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Event: 185

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Increase, Mutations

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
Increase, Mutations
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
mutation deoxyribonucleic acid increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Alkylation of DNA leading to heritable mutations KeyEvent Evgeniia Kazymova (send email) Open for citation & comment WPHA/WNT Endorsed
DNA alkylation -> cancer 2 KeyEvent Agnes Aggy (send email) Not under active development
DNA alkylation -> cancer 1 KeyEvent Arthur Author (send email) Open for adoption
RONS leading to breast cancer AdverseOutcome Evgeniia Kazymova (send email) Under development: Not open for comment. Do not cite Under Development
Increased DNA damage leading to breast cancer AdverseOutcome Allie Always (send email) Under development: Not open for comment. Do not cite Under Development
Oxidative DNA damage, chromosomal aberrations and mutations AdverseOutcome Brendan Ferreri-Hanberry (send email) Open for comment. Do not cite WPHA/WNT Endorsed
Deposition of energy leading to lung cancer KeyEvent Brendan Ferreri-Hanberry (send email) Open for citation & comment EAGMST Approved
Bulky DNA adducts leading to mutations AdverseOutcome Evgeniia Kazymova (send email) Under development: Not open for comment. Do not cite Under Development
DNA damage and metastatic breast cancer KeyEvent Agnes Aggy (send email) Under development: Not open for comment. Do not cite Under Development
Deposition of energy leading to cataracts KeyEvent Arthur Author (send email) Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
Mus musculus Mus musculus High NCBI
medaka Oryzias latipes Moderate NCBI
rat Rattus norvegicus High NCBI
Homo sapiens Homo sapiens Moderate NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
All life stages High

Sex Applicability

An indication of the the relevant sex for this KE. More help
Term Evidence
Unspecific High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

A mutation is a change in DNA sequence. Mutations can thus alter the coding sequence of genes, potentially leading to malformed or truncated proteins. Mutations can also occur in promoter regions, splice junctions, non-coding RNA, DNA segments, and other functional locations in the genome. These mutations can lead to various downstream consequences, including alterations in gene expression. There are several different types of mutations including missense, nonsense, insertion, deletion, duplication, and frameshift mutations, all of which can impact the genome and its expression in unique ways.

Missense mutations are the substitution of one base in the codon with another. This change is significant because the three bases in a codon code for a specific amino acid and the new combination may signal for a different amino acid to be formed. Nonsense mutations also result from changes to the codon bases, but in this case, they cause the generation of a stop codon in the DNA strand where there previously was not one. This stop codon takes the place of a normal coding triplet, preventing its translation into an amino acid. This will cause the translation of the strand to prematurely stop. Both missense and nonsense mutations can result from substitutions, insertions, or deletions of bases (Chakarov et al. 2014).  

Insertion and deletion mutations are the addition and removal of bases from the strand, respectively. These often accompany a frameshift mutation, as the alteration in the number of bases in the strand causes the frame of the base reader to shift by the added or reduced number, altering the amino acids that are produced if that number is not devisable by three. Codons come in specific orders, sectioned into groups of three. When the boundaries of which three bases are included in one group are changed, this can change the whole transcriptional output of the strand (Chakaroy et al. 2014). 

Mutations can be propagated to daughter cells upon cellular replication. Mutations in stem cells (versus terminally differentiated non-replicating cells) are the most concerning, as these will persist in the organism. The consequence of the mutation, and thus the fate of the cell, depends on the location (e.g., coding versus non-coding) and the type (e.g., nonsense versus silent) of mutation.

Mutations can occur in somatic cells or germ cells (sperm or egg).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

 Mutations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.

Somatic cells: The Salmonella mutagenicity test (Ames Test) is generally used as part of a first tier screen to determine if a chemical can cause gene mutations. This well-established test has an OECD test guideline (OECD TG 471, 2020). A variety of bacterial strains are used, in the presence and absence of a metabolic activation system (e.g., rat liver microsomal S9 fraction), to determine the mutagenic potency of chemicals by dose-response analysis. A full description is found in Test No. 471: Bacterial Reverse Mutation Test (OECD, 2016).

A variety of in vitro mammalian cell gene mutation tests are described in OECD’s Test Guidelines 476 (2016) and 490 (2015). TG 476 (2016) is used to identify substances that induce gene mutations at the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene, or the transgenic xprt (xanthine-guanine phosphoribosyl transferase) reporter locus. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. The only cells suitable for the XPRT test are AS52 cells containing the bacterial xprt (or gpt) transgene (from which the hprt gene was deleted).

The new OECD TG 490 (2015) describes two distinct in vitro mammalian gene mutation assays using the thymidine kinase (tk) locus and requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. The autosomal and heterozygous nature of the thymidine kinase gene in the two cell lines enables the detection of cells deficient in the enzyme thymidine kinase following mutation from tk+/- to tk-/-.

It is important to consider that different mutation spectra are detected by the different mutation endpoints assessed. The non-autosomal location of the hprt gene (X-chromosome) means that the types of mutations detected in this assay are point mutations, including base pair substitutions and frameshift mutations resulting from small insertions and deletions. Whereas, the autosomal location of the transgenic xprt, tk, or gpt locus allows the detection of large deletions not readily detected at the hemizygous hprt locus on X-chromosomes. Genetic events detected using the tk locus include both gene mutations (point mutations, frameshift mutations, small deletions) and large deletions.

The transgenic rodent mutation assay (OECD TG 488, 2020) is the only assay capable of measuring gene mutation in virtually all tissues in vivo. Specific details on the rodent transgenic mutation reporter assays are reviewed in Lambert et al. (2005, 2009). The transgenic reporter genes are used for detection of gene mutations and/or chromosomal deletions and rearrangements resulting in DNA size changes (the latter specifically in the lacZ plasmid and Spi- test models) induced in vivo by test substances (OECD, 2009, OECD, 2011; Lambert et al., 2005). Briefly, transgenic rodents (mouse or rat) are exposed to the chemical agent sub-chronically. Following a manifestation period, genomic DNA is extracted from tissues, transgenes are rescued from genomic DNA, and transfected into bacteria where the mutant frequency is measured using specific selection systems.

The Pig-a (phosphatidylinositol glycan, Class A) gene on the X chromosome codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Cells lacking GPI anchors, or GPI-anchored cell surface proteins are predominantly due to mutations in the Pig-a gene. Thus, flow cytometry of red blood cells expressing or not expressing the Pig-a gene has been developed for mutation analysis in blood cells from humans, rats, mice, and monkeys. The assay is described in detail in Dobrovolsky et al. (2010). Development of an OECD guideline for the Pig-a assay is underway. In addition, experiments determining precisely what proportion of cells expressing the Pig-a mutant phenotype have mutations in the Pig-a gene are in progress (e.g., Nicklas et al., 2015, Drobovolsky et al., 2015). A recent paper indicates that the majority of CD48 deficient cells from 7,12-dimethylbenz[a]anthracene-treated rats (78%) are indeed due to mutation in Pig-a (Drobovolsky et al., 2015).

Germ cells: Tandem repeat mutations can be measured in bone marrow, sperm, and other tissues using single-molecule PCR. This approach has been applied most frequently to measure repeat mutations occurring in sperm DNA. Isolation of sperm DNA is as described above for the transgenic rodent mutation assay, and analysis of tandem repeats is done using electrophoresis for size analysis of allele length using single-molecule PCR. For expanded simple tandem repeat this involved agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al. (2002) and Beal et al. (2015).

Mutations in rodent sperm can also be measured using the transgenic reporter model (OECD TG 488, 2020). A description of the approach is found within this published TG. Further modifications to this protocol have been made as of 2022 for the analysis of germ cells. Detailed methodology for detecting mutant frequency arising in spermatogonia is described in Douglas et al. (1995), O'Brien et al. (2013); and O'Brien et al. (2014). Briefly, male mice are exposed to the mutagen and killed at varying times post-exposure to evaluate effects on different phases of spermatogenesis. Sperm are collected from the vas deferens or caudal epididymis (the latter preferred). Modified protocols have been developed for extraction of DNA from sperm.

A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).

Please note, gene mutations that occur in somatic cells in vivo (OECD Test. No. 488, 2020) or in vitro (OECD Test No. 476: In vitro Mammalian Cell Gene Mutation Test, 2016), or in bacterial cells (i.e., OECD Test No. 471, 2020) can be used as an indicator that mutations in male pre-meiotic germ cells may occur for a particular agent (sensitivity and specificity of other assays for male germ cell effects is given in Waters et al., 1994). However, given the very unique biological features of spermatogenesis relative to other cell types, known exceptions to this rule, and the small database on which this is based, inferring results from somatic cell or bacterial tests to male pre-meiotic germ cells must be done with caution. That mutational assays in somatic cells may predict mutations in germ cells has not been rigorously tested empirically (Singer and Yauk, 2010). The IWGT working group on germ cells specifically addressed this gap in knowledge in their report (Yauk et al., 2015) and recommended that additional research address this issue. Mutations can be directly measured in humans (and other species) through the application of next-generation sequencing. Although single-molecule approaches are growing in prevalence, the most robust approach to measure mutation using next-generation sequencing today requires clonal expansion of the mutation to a sizable proportion (e.g., sequencing tumours; Shen et al., 2015), or analysis of families to identify germline derived mutations (reviewed in Campbell and Eichler, 2013; Adewoye et al., 2015).

Please refer to the table below for additional details and methodologies for measuring mutations.

Assay Name References Description OECD Approved Assay
Assorted Gene Loci Mutation Assays

Tindall et al., 1989; Kruger et al., 2015

After exposure to a chemical/mutagen, mutations can be measured by the ability of exposed cells to form colonies in the presence of specific compounds that would normally inhibit colony growth; Usually only cells -/- for the gene of interest are able to form colonies N/A
TK Mutation Assay

Yamamoto et al., 2017; Liber et al., 1982; Lloyd and Kidd, 2012

After exposure to a chemical/mutagen, mutations are detected at the thymidine kinase (TK) loci of L5178Y wild-type mouse lymphoma TK (+/-) cells by measuring resistance to lethaltriflurothymidine (TFT); Only TK-/- cells are able to form colonies Yes (No. 490)
HPRT Mutation Assay

Ayres et al., 2006; Parry and Parry, 2012

Similar to TK Mutation Assay above, X-linked HPRT mutations produced in response to chemical/mutagen exposure can be measured through colony formation in the presence of 6-TG or 8-azoguanine; Only HPRT-/- cells are able to form colonies Yes (No. 476)
Salmonella Mutagenicity Test (Ames Test) OECD, 1997 After exposure to a chemical/mutagen, point mutations are detected by analyzing the growth capacity of different bacterial strains in the presence and absence of various metabolic activation systems  Yes (No. 471)
PIG-A / PIG-O Assay

Kruger et al., 2015; Nakamura, 2012; Chikura, 2019

After exposure to a chemical/mutagen, mutations  in PIG-A or PIG-O (which decrease the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor protein) are assessed by the colony-forming capabilities of cells after in vitro exposure, or by flow cytometry of blood samples after in vivo exposure N/A
Single Molecule PCR

Kraytsberg & Khrapko, 2005; Yauk, 2002

This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies N/A
ACB-PCR

Myers et al., 2014 (Textbook, pg 345-363); Banda et al.,  2013; Banda et al.,  2015; Parsons et al., 2017

Using this PCR technique, single base pair substitution mutations within oncogenes or tumour suppressor genes can be detected by selectively amplifying specific point mutations within an allele and selectively blocking amplification of the wild-type allele N/A
Transgenic Rodent Mutation Assay

OECD 2013; Lambert 2005; Lambert 2009

This in vivo test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After in vivo exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype Yes (No. 488)
Conditionally inducible transgenic mouse models Parsons 2018 (Review) Inducible mutations linked to fluorescent tags are introduced into transgenic mice; Upon exposure of the transgenic mice to an inducing agent, the presence and functional assessment of the mutations can be easily ascertained due to expression of the linked fluorescent tags N/A
Error-Corrected Next Generation Sequencing (NGS) Salk 2018 (Review) This technique detects rare subclonal mutations within a pool of heterogeneous DNA samples through the application of new error-correction strategies to NGS; At present, few laboratories in the world are capable of doing this, but commercial services are becoming available (e.g., Duplex sequencing at TwinStrand BioSciences) N/A 

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

Taxonomic applicability: Mutations can occur in any organism and in any cell type, and are the fundamental material of evolution. The test guidelines described above range from analysis from prokaryotes, to rodents, to human cells in vitro. Mutations have been measured in virtually every human tissue sampled in vivo.

Life stage applicability: This key event is not life stage specific as all stages of life have DNA that can be mutated; however, baseline levels of mutations are seen to increase with age (Slebos et al., 2004; Kirkwood, 1989). 

Sex applicability: This key event is not sex specific as both sexes undergo mutations. Males have a higher mutation rate than females (Hedrick, 2007). 

Evidence for perturbation by a stressor: Many studies demonstrate that increased mutations can occur as a result of ionizing radiation (Sankaranarayanan & Nikjoo, 2015; Russell et al., 1957; Winegar et al., 1994; Gossen et al., 1995).  

Regulatory Significance of the Adverse Outcome

An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help

References

List of the literature that was cited for this KE description. More help

Adewoye, A.B. et al. (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", Nat. Commu., 6:6684. Doi: 10.1038/ncomms7684.

Ayres, M. F. et al. (2006),  “Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway”,  Genetics and Molecular Biology. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.

Banda M, Recio L, and Parsons BL. (2013), “ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver”, Environ Mol Mutagen. 54(8):659-67. Doi:10.1002/em.21808.

Banda,  M. et al. (2015), “Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation”,  Mutat Res Genet Toxicol Environ Mutagen. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003

Campbell, C.D. & E.E. Eichler (2013), "Properties and rates of germline mutations in humans", Trends Genet., 29(10): 575-84. Doi:  10.1016/j.tig.2013.04.005

Chakarov, S. et al. (2014), “DNA damage and mutation. Types of DNA damage”, BioDiscovery, Vol.11, Pensoft Publishers, Sofia, https://doi.org/10.7750/BIODISCOVERY.2014.11.1.

Chikura, S. et al. (2019), “Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society”,  Genes Environ.  27:41-5. Doi: 10.1186/s41021-019-0121-z.

Dobrovolsky, V.N. et al. (2015), "CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene. CD48-Deficient T-Lymphocytes from DMBA-Treated Rats Have De Novo Mutations in the Endogenous Pig-a Gene", Environ. Mol. Mutagen., (6): 674-683. Doi: 10.1002/em.21959.

Douglas, G.R. et al. (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", Proceedings of the National Academy of Sciences of the United States of America, 92(16): 7485-7489. Doi: 10.1073/pnas.92.16.7485.

Gossen, J.A. et al. (1995), "Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model", Mutation Research, 331/1, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(95)00055-N. 

Hedrick, P.W. (2007), “Sex: Differences In Mutation, Recombination, Selection, Gene Flow, And Genetic Drift”, Evolution, Vol.61/12, Wiley, Hoboken, https://doi.org/10.1111/j.1558-5646.2007.00250.x. 

Kirkwood, T.B.L. (1989), “DNA, mutations and aging”, Mutation Research, Vol.219/1, Elsevier B.V., Amsterdam, https://doi.org/10.1016/0921-8734(89)90035-0

Kraytsberg,Y. &  Khrapko, K. (2005),  “Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations”,  Expert Rev Mol Diagn. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.

Krüger, T. C., Hofmann, M., & Hartwig, A. (2015), “The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells”, Arch Toxicol. 89(12), 2429-43. Doi: 10.1007/s00204-014-1413-5.

Lambert, I.B. et al. (2005), "Detailed review of transgenic rodent mutation assays", Mutat Res., 590(1-3):1-280. Doi: 10.1016/j.mrrev.2005.04.002.

Liber, L. H., & Thilly, G. W. (1982),  “Mutation assay at the thymidine kinase locus in diploid human lymphoblasts”,  Mutation Research. 94: 467-485. Doi:10.1016/0027-5107(82)90308-6.

Lloyd, M., & Kidd, D. (2012), “The Mouse Lymphoma Assay. In: Parry J., Parry E. (eds) Genetic Toxicology, Methods in Molecular Biology (Methods and Protocols), 817. Springer, New York, NY.

Myers, M. B. et al., (2014), “ACB-PCR Quantification of Somatic Oncomutation”,  Molecular Toxicology Protocols, Methods in Molecular Biology. DOI: 10.1007/978-1-62703-739-6_27

Nakamura, J. et al., (2012), “Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system”, PLoS One.7(3): e33563. Doi:10.1371/journal.pone.0033563.

Nicklas, J.A., E.W. Carter and R.J. Albertini (2015), "Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line", Environ. Mol. Mutagen., 6(8):663-73. Doi: 10.1002/em.21953.

Norris, M.B. and R.N. Winn (2010), "Isolated spermatozoa as indicators of mutations transmitted to progeny", Mutat Res., 688(1-2): 36–40. Doi: 10.1016/j.mrfmmm.2010.02.008.

O'Brien, J.M. et al.(2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", Mutat. Res., 741-742:11-7. Doi: 10.1016/j.mrfmmm.2013.02.004.

O'Brien, J.M. et al. (2014), "Transgenic rodent assay for quanitifying male germ cell mutation frequency", Journal of Visual Experimentation, Aug 6;(90). Doi: 10.3791/51576.

O’Brien, J.M. et al. (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", Environ. Mol. Mutagen., 6(4): 347-355. Doi: 10.1002/em.21932.

OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2016), Test No. 476: In vitro Mammalian Cell Gene Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2009), Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, N° 103, ENV/JM/MONO 7, OECD, Paris.

OECD (2020), Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2016), Test. No. 490: In vitro mammalian cell gene mutation mutation tests using the thymidine kinase gene, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

Parry MJ, & Parry ME. 2012. Genetic Toxicology Principles and Methods. Humana Press. Springer Protocols.

Parsons BL, McKim KL, Myers MB. 2017. Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas. Environ Mol Mutagen. 58(7):466-476. Doi: 10.1002/em.22110.

Parsons BL. Multiclonal tumor origin: Evidence and implications. Mutat Res. 2018. 777:1-18. doi: 10.1016/j.mrrev.2018.05.001.

Russell, W.L. et al. (1957), "Radiation Dose Rate and Mutation Frequency.", Science, Vol.128/3338, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/science.128.3338.1546.

Salk JJ, Schmitt MW, &Loeb LA. (2018), “Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations”, Nat Rev Genet. 19(5):269-285. Doi: 10.1038/nrg.2017.117.

Sankaranarayanan, K. & H. Nikjoo (2015), "Genome-based, mechanism-driven computational modeling of risks of ionizing radiation: The next frontier in genetic risk estimation?", Mutation Research, Vol.764, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2014.12.003. 

Shen, T., S.H. Pajaro-Van de Stadt, N.C. Yeat and J.C. Lin (2015), "Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes" Front. Genet., 6: 215. Doi: 10.3389/fgene.2015.00215.

Singer, T.M. and C.L. Yauk CL (2010), "Germ cell mutagens: risk assessment challenges in the 21st century", Environ. Mol. Mutagen., 51(8-9): 919-928. Doi: 10.1002/em.20613.

Slebos, R.J.C. et al. (2004), “Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers”, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.559/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2004.01.003. 

Tindall, R. K., & Stankowski Jr., F. L. (1989),  “Molecular analysis of spontaneous mutations at the GPT locus in Chinese hamster ovary (AS52) cells”, Mutation Research, 220, 241-53. Doi: 10.1016/0165-1110(89)90028-6.

Waters, M.D. et al. (1994), "The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis", Mutat. Res., 341(2): 109-31. Doi: 10.1016/0165-1218(94)90093-0.

Winegar, R.A. et al. (1994), "Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice", Mutation Research, Vol.307/2, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(94)90258-5. 

Yamamoto, A. et al. (2017), “Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells”, Genes and Environment. 39: 22. Doi: 10.1186/s41021-017-0082-z.

Yauk, C.L. et al. (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus", Mutat. Res., 500(1-2): 147-56. Doi: 10.1016/s0027-5107(02)00005-2.

Yauk, C.L. et al. (2015), "Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays", Mutat. Res. Genet. Toxicol. Environ. Mutagen., 783: 36-54. Doi: 10.1016/j.mrgentox.2015.01.008.

Yeat and J.C. Lin. 2015. Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes. Front. Genet., 6: 215. Doi: 10.3389/fgene.2015.00215.