United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
Research and Development
EPA-600/S9-84-016 Jan. 1985
&EPA Project Summary
Workshop Proceedings:
Approaches for Improving the
Assessment of Human Genetic
Risk—Human Biomonitoring
A workshop entitled Approaches for
Improving the Assessment of Human
Genetic Risk: Human Biomonitoring
was held in December 1982 to identify
the types of experimental approaches
required to eliminate some of the as-
sumptions and uncertainties of mutagen-
icity risk assessment. The approaches
identified for using biomonitoring data
as a basis for building bridges between
experimental mammals and humans are
discussed in the full workshop proceed-
ings in order to provide direction for the
future research required to improve the
scientific basis for mutagenicity risk
assessment. Emphasis was placed on
practical ways to obtain useful data for
estimating mutation induction. The
workshop participants analyzed avail-
able techniques, their applicability, their
limitations, and possible methods for
their improvement. Discussions were
limited to approaches to identify muta-
tions. The impact of increases in
mutation frequency on the incidence of
human genetic disease was not con-
sidered to be within the scope of the
workshop.
This Project Summary was developed
by EPA's Office of Hearth and Environ-
mental Assessment, Washington, DC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
A considerable genetic disease burden
has been recognized in the human popu-
lation, and it is estimated that perhaps
10% of all human disease has a significant
genetic component. Humans are exposed
to a large and increasing number of
chemical substances, some of which have
mutagenic effects in other organisms and
may pose a genetic risk to people. Because
of the adverse consequences of mutation
induction including genetic diseases and
perhaps cancer, much effort has gone
into designing methods for detecting
mutagenic agents. Recently, combina-
tions of tests that are quite effective at
identifying mutagenic chemicals have
been developed. However, these tests are
not useful in monitoring humans for
heritable mutations, and, thus, the mag-
nitude of the contribution that chemical
mutagens may make to human genetic
disease is highly debated.
Concerns about the ability of man-
made chemical substances to alter the
environment led to the passage of federal
laws to protect against such effects. All of
these laws require a consideration of
adverse health effects in arriving at
regulatory decisions. Some, such as the
Toxic Substances Control Act, require
that specific effects of chemical sub-
stances, including mutagenicity, be con-
sidered in light of the benefits provided by
those chemicals in order to ensure that
human exposure does not result in an
unreasonable risk. This means that the
extent of the potential risk must be
quantified before decisions are made.
The task of quantifying potential muta-
genic risks associated with exposure to
chemical mutagens is complex, and many
assumptions must be made. Extrapola-
tions must be made between species if
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animal data are used to estimate human
genetic risk or between tissues in order to
use data from somatic cell biomonitoring
to estimate heritable genetic risk. In addi-
tion, uncertainties about the exposures
that humans receive and extrapolations
from effects at high experimental levels
to the types of effects expected from
much lower environmental levels further
compound the problems. Thus, quantita-
tive mutagenicity risk assessments are
not scientifically rigorous, because the
data base needed to support the extrapo-
lations is not adequate.
The workshop to which the full report is
addressed was convened to discuss both
direct and indirect methods for biomoni-
toring exposed populations, the biomoni-
toring assays available for mammalian
experimentation, the identification of
human populations exposed to chemical
mutagens, and approaches toward im-
proving mutagenicity risk assessment.
Methods for Biomonitoring
Exposed Populations
Methods for monitoring human popula-
tions to assess genetic risk may be
classified as direct or indirect, depending
on whether extrapolations are necessary
for estimating an effect in humans.
The direct method involves the search
for mutational effects in human popula-
tions exposed to a potential mutagen. It
can be used when there is a large
population of children of exposed persons
available for study. The observations that
might be made on children are of three
types: morphological; cytological; and
biochemical. Morphological observations
include the frequency of dominant muta-
tions, congenital defects and stillbirths,
altered physical growth and development,
and reduced survival. The cytological data
include scoring for an array of chromo-
somal abnormalities. The biochemical
approach involves a search for mutant
proteins not present in either parent.
Because most genetic diseases involve
protein alterations, the biochemical ap-
proach yields less ambiguous results than
the morphological and cytological ap-
proaches. However, this method requires
a higher level of technology.
Until recently, the biochemical ap-
proach in humans and experimental
animals has employed one-dimensional
electrophoresis and quantitative enzyme
level determinations. The development of
two-dimensional polyacrylamide gel elec-
trophoresis (2-D PAGE), however, has
permitted the separation of proteins on
the basis of both charge and size on a slab
gel; as many as 1000 different poly-
peptides contained in a single cell-type
can now be detected. Although not all of
these can be scored unequivocally for
genetic variation, at least 200 polypep-
tides potentially suitable for monitoring
can be identified from the components of
a venous blood sample, and computer
algorithms for both the enhancement of
these images and their scoring are under
development.
These developments may dramatically
improve the monitoring of human popula-
tions for genetic damage. There are
limitations, however. The population size
required for an adequate test of an altered
mutation rate is massive in cases of low-
level exposures. Because of this require-
ment and the fact that most exposures to
mutagens will involve low dosages, this
method is expensive and will be of limited
value scientifically in routine biomonitor-
ing of small groups. However, with appro-
priate exposures, it may be useful in a
coordinated effort pooling offspring from
several high-risk groups.
Despite the technical difficulty, studies
of human germinal mutation rates are
essential in order to understand the
increases in transmitted genetic damage
following exposure to mutagens and as
part of the basis for extrapolating from
animal studies to humans. Thus, it is
essential that at least one comprehensive
study of a sufficiently large or appropri-
ately pooled "worst case" population be
conducted in conjunction with observa-
tion on a variety of "presumptive" indi-
cators of mutation.
In an indirect approach, tissue samples
from exposed persons are analyzed for
genotoxic damage, or body fluids are
tested for the presence of mutagens.
Extrapolations from tissue to tissue are
then made in order to predict the risk of
genetic disease in future generations.
Available methods involve the study of
genetic endpoints, including mutations
and chromosomal aberrations, or deter-
minations of chemical interactions with
DNA.
At least six indirect tests can be per-
formed on germinal tissue from human
populations. Four of these measure ef-
fects on sperm cells, and the remaining
approaches utilize other cytological tech-
niques. Most of the tests for endpoints
that may have a genetic basis are not yet
well characterized genetically. In addition,
not all of these tests are applicable to
females because of the inability to study
ova. This may be a major limitation, since
studies restricted to male germ cells may
overestimate the true risk for the entire
population. Several genotoxic endpoints
can be measured in somatic tissue and
the information generated from such
measurements can be used to indirectly
monitor human populations for heritable
genetic damage. Some of the tests meas-
ure gene mutations or chromosomal
aberrations, while the remainder meas-
ure other endpoints indicative of geno-
toxic damage. Most of these tests can be
conducted in both humans and experi-
mental animals, providing a means for
correlating epidemiological and clinical
data with respect to adverse health
outcomes. Because these tests monitor
in vivo events, they offer several advan-
tages: (i) they detect genotoxicity from
agents whose in vivo effects are depend-
ent upon metabolic or pharmacokinetic
factors; (ii) they potentially are able to
determine the effects of complex mix-
tures; and (iii) for humans and animals,
they may detect heterogeneity for indi-
vidual susceptibilities to genotoxicants
However, for purposes of quantifying
heritable genetic risks, the various tests
using somatic tissues are limited. The
most obvious restriction is that they arc
performed with somatic tissue. Thus
tissue to tissue extrapolations must b<
made in order to make predictions o
transmissible genetic risk. Additionally
for risk assessment purposes, mutationa
rates rather than mutant cells are o
interest, but in somatic cell tests, it i
mutant frequencies rather than muta
tional rates that are quantified. It woul
be difficult to quantify the latter, becaus
little is known about in vivo cell genera
tions or cell kinetics. It is also difficu
with somatic cell tests to define th
genetic basis of the phenotypic change
at the somatic cell level. Although th
difficulty has 'been overcome for TGr 1
lymphocyte and mutant HbG tests, sever
potentially useful somatic tests have bee
abandoned because of the presence
"phenocopies."
Several approaches are available f
studying the frequency of chromosom
aberrations in peripheral blood lymph
cytes, bone marrow cells, and germ eel
Such cytogenetic studies allow compai
sons between effects in somatic cells ai
effects in germ cells, as well as compt
isons between species. Chromosorr
aberrationsprovide unequivocal eviden
of genetic damage and thus constitute
relevant endpoint for reproductive h<
ards. Furthermore, many carcinoge
have been shown to be clastogei
Generally accepted principles for t
conduct of tests and the scoring of resu
have been developed. Considerable
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search has been conducted to assess
spontaneous frequencies, the clastogenic
effect of physical and chemical agents,
and to define the technical variables in
the techniques. The limitations of cyto-
genic analysis are that it is labor intensive
and requires a high level of experience for
accurate scoring. In addition, the data
base on interindividual variation, persis-
tence of lesions, and the sensitivity of
peripheral blood lymphocytes to various
classes of chemicals is relatively small.
Micronucleus tests are also available; the
most common procedure involves the
scoring of micronuclei in the polychro-
matic erythrocytes (PCEs) in mammalian
bone marrow. However, because of the
short lifespan of PCEs (approximately 24
hr) and the requirement for bone marrow
samples, this approach is largely restricted
to tests that use acute exposure regimes
in experimental mammals.
Although it is possible to perform
somatic cell biomonitoring tests on sever-
al different cell-types, the difficulty of
obtaining most tissues necessitates that
peripheral blood will be the main source
of tissue for human biomonitoring studies.
Blood tissue is readily available, and thus
somatic cell systems offer the key advan-
tage of permitting the collection of data
from small populations of individuals.
However, measurements of mutational
events in somatic cells also have defici-
encies. Among these are the limited data
base for chemicals, the insensitivity of
some of the endpoints as indicators of
genotoxic damage, and the lack of evi-
dence for a correlation between elevated
levels of mutations in somatic cells and
an increased risk for adverse health
effects (including the lack of appropriate
bridging models to predict heritable
effects).
Various endpoints that potentially indi-
cate mutagenesis can be detected in both
somatic and germinal tissue. These in-
clude sister chromatid exchanges (SCEs),
chemical interactions with DNA, and DNA
repair. Of these three approaches, the
detection of SCEs is at the most advanced
stage of development. Difficulties are
encountered in all of these methods,
however, because of varying replication
rates and repair capabilities in different
cell-types and because of the restriction
of germ cell measurements to males.
Furthermore, positive findings with these
tests cannot be equated with an increase
in the frequency of mutations. Nonethe-
less, the measurement of SCEs in peri-
pheral lymphocytes is a relatively easy
and sensitive test, and several sensitive
< techniques are being developed for meas-
uring DNA damage. These approaches
may be used to provide information on
internal dosages resulting from human
exposures to chemical substances, and
as such may be employed to provide a
common denominator for tying human
biomonitoring and animal testing meth-
ods together.
Much remains to be learned about
specific DNA damage and its implications
for mutagenesis and carcinogenesis, and
simple models can prove valuable in
making the necessary first steps to im-
prove genetic risk assessment. For initial
studies with experimental animals, total
DNA alkylation may serve as a useful
indicator of dose. Eventually, the specific
type of alkylation product, rate of specific
adduct repair, amount of cell replication,
and the probability of mispairing of
specific adducts needs to be considered.
Although we are presently incapable of
accomplishing this goal, the methodology
for conducting such studies is developing
rapidly.
Biomonitoring Assays
Available for Mammalian
Experimentation
A number of direct and indirect methods
applicable only to animal experimentation
are described in this section of the full
report. These methods are useful for
defining intertissue relationships and for
making comparisons with human data in
order to strengthen the basis for extrapo-
lating between species.
Whole mouse tests for putative heri-
table gene mutations are generally con-
sidered the most valid experimental
approaches for making quantitative
mutagenicity risk assessments. Among
these are the morphological and biochem-
ical specific locus tests and tests for
dominant mutations causing skeletal
defects or cataracts. Other available tests
score for chromosomal aberrations; these
tests include the heritable translocation
test, dominant lethal test, and X chromo-
some loss test. All of these tests, except
perhaps the dominant lethal test, which
cannot be shown to respond only to
mutagenic events, may be used for quan-
tifying genetic risk. An indirect estimation
of heritable genetic effects in mice can be
performed using the mouse spot test.
Although the close biological and evo-
lutionary relationship that exists between
humans and other mammals is the basis
for estimating heritable human genetic
risk from mouse and rat data, there are
several limitations associated with using
studies in animals for predicting human
responses. One is the difficulty of ac-
counting for differences in metabolism,
repair, and cell cycle kinetics. Another is
the need to extrapolate from high acute
dosages, after .involving long sterile per-
iods, to dosage levels that would be more
typical of human exposures. Another
limitation in essentially all assays for
mutagenesis in germ cells is the shortage
of information on females; the great
majority of the available information
comes from males. Consequently, many
assumptions must be made in attempting
to project human genetic risk. This leads
to the inescapable conclusion that there
is no substitute for genetic data from
humans to calibrate the experimental
systems for risk assessment purposes.
Identification of Human
Populations Exposed to
Chemical Mutagens
Considerable effort will be required to
collect human data for assessing genetic
risk for just one chemical substance, and
all sources of information ought to be
drawn upon to select the appropriate
human population for study. A number of
populations at greatest risk should be
identified for potential studies. One is the
children of cancer chemotherapy patients.
There are other analogous populations as
well, and careful thought should be given
in the design of a program to identify and
select the most appropriate ones. Informa-
tion from these studies would be useful
for defining the extent of genetic hazard
and in validating the animal models as
predictors of human risk.
Approaches for the
Improvement of Mutagenicity
Risk Assessment
Many tests are available for identifying
chemical mutagens. Data from combina-
tions of tests provide a basis for making
qualitative assessments of the ability of
chemical substances to cause gene muta-
tions, chromosomal aberrations, and
other effects that are indicative of inter-
action with DNA. However, only a few
tests (i.e., heritable gene mutation and
heritable translocation tests in mice) can
be used by themselves for quantitatively
assessing genetic risk, but they are not
routine tests, and they cannot be used to
estimate human genetic risk directly. By
default, genetic risk must be assessed
qualitatively for most chemical substances.
For decision-making by the federal
government it is no longer adequate
merely to assess genetic risk quantita-
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tively; quantitative assessments are
needed to balance the risk associated
with exposure to a chemical against the
benefit of its use. Because our under-
standing of interorgan relationships is
inadequate, human monitoring data
cannot be used for such purposes. Many
assumptions have to be made before
genetic risk can be estimated quantita-
tively, and the assessments are not
scientifically rigorous. Because of practi-
cal and legal considerations, it is im-
portant to make optimal use of all sources
of information in future genetic risk
assessment efforts and to develop the
science to a point where rigorous assess-
ments can be made.
A. Bridging Human
Biomonftoring Endpoints with
Animal Experimentation
(Defining the Relationships)
Each test system has advantages and
limitations for assessing genetic risk to
humans. It is only by determining how the
endpoints measured in these tests relate
to events that occur in humans that full
advantage can be taken of each. Compar-
ative experimentation involving different
endpoints, test systems, and chemicals is
required to build intellectual bridges
between the systems.
Studies in mice can be used by them-
selves to predict mutagenic effects in
humans, because the same range of steps
between external exposure and produc-
tion of mutant offspring occurs in all
mammals. Measurements of somatic cell
and germ cell events should be performed
in mice in such a way that the relation-
ships between biomonitoring markers and
health outcomes of concern (i.e., genetic
disease, cancer, and birth defects) can be
determined. Such an approach will pro-
vide a basis for using biomonitoring
endpoints to predict adverse health
effects. It will also enable comparisons to
be made between different health out-
comes in order to determine their sensi-
tivity of expression and predictability. It is
necessary to conduct at least one study in
such a manner so that the data generated
in an experimental animal study can be
compared to the results obtained from
similarly exposed humans. Until this is
done, the applicability of animal data for
human risk assessment will be unknown.
B. Other Types of Testing
Needed
Because it is impossible to study genetic
damage in more than a very few human
4
populations, it will be necessary to rely
heavily on animal experimentation and
short-term biomonitoring tests to predict
human risk. This requires bridge building
between human heritable mutagenicity
data, other human biomonitoring data,
and animal heritable mutagenicity and
biomonitoring data. Although the specific
models that can be used to make bridges
remain controversial, there is some agree-
ment that ratios (or parallelograms as
they are sometimes called) involving
dosimetry should be further explored. As
such studies are conducted, knowledge
of biological processes rather than statis-
tical models can start to drive the risk
assessment procedure. Flexibility should
be maintained so that the risk assessment
process works differently when different
amounts of data are available and so that
the level of sophistication can be in-
creased as better data become available.
In addition, the effects caused by a
chemical in one cell-type might not predict
its effects in a different cell-type. Thus, it
is important to consider cellular specific-
ities of certain chemicals. Attention also
should be paid to the homeostatic mech-
anisms of humans and experimental
animals that may be important with
respect to disease outcomes, since the
stage of the cell cycle, level of differenti-
ation, and location in the body all affect a
tissue's response to toxic insults.
Need for Coordination of Efforts
To ensure maximum efficiency in col-
lecting relevant data, it is desirable to
search systematically for information on
existing efforts related to genetic risk
assessment rather than to attempt to set
up overlapping independent studies. Al-
though effective collaboration among
agencies is still sporadic, there are some
promising developments. For example,
the coordination between the U.S. Envi-
ronmental Protection Agency and the
National Toxicology Program (NTP) to
obtain dosimetric information on chemi-
cals being tested in the mouse specific
locus tests should enable existing NTP
studies to better be used for genetic risk
assessment. It now seems appropriate
that an oversight committee be formed
for guidance on needs in human biomoni-
toring and to facilitate the coordination of
efforts in genetic risk assessment.
The research effort required to answer
major questions in genetic risk assess-
ment must be cumulative rather than
episodic, and funding for these efforts
should endure over many years. With
proper support, an oversight committee
could help to ensure that this is accom-
plished. Investigators studying exposure
or genotoxic damage in different tissues
and different organisms must somehow
integrate and focus their efforts. More
effective sharing of valuable materials
would certainly be useful in this respect.
Within the purview of an oversight com-
mittee, a repository for biological mate-
rials obtained from animals and humans
that have been exposed to putative
mutagens should be established.
A series of regularly scheduled work-
shops should be inaugurated to facilitate
collaboration. These workshops should
include investigators using the biological
materials in the repository and should
provide for a cumulative review, compari-
son of results, and the identification of
research needs.
It may be worthwhile to consider the
selection of a few key compounds for
concentrated, long-term efforts in genetic
risk assessment. Since a long-term effort
to collect data on chemical mutagenesis
in human germ cells is a major under-
taking, with important implications for
genetic risk assessment, it should not be
undertaken lightly. There are other issues
that could be considered by an oversight
committee.
Efforts should also be coordinated
among government agencies, industrial
concerns, and the academic community
Some preliminary efforts are underway
and it is hoped that these efforts will b<
expanded.
The final section of the full report con
tains an extensive reference list docu
menting scientific opinions expressed b
participants in the workshop.
U. S. GOVERNMENT PRINTING OFFICE: 1985/559-111/107<
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This Project Summary was prepared by staff of the Office of Health and
Environmental Assessment, Washington, DC 20460.
John R. Fowle, III, is the EPA Project Officer (see below).
The complete report, entitled "Workshop Proceedings: Approaches for Improving
the Assessment of Human Genetic Risk—Human Biomonitoring," (Order No.
PB 85-103 018; Cost: $10.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC 20460
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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