Genetic toxicology is part of applied toxicology and is the field that investigates whether a chemical (xenobiotic or natural) has the potential to cause damage to the genetic material. Genotoxicity can occur via either via a direct mechanism (mutation of structure or content) or via an indirect mechanism (cellular process-related). The effect of a genotoxin is a change to the cellular DNA which may or may not become permanent. If such a change can happen in somatic cells, the risk is potential cancer whereas if the change can happen in the germline, then the risk is a heritable mutation.
Genetic toxicology assays have been in use since the early 1970s and over that period of time, they have been developed, evolved, been used, replaced etc as the understanding of the biology driving mutation and cancer development has increased. As no single assay can detect all types of mutation, a battery of tests has been developed and curated over time leading to the standard battery used today.
As genetic toxicology crosses industry boundaries and each industry has its own regulatory framework, strategies can differ however international effort is continual to maintain standard protocols and introduce improvements (when required) through soliciting international expert opinions coupled with a requirement for those working groups to reach full consensus prior to updates being published.
The Organisation for Economic Co-operation and Development (OECD) is a group of member countries where, for the purposes of regulatory testing of chemicals (performed to Good Laboratory Practice [GLP]), has a unique agreement called the Mutual Acceptance of Data (MAD). The principles of the MAD agreement are that test data generated to GLP in any member country are fully acceptable in any other member country. This agreement has a genuinely positive 3Rs impact (reduce, refine, replace) by facilitating a “Tested Once only” philosophy.
The OECD Guidelines for the Testing of Chemicals is split into 5 sections, being: 1) Physical-Chemical properties; 2) Effects on Biotic Systems; 3) Environmental Fate and behaviour; 4) Health Effects; 5) Other Test Guidelines. Section 4 contains the Genotoxicity Assay Test Guidelines for the assays that have gained international acceptance and are utilised in the various testing strategies employed throughout multiple industries globally.
The OECD began the Genetic Toxicology Test Guideline program in 1983 and reviews the status and applicability of those guidelines and considers new additions to the program on an annual basis through its Working group of National Co-ordinators of the TG program (WNT) body.
In support of the OECD Test Guideline program, several international organisations form ad-hoc working parties to facilitate the development of new methods, assess applicability and drive validation efforts to prepare a method for consideration by the OECD WNT. Examples of these bodies include the Health and Environmental Sciences Institute-Genetic Toxicology Technical Committee (HESI-GTTC) and the International Workgroups on Genotoxicity Testing (IWGT).
In vitro bacterial mutation
The principle of the Bacterial Reverse Mutation Test (better known as the Ames Test) is that the assay detects mutations which restore the functional capacity of the bacteria to synthesize an essential amino acid. By withholding the amino acid required by the parent strains (strains of Salmonella typhimurium and Escherichia coli), mutation frequency can be determined by growth of bacteria that have gained mutation during the exposure period of the assay whereas parental strain(s) die. The different bacterial strains used each contain a different mutation such that by testing against multiple strains can reveal if the chemical causes mutation and if so, which type of mutation.
The Ames Test has been in use since the late 1960s / early 1970s and now has are vast libraries of data and literature available on its performance. It has been in the standard battery for decades and when that battery was reduced to two assays from three, the Ames Test was retained as the gene mutation assay. It is widely used for regulatory strategies and also for screening programs due to its effectiveness for detecting mutations but also for its quick turn-around from chemical exposure to scoring of the bacterial plates.
In vitro mammalian cell mutation
The OECD Test Guideline for mammalian gene mutation was first adopted in 1984 (OECD TG 476). As the original Guideline was for mammalian cells, it covered the HPRT assay and the TK assay (as well as the XPRT assay). The Guideline was revised in 1997 and then during the review of the Genetic Toxicology Test Guidelines in 2015, it was split into two such that OECD TG 476 was specifically for the HPRT / XPRT assay and the TK assay had a new guideline assigned to it (OECD TG 490).
Both OECD TG 476 and OECD TG 490 are forward-mutation assays. The assays work by exposing a known number of cells to the test chemical for a defined period of time and then giving the cell culture time to recover and for any induced mutations to become fixed in the cellular population. Selection is then completed by the inclusion of chemicals that are toxic to cells that have retained functional reporter genes resulting in only mutant cells remaining. This then allows a mutation frequency to be calculated.
The HPRT assay (OECD TG 476) uses the Hprt gene as its reporter gene. Hprt is X-linked such that the assay can detect mutational events (such as frameshift, base pair substitutions, deletions and insertions) but detection of chromosomal deletions are not within its realm. The less commonly used XPRT assay is an autosomal reporter gene, therefore detection of chromosomal events (e.g.: large deletions, mitotic recombination) is possible. The HPRT assay can be performed on several different cell types but the XPRT assay can only be performed on the AS52 cell line.
The TK assay (OECD TG 490) uses the Tk gene as its reporter gene. There are two cells lines, one murine (L5178Y tk+/-3.7.2C) and one human (TK6 tk+/-). In both cell lines, the Tk gene is autosomal and heterozygous therefore loss of the single functional copy of the tk gene results in cells that, when exposed to the selective chemical, are purged from the population leaving only mutant cells behind.
In vitro chromosome damage
When it comes to assessing chromosomal damage, the In Vitro Micronucleus (IVMN) assay (OECD Test Guideline 487) has become the assay of choice over the In Vitro Chromosomal Aberration assay (OECD Test Guideline 473). The primary reason for this is that the chromosomal aberration assay will not detect aneupoidy whereas the micronucleus assay will.
The micronucleus assay can be performed in vitro or in vivo with the assay detecting lagging chromosomal material during the process of nuclear division. By arresting mitosis post nuclear division (karyokinesis) but prior to cellular division (cytokinesis), acentric chromosome fragments or whole chromosomes that do not get withdrawn to the daughter nuclei are encapsulated in nuclear membrane and can be observed as micronuclei in the cytoplasm. Centromere-negative micronuclei indicate clastogenicity and centromere-positive micronuclei indicate aneugenicity.
The chromosome aberration assay can also be performed in vitro or in vivo with the assay detecting chromosome or chromatid structural aberrations. Although polyploidy can be caused by aneugens, other cellular factors can also cause polyploidy so this assay is not designed to detect numerical aberrations. By blocking cells at mitosis, the chromosomal material can be stained and viewed microscopically thus allowing an investigator to score the number of chromosomes present and to count the number of aberrations within a defined number of appropriate metaphases.
In vitro mode of action
Determining whether a mode of action is clastogenic (chromosomal breakage), aneugenic (chromosomal loss) or non-DNA reactive (such as cellular stress pathways like oxidative stress or unfolded protein stress) could be a critical answer. The In Vitro Micronucleus assay indicates the occurrence of a genotoxic effect on chromosomal integrity but it can also yield mis-leading positive results should the test chemical trigger cellular stress pathway(s).
There are three methods for determining mode of action. Historically, the use of fluorescence in-situ hybridisation (F.I.S.H.) and the marking of centromeres has been the gold standard method with a centromere-negative result indicating a clastogenic mechanism whereas a centromere-positive result indicates a aneugenic mechanism. Expanding on that knowledge, the development of ToxTracker® and MultiFlow® have opened the option to investigating the mode of action as these assays can reveal the above two mechanisms as well as others (oxidative stress, p53 activation, protein folding errors, phospho-histone H3, γH2AX etc).
The predictive capacity of computational modelling has been utilised for genotoxicity for some time with both expert systems and knowledge-base systems available. Some are freely available, some are subscription services or available through contract research organisations. Examples include Derek Nexus, LeadScope, TIMES, CASE ultra, SARAH Nexus, VEGA, Toxtree, AMBIT, USEPA T.E.S.T, OECD QSAR Toolbox