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Commentary
A Return to the Origin of the EMGS: Rejuvenating the Quest for
Human Germ Cell Mutagens and Determining the Risk to Future
Generations
Francesco Marchetti ,*George R. Douglas, and Carole L.Yauk
Environmental Health Science Research Bureau, Health Canada, Ottawa, Ontario, Canada
Fifty years ago, the Environmental Mutagen Society
(now Environmental Mutagenesis and Genomics
Society) was founded with a laser-focus on germ cell
mutagenesis and the protection of “our most vital
assets”—the sperm and egg genomes. Yet, five
decades on, despite the fact that many agents have
been demonstrated to induce inherited changes in
the offspring of exposed laboratory rodents, there is
no consensus on whether human germ cell muta-
gens exist. We argue that it is time to reevaluate the
available data and conclude that we already have
evidence for the existence of environmental expo-
sures that impact human germ cells. What is missing
are definite data to demonstrate a significant
increase in de novo mutations in the offspring of
exposed parents. We believe that with over two
decades of research advancing knowledge and
technologies in genomics, we are at the cusp of
generating data to conclusively show that environ-
mental exposures cause heritable de novo changes
in the human offspring. We call on the research
community to harness our technologies, synergize
our efforts, and return to our Founders’original
focus. The next 50 years must involve collaborative
work between clinicians, epidemiologists, genetic
toxicologists, genomics experts and bioinformaticians
to precisely define how environmental exposures
impact germ cell genomes. It is time for the research
and regulatory communities to prepare to interpret
thecomingoutpouringofdataanddevelopa
framework for managing, communicating and
mitigating the risk of exposure to human germ cell
mutagens. Environ. Mol. Mutagen. 61:42–54,
2020. ©2019 Her Majesty the Queen in Right of Canada
Key words: mutation; germ cell; transgene; test guideli ne; heritable; nextg eneration sequencing
ROOTS OF EMGS IN GERM CELL MUTAGENESIS
Fifty years ago, the Environmental Mutagen Society
(now Environmental Mutagenesis and Genomics Society,
EMGS) was founded with the goal of identifying and elim-
inating agents from the environment that cause mutations
in germ cells to protect the health of human populations
(DeMarini 2020). Soon after, a committee was established
to formalize and publicize the fundamental role and mis-
sion of the EMGS (Committee 17 1975). Most of the con-
ferences and publications from this period emphasized the
health hazards of germ cell mutations (e.g., Workshop on
“The Evaluation of Chemical Mutagenicity Data in Rela-
tion to Population Risk, Research Triangle Park, North
Carolina, April 26–28, 1973”; Environmental Health Per-
spectives, Vol. 6, December 1973). Despite the main
emphasis on germ cell-mediated genetic effects, there was
one paper at the workshop (Ames 1973) that profoundly
changed the direction and intent of mutagenicity testing
from a germ cell orientation to the prediction of carcinoge-
nicity. As a result of the ground-breaking invention of the
Ames assay and the demonstrated high degree of correla-
tion between mutagenesis and carcinogenesis, the vast
majority of genetic toxicology test development and appli-
cation became centered on the relationship between muta-
tion and cancer.
Notwithstanding the increased emphasis on cancer, germ
cell mutation has remained an endpoint of regulatory con-
cern. Numerous reports have been published and regulatory
practices promulgated related to the application and use of
germ cell mutagenicity data (see review by Singer and
Yauk 2010). Indeed, there are examples of regulatory
assessment of germ cell data at a frequency similar to that
for the regulatory assessment of cancer data (Yauk et al.
2015a). Furthermore, there is renewed interest in the
Reproduced with the permission of the Minister of Health Canada
Grant sponsor: Health Canada.
*Correspondence to: Francesco Marchetti, Environmental Health Science
and Research Bureau, Health Canada, 50 Colombine Driveway, Ottawa,
ON K1A 0K9, Canada. E-mail: francesco.marchetti@canada.ca
Received 29 July 2019; Revised 21 August 2019; Accepted 28
August 2019
DOI: 10.1002/em.22327
Published online 31 August 2019 in
Wiley Online Library (wileyonlinelibrary.com).
Environmental and Molecular Mu tagenesis 61:42^54 (2020)
©2019 Her Majesty t he Queen in Right of Canada
regulatory assessment of germ cell hazard and risk because
of the wide international implementation of the Globally
Harmonized System (GHS) of classification and labelling
of chemicals (United Nations 2017), which considers germ
cell mutagenicity for hazard classification.
This commentary provides a brief summary of the issues
surrounding the use of germ cell mutagenicity data in regu-
latory hazard and risk assessment, the application of new
facilitating technologies, and the prospects for a germ cell
genotoxicity renaissance.
IMPACT OF GERM CELL MUTATIONS
The role of mutations in the carcinogenic process is
well established and thus, mutagenicity testing has been
routinely conducted in order to identify agents that are
capable of inducing mutations and may initiate cancer
(Heflich et al. 2020). In this context, it should be noted that
mutation is used a proxy for the ability of a compound to
induce cancer and not as a direct determinant of cancer
development. Also, although somatic mutations are associ-
ated with a broad spectrum of human diseases (Erickson
2014; Goschalk et al., 2020), there is still limited evidence
that a somatic mutation induced by an environmental expo-
sure may result in an adverse health effect other than cancer.
The impact of mutations in germ cells is dramatically
different. A single mutation in a germ cell will produce an
individual where every cell in the organism contains that
mutation. Thus, the chance of that mutation impacting the
health of the affected individual is much larger and it is not
restricted to cancer. Indeed, the Human Gene Mutation
Database (Stenson et al. 2017) currently catalogues over
260,000 different adverse mutational events affecting
approximately 10,000 genes (accessed June 2019).
Although point mutations represent the largest group of
mutations, genomic changes affecting a few to a few thou-
sand base pairs, translocations and complex chromosomal
aberrations are also observed. The database is a critical
resource to gather insights into the landscape of mutations
that are linked to human diseases, and while it provides no
information on the causes of the mutations, it would be
hard to imagine that not a single one of those mutations
was induced by an exogenous factor.
Although germ cell mutations are relatively rare at the
individual level, they can have a profound impact when
considered at the population level. It was recently estimated
that de novo mutations contribute to developmental disor-
ders in as many as 400,000 births per year (Deciphering
Developmental Disorders Study 2017). At the population
level, even a relatively small increase in mutation rate cau-
sed by an environmental exposure can have astounding
consequences. For example, an analysis that considered
only the spectrum of mutations that are expected to be
induced by tobacco smoke and the roughly 700 genes that
have been associated so far with intellectual disability
showed that even a modest 25% increase in tobacco-
induced sperm mutations would lead to over 500,000
affected children per generation across the global popula-
tion (Beal et al. 2017). The economic consequences of
these tobacco-induced de novo mutations for the health
care system can be easily estimated in the dozens of billion
US dollars.
The above example provides just a glimpse of the socie-
tal and economic impacts of environmentally induced de
novo mutations and of the importance of identifying and
eliminating exposure to environmental agents that can
induce these effects.
THE ENIGMA OF THE EXISTENCE OF HUMAN GERM CELL
MUTAGENS
Despite many decades of research, regulatory decisions
basedongermcellmutageniceffectsarestymiedbythelack
of even one established, and widely acknowledged, human
germ cell mutagen. As noted by DeMarini (2012), this
enigma may stem from the lack of an organization that is
specifically tasked with assessing the available data to deter-
mine whether the weight of evidence is sufficient to conclude
that an agent is a human germ cell mutagen (e.g., similarly to
what the International Agency for Research on Cancer
(IARC) does for carcinogens). We wholeheartedly endorse
this assessment and argue that we already have sufficient data
to say that human germ cell mutagens exist and that the cur-
rent notion to the contrary is not grounded in facts. Below,
we briefly discuss the evidence that substantiates the exis-
tence of human germ cell mutagens.
Over the past 80 years, a large body of literature has
accumulated on the existence of many germ cell mutagens
in animals. These studies demonstrate that exposure of lab-
oratory rodents to genotoxic agents spanning different
modes of action not only results in the induction of genetic
damage in the germ cells themselves, but also in the trans-
mission of de novo genetic changes to the offspring.
Several reviews have been published on agents that cause
genetic damage in rodent germ cells resulting in the induc-
tion of chromosomal structural aberrations (Marchetti and
Wyrobek 2005), aneuploidy (Pacchierotti et al. 2019),
gene mutations (Marchetti et al. 2018b), dominant lethal
mutations (Green et al. 1985) and inherited mutations
(Russell 2004). The information contained in these reviews
is sufficient to identify 84 agents as rodent germ cell muta-
gens (Table I), which is likely a large underestimate of the
true number of agents that are known to be genotoxic in
rodent germ cells. Thirty-one of the mammalian germ cell
mutagens have been shown to significantly increase
inherited genetic changes in the offspring of exposed
males (Table I). Thus, it is unquestionable that many envi-
ronmental agents when tested under controlled laboratory
Environmental and Molecular Mutagenesis. DOI 10.1002/em
43Quest for Human Germ Cell Mutagens
conditions damage the genetic material of rodent germ
cells and result in the transmission of genetic defects to
the offspring. Is it realistic then to think that none of these
agents possess these characteristics when humans are
exposed? We believe that the answer to this question is an
unequivocal “no”and that there are many factors that are
TABLE I. Mammalian Germ Cell Mutagens
a
Agent Germ cells
b
Embryo
c
Offspring
d
Agent Germ cells
b
Embryo
c
Offspring
d
Acrylamide M, R M M 7,12-Dimethylbenz[a]anthracene M M
Ionizing radiation H, M M H
e
, M Busulfan M M
Tobacco smoke H, M M H
f
, M Carbendazim DH, SH M
Benzo(a)pyrene M M M Chemotherapy cocktails H, M M
Bleomycin M M M Colchicine CH, DH, M M
Chlorambucil M M M Di-(2-ethylhexyl)-phthalate M M
Cyclophosphamide M M M Diepoxybutane M M
Ethyl methanesulfonate M M M Ethanol H, M M
Ethylene oxide M M, R M Hycantone methanesulfonate M R
Etoposide CH, H, M M M Nocodazole M M
Glycidamide M M M Taxol M M
Isopropyl methanesulfonate M M M Vinblastine sulfate CH, M M
Melphalan M M M 6-Mercaptopurine M
Methyl methanesulfonate M M M Captan M
Mitomycin C M M M Chloramphenicol M
N-ethyl-N-nitrosourea M M M Colcemid M
N-methyl-N-nitrosourea M M M Di-2-ethyl-hexyladipate M
Procarbazine M M M
Dihydroergotoxin
methanesulfonate M
Triethylenemelamine M M M Dimethylmyrelan M
Trophosphamide M M M Ergotamine tartrate M
1-(2-Chroethyl)
3-cycloehxyl-1-
nitrosourea R M Forfestrol tetrasodium M
1,3-Butadiene M M Fotrin M
Chlormethine M M Hexamethylphosphoramide M
Dacarbazine M M Methyl mercury chloride SH, M
Diethyl sulfate M M Methysergide hydrogen maleate M
Ifosfamide M M Mitomen M
n-Propyl methanesulfonate M M
N-methyl-N0-nitro-N-
nitrosoguanidine M
ThioTEPA M M Rubratoxin B M
Trimethyl phosphate M M Saccarin M
N-propyl-N-nitrosourea M M Triflupromazine M
Trichlorfon M H
g
Tris(2-methylazidiniyl)phosphine
oxide M
1,2-Dibromo-3-chloropropane M M 4-Nitroquinoline-1-oxide M
Acrylonitrile H Griseofulvin M
Air pollution M, HG Hydroquinone M
Amsacrine M Hydroxyurea M
Benomyl M Merbarone M
Benzene H Nitrilotriacetic acid M
Chloral hydrate M Orthovanadate M
Diethylstilbestrol M Podophyllotoxin CH
Econazole M Teniposide M
Epirubicin hydrochloride M Thiabendazole M
Fenvalerate H Vincristine sulfate CH
a
Agents reported as having positive results in the following papers: Green et al. (1985); Russell (2004); Marchetti and Wyrobek (2005); Marchetti et al.
(2018a); Marchetti et al. (2018b); Pacchierotti et al. (2019). For each agent, the species used in the studies is reported: CH (Chinese hamster); DH
(Djungarian Hamster); H (Humans); HG (Herring gull); M (Mouse); R (Rat); SH (Syrian Hamster).
b
Includes results obtained with the cytogenetic or FISH analyses of either sperm or oocytes.
c
Includes results obtained with the dominant lethal assay.
d
Includes results obtained in rodents with the specific locus test, heritable translocation assay in F1 males or tandem repeats and studies with human families.
e
Dubrova et al. (1996, 2002, 2006).
f
Secretan et al. (2009).
g
Czeizel et al. (1993).
Environmental and Molecular Mutagenesis. DOI 10.1002/em
44 Marchetti et al.
responsible for the apparent lack of human germ cell
mutagens.
Undoubtedly, there are many experimental variables that
make the chance of identifying rodent germ cell mutagens
more likely than doing the same in humans. First, studies
in laboratory animals are generally conducted at dose levels
that seldom occur in human populations. These studies use
primarily acute or subacute regimens rather than the
chronic exposure that is more common in humans. Acute
exposures use much higher doses than those that can be
applied under chronic conditions and are more likely to
result in a measurable effect because the amount of DNA
lesions induced at one time can overwhelm the DNA repair
defense mechanisms (O’Brien et al. 2015; Yauk et al.
2015b). Second, these experiments generally use animals
with a homogenous genetic background that is not repre-
sentative of the genetic diversity present in the human pop-
ulation. A genetically homogenous population reduces the
variability in the response and increases the statistical
power of detecting an effect. Third, exposure parameters
and experimental conditions can be very carefully con-
trolled in the laboratory to significantly reduce confounding
factors that may affect the response. Indeed, major chal-
lenges of conducting studies on the effects of environmen-
tal chemicals in human populations are the precise
characterization of the exposure and the identification of a
true control population. Fourth, technological limitations
have contributed to the challenges of identifying human
germ cell mutagens as the most common methods used for
investigating induced mutations in rodent germ cells
(e.g., the specific locus test [SLT]; Russell et al. 1998) are
not applicable to humans. Thus, both technological and
methodological difficulties associated with human studies
may be in part responsible for the current lack of consensus
on the existence of human germ cell mutagens.
Despite these caveats, rodent germ cell mutagens are pri-
mary candidates for human germ cell mutagens and,
indeed, there are several examples of agents that induce
effects in both rodent and human gametes. In his commen-
tary, DeMarini (2012) identified ionizing radiation, chemo-
therapeutic agents, tobacco smoking and air pollution as
potential human germ cell mutagens. The latter two expo-
sures constitute complex mixtures that contain dozens of
potential candidate germ cell mutagens, the most notable
being genotoxic polycyclic aromatic hydrocarbons (PAHs)
like benzo[a]pyrene (BaP). As briefly summarized below,
the available human data for these agents do indeed support
their categorization as human germ cell mutagens.
Since the pioneering work of Russell et al. with the SLT
(reviewed in Russell et al. 1998), ionizing radiation has
been one of the best characterized and most studied rodent
germ cell mutagen. In the early 90s, Dubrova et al. (1996)
reported increased numbers of mutations in tandem repeat
DNA sequences (minisatellites) in the offspring of men
who had been exposed to ionizing radiation during the
Chernobyl accident. These results were highly controversial
at the time because of criticisms on the selection of concur-
rent controls. However, follow up work with better mat-
ched control populations and longitudinal study designs
confirmed the initial findings (Dubrova 2002; Dubrova
et al. 2002, 2006) and were further supported by studies
with laboratory rodents demonstrating that ionizing radia-
tion induced the same types of mutations under controlled
laboratory conditions (Dubrova et al. 1993, 1998). The
findings of Dubrova et al. were also criticized because of
the type of mutations that were measured. It was thought
that alterations in minisatellite sequences had no health
impact and, thus, were of little significance for human
health. Since then, we have a much better understanding of
how duplications and deletions in noncoding genomic
regions (including tandem repeats) can impact human
health (Mirkin 2007; Mori et al. 2013; Lupski 2015). In
light of today’s knowledge and understanding that point
mutations and genomic alterations in coding genes repre-
sent only a portion of the possible genetic changes with
health-related implications, we contend that a re-evaluation
of the original results of Dubrova et al. leads to the conclu-
sion that these findings can be considered as evidence
supporting the induction of heritable de novo mutations in
humans by ionizing radiation.
Chemotherapy cocktails generally contain chemicals that
are known to be genotoxic and have been shown to induce
both chromosomal abnormalities (reviewed in Wyrobek
et al. 2005) and tandem repeat mutations (Vilarino-Guell
et al. 2003; Glen et al. 2008; Glen and Dubrova 2012) in
rodent sperm. Studies in cancer patients undergoing chemo-
therapy have also shown significant increases in the fre-
quency of sperm carrying either aneuploidy or structural
chromosomal abnormalities (Robbins et al. 1997; Frias
et al. 2003). Although in the majority of cases these
increases were temporary with the frequencies of sperm
with chromosomal abnormalities returning to baseline
levels with increasing time from the end of chemotherapy
(Martin et al. 1997; Robbins et al. 1997; Frias et al. 2003),
a few studies reported significantly higher levels of abnor-
mal sperm many years after chemotherapy (De Mas et al.
2001; Tempest et al. 2008). These results suggest a perma-
nent effect of the chemotherapeutic cocktails on human
stem cell spermatogonia. Importantly, these effects mirror
those observed in the sperm of laboratory animals and
demonstrate that when exposure levels are comparable,
similar effects are seen in both rodent and human germ
cells. Thus, for chemotherapeutic agents there is clear evi-
dence that results obtained in rodent germ cells are predic-
tive of similar effects in human germ cells.
Tobacco smoke is one of the most widespread environ-
mental exposures in human populations. The detrimental
effects of tobacco smoking have been known for many
decades, but in recent years there has been a renewed
appreciation for its effects in the germ cells of smokers and
Environmental and Molecular Mutagenesis. DOI 10.1002/em
45Quest for Human Germ Cell Mutagens
their potential consequences for the offspring. A recent
review on the effects of tobacco smoking identified
increases in DNA strand breaks, aneuploidy and mutations
as some of the genetic changes that are present in the
sperm of tobacco smokers (Beal et al. 2017). Also, studies
in both rodents (Yauk et al. 2007; Marchetti et al. 2011)
and humans (Linschooten et al. 2013) have consistently
reported increases in tobacco-induced tandem repeat muta-
tions. Perhaps surprisingly, there are yet no human data
showing an increase in de novo mutations in the offspring
of smokers, although whole genome analysis of cohorts of
families with smoking fathers was identified as a priority
research area (Yauk et al. 2013). Nevertheless, support for
the hereditary effects of tobacco smoke can be found in the
IARC’s assessment that preconception paternal smoking is
causally linked to an increased risk of childhood leukemia
in the offspring (Secretan et al. 2009).
Among the four potential human germ cell mutagens
proposed by DeMarini, air pollution is the one with the
more limited data in humans. However, multiple studies in
rodents (Somers et al. 2002; Yauk et al. 2008) and wildlife
species (Yauk and Quinn 1996; Yauk et al. 2000) have
consistently shown the impact of air pollution on the germ
cell genome, including the demonstration that the muta-
genic effects of air pollution in germ cells reside with par-
ticulate matter (Somers et al. 2004). Importantly, all these
studies of environmentally exposed animals resulted in her-
itable tandem repeat mutations induced by ambient levels
of air pollutants. Even in the absence of direct evidence in
humans, the available animal data would be considered,
when evaluated within an IARC style assessment, to be
sufficient to conclude that air pollution is likely to be a
human germ cell mutagen.
PAHs are exemplary mutagens in tobacco smoke and air
pollution. A large body of literature demonstrates that BaP
is highly mutagenic to adult rodent germ cells. Studies
clearly show that BaP causes both tandem repeat and gene
mutations in rodent spermatogonial stem cells and differen-
tiating spermatogonia (Olsen et al. 2010; Verhofstad et al.
2011; Xu et al. 2014; O’Brien et al. 2016a, b; Rowan-
Carroll et al. 2017). Moreover, a recent study applying
modern genomic technologies provides clear evidence that
BaP induced de novo mutations (both single nucleotides
and large duplications) in the offspring of exposed male
mice (Beal et al. 2019). These data also lend weight to the
relevance of tandem repeat mutations as biomarkers of
potential mutagenicity in other locations in the genome. In
parallel with this strong evidence of germ cell mutagenicity
in rodents, PAH DNA adducts are detected in human
sperm samples of individuals exposed to air pollution (Jeng
et al. 2015; Oliveri Conti et al. 2017). Sperm of human
smokers exhibit high levels of BaP DNA adducts as well
as increased burdens of bulky adducts and oxidative lesions
in general (reviewed in Beal et al. 2017). Therefore, the
presence of BaP and other bulky PAH adducts in human
sperm, and strong evidence supporting that BaP induces
mutations in rodent sperm that are transmitted to their off-
spring, strongly imply that BaP and PAHs are likely to
cause germ cell mutagenicity in humans.
Evidence for the induction of genetic damage in
human germ cells also comes from many studies that
have reported significant increases in genetic abnormali-
ties in the sperm of workers occupationally exposed to
benzene (Xing et al. 2010; Marchetti et al. 2012), acrylo-
nitrile (Xu et al. 2003) and fenvalerate (Xia et al. 2004)
among others. When the available human germ cell data
are evaluated as a whole, the unavoidable conclusion is
that we already have sufficient data to say that human
germ cell mutagens exist. Admittedly, what is missing is
the demonstration that some of these effects observed in
human germ cells are transmitted to the next generation.
One possible such an example is the reported case of a
cluster of Down syndrome cases in a human population
linked to environmental exposure to trichlorfon (Czeizel
et al. 1993). However, this finding is outweighed by the
current failure of epidemiological studies in children of
atomic bomb (Schull 2003) and cancer (Mulvihill 2012)
survivors to demonstrate an increase in inherited diseases
in the offspring. Although these human epidemiological
studies have shortcomings (discussed in detail in Schull
2003; DeMarini 2012; Mulvihill 2012), data demonstrat-
ing the induction of de novo mutations in the offspring
of exposed parents will be necessary to change this
notion that there are no human germ cell mutagens. As
discussed later, we believe that, as a scientific commu-
nity, we are at the cusp of applying the latest genomic
approaches to demonstrate transmission of environmen-
tally induced de novo mutations to human offspring and
conclusively demonstrate the existence of human germ
cell mutagens.
ARE HUMAN GERM CELL S FUNDAMENTALLY DIFFERENT
FROM RODENT GERM CELLS?
The results obtained in rodents may not be predictive of
similar effects in humans if there are fundamental differ-
ences between human and rodent spermatogenesis or if
human germ cells have a more efficient DNA damage
response than rodent germ cells. However, there is no solid
evidence for either of these two possibilities. An extensive
description of the process of spermatogenesis in mamma-
lian species, including DNA repair competency, is outside
of the scope of this commentary and the reader is directed
to a few comprehensive reviews on the topic (Olsen et al.
2005; Amann 2008; Hermo et al. 2010). Here, we aim to
succinctly argue that there are no physiological differences
in spermatogenic processes that can be invoked to cause
differences in the response of humans and rodents to envi-
ronmental germ cell mutagens.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
46 Marchetti et al.
The process of spermatogenesis has been studied in
detail in humans and in many laboratory animals. Sper-
matogenesis is remarkably similar among mammalian spe-
cies with the major difference being the time necessary to
produce mature sperm from stem cell spermatogonia,
which is species specific (Adler 1996). Furthermore, many
of the genes that are known to be essential for spermato-
genesis are conserved across species (Bonilla and Xu 2008;
White-Cooper and Bausek 2010) and defects in a particular
gene can produce the same phenotype in multiple species
(Vangompel and Xu 2011). Also conserved across species
is the fact that the DNA repair capacity is maximum in
spermatogonia and gradually disappears during the
postmeiotic phase of spermatogenesis (Olsen et al. 2005).
Data in rodents clearly show that this period is most vul-
nerable to the induction of genetic lesions that are retained
in the fertilizing sperm and that result in the formation of
de novo genetic defects in the developing embryo
(Marchetti and Wyrobek 2005).
The apparent lack of human germ cell mutagens cannot
be ascribed to a higher resistance of human germ cells to
the detrimental impact of genotoxic agents. As already dis-
cussed, exposure to comparable doses of ionizing radiation
or chemotherapeutic agents induce similar effects in both
rodent and human sperm. Furthermore, it is broadly
accepted that aging is a human germ cell mutagen. An
increase in the risk of having a child with a genetic syn-
drome as function of paternal age has been known for
decades (Crow 2000). Studies with next generation
sequencing (NGS) have unequivocally shown the presence
of a paternal age effect for the induction of de novo muta-
tions that are transmitted to the offspring (Kong et al.
2012; Francioli et al. 2015; Rahbari et al. 2016). More
recent studies have also demonstrated an increase in de
novo mutations, albeit smaller than the paternal effect, as
function of maternal age (Maretty et al. 2017; Gao et al.
2019). In these genomic studies, the stronger paternal age
effect is attributed to the continuous replication of stem cell
spermatogonia throughout the male’s reproductive life and
to the possibility of introducing an error during DNA repli-
cation. Interestingly, the potential contribution of an envi-
ronmental exposure in the paternal age effect is seldom
considered. For example, a study that analyzed the paternal
age effect in three human families with multiple children
showed that the slope of the paternal age effect varied
greatly among the fathers (Rahbari et al. 2016). While dif-
ferent baseline efficiencies in DNA repair activities could
contribute to this variability, so could differential exposure
of the fathers to an environmental mutagen. Finally, analy-
sis of the types of de novo mutations transmitted to human
offspring show clustering of mutations (Francioli et al.
2015) as observed in the offspring of mutagen-exposed ani-
mals (Adewoye et al. 2015; Beal et al. 2019) and signatures
of mutational processes that point to DNA damage as the
source of de novo mutations (Gao et al. 2019). It is difficult
to imagine a process by which the DNA repair machinery
of human germ cells is able to distinguish between lesions
that are induced by endogenous processes vs. those induced
by exogenous agents and are able to completely repair the
latter but not the former.
In summary, there is little biological plausibility to sup-
port the notion that the response of human germ cells to an
environmental insult is fundamentally different from that
occurring in rodent germ cells, which would negate the
possibility that established rodent germ cell mutagens
would also be human germ cell mutagens.
TECHNICAL LIMITATIONS OF AVAILABLE GERM CELL
MUTATION TESTS
The search for human germ cell mutagens has been ham-
pered by the lack of efficient test methods to detect the
impact of environmental agents on the germline. Even in
laboratory rodents, the study of germ cell mutagenesis has
suffered from the limitations of available tests, such as the
need for an exceedingly large number of animals. A prime
example is the SLT developed in the late 1950s at Oak
Ridge National Laboratory. For many decades, it was the
only available test to detect de novo mutations in the
offspring of exposed mice. The SLT was instrumental in
establishing occupational exposure limits for radiation
workers (Neel and Lewis 1990) and made fundamental
contributions to the elucidation of the varying sensitivity of
the different phases of spermatogenesis to environmental
exposure (Russell 2004). However, a typical experiment
required the analysis of several hundred thousand animals,
something that would not be considered remotely feasible
today, both for financial and ethical considerations. Other
available tests, although not requiring as many animals as
the SLT, did not provide information on the heritability of
mutations in live animals (i.e., the dominant lethal test) or
assayed only on a specific type of genetic damage
(i.e., reciprocal translocation test; results with both tests
reviewed in Marchetti and Wyrobek 2005).
Over the years, a variety of tests have been developed to
assess different types of genetic damage in germ cells and
applied to the question of environmentally induced germ
cell mutagenesis. Corollary assays from some of the current
rodent germ cell tests are available for use with human
sperm and have allowed a direct comparison of the
response to environmental insult across species. An in-
depth review of the strengths and limitations of these tests
is provided in the 2013 IWGT report on germ cell testing
(Yauk et al. 2015a). Here, we specifically highlight two
approaches that represented important breakthroughs in the
field of germ cell mutagenesis.
As discussed, the analysis of tandem repeat mutations in
sperm has been instrumental in demonstrating consistency
of effects across species and contributed to the overall
Environmental and Molecular Mutagenesis. DOI 10.1002/em
47Quest for Human Germ Cell Mutagens
weight of evidence that ionizing radiation, chemotherapeu-
tic drugs, tobacco smoking and air pollution are human
germ cell mutagens. The analysis of tandem repeat muta-
tions provided, for the first time, an opportunity to study
heritable mutagenesis in humans using a reasonable and
manageable population size. Furthermore, the assay can be
conducted in any species, including wild animals, and is
equally applicable to the analysis of sperm of exposed
fathers and their children enabling a direct demonstration
of the heritability of these mutations. Unfortunately, uncer-
tainties over the impact of these types of mutations for
human health and a technically demanding protocol has
hampered the broad acceptance of tandem repeat assays
among the regulatory and research communities. However,
extensive efforts are ongoing to develop advanced genomic
approaches (e.g., long-read sequencing; Chaisson et al.
2019; Levy-Sakin et al. 2019) for rapidly and efficiently
characterizing structural variation in the genome and it is
envisioned that these methods will become important tools
for analyzing environmentally induced structural variation
in the germ cell genome (Salk and Kennedy 2020).
A breakthrough for germ cell mutagenicity for regulatory
testing also occurred at the end of the 20th century when
transgenic rodent (TGR) models were developed (reviewed
in Lambert et al. 2005). These models have bacterial
reporter genes integrated into their genome that can be
recovered from any tissue and used in an in vitro assay to
measure mutations that were induced in vivo. The use of
TGR models for mutagenicity testing was later codified in
an Organisation for Economic Co-Operation and Develop-
ment Test Guideline (OECD 2013). TGR models provided
the opportunity to conduct in vivo mutagenicity testing
using a reasonable number of animals, in line with other
well-established genotoxicity tests for somatic cells, and to
directly compare the response in germ cells with that in
somatic tissues. This is particularly important because germ
cell mutagenicity is a health hazard criterion in the GHS
classification and labelling of chemicals (United Nations
2017) and has resulted in an increased demand for testing
in this area. The use of TGR models for germ cell muta-
genesis was recently summarized together with recommen-
dations on proper experimental design for integration with
somatic tissue mutagenicity testing (Marchetti et al.
2018a, b).
A major limitation of existing germ cell methods is the
inability to account for postfertilization events. There is a
wealth of data showing that the manifestation of damage
induced in postmeiotic germ cells into a fixed genetic
change, whether point mutation or chromosomal aberra-
tion, is not completed until after the sperm fertilizes the
egg (Marchetti and Wyrobek 2005). There is also an
example of a meiotic DNA lesion that does not manifest
itself as a permanent genetic abnormality until after fertili-
zation (Marchetti et al. 2015). These data point to the
importance of the perifertilization period in determining
the amount and types of lesions that originate de novo
genetic changes, and in particular, of the role that an effi-
cient DNA repair system in the fertilized egg can play in
fundamentally mediating how much of the sperm DNA
lesions are correctly repaired (Marchetti et al. 2007;
Derijck et al. 2008). We believe that recent developments
in genomic methodologies (described later) are well posi-
tioned to fill this key research gap and also address
another current limitation of germ cell tests; i.e., the lack
of efficient and practical methods to analyze the induction
of mutations in female germ cells that has resulted in
a paucity of experimental data on female germ cell
mutagenicity.
RISK ASSESSMENT OF GERM CELL MUTATIONS OVER THE
NEXT 50 YEARS OF THE EMGS
During the first 50 years of the EMGS, the mutagenic
activity of chemicals has been evaluated primarily in a
yes/no fashion. However, a paradigm shift is ongoing in
genetic toxicology to move away from a purely qualitative
analysis of genotoxic responses (Dearfield et al. 2017). The
proposed path forward implements refined dose–response
modeling approaches to establish point of departure (PoD)
metrics for potency comparisons, with the long-term vision
of using mutation as a PoD in human health risk assess-
ment (Johnson et al. 2015; Wills et al. 2016). The chal-
lenges of this paradigm shift and the necessary
improvement in methods to measure mutagenicity, and
how to refine risk assessment approaches to encourage
uptake by regulatory bodies, are discussed in Heflich
et al. (2020).
This paradigm shift has important implications for the
risk assessment of germ cell mutations and the need to
conduct germ cell mutagenesis testing. A general assump-
tion in risk assessment is that evaluations based on
somatic cell mutation assays protect the germline by
default. Historical and experimental data support that
somatic cell mutation assays predict putative mutagenic
effects in the germline. Indeed, there is currently no evi-
dence for a unique germ cell mutagen, although caution
should be exercised in drawing broad conclusions because
chemicals that are negative in somatic cells are rarely
tested in germ cells (Yauk et al. 2015a). This new empha-
sis on dose–response modeling requires that reliance on
somatic mutagenicity tests to predict effects in germ cells
now extends also to establishing PoDs that protect the
germline as well. However, we already know that this is
incorrect. Several examples exist demonstrating germ cell
effects at doses that do not elicit adverse effects in somatic
cells; thus, PoD based on somatic endpoints may underes-
timate the risk to the germline. For example, Witt et al.
(2003) reported strong dominant lethal effects in mice
exposed to N-hydroxyacrylamide while no significant
Environmental and Molecular Mutagenesis. DOI 10.1002/em
48 Marchetti et al.
effects could be detected using the bone marrow micronu-
cleus (MN) analysis, one of the most commonly con-
ducted tests for regulatory purpose. Similarly, Marchetti
et al. (2011) showed induction of tandem repeat mutations
in the sperm of mice exposed to either mainstream or side-
stream tobacco smoke at doses that did not increase MN
frequencies in bone marrow. Finally, a review of the gen-
otoxicity of acrylamide showed that the response in germ
cells is stronger than the response in somatic cells
(Dearfield et al. 1995).
It could be said that the above examples suffer from the
shortcoming of comparing different endpoints in germ cells
vs. somatic cells and, thus, the observed differential
responses are not a true indication of an increased sensitiv-
ity of germ cells to those agents, but simply reflect method-
ological differences in the sensitivity to detect an effect.
However, even when the same endpoint is analyzed, there
are still four cases of chemicals that failed to induce MN in
the mouse bone marrow, while significantly increasing their
frequency in spermatids (Cliet et al. 1993).
While recognizing the potential impact of induced
germline mutations on human populations, national and
international efforts have concentrated on the identifica-
tion of germ cell mutagen hazards, eliminating them, or
otherwise reducing their potential exposure to human
populations (Committee 17 1975; IPCS 1985; World
Health Organization 1986). Efforts also extended to
identifying epidemiologic associations between human
male exposures and mutation-based diseases in offspring
(Narod et al. 1988) to identify potential human germline
mutagens. In recognition of such potential hazards,
national and international regulatory authorities have
established guidelines and regulations that include the
goal of identifying and controlling human exposure to
germline mutagens (Cimino 2006).
Paramount among such regulatory schemes is the inter-
national adoption of the GHS of Classification and Label-
ling of Chemicals (United Nations 2017) by most
industrialized nations. The GHS includes human germ cell
mutation as a toxicological endpoint equal to other end-
points such as cancer and reproductive effects, thus, indi-
cating its universal international acceptance. While such
regulatory schemes do establish a basis for germline hazard
identification, they do not provide a clear path to the actual
estimation of the human risk in terms of induced mutation
rate in offspring associated with such hazards. Various
germ cell risk estimation methods have relied primarily on
the use of animal studies and some form of quantitative
extrapolation to a related class of human genetically based
disease (reviewed extensively in Yauk et al. 2015a).
There are limitations to the currently available
approaches for quantitative assessment of germ cell risk
from chemical exposures. For example: (1) animal studies
do not directly measure or quantitate effects in humans,
while current methods do not routinely assess
environmentally caused de novo mutations in specific
human target genes, or groups/categories of genes; (2) there
are no accepted, or validated, allometric and exposure fac-
tors for extrapolation of animal germline mutation data to
individual humans, or human population groups, and there
is insufficient information on the means to extrapolate
mutation data in animal genes to analogous human genes
and (3) there is no accepted de minimis risk level for the
estimation of unacceptable exposure levels for germline
mutation effects to offspring.
Despite such historically based shortcomings, we are
now at a turning point facilitated by the accelerated devel-
opment of new genomic technologies that will finally
enable us to make significant advances in the application of
genetic risk assessment in both the academic and regulatory
contexts.
THE FUTURE: NEXT GENERATIONSEQUENCING
Studies on the heritable effects of environmental agents
in humans have historically relied on measuring phenotypic
endpoints rather than detecting the underlying genetic
cause (DeMarini 2012). As not all de novo mutations have
a discernable phenotypic consequence, these approaches
were limited to measuring only dominant effects in early
life with a resulting loss of statistical power. In addition,
such approaches cannot quantify the impact of recessive
mutations on health until subsequent generations, or muta-
tions that contribute to multigenic disorders. The advent of
NGS approaches, together with great gains in bioinformat-
ics pipelines to handle, analyze and validate sequenced
data, are allowing the analysis of mutations over the entire
genome and are promising to revolutionize the way germ
cell mutagenicity is investigated. NGS complements other
genomic approaches such as array comparative genomic
hybridization to capture a variety of genomic changes that
can impact human health (Carvalho and Lupski 2016).
NGS approaches can also provide critical mechanistic
information on the types of mutations that are induced and
increase the confidence that these de novo mutations are
indeed the consequence of an exogenous exposure. Impor-
tantly, these approaches are feasible with a relatively small
number of samples and are equally applicable to laboratory
animals as well as human families (Webster et al. 2018). In
fact, experiments conducted in laboratory animals can
inform studies in human families on the proper experimen-
tal design, power of analysis and confounding factors.
There are already several examples of the use of these
genomic approaches for germ cell mutagenesis studies in
rodents. Adewoye et al. (2015) reported significant induc-
tion of copy number variants (CNVs) in the offspring of
irradiated animals. Interestingly, both irradiated spermato-
gonia and sperm caused similar levels of CNVs in off-
spring. In the same study, although there was evidence for
Environmental and Molecular Mutagenesis. DOI 10.1002/em
49Quest for Human Germ Cell Mutagens
clustering of mutations after irradiation of male germ cells,
the authors could not detect an overall significant increase
in de novo single nucleotide variants (SNVs). However, a
recent study (Beal et al. 2019) reported significant increases
in CNV gains and SNVs in the offspring of male mice
exposed to BaP with evidence of clustering of mutations.
The study also demonstrated that there was a significant
increase in constitutional SNVs (i.e., present at fertilization)
when spermatogonia were exposed to BaP, while there was
a significant increase in embryonic SNVs (i.e., originating
after fertilization) when postmeiotic germ cells were
exposed. Importantly, the types of mutations observed in
the offspring matched the expected mutational signature of
BaP and were consistent with the pattern of mutations
observed in the sperm of exposed males, conclusively dem-
onstrating that the increase in de novo mutation was linked
to the paternal exposure to BaP. Finally, there is also evi-
dence that exome sequencing may be sufficient to detect
significant increases in de novo mutations (Masumura et al.
2016a, b). These studies found that N-ethyl-N-nitrosourea
(ENU) significantly increased de novo mutations in the
exomes of offspring from exposed mice and that the
inherited mutations exhibited a spectrum characteristic of
ENU-induced mutations.
Together, these rodent studies: (1) provide foundational
examples for the application of genomic technologies for
investigating the induction of heritable mutations following
exposure to environmental mutagens; (2) demonstrate that
different mutagenic agents can differentially affect various
types of genetic changes; (3) provide support for the herita-
bility of mutations observed in sperm; and (4) confirm the
important role of postfertilization events in determining the
genetic load that is transmitted to the offspring.
Application of NGS to human families to study the
underlying causes of de novo mutations has focused mostly
on measuring the increase in de novo mutations as function
of paternal or maternal age. These studies are nevertheless
important because they provide information on the baseline
levels of de novo mutations and how they change as a
function of age. Clearly, paternal age is a critical con-
founding factor that must be controlled for in any study
assessing the impact of environmental exposure in the
human population. Indeed, a recent statistical power analy-
sis showed that controlling for the paternal age effect and
modeling family-to-family variability significantly reduced
the number of families needed to detect an increase in de
novo mutations (Webster et al. 2018). The analysis also
demonstrated that inclusion of families with multiple chil-
dren confers additional sensitivity to detect an effect. For
example, sequencing of just 6 four-child families per study
group provides an 80% power to detect a 30% increase in
de novo mutations (Webster et al. 2018). These data show
the feasibility of designing studies to evaluate the effects of
environmental exposures in human populations that do not
require large cohorts and significant financial investment. It
is easy to envision that these types of studies will become
common over the next few years and will provide funda-
mental data on the susceptibility of human germ cells to
environmental agents. One such an example is a recent
study that demonstrated a significant correlation between
levels of paternal exposure to dioxin and incidence of de
novo SNVs in their offspring (Ton et al. 2018).
THE NEXT 50 YEARS: A CALL TO ACTION
Fifty years ago, the founding members of the EMGS
would not have predicted that half a century would pass and
we would still be waiting for the demonstration of the exis-
tence of human germ cell mutagens. Yet, it is a testament of
the rightfulness and importance of the original mission of
the Society that the pursuit of that goal has survived. During
these 50 years, incredible progress has been made in our
understanding of the mechanisms of germ cell mutagenesis
and in the tools that we have at our disposition for fulfilling
the original mission of our society. It is because of the
relentless effort of a small number of EMGS members who
have kept the germ cell mutagenesis flame alive that we are
experiencing now a renaissance of the field.
Challenges still lay ahead. First and foremost, germ cell
mutagenesis is “threatened”by the current push to limit,
reduce and replace in vivo studies and the increased empha-
sis on in vitro and computational approaches to assess the
toxicological properties of chemicals. Under this paradigm,
germ cells risk may be marginalized as in vitro germ cell
systems are still in their infancy, with a consequent reliance
on somatic systems to predict effects in germ cells. Further-
more, even if in vitro germ cell systems would be fully
developed, they would just provide demonstration that a
chemical can affect the germ cell genome while contribut-
ing little to the ultimate goal of germ cell mutagenesis: the
demonstration of the heritability of environmentally
induced de novo mutations.
We now have the tools and are closer than ever before
to investigating the best possible experimental system:
ourselves. The continuing decline in the costs of NGS
approaches and the availability of new technologies
that are directly applicable to humans and that enable
the detection of rare mutations among heterogeneous
populations of cells (Salk et al. 2018) makes it inevitable
that many more agents will be demonstrated to induce
mutations in human germ cells and increase adverse health
effects in the offspring. These error-reduced sequencing
technologies can be applied in any species/tissue and are
poised to revolutionize genotoxicity testing in general
(Salk and Kennedy 2020). Such technologies provide
more opportunities for any laboratory in the world to con-
duct germ cell mutagenicity assessment. It is our hope
that with the GHS strategy, and the European Union regu-
lation for the Registration, Evaluation, Authorisation and
Environmental and Molecular Mutagenesis. DOI 10.1002/em
50 Marchetti et al.
Restriction of Chemicals requiring germ cell mutation assess-
ment, in parallel with our increasing knowledge of the major
prevalence of human genetic disorders that result from de
novo mutations and the impact of even small increases in
mutation rates on population health, more laboratories around
the world will begin to contribute data to increase our under-
standing of candidate human germ cell mutagens.
We predict that we will soon have data demonstrating an
increase in de novo mutations in the offspring of exposed par-
ents. A great challenge is to be sufficiently prepared to inter-
pret the coming outpouring of data and, together with the
regulatory community, develop a framework for managing,
communicating and mitigating the risk of exposure to human
germ cell mutagens. As indicated by DeMarini (2012), it is
time for the applied genetic toxicology community to formu-
late a plan to respond to the demonstration of environmentally
induced de novo mutations in the human population. We add
our voices to his call for the establishment of an international
body that is tasked with the mandate of reviewing the avail-
able data and formally declare whether there is sufficient evi-
dence for supporting the existence of human germ cell
mutagens and evaluate the risk of exposure to these agents for
the health of human populations.
ACKNOWLEDGMENTS
We thank Drs Marc Beal and Vinita Chauhan of Health
Canada for providing comments on the manuscript before
it was submitted. Funding for this work was provided by
Health Canada’s Chemicals Management Plan and the
Genomics Research and Development Initiative.
AUTHORCONTRIBUTIONS
All authors contributed to the writing of the manuscript
and all authors agree to its publication.
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Accepted by—
R. Heflich
Environmental and Molecular Mutagenesis. DOI 10.1002/em
54 Marchetti et al.
