Guidance

The use of biomarkers in genotoxicity risk assessment

Published 17 April 2025

Introduction

This guidance statement covers in vivo biomarkers of genotoxicity that can be used for human carcinogenicity risk assessment. Readers are also referred to the Committee on Carcinogenicity (COC) Guidance Statement G04 ‘The Use of Biomarkers in Carcinogenic Risk Assessment’ (COC, upcoming publication) which outlines relevant in vivo biomarkers specific to the carcinogenic process, and those of more general toxicological relevance to humans.

A biomarker is defined as “any substance, structure or process that can be measured in an organism or tissue, related to a specific exposure or effect or which can influence the incidence of the effect” (Choi and others, 2015); biomarkers are not necessarily a measure of disease (Ladeira and Smajdova, 2017).

Biomarkers can provide valuable information to aid chemical risk assessment processes and are used as investigative tools in human studies that aim to evaluate carcinogenic hazards and risk (COC, upcoming publication). Such human studies often use human biomonitoring (HBM), a scientifically robust approach for assessing chemical exposures from all routes into the body and from all external sources, providing evidence that exposure and/or uptake have occurred through the measurement of biomarkers of exposure and effect (Ladeira and Smajdova, 2017). Probably the most well-developed use of HBM has been in occupational settings where exposures to a chemical of particular concern might be relatively high. Here, routine HBM might be more informative about risk than air monitoring, particularly where skin uptake is an important contributing exposure pathway, and various types of reference values used for risk management might exist for the chemical of concern. In the general population, HBM is often used to inform on exposure to a chemical of particular concern and also, for changes over time (increase or decrease) for substances of interest related to industrial or consumer usage to existing or newly introduced substances (Bevan and others, 2017).

This guidance statement focuses on the detection of biomarkers of genotoxicity, that is, the induction of DNA damage, mutation, or both (Smith and others, 2016) in humans. The COM ‘Guidance on a strategy for genotoxicity testing of chemicals’ defines genotoxicity as the “interaction with, or damage to, DNA and/or other cellular components which regulate the fidelity of the genome”. Further, “it is a broad term that, as well as mutation, includes damage to DNA such as the production of DNA adducts, by the chemical itself or its metabolites. Cells have the capacity to protect themselves from such potentially lethal or mutagenic genotoxic effects by many repair processes and therefore many genotoxic events do not become evident as mutations. However, the capacity to damage the genome (genotoxicity) is an indicator of potential mutagenicity. Thus, some methods that measure genotoxicity do not provide direct evidence of heritable mutation” (COM, 2021).

The current risk assessment paradigm relies on the differentiation of carcinogens into 2 broad classes, based on their mechanism of chemical carcinogenicity: genotoxic and non-genotoxic carcinogens. Genotoxic carcinogens initiate the process of chemical carcinogenesis by damaging DNA and acting as mutagens. Non-genotoxic carcinogens promote carcinogenesis without binding, damaging or interacting directly with DNA. They act via many modes of action (MoA) including: causing cytotoxicity; binding to receptors such as oestrogen, androgen, aryl hydrocarbon, peroxisome or constitutive active receptors; suppressing immune system; increasing oxidative stress; or inhibiting DNA damage repair, that is, they do not act as a ‘traditional’ initiator in the development of carcinogenesis.

It is accepted that ‘no observed adverse effect levels’ (NOAELs) exist for non-genotoxic carcinogens and threshold values can therefore be estimated. For risk assessment purposes, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites have generally been considered to represent risk factors at all concentrations since even one or a few DNA lesions may, in principle, result in mutations and, thus, increase tumour risk, that is, there is no threshold of toxicity (Hartwig and others, 2020). The COM has, however, published guidance on possible threshold modes of genotoxicity which can include:

• involvement of non-DNA targets (for example, aneugen inhibition of microtubules)

• the contribution of protective mechanisms (for example, repair of DNA adducts formed from many low molecular weight alkylating agents)

• overload of detoxication pathways (for example, paracetamol) (COM, 2010)

MoA data is therefore key in establishing the genotoxic or non-genotoxic nature of a carcinogen.

Biomarker types and their use in risk assessment

The COC document ‘The Use of Biomarkers in Carcinogenic Risk Assessment’ broadly characterises biomarkers as those of exposure and those of effect, whilst recognising that the distinction between the two is not always clear-cut. Biomarkers in the context of carcinogenicity can mean proof of exposure to a carcinogen, detection of a reaction product or an indication that a preliminary genotoxic event or actual DNA damage has occurred (COC, XX).

Biomarkers of exposure are defined as “an exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some macromolecule or cell that is measured in a compartment within an organism” (Laideira and Viegas, 2016). DNA adducts or lesions can be considered as relevant examples of biomarkers of exposure. Biomarkers of exposure are further divided into those reflecting ‘internal dose’ and those reflecting ‘effective dose’. The concentration of a chemical (or metabolite) in blood following exposure is a basic measure of the internal dose, a proxy of the chemical (or metabolite) at the target site. The effective dose is a more accurate measurement of the exposure levels associated with the target molecule, structure or cell itself (Laideira and Viegas, 2017).

Biomarkers of effect are defined as “a measurable biochemical, physiological, behavioural or other alteration within an organism that, depending upon the magnitude, can be recognised as associated with an established or possible health impairment or disease” (IPCS, 1993; Jeddi and others, 2021), for example measures of chromosome damage that are related to carcinogenicity.

Other types of biomarkers exist, for example biomarkers of susceptibility, that were initially introduced as interpretative aids to epidemiological investigations of chemically induced carcinogenesis. Biomarkers of susceptibility are defined as “an indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a chemical” (Manno and others, 2010).

From a genotoxicity risk assessment perspective, biomarkers of exposure and effect cannot easily be separated in the same way as for other types of risk assessments and are therefore considered as one group in the text below. A number of in vitro, in vivo and ex vivo assays have been developed to assess biomarkers of genotoxicity to evaluate the impact of environmental and/or occupational exposures on “genetic (in)stability” (Ladeira and Smajdova, 2017). Of these, a number of currently used in vivo biomarkers of genotoxicity are discussed below.

Biomarker validity

As stated in COC Guidance Statement G04 ‘The Use of Biomarkers in Carcinogenic Risk Assessment’ (COC, upcoming publication), “Biomarkers must be appropriately characterised and validated before conclusions are drawn from their use. Particular emphasis may be placed on the early events (both mutagenic and non-mutagenic) in the carcinogenic process.” The document outlines the general criteria used for the evaluation of the most suitable exposure biomarkers (EB) and matrix (M) for the current European initiative, HBM4EU for carcinogens and non-carcinogens (Vorkamp and others, 2021). These considerations of specificity, biological sensitivity, half-life, stability, matrix availability and sample collection, and measurement validity also apply to biomarkers of genotoxicity. The validity requirements for in vivo biomarkers used in animal studies are the same as those for human studies.

In relation to biomarkers, the Strengthening the Reporting of OBservational studies in Epidemiology – Molecular Epidemiology (STROBE-ME) initiative (Gallo and others, 2012) provides standardised guidelines and a ‘checklist’ for the reporting of biomarker and molecular epidemiology studies (see STROBE-ME: an extension of the STROBE statement, viewed January 2024). An extension to STROBE is the STrengthening the REporting of Genetic Association studies (STREGA) which includes additions to 12 of the 22 items on the STROBE checklist that are important to consider in genetic association studies (Little and others, 2009).

Types of DNA damage

DNA is highly susceptible to modification following exposure to a wide range of environmental and endogenous chemicals. The consequent DNA damage is seen as being a key driver of disease onset and progression through mutagenesis (Chatterjee and Walker, 2017). DNA damage leading to mutational events can be subdivided into 3 categories:

• gene mutations

• structural chromosomal aberrations (clastogenicity)

• numerical chromosomal aberrations (aneugenicity)

Clastogenic substances induce structural chromosomal aberrations through breaks in DNA. Aneugenic substances induce numerical chromosomal aberrations through interactions with cellular targets other than DNA, such as proteins involved in the spindle controlling the segregation of chromosomes during mitosis or meiosis (European Food Safety Authority (EFSA), 2021).

Examples of DNA damage include DNA adducts (a molecule bound covalently to DNA) including DNA alkylation, DNA strand breaks (breaks in the phosphodiester bonds), and DNA–DNA or DNA–protein crosslinks. DNA damage by itself is not a mutation and generally does not alter the linear sequence of nucleotides (or bases) in the DNA, whereas a mutation is a change in the DNA sequence and usually arises as the cell attempts to repair the DNA damage (Shaughnessy and DeMarini 2009). DNA damage can be spontaneous in origin through errors of nucleic acid metabolism or can be induced by exogenous or endogenous agents. Examples of exogenous agents that are also generated endogenously include formaldehyde and acetaldehyde, and these can result in a ‘background’ level of DNA damage which may drive background or spontaneous mutation processes (Nakamura and others, 2000; Swenberg and others, 2011; Pottenger and others, 2019).

The different types of DNA adducts formed are governed by the structure of the reactive chemical, the nature of the electrophiles, and also the ability of the compounds to intercalate with DNA. As such, adduct formation can ‘favour’ specific nucleophilic sites of the DNA bases, including the C8 atoms of guanine and adenine and the endocyclic and exocyclic N and O atoms of the nucleobases. Prominent markers of oxidative stress include 7,8-dihydro-8-oxoguanine (8-oxo-Gua), formed following the oxidation of the C8-atom of guanine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-Gua), which are prominent markers of oxidative stress and genotoxic potential. In addition, the methylation of the C5-methyl group of cytosine is considered an important marker of epigenetics (Xu and Gao, 2020). Examples of endogenous DNA lesions include the apurinic (AP) site where a weakened glycosidic bond leads to loss of a purine base. AP sites have been reported as being highly mutagenic if present during DNA replication (Neto and others, 1992; Schaaper and Loeb, 1981) and the DNA oxidation products N7-HEG, N7-(2-oxoethyl)guanine (N7-OEG), and 8-oxoG (Boysen and others, 2010, Swenberg and others, 2011, Nakamura and others, 2014) that result from lipid peroxidation products or reactive oxygen species (ROS) (Pottenger and others, 2019).

It has been reported that, once formed, chemical DNA adducts can interfere with DNA replication and transcription which may lead to programmed cell death, mutagenesis and genomic instability (Hartwig and others, 2020). Where such changes occur, this could lead to cancer in somatic cells or to reproductive toxicity in germ cells. To maintain DNA integrity, DNA adducts are usually repaired by various DNA repair systems, DNA replication of damaged DNA can be prevented by cell cycle control mechanisms, or apoptosis can be induced where DNA is heavily damaged. Adaptive mechanisms can, however, allow for replication when low levels of DNA damage are present. In addition, mutations are possible through the direct integration of incorrect DNA bases in the course of replication. If replicated, DNA containing adducts can cause point mutations in the daughter strands, at the site of adducts (Hartwig and others, 2020).

The presence of apurinic/apyrimidinic (AP) or abasic sites, which have a missing base, are considered to be evidence of the direct induction of DNA damage. In addition, DNA adducts such as the methylated O6-methylguanine are reported to induce direct miscoding during DNA replication. Hartwig and others (2020) reported that the disturbance of DNA repair mechanisms can occur at very low, environmentally relevant, exposure levels to some chemicals including arsenic and its metabolites. Indirect impairment of genetic stability may also occur due to a shortening of the repair time window as a consequence of cell division and growth promotion.  

DNA adducts formed following exogenous or endogenous exposure can be linked to exposure to a specific chemical as they retain chemical-specific information related to their source. Although DNA adducts have variable stability, many are stable and measurable as nucleotides in numerous biological matrices, making them valuable for use as biomarkers of exposure. Mutations, however, are considered to be biomarkers of genotoxic effect as they represent a heritable change in the primary DNA sequence (Stice and others, 2019).

Measurement of DNA damage

In their overarching strategy, the COM currently recommends assessment of 3 types of DNA damage, namely gene mutations, clastogenicity and aneugenicity, to determine the potential genotoxicity of chemicals in vivo (COM, 2021). In terms of human biomarkers of genotoxic effect, there are a number of genotoxicity and mutational endpoints that have been studied in humans. These include:

• micronuclei (MN) in lymphocytes

• Pig-a mutation in erythrocytes

• DNA strand breaks in lymphocytes

It should be noted that the type of genotoxicity endpoint measured might be determined by the matrix being sampled; whilst frozen blood can be used for adduct analysis by mass spectrometry (MS) and other genomics methodologies, fresh blood is required for the Pig-a mutation analysis and cultured lymphocytes for MN determination. Other tissues that may be utilised for the measurement of DNA damage include buccal mucosal tissue (Thomas and others, 2009) and, more recently, urine (Cooke and others, 2018).

DNA adducts

The measurement of DNA adducts can indicate both exposure to toxicological agents and the potential susceptibility for disease development (Pottenger and others, 2019). The appropriate interpretation and application of DNA adduct data to inform carcinogenicity assessment decisions have been debated for some time. This debate has increased more recently due to the development of new, highly specific, and sensitive analytical methods. It should be appreciated that DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information that establishes links between DNA adducts (for example, type of adduct, frequency, persistence, type of repair process) and accepted outcome measures (for example, dosimetry, toxicity, mutagenicity, genotoxicity, and tumour incidence) to inform characterisation of the mode of action.

DNA adducts generally occur at very low levels in humans (< 1 adduct per 10⁷ nucleotides) meaning that analysis needs to be highly sensitive and specific. The major techniques that have been developed to measure DNA adducts include 32P-postlabeling (Randerath and others, 1981; Randerath and others, 1985; Shibutani and others, 2006; Pfau and others, 1993; Phillips, 2013), antibody-based immunoassay/immunohistochemistry (IHC) (Pratt and others, 2011; Zhang and others, 2002; Leuratti and others, 2002; Maatouk and others, 2004), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) (Liu and Wang, 2015; Tretyakova and others, 2013; Dizdaroglu, 1993; Brown, 2012; Guo and others, 2017; Dizdaroglu and others, 2015).

Although a very sensitive method (one adduct per 10¹⁰ nucleotides) requiring only small amounts of DNA (1-10 µg), 32-P postlabelling is difficult to perform, gives uncertain structural information about the adduct and is not quantitative. Some epidemiological and rodent studies have used the technique to show that many genotoxic chemicals undergo metabolism and covalently adduct to DNA in many organs, however, the assay did not provide equivocal data linking chemical exposures to DNA adducts and cancer risk (Yun and others, 2020).

The sensitivity of immunodetection methods depends on the affinity of the antibody, however a detection limit of around one adduct per 10⁸ nucleotides has been reported in conjunction with fluorescence or chemiluminescence spectroscopy (Divi and others, 2002; Mumford and others, 1996). In addition, immunohistochemical methods allow visualisation of DNA adducts in specific cell types of a tissue (Poirier and others, 2000; Santella, 1999). Specificity can be an issue with immunodetection methods due to the potential for cross-reactivity with DNA adducts of similar structure or cellular components. In addition, structural identification is not possible and the method is only semi-quantitative (Yun and others, 2020).

GC-MS with electron impact ionization, and more recently, negative ion chemical ionization can be used to measure DNA adducts (primarily oxidized DNA bases), allowing the corroboration of adduct structures from the MS fragmentation spectra (Sangaraju and others, 2012; Guo and others, 2016). A derivatisation process is needed for most DNA adducts however, which can introduce artifacts, particularly for oxidized DNA base measurements (Yun and others, 2020).

The predominant platform for DNA adduct analysis currently is liquid-chromatography-electrospray-ionization-mass spectrometry (LC-ESI-MS), which can measure many DNA adducts which would otherwise undergo thermal decomposition by gas-chromatography-mass spectrometry (GC-MS) (Fenn and others, 1989; Juraschek and others, 1999; Shen and others, 1992; El-Faramawy and others, 2005). The methodology provides very high sensitivity and selectivity, particularly in conjunction with ion trap and high-resolution accurate mass spectrometry (HRAMS). Levels of detection as low as one per 10¹¹ nucleotides have reported using a hybrid Orbitrap MS (Yun and others, 2020).

A comprehensive analysis of DNA adducts (DNA adductomics) enables the totality of DNA damage in the human genome to be assessed and can be used to establish mechanisms between chemical exposures and disease outcomes (Balbo and others, 2014; Villalta and Balbo, 2014). However, the technology is still relatively new and still developing. In addition, the screening and identification of DNA adducts in humans is analytically challenging due to the low levels of DNA adducts formed with environmental genotoxicants or endogenously produced electrophiles, which has been reported to range between one adduct per 10¹⁰ to one adduct per 10⁸ (Guo and Turesky, 2019). Chemical specific DNA adducts measured in target tissues have been identified following exposure to aflatoxin B1 (AFB1) and aristolochic acid (AA-I) in humans which has provided a mechanistic understanding of the causal roles for these chemicals in the development of liver and upper urothelial cancers, respectively (Pottenger and others, 2014).

Toxicogenomic signatures

The toxicogenomic approach brings together the knowledge gained from toxicology, genomics and bioinformatics. In relation to genotoxicity, toxicogenomic studies utilise comprehensive gene expression data (determined using transcriptomics) to identify gene expression signatures that strongly correlate with genetic toxicity. Additionally, toxicogenomic techniques monitor, for instance, functionally relevant changes in the genome (genomics), global protein and/or post-translational modifications (proteomics) or metabolites (metabolomics) (Alexander-Dann and others, 2018).

Studies to date have reported specific toxicogenomic signatures using rodent and human cell lines in vitro, and rodent models in vivo, to predict carcinogenicity in rodents and/or in humans. In addition, the differentiation of genotoxins versus non-genotoxins, aneugens versus clastogens, carcinogens versus noncarcinogens, and genotoxic carcinogens versus non-genotoxic carcinogens using toxicogenomic signatures has also been reported (Beedanagari and others, 2014). At present, there is no consensus on how to use toxicogenomic signatures for genotoxicity testing, which may be due to the diverse test models being used to derive them.

Micronuclei formation

The human lymphocyte MN assay has been used to study genotoxic events in humans for several decades and it has been reported that an increased incidence of MN formation can be used as a reliable biomarker for genotoxic damage in vivo, detecting both clastogenic and aneugenic substances (Bonassi and others, 2007). MN are defined as either a broken chromatid/chromosome fragment (1/10^th to 1/100^th the size of the nucleus) or, more rarely, as a whole chromatid/chromosome that remains outside of the nucleus after cell division. The former are formed due to unrepaired or misrepaired DNA breaks and the latter due to deficiency in chromosome segregation (Iarmarcovai and others, 2008).

The potential increase in MN frequency, measured using the in vivo bone marrow or blood (MNviv) assay, has been utilised for the biomonitoring of occupational and environmental exposures to genotoxic agents for many years. The most frequently investigated occupational group has been hospital personnel as they can be frequently exposed to genotoxic agents including, radiation, cytostatic drugs, anaesthetic gases and formaldehyde. Within the chemical industry, studies have evaluated the in vivo genotoxic potential from occupational exposure to styrene, vinyl chloride monomers, butadiene and ethylene oxide, by measurement of MN frequency. Other industries reporting use of MN frequency for biomonitoring include miners exposed to metals, radon and coal dust, and agricultural workers exposed to plant protection products (Nersesyan and others, 2016). Environmental studies using MN frequency evaluations include assessment following exposure in air or water to arsenic, cadmium, lead and PAHs. MN frequency has also been used as a measure of genotoxic effect and genotoxic exposure in school-aged children to support adductomics screening (Carlsson and others, 2017). Other examples of biomarkers of effect to chemical genotoxicants include; an increased frequency of MN formation in cervical cells of women smokers when compared to never smokers (Nersesyan and others, 2020); an increased MN frequency in patients with chronic kidney disease (Fenech and others, 2021); significantly increased frequencies of chromosomal aberrations in type 2 diabetic subjects (Fenech and others, 2021); a significant association between MN frequency and occurrence of chronic obstructive pulmonary disease (COPD) and/or lung cancer (Asanov and others, 2021).

Other biomarkers of genotoxicity and chromosomal instability that can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay include nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) (Fenech and others, 2011; Zhang and others, 2015).

DNA strand breaks

The in vivo comet assay (OECD TG 489: In vivo mammalian alkaline comet assay) is widely used as a biomonitoring tool to detect primarily DNA damage, in the form of single and/or double strand breaks, that may lead to gene mutation and/or chromosome aberrations; the assay can also be adapted to measure DNA repair (COM, 2021). Strand breaks within the DNA can arise from a number of processes, including: the direct modification of DNA by chemical agents or their metabolites; from the processes of DNA excision repair, replication, and recombination; or from the process of apoptosis (Chatterjee and Walker, 2017). The assay detects transient genetic damage to the DNA which is not a fixed change as they may be repaired. Most uses of the in vivo comet assay have been with white blood cells and detection levels of between a few hundred breaks per cell and a few thousand have been reported, encompassing levels of damage that can be repaired and tolerated by human cells (Chatterjee and Walker, 2017).

The comet assay has been used to quantitate DNA damage in populations following both environmental and occupational exposures to genotoxic chemicals. For example, human populations exposed to air pollution comprising PAHs, ozone, benzene, heavy metals, (ultra)fine particulate matter (PM), and passive smoking were reported to have increased levels of DNA damage. Occupational studies are more frequently reported and cover a wider range of exposure types. Increased DNA damage has been reported in workers exposed to: volatile organic compounds (benzene, toluene, xylene, styrene, vinyl chloride, PAH, and so on), traffic fumes, diesel exhaust, silica, and adhesives, asbestos and mineral fibres, metals (welding fumes, arsenic, boron, chromium, nickel, aluminium, cobalt, lead, cadmium, mercury), pesticides and in medical workers. Where evaluated, DNA damage measured using the comet assay was also closely correlated with MN formation and sister chromatid exchanges. The assay detects a very early measure of a recent response to the effect of genotoxic chemicals on DNA integrity (Collins and others, 2014).

A potential biomarker for double-stranded DNA breaks that is also being developed is the Ser139-phosphorylated histone H2AX (γ-H2AX) which is rapidly produced as a first step in recruiting and localising DNA repair proteins (Suzuki and others, 2020). It has also been reported that γ-H2AX accumulates in response to other types of DNA stress and has been explored as a biomarker for predicting bladder carcinogens, with both genotoxic and non-genotoxic mechanisms (Suzuki and others, 2020).

Gene mutation

In vivo measurement of DNA damage has been well established. However, as DNA damage may be repaired, a more informative end point to assess genotoxicity in vivo could be to measure mutations, as these are irreversible changes. This would require very sensitive human specific in vivo mutagenicity test systems to be developed.

The Pig-a mutation assay is well established in rodents (Lemieux 2011, Dobrovolsky and others, 2014) and measures mutations through loss of glycophosphatidylinositol (GPI) linked cell surface proteins. OECD test guideline 470, ‘Mammalian Erythrocyte Pig-a Gene Mutation Assay’, has been published (OECD, 2022). Recent developments have been made to optimise a human erythrocyte Pig-a assay for biomonitoring purposes, with a limited number of studies identifying a similar low background mutation frequency in the erythrocytes of the patients studied (Cao 2016, Dobrovolsky 2011, Dertinger 2015, Lawrence and others, 2020; Lawrence and others, 2023).

The minimally invasive nature of Pig-a testing (pin-prick sampling optional) offers the potential of the assay to be used as a human biomonitoring tool, assessing the risk of environmental, occupational and pharmaceutical exposures. The paucity of mutant red blood cells in healthy volunteers enhances the ability to identify mutagenic exposures above a very low background level and repeat sampling may permit the analysis of long term, chronic exposure.

A limited number of studies have evaluated the potential use of this mutation test as a sensitive biomarker for disease and as a biomonitoring tool to evaluate any relationship between Pig-a mutations and carcinogen exposure, which is poorly understood. It has been demonstrated that oesophageal cancer patients had elevated levels of GPI-deficient erythrocytes compared to non-neoplastic controls (Haboubi, 2019) suggesting a link between circulating mutations in blood cells and internal cancer risk. One study has shown patients with inflammatory bowel disease (IBD) treated with azathioprine over prolonged periods had increased levels of Pig-a mutant erythrocytes (Cao and others, 2020a). In terms of studying human mutation levels with a view to human biomonitoring, the human Pig-a assay has also revealed mutational links to diet, medication and age (Lawrence and others, 2020) and exposure to heavy metals such as lead (Cao and others, 2020b).

Strategic uses of human biomonitoring

There are a number of well-established and ongoing national and international human biomonitoring surveillance programmes. In Europe there are the German Environmental Survey (GerES, Germany), the Flemish Environment and Health Study (FLEHS, Belgium), the French National Survey on Nutrition and Health (ENNS, France), BIOAMBIENT.ES (Spain), Program for Biomonitoring the Italian Population Exposure (PROBE, Italy), Human Biomonitoring Project (CZ-HBM, Czech Republic). The US has the National Health and Nutrition Examination Survey (NHANES), Canada the Health Measures Survey (CHMS) and Asia the Korea National Survey for Environmental Pollutans in the Human Body (KorSEP) (Ladeira and Smajdova, 2017). All programmes typically use well-established biomonitoring techniques (for example biomarkers which are known to reflect exposure to the chemical of interest, standardised sampling methods and verified analytical techniques) to collect information on population exposures to environmental hazards that are known to be significant to public health. As biomonitoring does not generally determine exposure sources and routes of exposure, environmental monitoring remains crucial (Ladeira and Viegas, 2016).

A biomonitoring equivalent (BE) is an estimated concentration or range of concentrations of an environmental chemical in humans that is consistent with existing health-based guidance values such as the Tolerable Daily Intake (TDI) or reference dose or concentration (RfD, RfC). It provides a way of interpreting biomonitoring data in the context of these values (Hays and others, 2008). It is envisaged that they will be useful for understanding and prioritising risk management practices and will enable the available biomonitoring data to be utilised more fully. However, to date, there is limited information on the use of BEs for estimating chemical exposure in the context of carcinogenesis (Faure and others, 2020).

Human biomonitoring guidance values (HBM-GVs) are being derived by the European Human Biomonitoring Initiative referred to as HBM4EU. There is currently a diversity in the derivation of health-based guidance values for both the general population and for occupational exposure. The HBM4EU initiative aims to increase confidence in HBM-GVs derived using a harmonised, systematic and generally accepted strategy for the derivation of HBM-GVs at the European level (Vorkamp and others, 2021).  

With time, and following validation, the biomarkers of genotoxicity discussed in this document that are currently generally used at a research level, may become incorporated into some of these existing schemes for risk assessment purposes.

Summary

Biomarkers can provide valuable information to aid chemical risk assessment processes and are used as investigative tools in human studies which aim to evaluate carcinogenic hazards and risk. Human studies often use HBM as this approach assesses chemical exposures from all routes and sources through measurement of biomarkers of exposure and effect. Biomarker detection should not be considered as a measure of disease.  

This guidance statement focuses on the detection of biomarkers of genotoxicity, that is, the induction of DNA damage, mutation, or both, in humans.

DNA is highly susceptible to modification following exposure to a wide range of environmental and endogenous chemicals. In general, from a genotoxicity risk assessment perspective, biomarkers of exposure and effect cannot easily be separated. However, the DNA adducts formed following exogenous or endogenous exposure can be linked to exposure to a specific chemical as they retain chemical-specific information related to their source and, as such, are valuable for use as biomarkers of exposure. Mutations are considered to be biomarkers of genotoxic effect as they represent a heritable change in the primary DNA sequence.

A limited number of genotoxicity and mutational endpoints have been studied in humans including lymphocyte micronuclei (MN), Pig-a mutation in erythrocytes and DNA strand breaks in lymphocytes.

Developing approaches include DNA adductomics, a comprehensive analysis of DNA adducts that enables the totality of DNA damage in the human genome to be assessed, which can be used to establish mechanisms between chemical exposures and disease outcomes.

Following appropriate validation of currently used biomarkers of genotoxicity, it might be possible for these to be used more strategically and practically as part of established BM schemes.   

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