the centromeres appear separated from the rest
of the chromatids, and this separation may be more pronounced in
one chromatid than in the other. This phenomenon should not be
mistaken for a gap. Quite frequently, two acrocentric mouse
chromosomes may appear in close juxtaposition at their centromeric
ends and, thus, mimic a Robertsonian translocation. True
centromeric fusions to Robertsonian translocations are very rare
events. Chromatid breaks in the centromeric region lead to whole
arm exchanges, which should be clearly distinguished from short-arm
association due to preparational artifacts.
It is important that only cells with the complete number of
centromeres are included in the scores. If cells carrying an
aberration, but lacking some of the chromosomes, were included in
the scoring, it would be biased in favour of aberration-carrying
cells. If normal cells with one or two fewer chromosomes were
included in the scores, it would be biased against aberrations,
since the lost chromosomes might have been aberrant.
Artifacts can also obscure the micronucleus scores. Granules
shed by mast cells have already been described. If they lie on
PCEs, they can be mistaken for micronuclei. Granular or fibrillar
structures in or on PCEs can be discriminated from micronuclei by
their irregular appearance. True micronuclei are round or, on rare
occasions, oval or half-moon shaped, but always with a sharp
contour and evenly stained. Most artifacts can be recognized as
such by focusing up and down. If the particles show a ring of
reflection when out of focus, they are artifacts.
184.108.40.206. Comparison of test sensitivity
The two methods, metaphase analysis and micronucleus test, are
described, in most cases, as if they were equally sensitive.
Accumulating evidence supports the theoretical expectation that
metaphase analysis is more sensitive (Kliesch & Adler, 1983). It
is certainly more time consuming. However, it should also be more
exact, because all types of aberrations are scored. As stated
earlier, micronuclei reflect basically acentric fragments.
Equating the frequency of acentric fragments at metaphase with the
frequency of micronuclei in simultaneous experiments demonstrated
that, for an expected frequency of micronuclei, the observed
micronucleus frequencies were always below the expected, but
without any particular pattern of reduction. Thus, it has to be
assumed that not every fragment forms a micronucleus. Other
possibilities are that it can be maintained in the main nucleus, or
that it can be lost from observation because of cell death.
Furthermore, the micronucleus, as such, can be expelled with the
main nucleus. On the other hand, the micronucleus test can reveal
chemicals that disturb the function of the spindle, such as
colchicine or related spindle poisons, or chemicals that
predominantly act on tubular proteins rather than DNA, thus
inducing aneuploidy instead of structural chromosomal aberrations.
For the reasons mentioned above, the two tests cannot be regarded
as true alternatives. Thus, even though metaphase analysis
requires a higher degree of skill and is more time consuming than
the micronucleus test, the extra effort would seem justified.
220.127.116.11. Application of the method to other tissues
Chromosomal aberrations can be evaluated in most tissues of
treated experimental animals, whether somatic or germinal, e.g.,
lymphocytes, spleen, or liver (after stimulation by partial
hepatectomy or in vitro) (Dean, 1969), ascites tumours (Adler,
1970), early cleavage stages of embryos (Brewen et al., 1975),
embryonic tissues (Adler, 1981), spermatogonial mitoses (Adler,
1974), and meioses of spermatocytes and oocytes (Adler, 1982; Adler
& Brewen, 1982). However, because of the increased effort needed
to examine the preparations, the use of these tissues is, at
present, confined to special studies.
Similarly, micronuclei can be counted in the cells of various
tissues, despite the presence of the main nucleus. Reports on the
application of the micronucleus test to rat liver hetaptocytes
(Tates et al., 1980), mouse embryonic liver and blood erythrocytes
(Cole et al., 1979), and rat spermatids (Lähdetie & Parvinen, 1981)
have been published.
It is frequently stated that in vivo methods lack the
necessary sensitivity compared with in vitro studies. This
argument is more than offset by the considerable advantage that an
in vivo study is much closer to the human situation, which is the
ultimate concern of these studies. In vivo metabolic processes
such as activation and detoxification have to be stimulated in
vitro by relatively crude enzymatic preparations. Thus, negative
results in vivo may be more relevant than positive results in
vitro, even with mammalian cell preparations. The resolution of
these discrepancies requires careful additional studies on the
metabolism of the chemical. Whether the problem is resolved
depends on an understanding of whether or not the test compound or
one of its metabolites reaches a target organ in significant
amounts and what is the half life of the molecule at the target
site. It is also necessary to establish that the same metabolite
occurs in the in vitro and in vivo situation. If similar
information can be obtained from studies on human beings, then
sound grounds for a decision on whether or not a particular agent
poses a genotoxic danger have been established.
2.9. Dominant Lethal Assay
The term dominant lethal is used to describe a genetic change
in a gamete that kills the conceptus early in development. Any
induced changes that affect the viability of the germ cells
themselves or render the gametes incapable of participating in
fertilization are excluded. The pioneering studies of Hertwig
(1935), Brenneke (1937), and Schaefer (1939) had already indicated
that litters sired during the pre-sterile period of irradiated male
mice were found to be of reduced size. Since there was no effect
on sperm mobility and since the number of fertilized eggs was
normal, it was concluded that the reduced litter size was due to
death of embryos after ferilization. The observation of various
nuclear and chromosomal abnormalities in fertilized ova led to the
conclusion that embryonic death was caused by chromosomal
abnormalities, induced by irradiation in spermatozoa. The dominant
lethal assay was used as an indicator of radiation-induced
mutations by Kaplan & Lyon (1953), W.L. Russel et al. (1954), and
recommended for mutagenicity testing by Bateman (1966).
To study the cytogenetic basis of chemically-induced dominant
lethals, Brewen et al. (1975) collected fertilized ova from females
mated to young adult male mice after treatment with methyl
methanesulfonate (MMS). The ova were collected from day 1 to day
23 after injection. The types of aberrations observed were
predominantly double fragments (presumably isochromatid deletions),
chromatid interchanges, and some chromatid deletions, as well as a
shattering effect of the male chromosomal complement at 100 mg
MMS/kg body weight during the peak sensitivity of dominant lethal
induction. These data strongly suggest that chromosomal
aberrations observed at the first cleavage division of zygotes are
the basis of MMS-induced dominant lethality. In general, it can be
concluded that most dominant lethals probably result from multiple
chromosomal breaks in the germ cells.
A basic problem of chemical mutagenesis is that it is not
possible to measure directly the genetic effects of chemicals in
human germ cells. Therefore, there is no alternative to using the
data obtained in studies with mammals, particularly the mouse, to
predict the induction of mutations in human beings. The general
belief that the human gonads are well protected (Neel & Schull,
1958) was one important reason for a 20-year delay between the
discovery of the induction of mutations by chemicals and the
development of research programmes for mutagenicity testing.
The effectiveness of the blood-testis barrier (Setchell, 1970)
was sometimes wrongly used as an argument to conclude that the
dominant lethal assay is insensitive. In fact, the dominant lethal
assay is one of the few test systems that provides information
about compounds that are able to cross the blood-testis barrier.
Such information is of great importance in the assessment of the
possible mutagenic hazard of a chemical.
When a chemical substance has penetrated the blood-testis
barrier, it might be subjected to the enzymatic activation
processes in the various tissues of the gonad. The compound can
also be detoxified. After these modifying processes, the chemicals
or their metabolic products may interact with the DNA. The
resulting damage may be subjected to different DNA repair processes
and, finally, a sperm develop that may or may not carry a mutation.
In addition, Generoso et al. (1979) demonstrated that the yield of
chemically-induced dominant lethal mutations in male mice depends
on the genotype of the female, and this difference between females
of different strains may be caused by differences in the activity
of the repair enzymes. Though the possibility cannot be ruled out
that the induction of a repair enzyme was stimulated by the
chromosome lesions themselves, it seems more likely that the repair
enzyme existed in the egg prior to sperm entry.
2.9.2. Procedure for male mice
The method consists essentially of sequential mating between
treated or untreated male mice and untreated females. Mating
usually occurs at night, and conceptions can be recognized the
following morning by the presence of a vaginal plug. This plug is
a convenient means of timing a pregnancy. Pregnant females are
sacrificed on the 14 - 16th day of pregnancy. The corpora lutea,
representing the number of ova shed, are counted. The uterine
contents are scored for early and late deaths and living fetuses.
The induction of dominant lethals is determined by the increase in
pre- and postimplantation loss of zygotes in the experimental group
over the loss in the control group. This simple procedure is an
essential advantage of the dominant lethal assay.
The testis of the adult male contains the complete sequence of
maturation stages of the germ cells, from the stem-cell
spermatogonia to the spermatozoa passing out of the testis into the
epididymis. The timing of this sequence has been determined for
the mouse by Oakberg (1960). The earliest time at which specified
cells reach the ejaculate is:
1 - 7 days spermatozoa
8 - 21 days spermatids
22 - 35 days spermatocytes
36 - 41 days differentiated spermatogonia
more than 42 days AS (stem cell) spermatogonia.
During the first day after conception, fertilized eggs remain
aggregated in the cumulus of follicle cells. By the 4th day, a
blastocyst has developed. The blastocyst passes from the oviduct
into the uterus. It is the most advanced stage to which a
fertilized egg can develop without implantation. Implantation
occurs on the 5th day. By the 10th day, organogenesis is complete
and, during the following 10 days, the existing structures
differentiate, and the embryo completes its development (Bateman &
Epstein, 1971). Dominant lethals include the loss of fertilized
eggs before and after implantation.
The various germ-cell stages have different sensitivities to
the induction of dominant lethals by chemical mutagens, and the
germ-cell stage assayed depends on the interval between treatment
and mating. The frequency of dominant lethals can change
drastically in a 24-h mating interval (Ehling et al., 1968).
Therefore, it is essential to use a sequential mating schedule of
only a few days. An overview of the differential induction of
dominant lethals has been made by Ehling (1977).
The recommended test systems for mutagenicity screening are
generally based on the expertise and the facilities of a given
laboratory. The procedure given here for the dominant lethal assay
is based on a collaborative study involving 9 laboratories (Ehling
et al., 1978). The comparative testing of substances using the
same method in several laboratories was designed to identify
criteria that are critical for the optimal conduct of the dominant
lethal assay. From these studies, it was concluded that, while
certain test conditions could be standardized for improving the
reproducibility of results obtained in different laboratories,
other test conditions were matters for establishment in individual
laboratories, depending on preferences and conditions.
18.104.22.168. Standard and test conditions
(a) The mating period should be short enough to provide
information about the action of a chemical mutagen on
a specific germ cell-stage. For screening purposes,
where high fertilization rates are expected, a 4-day
mating period is recommended.
The reason for this recommendation is the 4-day
estrous cycle in female mice. In addition, the
incidence of conceptions should be approximately
equally distributed over all days, and only a few
females conceive on days 5, 6, and 7 during a weekly
(b) The total test period should cover the whole
spermatogenic cycle, i.e., at least 12 consecutive
mating intervals of 4 days each. Limiting of the
dominant lethal assay to certain "critical" mating
periods of high sensitivity is only permissible in
repeat tests, or if the parts of gametogenesis
concerned, e.g., spermatogoniogenesis or
spermatocytogenesis, are known from previous studies.
The reason for this recommendation is that chemicals
of unknown mutagenic action can induce mutations in a
very specific stage of gametogenesis. After long-term
treatment, however, either a 4-day or a 7-day mating
interval following treatment can be used (Anderson et
(c) The preferred ratio of mating is 1 female to each male.
The reason for this recommendation is the high
conception rate of females when mated 1:1. In
addition, using a 1:1 mating mode, the results of
each female can be directly associated with a certain
male. However, other mating modes, e.g., 2 females
to each male, are widely used and acceptable,
providing a highly fertile strain of mice is used
(Anderson et al., 1983).
(d) Dose levels should be calculated in terms of mg/kg
body weight. The dose volume is adjusted according
to the weight of the animals, and administered by
the appropriate route.
(e) The allocation of the animals to the various treatment
groups must be based on a statistically randomized
(f) Results obtained from sick animals or those that died
during the course of the trial should not be included
in the evaluation, but should be reported.
(g) The sensitivity of the chosen mouse strains should be
regularly checked using a standard dose of a known
mutagen (section 22.214.171.124, recommendation (b)). The
results of these studies should be documented.
126.96.36.199. Test conditions to be established by each investigator
(a) The following test conditions are matters for the
individual laboratory and are based on the experience
of the investigator: animal strain, age, and housing
conditions. A preliminary mating to check the
fertility of the animals may be necessary, and vaginal
plug evidence is useful for this purpose. The
spontaneous postimplantation losses depend, not only
on the genome and the housing conditions, but also on
the age of the females. The optimum age for the
genotype of females to be used must be determined.
This age is characterized by a maximum number of
corpora lutea and a minimum postimplantation loss.
(b) The suitability of an animal strain for the dominant
lethal assay has to be confirmed by using known mutagens
that produce specific effects at different germ-cell
stages. According to experience gained in a coordinated
study, the induction of dominant lethals following the
intraperitoneal injection of 20 mg MMS/kg body weight or
40 mg cyclophosphamide/kg body weight is an indicator of
the suitability of a particular mouse strain.
(c) If vaginal plug data are not used to determine the timing
of the pregnancy, the autopsy of the females is best
carried out a fortnight after the middle day of the mating
2.9.3. Dominant lethals in female germ cells
Females are less suitable than males for the screening of
potential mutagens. The treatment of a female with a chemical
mutagen could interfere with the hormonal status and thereby the
competence of the animal to carry pregnancies to full term. The
treatment could also interfere with the implantation or affect the
cytoplasm of the ovum in such a way that the chances of it being
fertilized are reduced or the cleavage divisions interfered with.
The mutagen could also affect the ovulation rate. All these
factors are extremely important for the interpretation of dominant
lethal studies in the female (Bateman & Epstein, 1971). There is
also the simple technical point that, while the mutational response
of a male can be analysed by mating it with several females at
different times, the response of a female can be studied only in a
However, for some compounds, it may be desirable to test the
induction of dominant lethals in female mice. This will
necessitate proving that the ova were fertilized. On the basis of
this knowledge, Generoso (1969) developed a method for the
calculation of the dominant lethal frequency in females.
2.9.4. Data processing and presentation
The number of animals necessary for mutagenicity testing with
the dominant lethal assay depends on the genotype of mice and the
quality of the animal husbandry. In simulation runs on a computer,
the sample sizes have been determined for NMRI-Kisslegg and
(101xC3H)F1 mice. The data for simulation runs were taken from a
total of 7000 untreated control animals. If a type 1 error of
a = 0.05 is assumed, together with an equally large type 2 error of
B = 0.05, then the sample sizes required for different alternative
hypotheses are given in Table 1 (Vollmar, 1977).
The presentation of the data should contain all the information
required to assess the test design and to evaluate the results.
The following items should be stated:
number of females paired (absolute);
number of females with implantations (absolute and in %);
number of implantations (absolute and per female);
live implants (absolute and per female); and
dead implants = postimplantation loss (absolute and per
Table 1. Samples sizes for dominant lethal assay
in the male mousea
Mutagenic effectb Genotype
(%) NMRI-Kisslegg (101 x C3H)F1
10 70 45
15 27 19
20 22 15
a From: Vollmar (1977).
b Lowering of the probability that a live implant
will arise from an ovulated oocyte by %.
In some strains, corpora lutea counts pose difficulties. Since
the knowledge of the number of corpora lutea is not absolutely
necessary for the evaluation of induced mutagenicity in the
dominant lethal assay on male animals, this count can be dispensed
with, even though, depending on the quality of the counts, this
entails a loss of information. However, for certain methods of
evaluation, the corpora lutea count is indispensable. It is also
necessary for the dominant lethal assay on female mice, because,
for example, of the possibility of induced superovulation (Russell
& Russell, 1956).
If the corpora lutea count has been obtained, it should be
stated (absolute and per female) as should the preimplantation loss
derived from the difference between the number of corpora lutea and
the number of implantations (absolute and per female). Although
the postimplantation loss is the most important criterion for the
dominant lethal assay, calculations of the frequency of dominant
lethals should not be based on the rate of postimplantation losses
only. Otherwise, a sterile phase, induced by a highly potent
mutagen as a result of cytotoxic effects or a 100% preimplantation
loss due to a genetic cause (Kratochvilova, 1978), could be
The following formal relationship exists between the
postimplantation and the preimplantation losses:
DI < I, with I = CL - PL
(DI = dead implants = postimplantation loss; I = number of
implantations; CL = corpora lutea; PL = preimplantation loss).
This means that after a high preimplantation loss, the maximum
possible postimplantation loss decreases automatically, since the
number of corpora lutea can be assumed to be fixed when the male
mice were treated.
The calculation of dominant lethals comprises principally the
pre- and postimplantation losses. These losses are expressed as
the mean number of live implants per female. A good approximation
for the induced dominant lethal frequency can be obtained by using
the formula of Ehling et al. (1968), which is based on the number
of live implants:
Frequency of dominant lethals (FL) =
live implants per female of the test group
1 - ---------------------------------------------
live implants per female of the control group
or the percentage of the dominant lethals (FL%) =
live implants per female of the test group
1 - --------------------------------------------- x 100
live implants per female of the control group
This formula has the advantage that the spontaneous lethal
rate, which is independent of the treatment and is specific for
each mouse strain, is eliminated so that the proportion of lethals
induced by the treatment is directly evident. In addition, if the
treatment is effective, this calculation should give a zero value,
but the sample size will result in statistically-insignificant
deviation from zero in positive and negative directions. The minus
deviations are a good indicator of the biological variability of
A drawback of this formula is that it is not derived from the
individual values of the females and does not incorporate
interindividual variability, which is obligatory for statistical
analysis. Furthermore, this kind of computation presupposes a
target model with Poisson distribution, which cannot be assumed in
every case. It should be noted that the proposed calculation can
only be used for the evaluation of treated male mice. The
calculation of the induction of dominant lethals in females is
based on the numbers of corpora lutea (Russell & Russell, 1956).
For statistical evaluation, it must be decided which biological
model is to be used and which statistical criteria are appropriate.
For biometric analysis, 5 variables are available at 3 levels
(Table 2). All these variables are integer frequencies and must be
rated as discrete variates.
The female should be chosen as the sample unit. Prior to the
actual analysis for mutagenic effects, the death rate of the male
animals (if required) and the fertilization rate of the females
(obligatory) must be ascertained. If there is a significant
difference between groups, the analysis of any mutagenic action
will be limited, and ambiguous conclusions may result.
The Wilcoxon test, modified by Krauth and recommended by
Vollmar (1977) is an appropriate procedure for the statistical
analysis of the dominant lethal assay. According to Vollmar,
fertilization rate is tested by the exact Fisher-Yates test and the
quotients I/CL, LI/CL or DI/CL by a separate linear rank test for
each mating interval.
Table 2. Variables for biometric analysis
Level Variable Abbreviation
I Number of corpora lutea per female CL
II Number of implantations per female I
Number of preimplantation losses per female PL
III Number of live implants per female LI
Number of dead implants per female DI
Haseman & Soares (1976) have compared different statistical
test procedures by computer simulations of the dominant lethal
assay. They concluded that the Chi-squared test, frequently used
for the analysis of the dominant lethal data, may seriously
exaggerate the level of significance and should not be used. The
inappropriateness of the underlying Poisson or binominal models
appears to have little effect on the validity of analysis of
variance procedures based on transformed fetal death data. It can
be concluded that, until a satisfactory parametic model can be
established, nonparametic procedures are to be preferred.
Two different approaches are used to determine the frequency of
dominant lethal mutations in male mice. One is based on
postimplantation death, the other on both pre- and postimplantation
loss. The index of dominant lethality based on postimplantation
death alone was advocated by Bateman (1958), Epstein & Shafner
(1968), and Searle & Beechy (1974). Support for this index comes
from irradiation experiments in which Searle & Beechey (1974) found
that the decrease in implantations per female was mainly due to
failure of fertilization. This finding cannot, however, be
generalized to apply to chemically-induced dominant lethals.
Results of MMS studies clearly demonstrate that 100%
preimplantation death of fertilized ova can be observed in the
mating interval, 9 - 12 day post-treatment (Kratochvilova, 1978).
The postimplantation index underestimates even the frequency of
radiation induced dominant lethal mutations, as has already been
pointed out by L.B. Russell (1962). Certainly, for the detection
of a chemical mutagen, possible underestimation of the mutation
frequency should be avoided.
Calculations based on comparisons of treated and control groups
with respect to the ratio of living plus recently dead embryos to
corpora lutea, used by W.L. Russell et al. (1954), or with respect
to the number of live embryos per females (Ehling et al., 1968),
include the pre- and postimplantation losses. The disadvantage of
such calculations of dominant lethal frequency is that the formula
does not differentiate between preimplantation loss and
unfertilized ova. However, no formula, by itself, can achieve such
a differentiation. For the exact determination of dominant lethal
frequency, it is necessary to determine the rate of fertilization
of ova (Kratochvilova, 1978). It should be mentioned that a
decreased frequency of fertilization is also an indication of a
A critical review of the mutagenicity of 20 selected chemicals
in the dominant lethal assay was published by Dean et al. (1981).
Approximately 300 publications were scrutinized, and data from 130
of these were selected for inclusion in the review. This report
contains a concise tabulation of the most relevant data and a
detailed review of each individual chemical including the lowest
dose to induce dominant lethals and the highest dose with no
significant dominant lethality. The review also contains data for
the induction of dominant lethals in rats.
The protocol described in this paper is based on the experience
with the mouse. However, the factors that are important for the
optimal test procedure are likewise essential for testing dominant
lethals in other species. Species differences for estrous cycle
and embryonic development have to be taken into account for the
adoption of this protocol for other species.
3. LABORATORY FACILITIES AND GOOD LABORATORY PRACTICE
In order to conduct the procedures described in section 2
according to acceptable scientific standards, certain minimum
levels of laboratory facilities and equipment are essential. The
design of the facilities and their complexity varies with the type
of study to be performed but, in all cases, they are governed by
the need to ensure adequate control of cleanliness and sterility,
safety, and accuracy and reproducibility of experimental results.
The need for national and international authorities to regulate
the manufacture, transport, and use of chemical substances has led
to the introduction of legislation to control these activities.
Included in this legislation are requirements for the toxicological
testing of chemicals that, in most cases, include testing for
possible mutagenic and carcinogenic hazards. In order to
establish acceptable standards of quality and reliability of the
toxicological data submitted to the regulatory authorities, various
bodies have published codes of Good Laboratory Practice. The
application of Good Laboratory Practice (GLP) in genetic toxicology
testing laboratories is described in section 3.3.
3.2. Laboratory Facilities and Equipment
Regardless of whether laboratories are designed for conducting
only the minimum of in vitro mutagenicity studies or for carrying
out an extensive programme of in vitro and in vivo genetic
toxicology testing, the same basic principles of laboratory design
apply. For example, separate areas should be provided for
microbiology, tissue culture, cytogenetics and, where necessary,
for Drosophila testing and plant studies. They will usually be
situated in the same building and should be conveniently served by
a common glassware washing and sterilising facility. Chemistry and
biochemistry laboratories should be available nearby to provide
analytical support, e.g., for confirming the stability, purity,
etc., of test compounds or for conducting associated metabolic
studies. Where in vivo studies are to be carried out, animal
facilities should either be housed in a different building or, at
least, have a separate entrance to that of the microbiology and
tissue culture laboratories. Animal-holding rooms may be required
in the main laboratory complex for cytogenetic studies and for
providing material for microsomal enzyme preparations.
Particular attention should be paid to environmental conditions
within the laboratory. Temperature and humidity should be
controlled within strictly-defined limits appropriate to the
techniques being carried out. Ventilation should be adequate with
a given number of air changes, e.g., 8 - 10/h, but draughts and
direct-intake of external unfiltered air should be avoided to
minimize the introduction of dust and contaminating microorganisms.
The design of individual laboratories should be based on the
provision of adequate bench space for the number of staff to be
employed and adequate room for the equipment and storage of
materials. The safety of staff should be a prime consideration.
Access to areas where studies are conducted should be limited to
those directly involved in the testing. Staff should be fully
aware of the hazards of working with carcinogenic and mutagenic
chemicals, particularly with the safe disposal of chemical waste,
and appropriate safety cabinets, protective clothing, and washing
facilities should be provided. Such practices as mouth-pipetting
should be prohibited, and an area should be specifically designated
for weighing mutagens and carcinogens and for the preparation of
stock solutions. A high standard of cleanliness should be
encouraged in all working areas, and the design of working
surfaces, storage areas, ventilation systems, etc., should be aimed
at making clean and sterile working practices an easily-attainable
Certain items of equipment are common to most types of testing
laboratories. Refrigerators should be capable of safe storage of
flammable solvents, while deep freezes are required to be suitable
for the long-term storage of materials at low temperature (-70 °C).
In areas where the main electricity supply is unreliable, some form
of emergency power generation is advisable. A competent glassware
washing and sterilizing facility is essential for experimental work
of acceptable quality. In addition to properly-trained personnel,
a supply of high-quality distilled water and a suitable non-
residual detergent are necessary to provide clean glassware.
Equipment such as autoclaves and hot-air sterilizers should be
of a design appropriate to the types of materials being sterilized.
3.2.1. Microbial laboratories
Two important factors in the design of laboratories for
bacterial or yeast assays are the prevention of contamination of
cultures by other microorganisms and the protection of staff
against exposure to hazardous test chemicals. Experimental
procedures should be conducted in appropriate biological safety
cabinets in which a curtain of filter-sterilised air protects the
worker from chemical exposure and the cultures from contamination.
Air from the cabinets should be extracted outside the building
through appropriate filters to prevent environmental contamination.
Incubators should have precise temperature control, and those used
for testing purposes should be in an area where the ventilation
system removes any hazardous vapours from volatile test chemicals,
when incubator doors are opened. Culture media may be either
purchased as ready-poured plates or prepared in the laboratory from
basic ingredients. In the latter case, a clean working area must
be available for pouring and drying plates. Either manual or
electronic devices are available for counting bacterial colonies.
A safe means of disposal of cultures should be provided, e.g., they
should be sealed in plastic or paper sacks in the laboratory and
3.2.2. Tissue culture laboratories
Laboratories involved in cell and tissue culture are even more
dependent on sterile procedures and working conditions than
microbial laboratories. Even a small initial microbial
contamination can rapidly spread to other cultures and easily
destroy a number of experiments and many weeks work. Although the
incorporation of antibiotics into the culture medium serves to
limit many bacterial infections; contamination with yeast and fungi
presents serious problems in inadequate working conditions. The
incidence of contamination can be significantly reduced by
conducting all manipulations of cell cultures in appropriate
biological cabinets. Culture media can be purchased in a ready-
prepared form or can be prepared and filter-sterilized in the
laboratory. Liquid nitrogen storage flasks are necessary for
keeping stocks of cell lines. For many types of experiments, cells
are cultured in Petri-type dishes and incubators in which a 5%
carbon dioxide (CO2) atmosphere can be maintained are required.
The safety precautions described above (section 3.2, 3.2.1) for
handling and disposing of material containing hazardous chemicals
also apply to tissue culture procedures.
3.2.3. Facilities for other procedures
The main requirement for cytogenetic studies on mammalian cells
or plant material is for high magnification and resolution
microscopes with good quality lenses. Microphotography equipment
is useful for recording purposes, and a dark-room facility is
required for unscheduled DNA synthesis studies.
One of the major advantages of test systems using plants is
that many of them can be performed with relatively simple
facilities and equipment. For example, root tip chromosome studies
in Allium bulbs require little more than a thermostatically
controlled water bath, basic glassware, and a suitable microscope,
while seeds of Vicia, Hordeum, and Allium can be germinated on
moist filter paper or in a suitable aqueous medium (section 2.6).
Specific locus studies in species such as Tradescantia, Zea, and
Hordeum can be conducted either in controlled environmental
chambers, conventional greenhouses or, where climatic conditions
are suitable, outdoors. In these cases, some form of control over
temperature, light conditions, and humidity is important.
In general, Drosophila studies, also, only require relatively
simple equipment. Stock cultures are maintained in glass bottles,
smaller vials are used for the experimental procedures, and they
need to be kept in a constant temperature room. A means of
anaesthetising the flies is required; examinations are carried out
using a low power microscope, and it is useful to have a separate
area for the preparation of Drosophila medium.
The maintenance of laboratory animal facilities for breeding
and experimentation purposes is an extremely expensive part of a
toxicology laboratory, and it is probably uneconomical to maintain
breeding colonies of rodents wholly for use in in vivo
genotoxicity studies. They are usually available from adjoining
conventional toxicology laboratories or from commercial suppliers.
An animal-holding area to house animals during dosing and
dissection is useful for cytogenetic studies, but should be
isolated from laboratories undertaking sterile procedures. In
addition, the area used for dosing and holding animals during
studies should be of a design suitable for housing in safety,
animals dosed with genotoxic chemicals. Dominant lethal studies
require more extensive animal facilities and should be conducted in
a conventional toxicology animal unit.
3.3. Good Laboratory Practice in Genetic Toxicology
3.3.1. Origins and nature of GLP
In the years following 1970, several national governments
enacted legislation to control the way in which chemical substances
were manufactured, traded, and used. In general, the laws placed
the responsibility of testing chemicals to detect potential hazards
for man and the environment on the manufacturers, as a prerequisite
of their being allowed to market the chemicals. The present-day
codes of Good Laboratory Practice (GLP) have arisen as a result of
The reliability of the data was of crucial importance to the
governmental agencies charged with administering these regulations,
and with assessing the risks on the basis of the experimental
evidence submitted to them. The US Food and Drug Administration
(US FDA) reacted to finding that some of the data being presented
were of poor quality and unreliable by publishing a code of
practice (Federal Register, 1978), to which all laboratories
generating data for submission to this authority were expected to
adhere. Conformity with the code was to be monitored by inspectors
from the US FDA. Other regulatory authorities, similarly placed,
followed in promulgating codes of their own. These authorities
included the US Environmental Protection Agency, concerned
separately with industrial chemicals and with pesticides, and a
number of departments of other national governments.
Trade in chemicals is international and the proliferation of
national and departmental codes reflecting different criteria for
the acceptability of data would have hindered it. Thus, the work
of the Organisation of Economic Cooperation and Development (OECD)
in developing its Principles of Good Laboratory Practice in
May 1981 (OECD, 1982) was significant as one element in a programme
to enable the mutual acceptance of experimental data among the
member nations. The OECD Principles are generic, i.e., they are
intended to be applied irrespective of the end-use proposed for the
material being tested (e.g., drug, pesticide, or industrial
chemical) and of the nature of the data being sought (e.g.,
determination of mammalian toxicity, or of ecotoxicity). Mutual
acceptability of study data would also depend on the studies having
been conducted in accordance with the OECD Test Guidelines
(standards relating to the scientific content of the tests) and on
the testing laboratory having been subject to inspection, in
accordance with OECD recommendations, by its national inspectorate.
Thus, data generated in one country in compliance with the OECD
principles and also subject to the other criteria mentioned, should
be accepted by all the member states.
Acceptance and recommendation of the Principles of GLP
established by the Council of the OECD is not legally binding for
the member countries. The various regulatory authorities, within
their own countries, are subject to different legislative and
administrative systems. The OECD Principles have therefore to be
seen as guidelines to be incorporated by each country into its own
legislation and adapted according to its special needs. To date,
this has been the case when countries or regulatory bodies have
issued definitive codes of GLP. However, differences in
requirements reflected in existing codes of GLP are not serious,
and there is a consensus that adherence to the OECD Principles
would ensure the integrity of experimental data. The US FDA rules
(US FDA, 1979, 1982) remain the most comprehensively documented of
the existing codes.
GLP sets a standard for the management of scientific
experimentation so that laboratory management and scientists are
able to assure, and regulatory authorities to assess, the integrity
and quality of the data generated. As defined by the OECD, it is
concerned "with the organisational processes and environmental
conditions under which laboratory studies are planned, performed,
monitored, recorded and reported". Owing to their origin, codes of
GLP relate only to studies made for regulatory submission.
Although several codes of GLP exist, the underlying concepts are
common to them all; moreover, scientists will recognize that the
majority of the requirements are those that have traditionally been
regarded as the basis of all sound scientific investigation.
The objectives of GLP are met by ensuring that:
(a) responsibilities of personnel are properly delineated
(b) appropriate standards are defined for all resources
(staff, facilities, equipment, materials) and for the
conduct of work, and appropriate plans are defined
for studies; adherence to the set standards and plans
is monitored; and
(c) all relevant aspects of the running of a testing
laboratory or the conduct of a study are documented
and the records are kept so that a study can be
reconstructed and assessed in retrospect.
It is worth emphasising the importance of adequate
documentation in GLP.
A testing laboratory generating data for submission to
regulatory authorities must therefore establish formal systems for
fulfilling the requirements of GLP and must document these systems.
The salient points of GLP are described and commented on in the
following section. More detailed accounts can be found in the
Federal Register (1978, 1983), US FDA (1979, 1982), and OECD
3.3.2. GLP requirements
(a) Roles and responsibilities of personnel
Three principal and distinct roles can be discerned in relation
to the conduct of studies under GLP.
The Management of a testing laboratory carries the ultimate
responsibility for running the laboratory and ensuring that
compliance with GLP is maintained throughout. Management defines
the appropriate standards for all necessary resources including
laboratory facilities, equipment and supplies, personnel, and
methodologies, and ensures their timely and adequate provision. A
particular obligation of management is to appoint a Study Director
for each study, before the study starts, and to replace him
promptly, if necessary.
The Study Director is the "chief scientist" in overall charge
of the study. GLP requires this single point of control to avoid
ambiguities and conflicting instructions that might arise from a
diffusion of the responsibility among more than one individual.
The Study Director must agree to the approved protocol or plan of
the study and, thereafter, must ensure that the study is carried
out in accordance with the protocol and with GLP. He must obtain
authorization for, and document, all necessary deviations from the
protocol. He is responsible overall for the technical conduct of
the study and the recording, interpretation, and reporting of the
observations including unanticipated responses or relevant
unforeseen circumstances. The scientist appointed Study Director
must have training and experience appropriate to, and commensurate
with, his role in the study.
The third principal role identified in GLP is that of the
Quality Assurance Unit (QAU). This is a concept adopted from the
more traditional and familiar function of product quality control
in industry. The "product" of a testing laboratory consists of the
data that arise from its studies and the concern of the QAU is with
the authenticity and integrity of these data.
The primary responsibility of the QAU is to monitor, by direct
observation, the operation of the testing laboratory and the
conduct of studies, to ensure that these comply with the approved
standards and with GLP and, in the case of the study, that the
protocol is being followed. Thus, the QAU must carry out periodic
inspections of the facilities and equipment, the operation of the
relevant administrative systems and the actual conduct of the
experimental work. Surveillance of a study by the QAU must include
an audit of the final report. The findings of the QAU inspections
or audits are reported to Laboratory Management and, in respect of
studies, also, to the Study Director so that necessary corrective
action can be taken. It has been the policy of regulatory
authorities, when monitoring laboratories for GLP compliance, not
to look at the reports of QAU inspections in order to promote the
frankness and hence the effectiveness of the laboratories internal
monitoring. The QAU must maintain a "master schedule" giving
details of the identity and current status of studies conducted at
Personnel of the QAU report to management. They must be
entirely independent of staff engaged in carrying out the study
that is being monitored, i.e., they may not participate technically
and may not be subordinates of the Study Director (it follows that
the chief manager of a testing laboratory may not himself be a
Study Director). However, the QAU staff need not be dedicated
solely to the quality assurance role, e.g., a scientist
participating in one study can be assigned quality assurance
responsibilities in respect of another. If this expedient is used,
the quality assurance documentation must still be kept in the one
place in the testing laboratory. QAU personnel must be familiar
with GLP requirements and also be knowledgeable about the tests
being monitored, though the quality assurance function does not
extend to a scientific appraisal of the study and its results.
All staff concerned with studies must have qualifications,
training, and experience appropriate to the function to which they
have been assigned. This should not only be in respect of the
scientific discipline within which their contribution is made, but
also in respect of the requirements of GLP. When necessary,
appropriate training must be given, the level of which should take
into consideration the degree of supervision of the staff member.
The management of a testing laboratory must maintain a current job
description for every staff member together with a summary of any
training and experience received in relation to the job.
Laboratory accommodation must be of adequate size and be
suitably constructed and located for the experimental work that is
to be performed. In genotoxicity work, the safe containment of
hazardous materials, both chemical and biological, is an important
consideration but, generally, the requirements of personnel safety
are prescribed in legislation other than that of GLP. Two criteria
are of particular relevance to GLP. First, similar materials from
different studies must be sufficiently separated so that no
confusion between them can occur. Disciplined working methods,
e.g., adequate labelling of containers, will complement, but cannot
replace, adequate provision of bench space or incubator capacity to
achieve this. However, once laboratory facilities have been
provided, foresight is essential to avoid having too many studies
at the same stage simultaneously.
Different phases of studies must also be adequately separated
to prevent interference between them. Thus, areas devoted to
chemistry and formulation, where the test and control substances
are handled at high concentrations, should be separate from areas
where these materials are encountered at only low concentrations.
In the construction of laboratories, appropriate attention should
be given to ventilation and access so that the likelihood of cross-
contamination is minimized.
Similarly, separate areas are necessary for the storage of
contaminated glassware pending its disposal or cleaning. GLP also
requires appropriate areas to be made available for administration,
e.g., for "writing up", and further areas for the general
convenience of personnel such as for changing into and from
protective clothing. The requirement for archive space is noted
Use of equipment that is inappropriate, inadequately designed,
or faulty can lead to the generation of unreliable data.
Furthermore, equipment must be properly maintained and, where
relevant, calibrated. It should, therefore, be easily accessible
for inspection, cleaning, and servicing. These rules apply both to
equipment used to generate data (laboratory instrumentation) and
equipment used to maintain special environmental conditions.
Written instructions, i.e., Standard Operating Procedures
(SOPs) (see below), must be provided concerning procedures for the
use, cleaning, and routine care of the equipment. These should
include schedules for the items of routine maintenance and the name
of the person responsible for seeing that they are carried out.
Appropriate action in the event of breakdown must also be covered.
Much of this information will be available in the manufacturer's
literature, but, where necessary, the handbook must be
supplemented, e.g., to cover variations in technique peculiar to
the laboratory. This documentation must be freely accessible in
the area where the equipment is used.
Written records must be kept of all routine maintenance of the
equipment and also of non-routine events such as repairs after
breakdown. In the latter case, the nature and circumstances of the
defect and the remedial action taken must be recorded. One
instance of equipment malfunction could conceivably affect the data
from more than one study. Therefore, while routine equipment
maintenance records can be regarded as non-study specific and filed
chronologically, individual study records should enable equipment
used in the study to be identified and unscheduled events to be
(d) Standard operating procedures (SOP)
Under GLP, the routine methods of a testing laboratory, as well
as some administrative procedures that relate to the conduct of
studies, have also to be documented in the form of "standard
operating procedures" (SOPs). Written SOPs serve to ensure that
all staff are familiar with, and use, the same working methods;
thus, errors or loss of data arising from variability between
individuals is minimized. They can serve also as a documented
specification of the laboratory's procedures, helping evaluation of
study methods, or the monitoring of compliance for quality
assurance purposes. Documentation of the following procedures is
usually regarded as the minimal requirement:
Test and reference substances receipt, identification,
labelling, handling, sampling,
storage; confirming homogeneity
of test formulations, stability
under test conditions;
Equipment use, routine maintenance;
Records coding, indexing, or labelling
of studies and study-related
material; collection, handling,
storage, retrieval of data;
Laboratory operations special environmental
techniques, preparation of
Quality assurance conduct and reporting of
Working methods should be presented in SOPs in sufficient
detail to ensure the integrity of study data, judgement of their
adequacy being the prerogative of management. The degree of detail
should also be such that the SOPs can be understood and followed by
trained staff. The instructions should cover any work necessary,
preliminary to the main procedure, e.g., methods of sampling prior
to the application of a given test, and should extend to procedures
for the handling of data and records. Citation of published
literature is permissible to supplement the text of an SOP. Copies
of both the SOP and any supplementary document cited must be freely
available in the area where the procedure is carried out.
Adoption of a given standard procedure by a testing laboratory
and any changes made have to be approved by the laboratory
management and all changes must be formally documented. The
laboratory must then retain copies of the superseded SOPs with a
record of the dates of their implementation and replacement. It
has been claimed that too rigid documentation of standard methods
can present difficulties in scientific areas, such as genetic
toxicology, where techniques are undergoing rapid evolution, but
regulatory authorities have not excluded genotoxicity studies from
the requirement. Deviations (as distinct from significant changes)
from a documented SOP in the course of a study can be made on the
authorisation of the Study Director, provided that such departures
are noted in the experimental record, if not already anticipated in
the study plan.
(e) Planning conduct and monitoring of studies
Sound experimentation requires clear objectives and a
definition of how the objectives are to be attained. The design
and methods of the study can then be evaluated in relation to its
objectives to ensure that attainment of the latter is within the
proposed scope of the study. Few studies are entirely within the
compass of a single scientific discipline and involvement of all
the relevant professionals at the design stage is highly desirable.
For these reasons, the protocol or plan of a study under GLP must
be drawn up in writing before the study starts. The codes of GLP
itemize the information that the protocol must contain but,
fundamentally, it must state the objectives and, in detail, all of
the experimental design and methods that are to be used. Citation
of readily available documents such as SOPs is in order. The
protocol must be formally approved by the laboratory management
and, where appropriate, by the sponsor of the study and must be
agreed to, and signed by the Study Director. Where changes in the
protocol become necessary during the course of the study, these
must be justified and documented in a formal protocol amendment,
signed by the Study Director. Except as provided for in protocol
amendments, the conduct of the study must then follow the approved
protocol and any inadvertent derviations must be documented in the
The batch or sample indicator, as well as the chemical identity
of the test substance, will be given in the study protocol and
records, but it is also necessary to characterize the test
substance and authenticate the sample by appropriate chemical
tests. In this connection, the stability of the test substance
itself has to be known; moreover, if its stablity under the test
conditions, i.e., in the formulation media, is not known, exposure
of the test organism to the intended challenge cannot be assured.
The homogeneity, concentration, and stability of the test
formulation must therefore be determined.
In GLP, considerable importance is attached to the authenticity
of the records. Observations and data must be recorded directly
and indelibly, and any changes made to the original record must not
obscure the superseded entry. Each entry or change must show the
identity of the originator and the date, and the justification for
the changes must be included. The originals of data records,
termed the "raw data", have a special importance in GLP in that
they have to be preserved, though exact copies of the originals are
also acceptable (not, however, expurgated records transcribed from
the originals into clean notebooks.)
The conduct of studies under GLP calls, therefore, for a
disciplined approach to the making and recording of observations
and measurements. Responsibility for the accuracy and completeness
of the data resides formally with the Study Director, though he is
not expected to validate individual entries; rather, he should
assure that recording methods are adequate. Careful attention to
the design of the data-collection methods, e.g., the provision of
printed data sheets that incorporate prompts to help ensure
completeness of the entries and display them for easy review, can
complement valuably the proper training and supervision of the
operators. However, the correct recording of unanticipated events
or observations must not be overlooked.
The role of the QAU in monitoring the operation of the
laboratory facilities and the conduct of studies has already been
indicated. For repetitive, short studies such as genotoxicity
studies, the formal GLP requirement to inspect each phase of every
study can be satisfied by inspection on a random sample basis.
The final report of a study must be a complete presentation of
its objectives, the results of the observations made, and the
conclusions drawn from them. To enable accurate evaluation of the
conclusions to be made, the conditions under which the work was
done must be correctly described, including unplanned occurrences.
An additional requirement under GLP is that the report must be
audited against the raw data by the QAU. This is to ensure that
the methods and conditions of the study are correctly given and
that the results reported are an accurate reflection of the raw
data recorded. An evaluation of the interpretations placed on the
results by the scientists is beyond the remit of the QAU. The
report must include a statement signed by the QAU listing the dates
on which the inspections and audits of the quality assurance
programme (including the audit of the report itself) were made and
when the findings were presented to management.
The Study Director must sign and date the final report; this
signifies the termination of the study. Subsequent changes to the
report must be made formally, the amendment and its justification
being signed and dated by the person responsible.
Full evaluation of a completed study and its report could
necessitate reconstruction of part or all of the study and this
would require access to the experimental records. A suitable
archive for the preservation of the records must, therefore, be
provided. At the end of a study, the Study Director must ensure
the transfer to the archives of all the raw data and other relevant
documentation as well as the approved protocol and the final
report. Records that are not specific to any study, such as
records of staff training, of equipment maintenance, and superseded
SOPs, will also be stored in the archive.
Control and care of the archive and its contents must be vested
in a named individual, with access to the archive restricted to
authorised personnel. Storage of the material must be orderly with
appropriate indexing to facilitate its retrieval. The period of
time for which the records of a given study are retained should be
in accordance with the rules of the regulatory authority to whom a
submission based on the study will be made. This period cannot
easily be predicted but a requirement in excess of ten years should
be anticipated. Reasonable precautions, having regard to the
prevailing risks and climatic conditions, must be taken to ensure
that the stored records remain viable for this period.
3.3.3. Summary of resources and records needed
The resources and mechanisms that must be established and the
records that must be kept to comply with GLP are summarized in
Table 3. Resources and mechanisms that must be established and records
that must be kept to comply with GLP
Resource Provision Documentation
Personnel Adequate appropriate staff Responsibilities
Study Director Training, experience
Quality Assurance Unit
Facilities Laboratories (biological, Special conditions,
Archives procedures, indexing
Equipment Adequate capacity, Methods of use and
appropriate design Records of maintenance
Methodologies/ Scientific techniques
administrative Protocol development, approval
systems Test substance handling, SOPs indexing
Data collecting handling, storage
Records to be preserved
Personnel responsibilities, training and experience
SOPs: superseded editions, with dates
Master schedule of studies
Quality assurance reports
4. SELECTION, APPLICATION, AND INTERPRETATION OF SHORT-TERM TESTS
The procedures described in section 2 of this guide are those
that are generally accepted as suitable for testing chemicals for
mutagenic and putative carcinogenic activity. Some are more widely
used than others, and the purpose of this section is to offer
practical guidance on the use and interpretation of these tests on
the basis of current knowledge, experience, and acceptance. It
must be emphasized that there is no universal agreement on the best
test or combination of tests for a particular purpose, though there
have been attempts to harmonise the selection of the most
appropriate assays by national and international bodies such as the
Organisation for Economic Cooperation and Development. An expert
committee of the International Commission for Protection against
Evironmental Mutagens and Carcinogens (ICPEMC) is also considering
the question of the most effective combination of short-term tests
to detect mutagenic and carcinogenic chemicals. In addition, the
International Programme on Chemical Safety has organized a series
of international collaborative studies aimed at assessing the
performance of short-term tests. The results of the latest of
these, the IPCS Collaborative Study of Short-term Tests for
Genotoxicity and Carcinogenicity (CSSTT), has recently been
published (Ashby et al., 1985).
The objective of testing chemicals in these short-term
procedures is to provide an assessment of the possible mutagenic
and carcinogenic hazards associated with the release of the
chemicals into the human environment. No single test has yet been
devised that can achieve this objective with certainty. By the
judicious selection of a combination of assays, however, and by
strict adherance to certain minimum technical and scientific
criteria in their conduct, the possible genotoxic hazard of many
groups of chemicals can be assessed with a useful degree of
confidence. Such assessments are inevitably subject to errors,
varying in magnitude, that are influenced by, among other factors,
the suitability of the chosen assays for a particular class of
chemicals. Furthermore, assays that detect genotoxic activity do
not usually detect tumour promotors, hormones, and various other
factors that affect tumour formation.
The possible adverse consequences of human exposure to a
specific chemical will rarely be assessed from short-term tests
alone. Rather, the judgment is made from a total toxicology data
package that may include, depending on the nature of the chemical
or product, short- and long-term animal studies including tests for
reproductive effects, irritancy, sensitisation, neurotoxicity, and
data on the absorption, distribution, metabolism, and excretion of
the chemical. Data from some of these studies may also help
achieve a proper understanding of the significance of the results
of short-term tests.
Following the overall assessment of the possible human hazard
from exposure to a chemical, the potential risk associated with
human exposure is estimated by a regulatory process called risk
management. In this process, the potential hazard is balanced
against the likely extent of human exposure, the perceived benefit
of using the chemical, and other considerations. Risk management
involves non-scientific as well as scientific considerations and
should not be confused with hazard assessment, which is based on
the scientific evaluation of toxicological data.
4.2. Selection of Assays
Some 20 - 30 diffferent assays are referred to in the eight
subsections of section 2. The selection of the most appropriate of
these to meet a particular requirement is governed by a number of
factors. These include the type of genetic change to be detected,
the metabolic capability of the procedure in relation to the
structure of the chemical to be tested, the predictive value of the
assay in terms of mutagenicity and carcinogenicity, the available
expertise and facilities and, when appropriate, the legislative
requirements of regulatory authorities.
4.2.1. Detection of the major types of genetic damage
Chemicals that interact with DNA produce lesions that, after
the influence of various repair processes, may lead to genetic
changes at the gene level, e.g., gene or point mutations, small
deletions, mitotic gene conversion (e.g., in yeast), or various
microscopically-visible chromosome changes; assays are available to
investigate each of these events (section 1).
188.8.131.52. Gene mutations
The most widely used and most fully validated assays for
detecting chemically induced gene mutations are those using
bacteria (section 2.1). They are relatively simple to perform,
reproducible, and give reliable data on the ability of a chemical
to interact with DNA and produce mutations. It should be
remembered, however, that bacteria are very simple organisms, and
that a positive result in a bacterial assay does not necessarily
indicate that the compound will induce similar effects in animal
cells or other eukaryotes. Likewise, a negative result does not
invariably mean that the compound lacks mutagenic activity in
eukaryotic cells or in intact mammals.
In order to generate data on gene mutations in eukaryotic
cells, a choice of screening test systems is available, including
certain procedures with yeasts (section 2.2), cultured mammalian
cells (section 2.5), Drosophila (section 2.7) and, to a lesser
extent, some plant systems (section 2.6). Each of these has
certain advantages and disadvantages that will be further discussed
later in this section.
184.108.40.206. Chromosomal damage
As discussed in section 1, chromosomal aberrations are changes
in the structure of eukaryotic chromosomes. The simplest assays
for investigating clastogenic (i.e., chromosome-breaking) effects
are those involving either cultured mammalian cells (section 2.4)
or plant root tips (section 2.6). These tests can identify
chemicals capable of inducing chromosome damage, per se. In order
to investigate the ability of a chemical to produce chromosome
damage in the whole mammal, two well-established in vivo
procedures are available. Clastogenicity in somatic cells can be
studied in the bone-marrow cells of rodents dosed with the suspect
chemical, either by counting micronuclei in polychromatic
erythrocytes or by analysing chromosomes in metaphase cells
(section 2.8). Alternatively, chemicals that cause chromosome
damage in germ cells can be detected using the dominant lethal
assay (section 2.9).
There is increasing evidence that chemically-induced numerical
chromosome changes (i.e., aneuploidy) as well as being the cause of
much inherited disease, are associated with the carcinogenic
process. Among assays for detecting such chemicals, a system using
yeast is described in section 2.2. It is not yet clear, however,
how predictive this test is for effects on mammals.
220.127.116.11. DNA damage
Three of the procedures described in section 2 are generally
accepted as assays that respond to chemically-induced DNA damage.
One cellular response to such damage is the initiation of enzymatic
repair of the damage, which involves the synthesis of a new,
relatively short, strand of DNA. Such repair, referred to as
"unscheduled DNA synthesis" or UDS (to differentiate it from the
synthesis occurring during normal cell replication), is the basis
of the UDS assay in cultured mammalian cells (section 2.3).
Mitotic gene conversion in the yeast Saccharomyces cerevisiae,
involves the accurate transfer of small segments of DNA between
homologous chromosomes and is also regarded as a useful indicator
of primary DNA damage. The investigation of sister chromatid
exchange (SCE) in cultured mammalian cells also falls within this
category. Although the molecular mechanism of SCE formation has
still to be fully elucidated, it has been shown to be different
from the mechanism leading to chromosome breakage, and the SCE
assay is a useful method for detecting chemicals that interact with
and damage DNA.
4.2.2. Scientific validity
Before a short-term test can be used with confidence, it must
be shown to be a valid procedure for the purpose of detecting
genotoxic chemicals. The target cell of the assay, whether it is a
bacterium, a yeast, or an animal cell, must be fully characterized
both genetically and biologically to ensure that it will respond in
the expected fashion in the experimental system in which it is
being used. The second important factor is the experimental system
itself. The system must be capable of maintaining the target cell
in optimum experimental conditions while ensuring that the test
chemical has every opportunity of reaching the molecular target
(e.g., DNA) in the cell in its most reactive form. Third, the
assay must be shown to be "robust", i.e., it should be fully
reproducible so that data generated in different laboratories are
18.104.22.168. Genetic basis
The genetic basis of the target cells used in the bacterial and
yeast assays is described comprehensively in sections 2.1 and 2.2,
respectively. Guidance on the maintenance of the genetic integrity
of the suggested strains is also given. The details given in these
sections must be followed faithfully to ensure that the genetic
make-up of the test organisms meets the requirements of the
particular assay. For example, the Salmonella strains usually
used in bacterial assays respond to different types of mutagens,
and the range of strains selected must be capable of detecting
these different mutagens, e.g., frame-shift mutagens; base-pair
substitution mutagens. Similarly, the yeast strains described in
section 2.2 have been specially selected to respond to genetic
events and it is essential to confirm that the correct strains are
used. Similar principles also apply to Drosophila tests in which
properly maintained colonies of the correct strains must be used
With mammalian-cell assays, the situation is slightly
different. The cell types described in section 2 are, in general,
selected by tissue culture cloning techniques so that they meet the
requirements of the genetic change being investigated. For
example, CHO cells, used for cytogenetic assays (section 2.4) are
selected and cultured in a way that maintains the integrity of the
chromosome complement. Cells used in gene-mutation assays (section
2.5) must be sensitive to a particular type of induced mutation
(e.g., at the HGPRT locus), and cultures with a low spontaneous
mutation frequency are maintained.
22.214.171.124. Metabolic capability
Many carcinogenic/mutagenic chemicals are not able to interact
with DNA until they have undergone some degree of enzyme-mediated
biotransformation. In animals, including man, foreign chemicals
are subject to a series of modifying enzymic and non-enzymic
reactions aimed at detoxifying the chemical and altering it to
water-soluble forms suitable for elimination from the body. These
enzymic reactions are also capable of activating certain chemicals
to reactive molecules that can interact with DNA to produce
potentially harmful damage (section 1). The appropriate enzyme
systems are usually partially or completely inactive or absent in
bacteria, yeasts, and cultured mammalian cell systems and are
introduced in the form of an enzyme-rich, cell-free fraction of
mammalian liver (section 2.1).
An acceptable in vitro assay must, therefore, be shown to
have a metabolic capability appropriate to the chemical class
being studied and the experimental conditions must be designed
to allow the metabolic activation system to operate at an
optimum rate. Guidance on these factors is provided in
section 2. Although most mammalian cell types used in in
vitro tests retain some endogenous enzyme activity, it is
usually too low to activate the majority of carcinogens and
such tests are supplemented as described above. Some
carcinogens, however, have been shown to be poorly metabolized
by the conventional rat liver (S9) microsomal enzyme system,
but, under appropriate experimental conditions, can be
activated by enzymes endogenous to the cultured cell.
Technical modifications necessary to detect this type of
chemical usually involve a longer than usual incubation period
that allows the compound to be available to endogenous enzymes
for 18 - 30 h (Dean, 1985).
4.2.3. Predictive value
The ultimate goal of the short-term tests described in this
guide is to identify, with an useful degree of confidence,
chemicals that may be hazardous. As will be discussed later in
this section, such a goal is approached through a series of stages,
each of which leads to an assessment of the activity of the
chemical at that stage. Proving the safety of a compound is a much
more difficult undertaking, rarely performed, and never based on
short-term tests alone. In general, given the limited resources
available, it is usual to accept a substance as safe in practice in
the absence of evidence to suggest otherwise.
126.96.36.199. Mutagenic activity
The first stage in the evaluation of a chemical is to
investigate the ability of the chemical to interact with DNA and
produce a detectable change in the genetic material. Bacterial,
yeast, plant, Drosophila, and in vitro mammalian cell assays are
designed for this purpose. They have a high value in predicting
whether a chemical is a bacterial mutagen, is active in yeasts or
plants, or can induce genetic damage in insect tissues or isolated
animal cells. It cannot be predicted with a high degree of
certainty, from these assays alone, whether a chemical will produce
mutations in a mammal such as man. To provide an insight into the
activity of the chemical in the whole animal, in vivo procedures
are used in which the chemical is given to test animals by an
appropriate route and some means of detecting genetic changes is
applied (e.g., chromosome study in bone-marrow cells, dominant
lethal assay, or detection of mutagenic excretory products). The
predictive value of these in vivo assays is fairly high when
positive results are observed. For example, if a chemical produces
chromosome damage in rodent bone marrow it is usually assumed that
the chemical could present a human hazard, under particular
exposure conditions. Negative findings in a properly conducted
chromosome study in rodents are also frequently regarded as
indicating a low or negligible human hazard, even with a chemical
that induces chromosome changes in cultured cells (de Serres &
Ashby, 1981; ICPEMC, 1983a). However, certain chemicals, the
effects of which are confined to specific tissues, such as the
liver or gut, may not be detected using a bone-marrow assay. For
such chemicals, tissue-specific assays are being developed, e.g.,
unscheduled DNA synthesis in rodent liver (Mirsalis & Butterworth,
1980), and an assay for nuclear anomalies in gut tissue (Heddle et
al., 1982). There are few practicable procedures for investigating
gene mutations in animals and a negative bone-marrow study may have
poor predictive value for chemicals that have been shown to induce
only gene mutations in in vitro studies. These problems of
interpretation are discussed further in section 4.5.
The hazards associated with exposure to genotoxic chemicals
differ according to the cell type in which the genetic damage is
induced. Mutations in somatic cells are generally regarded as
presenting a hazard (e.g., carcinogenic) only to the individual in
which they occur. Germ-cell mutations, however, may have far-
reaching effects in future generations and it is important to be
able to predict whether a mutagenic chemical may present this
hazard. Unfortunately, the only practicable procedures currently
available for studying germ-cell genetic changes directly are
limited to chromosome damage. It is usually assumed, in the
absence of evidence to the contrary, that chemicals shown to induce
chromosome damage in mammalian germ cells may be able to cause
mutations in human germ cells. Negative results in such assays
with a chemical shown to be a clastogen in other tests may also be
highly relevant (section 4.5). Existing procedures, with the
exception of large-scale experimental animal studies such as
specific locus tests (section 1) cannot be extrapolated directly to
the induction of gene mutations in human germ cells.
188.8.131.52. Carcinogenic activity
As outlined in the Introduction (section 1), carcinogenesis
induced by genotoxic agents is a multi-stage process that includes
transport and metabolism of the chemical, interaction with the
critical target molecule (e.g., DNA), DNA repair and replication of
the lesion, and progressive development of the fixed lesion to form
a malignant cell. Long-term studies for carcinogenicity in
experimental animals do not necessarily reflect a realistic
situation as they only measure the ability of a test compound to
function as a complete carcinogen. In reality, a person may be
exposed to a combination of agents acting on different stages of
the carcinogenic process. Current in vitro tests cannot, of
course, mimic all these stages and are frequently assumed to detect
only the event leading to the initiation phase, i.e., the ability
to induce a mutagenic or clastogenic DNA lesion. The main value of
short-term tests, therefore, lies in their ability to identify
chemicals that may, under certain exposure conditions, either cause
cancer by a predominantly genotoxic mechanism or induce the initial
phase of the carcinogenic process. Carcinogenesis enhancers
(Clayson, 1981), including the so-called tumour promotors, will
usually escape detection in the conventional DNA-based assays. It
is apparent, from the complexity of the carcinogenic process
compared with the relative simplicity of short-term in vitro
assays, that, although such assays provide useful qualitative
information, considerable caution is required in their
interpretation in terms of human carcinogenicity. The predictivity
of the tests for detecting potential carcinogens is usually derived
from their performance in validation studies in which a range of
established carcinogenic and non-carcinogenic chemicals are tested
under controlled conditions (the designation "carcinogen" or "non-
carcinogen" is obtained from long-term studies in laboratory
animals or, more rarely, from human epidemiology data). For
example, the predictive value of the Salmonella/microsomal
bacterial assay (section 2.1) has been evaluated on a number of
occasions, and the accuracy with which it differentiates between
carcinogens and non-carcinogens varies between about 60 and 90%
depending, among other factors, on the nature of the chemicals
selected for the study (Rinkus & Legator, 1979). Technical factors
also contribute towards its accuracy and it is important when
considering data from bacterial assays (and other tests) that the
experimental protocol was appropriate to the chemical type being
tested (section 2.3.3). However, a properly-conducted bacterial-
mutation assay can give results of a useful predictive value, when
considered together with results from other tests.
The induction of structural chromosome damage is also a
property common to many carcinogenic chemicals (de Serres & Ashby,
1981), and recent studies have shown that some carcinogens that do
not induce mutations in bacterial systems are capable of causing
chromosomal damage in cultured mammalian cells (Dean, 1985). Thus,
as will be discussed in section 4.5, a combination of a bacterial
mutation test and a chromosome assay in cultured cells is
considered, by many, to have a higher predictive value for
carcinogenic activity than either test alone (e.g., Ashby et al.,
Assays that indicate DNA damage, such as the yeast assay for
mitotic gene conversion (section 2.3), SCE in animal cells (section
2.4), and unscheduled DNA synthesis have also provided valuable
predictive information on carcinogenic potential. Positive results
in these assays generally indicate that a chemical can interact
with DNA in a eukaryotic cell, though they do not prove whether or
not the lesion induced is capable of progression to a true somatic
mutation or a carcinogenic initiation event. Such assays, however,
provide useful supplementary evidence in constructing an overall
genotoxic profile of the possible adverse effects of a chemical.
Because of the physiological and genetic differences between
bacterial and eukaryotic cells, it is inevitable that some
chemicals will induce gene mutations in bacteria but not in
eukaryotes (and occasionally, vice versa). Assays for gene
mutations in mammalian cells (section 2.5), yeasts (section 2.2),
and recessive lethal mutations in Drosophila (section 2.7) are
extremely useful for detecting some classes of chemical
184.108.40.206. Relevance to chemical class
The experimental protocols outlined in section 2 were designed
to provide optimum experimental conditions for testing most types
of chemicals. Because of variation in chemical structure and
reactivity, it must be accepted that such protocols, particularly
for the in vitro tests, are, in reality, compromises, and that, in
many cases, the conditions are not necessarily the best for the
particular chemical being studied. The nature and rate of the
enzymic reactions that transform a pro-carcinogenic chemical to its
ultimate reactive form are dependant on the structure of the
chemical. The enzymes provided by the microsomal fraction usually
incorporated into in vitro tests, i.e., predominantly mixed-
function oxidases, are capable of activating most pro-carcinogens.
Experimental conditions, including the source (e.g., species,
tissue) and quantity of the microsomal fraction and the proportion
of co-factors may need to be adjusted to provide near-optimum
conditions for a particular chemical class. For example, the
standard Salmonella/microsomal assay can detect most aromatic
amines, polycyclic hydrocarbons, mono- and bi-functional alkylating
agents and mycotoxins, but must be modified to respond to some
nitrosamines, metallic salts, and many other compounds. A few
compounds, such as 1,2-dichloroethane, are activated by conjugation
with glutathione, in which case an exogenous metabolic system may
not provide optimum conditions for activation. In addition,
compounds activated by enzymes not active in the liver microsomal
preparation, e.g., those provided by intestinal flora, will not be
detected. These factors apply to all assays that are enriched with
exogenous metabolizing enzymes. Other confounding factors may also
influence the biotransformation of a pro-carcinogen. For some
chemicals, the residual endogenous enzymes in cultured mammalian
cells are more active than the added microsomal enzyme mixture
(section 220.127.116.11). Under appropriate conditions, the yeast
Saccharomyces cerevisiae contains stage-dependant mixed-function
oxidase activity that may also be more suitable for the activation
of some chemicals.
Although, as already mentioned, the standard protocols for the
in vitro assays represent compromises in experimental design, in
practice, reliable results can be obtained with most chemicals
using these protocols. For the interpretation of results, however,
an awareness of the possible influence of the structure of the
chemical is an important factor in deciding the adequacy of an
experimental protocol for that particular chemical.
4.2.4. Available expertise and facilities
Most short-term test data are generated in government,
academic, industrial, or contract laboratories by personnel having
considerable experience with the techniques (i.e., section 3). The
selection of assays to meet a particular need may, in some
respects, be based on the specific areas of expertise and
facilities available in a specific institute. For example, a
laboratory with a long history of research on Drosophila or fungal
genetics may choose to select these organisms in preference to, for
example, mammalian cells, for the study of gene mutation. Indeed,
in such situations more reliable data might be obtained from these
organisms, at least intially, if experience in, or appropriate
facilities for, mammalian cell culture techniques were lacking.
Similarly, a life-long experience with a particular set of
bacterial strains, e.g., Escherichia coli, may lead to their use
in preference to Salmonella typhimurium. Although, in principle,
some tests have distinct advantages over others for detecting the
same type of genetic change, a solid background of experience and
an adequately equipped laboratory is essential before attempting to
generate data from a "new" (i.e., to the laboratory) assay for
hazard assessment purposes.
4.3. Application of Assays
Under ideal circumstances, short-term tests are applied in such
a way that, beginning with an initial battery of two to four
assays, tests are selected in order to accumulate data on the
activity of a compound until a point is reached where an assessment
of the probable genotoxic hazard can be made with an acceptable
degree of confidence.
4.3.1. The phased approach
Some 80 - 90% of chemicals shown to be carcinogenic in
laboratory animals are capable of interacting with DNA and, under
appropriate experimental conditions, the majority of assays for
mutation will respond to most genotoxic chemical carcinogens. Some
assays of genetic damage respond better than others to various
classes of chemical carcinogens and mutagens; some carcinogens give
consistently negative results in standard assays for mutation-
induction, and other chemicals, shown to be non-carcinogenic in
laboratory animals are fairly strong mutagens. Since no single
assay has proved capable of detecting animal carcinogens with an
acceptable level of precision and reproducibility, it is usual
practice to apply the assays in "packages" or "batteries". For
practical purposes, testing is usually divided into two or three
phases or tiers (for review see Williams, 1980), though in many
cases, data from the first phase of testing provides sufficient
information for a provisional assessment of the genotoxicity of a
compound. The first phase, i.e., the basic screen, consists of a
battery of two to four assays designed to detect genetic activity
in the test material. The second and third phases consist of
supplementary assays selected to complement the phase 1 tests, to
establish whether genetic damage is induced in vivo and to provide
a basis for making an assessment of possible human hazard
associated with exposure to the material.
18.104.22.168. Phase 1 - the basic screen
The initial battery (i.e., the basic screen or the "base set")
consists of tests with an established broad data base generated
from extensive validation studies. One fairly comprehensive
package consists of a bacterial mutation assay, an assay for
chromosome changes, a test for DNA damage, and a eukaryotic gene-
mutation assay. Final selection may be influenced by the nature of
the material, e.g., drug, pesticide, industrial chemical, the
extent of its eventual distribution and use (section 4.3.2), the
objective of testing the material and, in some cases, the available
technical expertise in the testing laboratory.
Although a package containing four assays is sometimes
recommended for chemicals where extensive human exposure is
anticipated (Draper & Griffin, 1980; DHSS, 1981), the assessment of
a chemical is often begun with data from an initial battery of just
two assays (OECD, 1982b, 1984). These are usually a bacterial
assay using a range of tester strains of Salmonella typhimurium
(section 2.1) and a test for the induction of structural chromosome
aberrations. The latter may be a micronucleus test in rodent bone-
marrow cells (section 2.8) or, more often, a chromosome assay in
cultured mammalian cells (section 2.4). Providing full
consideration is paid to the physical and chemical properties and
the metabolic behaviour of test chemicals, few potential mutagens
or genotoxic carcinogens will escape detection in a combination of
a Salmonella/microsomal activation assay and an in vitro
mammalian cell chromosome assay (Ishidate & Odashima, 1977;
Ishidate, 1981; Ashby et al., 1985). It must be noted, however,
that it is inherent in the concept of in vitro screening that some
potentially harmful molecules will slip through the screen and that
some molecules active in the in vitro system will prove to be
inactive in vivo.
A recent international collaborative study sponsored by the
International Programme on Chemical Safety, was designed to
identify the assay most suitable to be used in parallel with the
Salmonella/microsomal activation assay in a two-test battery for
the detection of genotoxic chemicals. Eight carcinogenic
chemicals, chosen for the ambiguity of their results in bacterial
mutation assays, together with 2 carefully-chosen non-carcinogens,
were tested in a variety of in vitro assays (Ashby et al., 1985).
A number of assays performed extremely well in differentiating
between carcinogens and non-carcinogens in the group of ten. In a
final analysis, however, an in vitro mammalian cell culture assay
for chromosomal aberrations was selected as the most suitable
partner for bacterial tests on the basis of (a) performance in the
collaborative study, (b) their advanced state of technical
development and wide usage, and (c) a generally internationally
accepted broad data base.
It will be evident from the previous paragraph that a reliable
bacterial mutation test is widely regarded as a virtually
indispensible component of the first phase of testing and that the
second test will usually investigate chromosome changes. In some
cases, it may be appropriate and more convenient to study the
effects of a chemical on chromosome structure in vivo rather than
in cultured cells, and assays such as the micronucleus tests
(section 2.8) can be used in parallel with the bacterial assay.
Though the published data base for in vivo assays is not as
extensive as that for cell-culture procedures, the micronucleus
test (or a metaphase analysis of bone-marrow cells) has the
advantage of using an intact animal and this may provide a sounder
basis for hazard assessment. In laboratories where cell culture
facilities and laboratory animals are not readily available, it may
be necessary to generate chromosome data from plant material.
Although well-established techniques are available and a limited
number of studies have demonstrated some correlation between plant
chromosome changes and mammalian genotoxicity, additional
validation of plant systems is essential, before they can be used
to assess the potential effects of a chemical on man with any
degree of confidence (section 2.6).
As described above, data from a two-test base set can provide
reliable detection of most genotoxic chemicals. It must be
emphasized, however, that not all chemicals that provoke a positive
response in one or both of the tests are necessarily hazardous for
man (section 4.5.1). In addition, negative results in these assays
do not prove conclusively that the compound lacks genotoxic
activity in the intact mammal, including man.
22.214.171.124. Supplementary tests
Supplementary tests are conducted to complement, verify, or
assist in the interpretation of the results of the initial battery.
They may simply involve repetition of one of the initial assays
under different experimental conditions or, in other cases, a
completely different type of test.
The results of the basic screen provide information on the
ability of the test chemical to induce genotoxic effects in a
limited number of assays, e.g., mutation in bacteria and chromosome
aberrations in eukaryotic cells. In many instances, these may
provide sufficient data and, indeed, may be the only available data
on which to make a preliminary hazard assessment. Where, however,
a bacterial mutagen does not produce chromosome damage in the
mammalian cell assay, it may be useful to know if the genetic
activity is confined to bacterial cells, or if the chemical is also
active in eukaryotic cells. A variety of systems can be used to
answer this question including gene conversion or mutation in
yeasts (section 2.2), gene mutation (section 2.5), sister chromatid
exchanges (section 2.4) or unscheduled DNA synthesis (section 2.3)
in mammalian cells, or mutation in Drosophila (section 2.7). The
next stage in the assessment may be to determine whether a chemical
shown to be genotoxic in eukaryotic cells is also active in a
whole animal. In vitro clastogens can be investigated using
chromosome studies on the bone-marrow cells of rodents after dosing
with the suspect chemical. The in vivo activity of bacterial
mutagens can be further evaluated in studies of mutagenic products
in urine or body fluids from animals treated with the compound
(section 2.1) (Combes et al., 1984) or in the mouse coat Spot Test
(Fahrig, 1977; Russell, 1978). Techniques are also available for
investigating DNA repair (Waters et al., 1984) and sister chromatid
exchanges (Perry & Thomson, 1984) in treated animals. In some
cases, it may be appropriate to study the effect of a compound on
mammalian germ cells using either the dominant lethal assay
(section 2.8) or chromosome analysis of rodent germ cells (Adler,
1982; Brewen & Preston, 1982; Albanese et al., 1984).
In summary, an assessment of the possible genotoxic hazard is
generally carried out after each phase of testing, attempting to
answer the questions, a) is the compound mutagenic? (Phase 1); is
it active in mammalian cells? (Phases 1 and 2); is it active in
vivo? (Phases 2 and 3); does it present a hazard for man? (may be
considered after Phase 1, 2 or 3, depending on the nature of the
chemical) (sections 4.3.2, 4.5.2).
4.3.2. Nature and extent of potential human exposure
The selection of assays and, in particular, the extent of
testing required before assessing the potential hazard, depends on
the nature and eventual use of the chemical or product.
126.96.36.199. Limited or negligible distribution
There are chemicals for which environmental distribution is
severely limited and the chance of human exposure is unlikely, or
limited to small groups of people, the levels of exposure being
very low. In these cases, data from a base set of two assays are
often the only data available to those who have to determine how
such chemicals should be handled. For example, manufacturing
intermediates are usually handled by trained personnel using
established safe handling procedures and information on the
genotoxicity of such chemicals is of value in the design of safe
manufacturing processes. Specialized chemicals, that are usually
produced in small volumes and supplied for specific industrial or
research applications, may be tested in order to assess the safety
requirements in their transport or use. For materials of this
type, data from both base set assays are normally provided. Only
in rare cases is information from a single assay, e.g., a bacterial
mutation test, considered adequate, as for example, when screening
candidate pharmaceuticals, or dyestuffs, food additives, etc. The
results from simple bacterial assays may then be sufficient to
identify mutagenic structures in a series of analogues and thus set
priorities for further testing or further product development.
188.8.131.52. Medium distribution, limited exposure potential
Chemicals in this group are those to which some degree of human
exposure may be possible, but where environmental distribution is
restricted and only specific groups of individuals may be
inadvertantly exposed. Examples include solvents, paints,
adhesives, oil products, some pesticides, and other materials that
will generally be used in an industrial or commercial environment
and to which the general population is unlikely to be exposed.
They are chemicals that may be fairly widely used in an environment
where potential exposure can be controlled, but do not include
materials for domestic use, or those that are present in products
available to the general population or are released into the
For assessing the genotoxic hazard of such chemicals, results
from two-base set tests together with information on their
structural relationship with known carcinogenic and non-
carcinogenic chemicals and other basic toxicity data are normally
available. Such an assessement may require data from supplementary
assays, for example, to confirm negative results provided by the
base set or to determine if the genetic damage identified in the in
vitro assays can be detected in animals. However, this should not
be regarded as a complete evaluation of genetic toxicity.
184.108.40.206. Extensive distribution, intentional or unavoidable exposure
This group contains chemicals, materials, and products that
may be widely distributed in the environment and to which human
beings will almost certainly be exposed. Examples include:
pharmaceutical products, both those used for very specialised
treatments and those used by a relatively high proportion of the
population; chemicals that are an integral part of foodstuffs or
may appear as residues or contaminants in food; domestic and
agricultural pesticides; domestic chemicals of all kinds;
environmental contaminants including naturally occurring chemicals
in plants, soil etc.; combustion products; industrial effluents;
and many more.
Because human exposure to these materials is generally to be
expected, the objective of short-term tests (and, indeed, of all
toxicity testing) is to ensure, as far as possible, that exposure
does not present a potential or actual hazard. It is important to
keep in mind that the eventual assessment of genotoxicity is aimed
at deciding if the chemical presents either a carcinogenic hazard
or an adverse effect on germ cells with the possibility of
producing heritable genetic damage.
Initially, results of the base set are assessed, but data from
these assays are rarely sufficient for hazard assessment for
chemicals in this group (section 4.5.1). It is usual to conduct
supplementary assays to investigate other genotoxic effects such as
the induction of gene mutations or unscheduled DNA synthesis in
mammalian cells, and, when this genetic profile has been completed
(section 220.127.116.11), to assess the activity of the material in vivo.
At this stage, and with the help of data on the absorption,
distribution, metabolism, and excretion of the chemical and other
toxicological data, it is possible to conduct a reasonable
assessment of the potential of a substance for mutagenicity and
genotoxic carcinogenicity. The final assessment of carcinogenic
potential, however, usually requires the provision of data from
long-term cancer studies in laboratory animals.
4.3.3. Regulatory requirements
Many countries require the submission of testing data before
approving the marketing of certain types of products. However, the
requirements differ considerably between countries, and various
national and international bodies have attempted to harmonise
mutagenicity testing requirements by preparing guidelines.
Organizations such as the Organisation for Economic Cooperation and
Development (OECD) and the European Economic Community (EEC) have
published regulatory requirements or guidelines for mutagenicity
testing, though many countries continue to have their own
individual requirements. The specific assays required by
individual countries are often unclear and usually depend on the
nature of the chemical or product and the outcome of discussions
between the marketing company and the competent authority of the
country (ICPEMC, 1983b).
Two bodies whose authority extends beyond national boundaries
are the OECD (representing some 24 countries) and the EEC. Their
guidance and regulations, respectively, on mutagenicity testing are
very similar and, in practice, apply to the marketing of new
substances rather than existing chemicals. Both authorities relate
the extent of testing to the perceived degree of exposure and
distribution. For example, testing is only required on chemicals
that will be produced or imported in quantities of one tonne or
more per annum. Many types of product are excluded by these
authorities; medicinal and food products are often regulated by
individual countries rather than international bodies.
The OECD and EEC require a base-set of two tests for
mutagenicity, i.e., assays for bacterial mutation and for
chromosome damage, on all products that are not excluded from their
authority, with a requirement for supplementary assays when
exposure of relatively large numbers of people is unavoidable or
4.4. Acceptability and Reliability of Data
It is essential that data used to assess the genotoxicity of a
chemical are derived from studies designed to meet predefined
minimum technical and scientific criteria. One of the purposes of
the descriptions of assays contained in section 2 of this guide is
to define these criteria. The reliability of data, therefore, can
be confirmed by ensuring that the experimental protocol used to
produce the data conforms to the requirements for an acceptable
assay. In addition, evidence must be provided to show that the
investigator faithfully adhered to the protocol by accurate and
full recording of all experimental procedures, raw data,
mathematical calculations, etc., so that every step of each assay
can be audited by an independent observer. Such monitoring
procedures are described in detail in section 3.3.
4.5. Interpretation of Results and Significance for Human Hazard
Results of short-term tests are assessed with two distinct
types of hazard in mind: the carcinogenic activity of the chemical
and the possibility that the chemical may affect human germ cells
to produce heritable genetic changes.
4.5.1. General principles
Much of the data used in the assessment are generated from
relatively simple tests, often consisting of cultures of single
cells, and it must be emphasized at the outset that the behaviour
of a particular chemical may be dramatically different in a complex
organism such as man. In a simple bacterial mutation assay, the
chemical may be metabolically transformed by an auxillary
microsomal enzyme system and the reactive molecule thus generated
has simply to penetrate the bacterial cell wall to be readily
available to interact with DNA to produce genetic changes. In the
whole animal, however, the same chemical must be absorbed into the
body across a number of chemical and physical barriers, and must be
transported to the site where the appropriate metabolizing enzymes
are situated, where it may be activated or detoxified, before it is
in a form that can interact with DNA (some metabolism may also
occur in the gut). Even then, the DNA lesion is subject to
protective devices, such as DNA repair, before genetic changes are
expressed, and these protective factors differ between the
bacterial system and animal cells. Thus, in animals, a range of
physiological and biochemical factors that are different from those
in simple assays may influence the ultimate fate of the chemical,
either inhibiting or enhancing its potential toxicity.
The structure of the chemical and its possible fate in animals
are important factors in the interpretive process. Data from
studies on absorption, distribution, metabolism, and excretion are
generally only available for chemicals for which the possibility of
human exposure is relatively high, e.g., drugs, foods, and many
pesticides, and, even then, detailed metabolic data may not be
available. For other materials, the possible in vivo fate may be
inferred from the chemical structure by analogy with related
chemicals for which more information is available.
A further factor in the interpretation of short-term tests lies
in the correlation between positive and negative results in a
particular assay and known carcinogenic and non-carcinogenic
activity. This correlation is obtained from validation studies in
which the activity of known animal carcinogens and non-carcinogens
is established in the short-term test (Purchase et al., 1978). The
assays commonly used in the initial test batteries, e.g., bacterial
mutation tests and in vitro chromosome assays, are selected
because they have performed well in validation studies and
currently have a good predictive value for animal carcinogenicity
with many classes of chemicals (de Serres & Ashby, 1981; Ashby et
Thus, the important factors to be considered when interpreting
the findings of short-term tests are: (a) the predictive value of
the assays as demonstrated by their correlation with known
carcinogens and non-carcinogens; (b) the structure of the chemical
in relation to chemicals of known genotoxicity; (c) the known or
probable metabolic route of the chemical in the whole animal; and
(d) data from other toxicity studies.
In section 18.104.22.168, the significance of the results of
individual assays is discussed, but it must be emphasised that
assessment of human hazard should be based on combinations of
assays rather than on data from single tests. The interpretation
of data from batteries of short-term assays is described in section
22.214.171.124, together with the application of the phased approach to
assessing genotoxic hazard using supplementary tests.
126.96.36.199. Results of individual assays
For many years, assays using a range of tester strains of
bacteria have been the cornerstone of short-term tests for
genotoxicity. Positive results indicate, primarily, that the
chemical or one of its metabolites is capable of interacting with
DNA to produce mutations. In spite of the fact that many genotoxic
carcinogens produce mutations in bacteria, not all bacterial
mutagens are animal carcinogens and the interpretation of bacterial
assays in isolation, in terms of human hazard is not an acceptable
procedure. Data from at least two base set assays are usually
available before even a preliminary extrapolation is attempted,
for example, when establishing safe working practices in a
There is increasing evidence, particularly from a recent
collaborative study sponsored by the International Programme on
Chemical Safety (Ashby et al., 1985), that some carcinogens that
are negative or difficult to detect in bacteria induce genetic
changes in eukaryotic systems, such as cultured mammalian cells,
yeasts, or Drosophila, in the form of structural chromosomal
aberrations or gene mutations. Again, these assays are not usually
interpreted in isolation but only as part of an expanding data
Studies on whole animals are usually considered to be more
relevant to man than in vitro assays and, as a general rule, a
chemical that gives clear, unequivocal positive results in an in
vivo assay, such as chromosome damage in rodent bone-marrow cells
or the dominant lethal assay, is usually regarded as a possible
human mutagen or carcinogen.
188.8.131.52. Results from combinations of assays
It is usual practice to begin the assessment of the
genotoxicity of a chemical on the results of an initial battery of
at least two assays. One of these is almost invariably a bacterial
mutation assay and, in a two-test battery, the second is usually an
in vitro or in vivo chromosome assay. Data from other tests,
e.g., yeasts, UDS, etc., may also be available, and, where other
toxicity or pharmacokinetic studies have been conducted, the data
base is considered as a whole during the assessment of possible
The following sequence of assessment procedures is based, as an
example, on data available, initially, from a bacterial mutation
assay and a chromosome assay in cultured mammalian cells. For the
purpose of this exercise, it is assumed that the data have been
generated from reliable and acceptable protocols (section 4.4).
A. Chemicals clearly positive in both assays
Such findings demonstrate unequivocally that the chemical or a
metabolic derivative is capable of interacting with DNA to produce
genetic damage in both eukaryotic and prokaryotic cells. It is
thus classified as a genotoxic chemical and, unless, and until,
data from other toxicity studies or supplementary short-term tests
show that it is unlikely to be active in vivo, it is prudent to
regard it as potentially hazardous for man.
In some instances, chromosome data may be presented from plant
systems rather than mammalian cells, and the same principles apply.
However, it may be appropriate to confirm the plant data in a
mammalian-cell assay or other eukaryote (e.g., Drosophila or
yeast) at an early stage.
It may be prudent to designate a chemical as potentially
hazardous on the basis of these assays and this may indicate that
distribution and human exposure will be restricted. The potential
value of the chemical may be such that further testing designed
either to confirm this assessment or to determine the extent of the
potential hazard may be worthwhile. By no means will all such
chemicals be shown to be hazardous in subsequent testing.
Additional testing may be aimed at elucidating: (a) the nature of
the genetic change induced by the chemical in mammalian cells; (b)
the dependance of the chemical on metabolic enzyme activation for
its genetic activity; and (c) the behaviour of the chemical and the
genetic damage it induces in the intact mammal.
(i) Positive results only after metabolic activation
Such results indicate that reactive metabolites are generated
by microsomal enzymes, i.e., the compound is an "activation-
dependent" mutagen. As the chemical has been proved to be a
clastogen in vitro, the next step may be to carry out a chromosome
study in bone-marrow cells in rodents after dosing with the
chemical by an appropriate route (e.g., oral, intraperitoneal).
Either the micronucleus test or analysis of metaphase chromosomes
can be used. If the chemical is shown to produce chromosome damage
in vivo, there is little to be gained by any further testing and
it is usually regarded as having mutagenic or carcinogenic
potential for man. In rare cases, for example, mutagenic anti-
tumour agents, the benefits of using the drug may outweigh this
potential hazard and it may be useful to conduct a dominant lethal
assay or a cytogenetic analysis of germ cells in rodents to assess
the induction of heritable genetic changes.
Negative results in a properly conducted in vivo study may
alleviate most concern regarding the potential hazard of a chemical
and, with many chemicals, such negative results suggest that the
adverse chromosome effects shown in vitro are unlikely to occur in
the intact animal. (It should be remembered that, occasionally, a
negative result may be obtained in a bone-marrow chromosome study
because the compound or its reactive metabolite(s) did not reach
the target cell in the bone marrow.) However, the chemical is
still a mutagen and the decision to release it into the general
environment will usually be measured very cautiously against its
possible benefits; further testing may be judicious. Since sister
chromatid exchange or unscheduled DNA synthesis (UDS) are
mechanistically unrelated to chromosome breakage, it may prove
useful to establish the activity of the chemical in these in vitro
assays. If either yields a positive result, in vivo activity can
be investigated by conducting assays for SCE in bone-marrow cells
or UDS in hepatocyte cultures from treated rodents.
A detailed pharmacokinetic profile of the chemical may be
available and could provide evidence on the generation of reactive
metabolites in vivo. Such evidence may support the assumption that
the chemical is or is not putatively genotoxic for mammals.
(ii) Positive results in the absence of metabolic activation
Chemicals that produce mutations in bacteria without the need
for exogenous metabolic activation are either "direct-acting
mutagens" or, in rare cases, are activated by bacterial enzymes.
Most eukaryotes are capable of some degree of endogenous activation
(i.e., without the use of an auxillary metabolizing system) and
direct-acting chemicals are usually classified as such on the
results of bacterial assays. If there is an indication from the in
vitro assays that the incorporation of a metabolic activation
system eliminates or significantly reduces the mutagenic activity,
then it is possible that the microsomal enzymes serve to detoxify
the chemical. Some confirmation of this can be obtained from an in
vivo chromosome study, from the results of one of the other in
vivo tests for mutagenic activity (A(i)), or from the results of a
study of the metabolism of the chemical in vivo. Negative results
from a properly conducted in vivo investigation of a direct-acting
mutagen usually indicate that it is unlikely to pose a serious
B. Chemicals that produce gene mutations in bacteria but not
chromosome aberrations in mammalian cells
A chemical that produces mutations in bacteria with negative
results in the eukaryotic-cell test is classified as a bacterial
mutagen. The question then arises as to whether the mutagenic
activity is specific to bacterial cells. This may be investigated
by applying one or more of the other tests described in section 2.
Where mutagenic activity is established in a eukaryotic cell
system, interpretation of the data and the need for additional
tests follows the procedure outlined in A(i).
Occasionally, chemicals are encountered that produce mutations
in bacteria, but are clearly negative in other in vitro tests.
The assessment of such findings presents a number of difficulties.
Interpretation may be helped by conducting an in vivo cytogenetic
assay (in spite of the fact that the in vitro chromosome assay was
negative, the absence of chromosome aberrations in a bone marrow
study is valuable confirmatory evidence), by testing urine from
treated animals for mutagenic activity and by consideration of the
pharmacokinetics, the relationship of the structure of the chemical
to known genotoxins, and other toxicity data. However, the
observation of bacterial mutagenicity may be the only evidence that
the compound is genotoxic and a great deal of effort can be
expended in trying to elucidate its significance to human hazard.
Where other in vitro and in vivo tests fail to reveal mutagenic
activity, and where there is no evidence from pharmacokinetic and
conventional toxicity studies to suspect possible adverse effects,
then the finding of bacterial mutagenic activity in isolation,
particularly at high test concentrations, may not constrain the use
and distribution of most materials. Certain drugs, food chemicals,
and ubiquitous materials have been exempt from this view and their
use restricted pending long-term cancer studies in laboratory
C. Chemicals that produce chromosome aberrations in mammalian
cells but not mutations in bacteria
This pattern of results raises three important questions:
(a) has the chemical been tested in bacteria under an
appropriate range of experimental conditions, e.g.,
using a preincubation technique, variable levels of
metabolic activation (i.e., S9), and using an
adequate range of tester strains of bacteria;
(b) can the chemical induce mutations or UDS in cultured
(c) can the in vitro chromosome damage be reproduced in
Some chemical carcinogens (e.g., hexamethylphosphoramide) give
negative results in bacteria but are clearly mutagenic when tested
in mammalian cells (Ashby et al., 1985). It is often useful,
therefore, to check the mutagenicity in a mammalian-cell system and
if shown to be active, it can be assessed as described in A(i).
For small-volume chemicals (section 184.108.40.206), the information that
a chemical induces chromosome aberrations may be all that is
available to formulate guidance on its use and distribution. Where
it is important to determine its activity in vivo, a bone-marrow
cytogenetic assay using micronuclei counts or metaphase analysis is
the next logical step. Positive results in such a test confirm
genotoxic activity in vivo and, in most cases, are interpreted as
suggesting a possible hazard for man. Where the induction of
chromosome damage in cultured cells is the only indication of
genotoxicity, i.e. where other short-term tests in cultured
mammalian cells are negative, where there is no evidence of
chromosomal damage in animal studies and other toxicity studies do
not show any adverse effects, the chemical is unlikely to pose a
genotoxic hazard for man. As indicated in section B, certain drugs
and food chemicals may be exempt from this view and long-term
animal studies are desirable before the material is released for
D. Negative results in both assays
In assessing the significance of negative results in the basic
screen, it is essential to determine whether the results are a true
indication of lack of genotoxic activity by confirming that the
protocols used were appropriate for the type of material being
tested. For example, if the chemical is volatile, assays must be
conducted in sealed vessels to prevent erroneous negative results
caused by evaporation of the test chemical. Some chemicals, e.g.,
nitrosamines, require special experimental conditions to detect
mutagenic activity. The physical and chemical properties of the
test agents and the influence of protocol variables on the
performance of the assays are taken into consideration when
evaluating the significance of negative results in the basic
screen. For many chemicals of limited distribution and exposure,
the provision of reliable evidence of the absence of mutagenic
activity in the two initial assays is frequently considered to be
sufficient grounds for regarding the chemical as non-genotoxic.
Because of the existence of a small class of genotoxic agents that
are not detected in the two initial assays, it must be accepted
that a decision to permit widespread use and unlimited distribution
on the basis of negative results in these tests carries a
significant risk, and further testing in other assays may be of
E. Non-genotoxic carcinogens
The majority of carcinogenic chemicals have demonstrable
genotoxic activity. There are certain classes of chemicals,
including, for example, some metals, organochlorine compounds, and
estrogens, that are known to be carcinogenic in animals but fail to
elicit a positive response in assays for genotoxicity. There are
other compounds that are not in themselves complete carcinogens,
which are able to exacerbate certain stages of the carcinogenic
process (ICPEMC, 1982). Collectively, these latter compounds are
referred to as carcinogen enhancers (Clayson, 1981). At present,
there is no short-term test that has been sufficiently well
validated to be used with confidence to detect non-genotoxic
carcinogens and enhancers. Evidence is emerging that some of these
chemicals can induce numerical chromosome changes in eukaryotic
cells and, in some cases, structural chromosomal aberrations (Ashby
et al., 1985) and that they can be detected in modified forms of
certain assays for neoplastic transformation (Meyer, 1983).
However, these findings require confirmation in further validation
studies and it must be accepted that a proportion of this class of
chemical will escape detection in current toxicological practice.
F. Complex mixtures
The principles outlined in this guide apply to chemicals that
are pure compounds or relatively simple mixtures, formulations, or
solutions. The application of in vitro assays to more complex
mixtures, e.g., foods, crude industrial products, etc., may give
results that are unreliable because of the influence of such
factors as, for example, competition between components in the
mixture for enzyme sites in the activation system, presence in the
mixture of cytotoxic components that limit adequate testing, and
uncertainties regarding the concentrations of mutagenic components.
In vitro assays, therefore, must be used and interpreted with
caution and, where it is not possible to isolate and identify
mutagenic components, the emphasis should be on in vivo testing
(although it must be realized that problems similar to those
mentioned above may beset whole-animal assays).
4.5.2. Influence of the extent of exposure and distribution
The amount of toxicity testing that a material undergoes before
it can be released for specific or general applications or into the
environment is decided, to a large extent, by its perceived
distribution and the expected pattern of human exposure. For each
of the broad groups of materials considered here, evaluation of
genotoxic activity is based on the phased approach (section 4.3.1)
using as a starting point, data from a two-test base set. It must
be remembered that mutagenicity tests provide only part of an
overall package of toxicity data that should be available before
making a final assessment of human hazard.
This guide is concerned only with hazard assessment and it is
not within the objectives of the guide to describe methods for
estimating, in quantitative terms, the risk of adverse effects in
man following exposure to specific genotoxic chemicals. However,
in order to avoid confusion between the terms "hazard" and "risk",
brief definitions of risk estimation and risk management are
(a) Hazard assessment
The assessment of the possible hazard associated with exposure
to a genotoxic chemical is a purely scientific process and involves
an appraisal of experimental data in order to attempt to predict
the possibility and the nature of any adverse effect in man.
(b) Risk estimation
This is the second stage in the evaluation of a product or
chemical and is an attempt to derive a quantitative estimate of the
risk resulting from the use or release of the material, i.e., the
number or frequency of individuals in a population of a given size
who may exhibit a given adverse effect (e.g., cancer or heritable
mutations) under certain exposure conditions. Risk estimation is
almost always an uncertain undertaking. Quantitative data derived
from screening tests cannot be used as a basis for predicting the
potency of carcinogenic activity in animals or man (For additional
details see Bridges et al, 1979; Ehrenberg, 1979; Brusick, 1980;
Sankaranarayanan, 1982; ICPEMC, 1983d).
(c) Risk management
While hazard assessment and risk estimation are scientific
processes, risk management is a non-scientific decision-making
procedure (US NAS, 1983). Risk management attempts to balance the
perceived benefit of using or distributing the chemical or product
with the risk of adverse effects to individuals or to populations,
i.e., a risk-benefit equation. Where the benefits are regarded as
great, for example with a new, unique and valuable drug or
pesticide, an identifiable and measurable risk may be considered
acceptable. In situations where less toxic alternatives with
similar benefits are available, the risk associated with the
introduction of the new material would normally be unacceptable.
220.127.116.11. Pharmaceutical compounds
With few exceptions, pharmaceutical compounds that show
unequivocal mutagenic activity are identified and discarded by the
drug company at an early stage of development. Most major
companies involved in the development and manufacturing of drugs
use a three-stage development regime that includes some toxicity
testing at each stage. The first stage is an in-house toxicity
screen that is used primarily for the selection of candidate
compounds, i.e., those that show promising pharmacological
activity, for further development. Only very limited mutagenicity
testing is likely to be conducted at this time, and, in most cases,
will consist of a bacterial mutation test. Compounds that show
mutagenic activity will frequently be discarded at this early
stage. There are exceptions to this rule, for example, where a
compound or a group of compounds with unique pharmacological
activity may be considered of great potential benefit. Promising
candidates eventually reach a stage of development where it is
necessary to test their efficacy in human beings. Before these
clinical trials, the compounds undergo a second phase of testing,
i.e., the pre-clinical trial toxicity screen, the objective of
which is to assess the safety of the drug for use in small groups
of human volunteers. Assuming that a bacterial assay was conducted
in the primary toxicity screen, mutagenicity testing usually
consists, initially, of either a test for the induction of
chromosome aberrations in cultured cells or an in vivo assay in
rodent bone-marrow cells for micronuclei or metaphase chromosome
The interpretation of the findings from the initial
mutagenicity assays is greatly influenced by the pharmacological
and pharmacokinetic data generated during the development of the
drug. If clear negative results are obtained with a compound that
also shows no indication of interaction with macromolecules such as
DNA, then it may be regarded as safe enough, from the point of view
of genotoxicity, to proceed to clinical trials. Where there is any
doubt about the pharmacokinetics of the drug or its metabolites (as
is often the case at this stage of development), i.e., where the
possibility of DNA interaction cannot be excluded, then additional
testing is indicated. The supplementary tests are aimed at filling
in the gaps in the genotoxicity profile. For example, if an
analysis of metaphase chromosomes in bone-marrow cells from treated
rodents was not part of the initial testing, then this is usually
also carried out. Although the chemical failed to induce mutations
in bacteria, it may be tested in a mammalian cell assay for gene
mutations, or, perhaps, for recessive lethal mutations in
Drosophila or gene mutations in yeasts. Other tests that indicate
the induction of DNA damage may also provide useful data. The
pharmacological data available may also indicate the testing of
urine and other body fluids from treated animals for mutagenic
activity, using bacterial assays. Where the structure of the
chemical indicates that nitrosation products may be formed in the
human stomach, tests for the formation of such products and their
mutagenicity may be conducted (Kirkland et al., 1984).
Thus, before a pharmaceutical chemical undergoes clinical
trials in human volunteers, it is normally shown at least to be
incapable of inducing mutations in bacteria and chromosome damage
in mammalian cells. Where the structure or the pharmacokinetics of
the chemical suggest that genotoxic interactions are conceivable,
additional testing will usually include analysis of chromosome
aberrations in vivo, eukaryotic assays for primary DNA damage and
gene mutations and, where indicated, for chromosome changes in germ
cells (e.g., a dominant lethal assay) and for mutagenic metabolites
in urine or body fluids of treated animals. Assuming negative
findings in these assays and after considering the data from other
toxicity studies, the drug may then undergo clinical trials.
Except in the case of, for example, cytostatic or cytotoxic
drugs used in the treatment of serious, life-threatening diseases,
compounds that are shown to be genotoxic will rarely undergo
efficacy studies in man. Where there is sound evidence that a
drug, shown to be mutagenic in vitro, is rapidly detoxified in
intact animal studies, limited clinical trials may occasionally be
The first two stages of toxicity evaluation are conducted as
part of the development programme. After successful clinical
trials, a final toxicological evaluation is undertaken before the
drug is submitted for registration prior to marketing.
Registration is usually a responsibility of a government department
in the country in which a marketing permit is sought. The
genotoxicity data required for registration vary considerably
between individual countries though, in most cases, the assays
conducted before clinical studies in human beings comprise a
package that is acceptable for registration purposes. Some
authorities require data from a very specific series of assays,
but, in general, a package that includes properly conducted assays
for mutations in bacteria, chromosome aberrations and gene
mutations in mammalian cells, and an in vivo test for chromosome
aberrations (in somatic and/or germ cells) should satisfy most
authorities of the absence of a potential mutagenic hazard
providing that there is no contradictory evidence from other
This package of assays also provides some indication of the
carcinogenic hazard. However, for registration and marketing
purposes with a new drug, carcinogenicity is almost invariably
assessed from animal studies rather than predicted from in vitro
18.104.22.168. Chemical compounds in food
Although this section is primarily concerned with chemicals
that are added to natural food products to improve their keeping
properties, palatability or appearance, etc., it is pertinent to
summarize some other factors that contribute towards the mutagenic
activity of food (For review, see Knudson, 1982). Some edible
plants and their fruits contain compounds, e.g., pyrrolizidine
alkaloids, flavonoids, etc., that are mutagenic in in vitro
assays. A small number are also carcinogenic, but the majority
have not yet been tested for carcinogenicity. They are often
present in only minute quantities in the plant material and are
often destroyed when the plants are cooked or completely detoxified
by the gut flora. The contribution of mutagenic food components to
human cancer is not known. Another source of potential
genotoxicity is the fungal contamination of foods, e.g., aflatoxins
in mouldy groundnuts. Food may also contain residues of
pesticides, compounds absorbed from packaging materials, and other
chemicals. An additional contribution to genotoxic activity can
occur during cooking, and it has been demonstrated that pyrolysis
products, formed during the cooking of meat and fish at certain
temperatures, have significant mutagenic activity. Although
dietary factors are known to contribute towards the overall
incidence of cancer in man, the part played by naturally occurring
mutagens and pyrolysis products in human disease has yet to be
Most foods are complex mixtures of many hundreds of compounds
and evaluating their genotoxicity is far more difficult than
evaluating that of pure chemicals. Because of this, attempts to
investigate the genotoxicity of whole foods are usually undertaken
using intact animals, including Drosophila. However, the
fractionation of foods for mutagenicity testing purposes is
currently being explored (Rowland et al., 1984).
Artificial food additives include chemicals that either enhance
the natural flavour of foods, improve colour or appearance, or are
preservatives added to prevent bacterial spoiling or oxidative
degradation of food. Artificial flavouring materials are usually
identical in chemical structure to naturally occurring flavourings
and are either synthesized or purified extracts from natural
sources. A large number of natural and synthetic dyes have been
used to improve the appearance of food. Many synthetic dyes have
been removed from national and international lists of permitted
food colourants because of their mutagenic or carcinogenic
activity. Compounds commonly used to preserve foods include sodium
nitrite, a weak mutagen in in vitro tests, and antioxidants such
as butylated hydroxytoluene (BHT). Although the mutagenicity of
nitrite itself is unlikely to present a human hazard, it is able to
react with secondary amines in conditions found in the human
stomach to form carcinogenic nitrosamines.
The application of short-term tests for genotoxicity to food
additives follows the principles outlined earlier. Chemicals
proposed for use in foods are usually tested initially in a base
set of two assays and those that, for example, induce mutations in
bacteria or chromosome aberrations in mammalian cells are very
carefully evaluated before being used as either flavouring or
colouring materials. Chemicals that give negative results in these
assays usually undergo a second phase of tests in eukaryotic cells
for the induction of gene mutations, and, possibly, for the
induction of DNA damage, and for the induction of chromosome damage
in rodent bone marrow. Completely negative results in these assays
frequently allay concern regarding mutagenic potential with most
chemicals. However, additives that are structurally related to
known mutagens or carcinogens, and, in particular, chemicals
containing a secondary amine structure may be candidates for
additional in vivo testing, e.g., germ cell chromosome studies,
dominant lethal assays, body fluid mutation tests, etc. Negative
results also suggest that the chemical is unlikely to be
carcinogenic, but few new food additives are currently released for
general use without evidence of the absence of carcinogenic
activity in long-term animal studies.
22.214.171.124. Domestic chemical compounds
Cosmetics such as perfumes, hair dyes, sun screen oils, etc.,
household detergents and cleaning fluids, and a variety of other
chemical mixtures are considered under the general heading of
domestic chemical compounds. Because of their diverse nature,
there have been wide differences in the amount of toxicological
information available on these materials and the following examples
illustrate the need for caution when considering their safety in
domestic use. Several hair dyes of the substituted
phenylenediamine type have been shown to be mutagenic in in vitro
assays, and some of these have produced cancers in experimental
animals. Tris(2,3-dibromopropyl)phosphate is not strictly a
domestic chemical but enters the home in the form of a flame
retardent in clothing. Widely used to reduce the flammability of
children's clothing in particular, the compound was detected as a
bacterial mutagen initially, and was eventually shown to be
carcinogenic in long-term rodent studies. Fortunately, these are
relatively infrequent instances and would have been detected in the
base set of two assays now widely used to assess the genotoxicity
of new products.
Because these materials are sold for use in an environment
where human exposure is to be expected or intended, a complete
genotoxicity assessment is usual, and may begin with data from in
vitro assays in bacteria and mammalian cells. The sequence of
assessment phases described in section 126.96.36.199 is then followed.
New chemicals intended for domestic use will normally give
unequivocal negative results in tests for gene mutation in both
prokaryotic and eukaryotic cells, and for chromosome aberrations in
vitro. Where direct contact with the chemical is perceived, data
from an in vivo assay for chromosome breakage are usually
available. Following the principles developed earlier, positive
results in any of these assays may prevent the release of a
chemical for domestic use. Evidence from in vivo mutation
studies, pharmacokinetic data, or long-term animal studies may,
however, remove the concern caused by an isolated positive result
in an in vitro assay.
Exposure to pesticides may occur in a variety of different ways
including exposure of workers during manufacture, exposure during
the transport, formulation, or application of pesticides, and
exposure to residues in edible crops, soil, and water. Adverse
effects on man may result from either the compound itself, its
mammalian metabolites, plant and soil metabolites and, possibly,
from breakdown products in the environment. Unlike the chemicals
described previously as medicinal, food, and domestic chemicals,
pesticides are often dispersed widely in the environment and stable
materials, such as DDT, may remain as virtually permanent
contaminants at minute, though detectable concentrations.
Because of this potential for ubiquity, detailed information on
the toxicity, stability, and fate of pesticides in the environment
is mandatory in many countries, before they can be registered and
released for use. The use of pesticides, however, is virtually
indispensible for the successful production of most major crops,
and for the control of certain major insect-born diseases of man
and domestic animals. This, together with the fact that pesticides
are highly biologically-active molecules, requires a fine balance
to be set between the benefits accrued by using the pesticide and
its possible hazard to man or the environment.
Tests for mutagenicity form only a small part of the overall
package of data accumulated before a pesticide is released for use.
Short-term tests are usually carried out in parallel with the
development of a new pesticide. For example, bacterial mutation
data are normally available before the first limited field trials
to test the efficacy of candidate compounds are carried out so that
safe handling procedures can be formulated for both laboratory and
field researchers. The next stage in the development is usually a
more extensive field trial on the target crop grown under
commercial conditions, and another phase of toxicity testing,
including an assay for chromosome aberrations in mammalian cells,
precedes this stage. Unless there is evidence from other toxicity
studies or from chemical structure/pharmacokinetic considerations
that the chemical may be genotoxic, negative results in base set
assays frequently allow the pesticide to proceed through the
developmental and evaluation stages. Where, however, potential
genotoxicity is still suspected, supplementary tests, including
assays for gene mutations or primary DNA damage in eukaryotic cells
may be considered at this time.
The final phase of toxicity testing is carried out after the
development stage is completed and field evaluation has
demonstrated a potentially successful product. These tests are
usually designed to complete the toxicity package required by most
authorities responsible for the licencing of pesticides for use.
Results from a battery of short-term tests including the bacterial
and chromosome assays conducted during the development phase, an
assay for gene mutations in mammalian cells, and an analysis of
metaphase chromosomes of bone-marrow cells from rodents dosed with
the chemical, meet the requirements of most regulatory authorities.
However, different countries have different requirements, and
additional tests, for example, for aneuploidy or primary DNA
damage, may sometimes be required.
The finding of mutagenic activity in either of the two initial
short-term tests need not necessarily indicate that development of
a pesticide should be abandoned, though this is often the case. If
the potential value of the pesticide merits further development, it
is usually treated as a highly toxic material and handled
accordingly in subsequent field trials. Additional testing to
characterise the mutagenic activity and to determine its activity
in vivo may then be initiated. An assessment of the hazards
associated with a mutagenic pesticide will depend on data from in
vivo studies (e.g., bone-marrow cytogenetics and either germ-cell
cytogenetics or a dominant lethal assay), the metabolic profile of
the chemical, and data on its stability and rate of elimination or
degradation from the crop and the immediate environment. A final
decision on whether to continue large-scale development and
evaluation of a mutagenic pesticide may be delayed until data from
other biochemical and toxicological studies, including long-term
animal cancer studies are available.
Pesticides are often supplied and used in a variety of
formulations and in mixtures with other pesticides. It is usual,
therefore, to consider both the pure material and the specific
formulation, when testing pesticides and assessing the significance
of toxicity data.
The assessment of the hazards of residues of pesticides in
plants, soil, and water is usually based on analytical chemical
data. However, some pesticides, e.g., some atrazines, are
metabolized by plant enzymes to mutagenic products. Although these
metabolites can be analysed chemically, their mutagenic activity
can be detected by testing extracts of plants exposed to pesticides
in bacterial mutation assays.
Pesticides as a class contain two widely quoted examples of
ambiguity between mutagenic activity and carcinogenicity.
Dichlorvos (2,2-dichlorovinyl dimethyl phosphate), an
organophosphate insecticide, is a confirmed bacterial mutagen.
However, results from in vitro mammalian cell assays are either
negative or equivocal, and it does not produce mutations in vivo.
Comprehensive long-term cancer studies indicate that dichlorvos is
not a carcinogen. Pharmacokinetic and other biochemical studies
suggest that this compound is efficiently detoxified in animals, so
that, in spite of being a bacterial mutagen, it is still marketed
as a domestic and agricultural insecticide. The other example is a
class of insecticides including DDT and dieldrin known collectively
as organochlorine compounds. Both these chemicals induce tumours
in liver tissue in mice after prolonged exposure. Both have also
been subjected to comprehensive in vivo and in vitro mutagenicity
tests, and although isolated positive results appear in the
literature, a detailed analysis of the data suggests that these two
organochlorines are not genotoxic, i.e., do not cause adverse
effects as a direct result of a DNA lesion. The primary
carcinogenic growth appears to be confined to rodent liver, and
although the potential hazard has been debated at length for many
years, the significance of these findings for human health remains
Both these examples are given to illustrate the complexity of
the extrapolation of in vitro data to animal data to human hazard
and serve to emphasise the caution needed in some cases in the
assessment of genotoxic hazard from the results of short-term
188.8.131.52. Chemical compounds used in industry
Most industries use chemical compounds in one form or another.
The function of the chemical industry itself is to manufacture,
from primary sources such as oil, coal, and ore, chemicals that are
valuable commodities in everyday life or that are necessary
components in the manufacture of other products. The principal raw
materials undergo a series of processes to convert them initially
to base chemicals (e.g., inorganic compounds such as alkalis and
acids, and organic compounds such as olefin and aromatic
compounds), then to intermediates and finally to the finished
chemical product. These may be consumer products such as solvents,
etc., or are used by other industries in the manufacture of, for
example, paints, adhesives, drugs, and plastics.
The output of the chemical industry is enormous in both
quantity and diversity and the management of safety in the industry
is based on the principle of identifying and assessing the hazards
of exposure to particular chemicals, and then taking steps to
reduce or eliminate human exposure. It should be accepted that
many of the chemicals used in industry are dangerous to man and a
great deal of effort is expended in ensuring the safety of workers
by the introduction of safe handling procedures, protective
clothing, and enclosed industrial processes.
Exposure to chemicals is possible during manufacture, during
transport of material from one industry to another, and as a result
of environmental contamination. The amount of toxicity data
necessary to provide a sound assessment of the possible hazard of a
chemical is governed primarily by the extent of human exposure and
environmental distribution. For many of the chemicals used in
industry, human exposure is minimal and data from base set assays
are often regarded as providing sufficient information on the
potential mutagenicity or carcinogenicity of such chemicals to
allow the small groups of workers involved to be protected
accordingly. Materials that are produced in larger volumes and
that are transported in bulk or widely used in other industries may
require additional testing. Bulk products that are non-mutagenic
in the initial battery of tests may need to have these findings
confirmed in, for example, a eukaryotic assay for gene mutation or
primary DNA damage and an in vivo test for chromosome
aberrations, before assessing the genotoxic hazard. With large-
scale chemicals that are shown to be mutagenic in the base set, the
genotoxicity may need to be further characterised in supplementary
in vitro and in vivo assays. Further testing may involve detailed
studies of mutagenic activity in laboratory animals and long-term
cancer studies may be necessary before the potential hazard can be
fully evaluated and safe working conditions established.
Many chemicals used in industry are volatile and present a
different sort of hazard, for not only can such chemicals present
atmospheric contamination in the workplace, they may also escape
into the surrounding environment. In the modern chemical industry,
the hazards associated with toxic vapours are well recognised and
safe working practices are, in general, fully implemented, though
the toxicity of vapours is still a real hazard in some cottage
industries. When assessing the mutagenicity of volatile chemicals,
it is important to ensure that the experimental conditions were
appropriate, i.e., in vitro tests require the use of sealed
vessels to eliminate the loss of test material by evaporation, and,
ideally, an inhalation exposure regimen should be used in in vivo
The manufacture, use, and transport of chemicals used in
industry is strictly regulated by national and international bodies
responsible for industrial and environmental health. The role of
toxicity testing is described in detail in the guidelines of the
appropriate authorities such as the Organisation for Economic
Cooperation and Development (OECD).
Acentric chromosomal fragment lacking a
Acrocentric chromosome with the centromere close
to one end of the chromatids
Allele one of two or more alternate forms of
a gene at a specific locus on a
Anaphase stage of mitosis in which the
centromere divides and the chromatids
migrate towards poles of the cell
Aneuploidy addition or loss of one or more
chromosomes from the haploid (i.e.,
meieosis) or diploid (i.e., in
mitosis) number, i.e., 2n + 1,
2n - 2, etc.
Autosome any chromosome other than the sex
(i.e., X and Y) chromosomes
Banding techniques that result in
differentially-stained bands along a
chromosome, the pattern of banding
being characteristic for a particular
species and for specific chromosomes;
banding techniques are commonly used
to identify exchange of material
between chromosomes, e.g.,
Break damage to a chromatid or isochromatid
involving a discontinuity of the
chromosome greater than the width of
Bromodeoxyuridine (BrdUrd) base analogue that is incorporated
into DNA in place of thymidine and,
using suitable techniques, makes
possible the observation of sister
Budding and Fission morphological features of cell
division in yeast species
Centriole cellular component that divides into
two prior to mitosis allowing the two
daughter centrioles to migrate to
opposite ends of the cell forming
points of origin of the spindle
Centromere region at which sister chromatids are
held together; also known as the
kinetochore, it is the structure by
which chromosomes are attached to the
spindle; the centromere splits
longitudinally at anaphase allowing
the chromatids to move to opposite
Chromatid unreplicated chromosome or one half
of a complete chromosome with the
identical copy being its sister
Chromatid aberration structural aberration affecting only
one of the two chromatids of a
Chromosomal aberration structural aberration affecting both
chromatids of a chromosome; also
referred to as an isochromatid
Clastogen a physical or chemical agent that
induces chromosome breakage
Cross links covalent bonds between bases in
parallel DNA strands
Deletion chromatid or isochromatid aberration
in which part of a chromosome is
missing as a result of a break; the
deletion may be from the end of the
chromatid, i.e., terminal, or from
the middle of the chromatid, i.e.,
Dicentric a chromosome with two centromeres
Diploid the normal chromosome number of the
somatic cells of most higher
organisms; referred to as "2n", where
n = the haploid number
DNA deoxyribonucleic acid
Dominant mutant term applied to any mutant the effect
of which is detectable in the
Double-strand breaks rupture of both strands of the DNA
double helix at the same site
Endo-reduplication chromatid alignment is maintained in
a cell in which the chromosomes have
duplicated but the cell has failed to
cleave; a form of polyploidy
Erythroblast proliferating precurser of red blood
Extrachromosomal gene gene carried on an element outside
the nucleus, e.g., a mitochondrial
Gap non-staining region of chromatid not
larger than the width of the
Gene conversion recombination event within a gene
producing non-reciprocal product
Giemsa stain chromosome-staining solution
containing the dyes azure, eosin, and
Haploid chromosome number in the gametes; a
single set of the chromosomes;
referred to as the "n" number of
Hemizygous occurrence of genes in a haploid
condition in a normally diploid cell
or organism; as on the X-chromosome
of Drosophila males
Heterozygote a zygote derived from the union of
gametes, dissimilar in respect of the
quality, quantity, or arrangement of
Heteroallele diploid cell carrying two non-
identical alleles of a gene
Heterozygote diploid cells contain two complete
sets of chromosomes; the pairs of
equivalent chromosomes are called
"homologous" and are considered to be
structurally identical, at equivalent
loci, along the chromsome, alleles of
a gene occur which, in homologues,
serve the same function; sometimes,
the pairs of alleles are not
identical and, in such cases, the
cell is described as "heterozygous"
for the gene at that locus
Hoechst 33258(R) fluorescent dye used to demonstrate
chromosomes in which the DNA has been
treated with bromodeoxyuridine,
making observation of sister
chromatid exchanges under a
fluorescent microscope possible;
subsequent staining with Giemsa
allows observation of sister
chromatid exchange under a light
Homoallele diploid cell carrying two identical
alleles of a gene
Homologous see heterozygote
Homozygote a zygote derived from the union of
gametes identical in respect of the
quality, quantity, and arrangement of
Hyperdiploidy aneuploidy in which the chromosome
number is greater than 2n
Hypotonic solution with an ionic strength lower
than that of the cell contents; when
cells are placed in a hypotonic
solution, there is a net uptake of
water resulting in swelling of the
cell; hypotonic treatment of cells at
metaphase improves spreading of
chromosomes for microscopic
Idiogram (Karyogram) the arrangement of chromosomes (i.e.,
from a photograph or drawing) into
pairs and groups of pairs, usually in
order of decreasing size
Instars periods in larval development in
Drosophila; the larvae undergoes two
moults so that the larval period
consists of three stages: the first,
second, and third instars
Intercalation insertion of a molecule, e.g.,
adriamycin, between adjacent bases in
the DNA molecule
Interchange exchange of material between two
chromatids from different chromosomes
Intrachange exchange of material between sister
chromatids, i.e., on the same
chromosome, or exchange within one
Inversion chromosome rearrangement in which a
region between two breaks has been
inverted; "paracentric": the inverted
region is within one chromatid arm;
"pericentric": the inverted region
includes the centromere
Isochromatid aberration chromosome aberration affecting both
chromatids; chromosomal aberration
Karyotype the chromosome complement of a cell
or of a particular species
Lethal gene a gene the substitution of which, for
its normal allele, converts a viable
into a non-viable gamete or zygote;
may be dominant or recessive
Mating type in yeasts, mating occurs between
strains of opposite mating type,
i.e., a and alpha strains in
S. cerevisiae and h+ and h- in
S. pombe; the genetic event that
changes mating type from a to alpha
and vice-versa is called a "mating
Meiosis cell division in germinal cells
resulting in cells with the haploid
number of chromosomes
Metacentric chromosome with the centromere
approximately at the midpoint;
"submetacentric": centromere between
the centre and one end of the
Metaphase stage of mitosis at which the
chromosomes are condensed and aligned
on the equator of the spindle
Micronucleus small fragment of chromosome material
visible during interphase outside and
separate from the main nucleus; may
occur as a result of a chromosome
fragment or a whole chromosome that
detached from the spindle during
Minute very small fragment or minute ring of
chromosome material; may occur singly
or in pairs
Mis-sense a mutation producing a gene product
with a substituted amino acid
Mitogen an agent that stimulates resting
(interphase) cells to divide and
Mitotic index the proportion, usually expressed as
a percentage, of dividing cells in a
Mitosis stage of the cell cycle at which the
chromosomes condense, thus becoming
discrete structures when observed
microscopically; the chromosomes
align on the spindle and then
separate into chromatids that migrate
to opposite poles of the cell before
the cell cleaves to form two daughter
Mosaic a state in which a single individual
has cells of two or more different
Non-disjunction failure of chromosomes to separate
during mitosis or meiosis resulting
in daughter cells with additional and
Nonsense mutation producing a messenger RNA
molecule with a triplet not coding
for an amino acid, e.g., "amber" and
"ochre" are nonsense mutations
Normochromatic erythrocytes mature erythrocytes staining red-
yellow with Giemsa stain
Orcein chromosome-staining solution
Polychromatic erythrocytes young or immature erythrocytes
staining blue-red with Giemsa stain
Polyploidy cell containing more than the diploid
number (2n) of chromosomes in exact
multiples of the haploid number (n),
e.g., triploid = 3n, tetraploid = 4n,
Recessive mutant term applied to any mutant the effect
of which is detectable in the
homozygous or hemizygous condition
Ring chromosome rearrangement in which
fusion of ends of a chromosome
results in a ring structure either
with (centric) or without (acentric)
Sex chromosomes chromosomes that determine the gender
of an individual; in mammals, the X
chromosome signifies female gender,
and the Y chromosome indicates males;
diploid cells in normal females are
XX and XY in normal males
Single-strand breaks breakage of only one of the two
molecules (strands) in the DNA double
Sister chromatid exchange an apparently symmetrical exchange of
(SCE) material between sister chromatids
S-phase phase in the cell cycle during which
normal DNA synthesis occurs
Spermatogenesis development of the sperm from its
precurser cell; successive stages in
spermatogenesis are spermatogonia
(pre-meiotic), spermatocytes (meiotic
stages), spermatids, and spermatazoa
Spindle polymerized tubulin, radiating from
the centrioles formed early in
mitosis; chromosomes attach to the
central point (equator) of the
spindle at their centromeres and,
subsequently, move along the spindle
fibres during anaphase
Spindle poison agent such as colchicine, colcemid,
and vinblastine that prevents tubulin
polymerization and thus, chromosome
migration, resulting in an
accumulation of cells at metaphase;
used to arrest cells at metaphase for
SLRL Sex-linked Recessive Lethals:
recessive lethal mutations located on
sex chromosomes, i.e., the
X-chromosome of Drosophila
Suppressor mutation second site mutation that eliminates
the phenotype produced by a previous
Telocentric chromosome with the centromere at the
end of the chromatids
Telophase stage of mitosis during which the
cell cleaves to give two daughter
Translocation isochromatid rearrangement resulting
from an exchange of material between
Transposition transformation of genetic information
from one chromosome location to
another, e.g., in yeast cells
Vernier reading location of an object, e.g., a cell,
on a microscope slide given as values
on two scales (the X- and Y-axis) of
the microscopic stage
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