The yeast DNA deletion (DEL) recombination assay has been proposed as a simple and rapid method to measure the reversion frequency in the HIS3 gene through homologous intrachromosomal recombination [69], offering a high degree of both sensitivity and specificity to carcinogens.

Ku proposed adapting it, along with a toxicogenomics add-on for MOA determination (and possibly a confirmatory in vivo assay) as an alternative to the ICH S2 genotoxicity test guidelines, which include both in vivo and in vitro testing [7]. At that time, cell transformation assays represented the only in vitro alternative; however, they were inadequate and misleading, and the ICH test battery had been tested using large databases and found to have limited predictive power for “carcinogenicity outcomes, which have genotoxic relevance”. The argument was that beyond the initial test set used to develop the ICH battery, there was little actual predictive utility, as demonstrated by retrospective analysis of marketed drugs. The frequent occurrence of false positives in standard in vitro assays was also mentioned as a disadvantage. Therefore, a single in vitro test was proposed to detect mutations of carcinogenic relevance, which would be widely applicable to various test situations (including contaminants, industrial chemicals, drugs, and candidate biologics) and would mimic human Phase I and II metabolism. Therefore, and to additionally provide MOA information, the system should possess a genome highly like that of humans. Additionally, it should be amenable to high throughput. Several arguments supporting the association between DEL recombination in yeast and carcinogenesis, as well as the improved reliability of detecting true tumorigens [7], were put forward.

The system's strengths include its ability to detect direct- and indirect-acting carcinogens, aneugens, and a wide variety of DNA lesions. It is sensitive, specific, simple, and fast; with add-ons, it can also yield information on the mechanism of action (MOA). At 11 days, the assay length is intermediate.

Gardner, [9] Jaspersen emphasized that Saccharomyces cerevisiae is particularly well-suited for analyzing gene function due to its ease of manipulation (deletion, mutation, and tagging by PCR) through facile homologous recombination with short stretches of sequence homology. However, it is a disadvantage that the in vitro yeast DEL recombination assay is not a human or a mammalian system, and the results are therefore an extrapolation based on analogy. However, the metabolism is a good mimic for human Phase I and II metabolism.

Following the DEL recombination assay, transcriptomic analysis should be carried out to interpret the MOA, and potentially, an in vivo confirmatory assay could be carried out if the results are equivocal.

The EpiDerm™ tissue model [1012] consists of 3-dimensional normal human epidermal keratinocytes (NHEK) cultured on tissue culture inserts and is ECVAM validated and accepted under OECD test guidelines. A Mat Tek EpiAlveolar™ 3D tissue model has also been developed (Charles River, 2024) for the detection of fibrosis-causing agents. Fibrosis can lead to downstream cancer outcomes in an epigenetic fashion; therefore, this represents another viable transformation test method.

Due to their ability to control all facets of the experiment, these systems offer the advantages of in vivo tests while avoiding associated problems, such as uncertainty about whether the toxicant has reached the target organ and at what concentration. Some researchers[13] have grown human bronchial epithelial cells (HBEC) at the air-liquid interface, but without the addition of other cell types, such as immune cells (macrophages), to study the toxicity of indoor air particulate matter. The addition of multiple cell types, such as goblet cells, a secretory cell type of the respiratory airway, or Langerhans/dendritic cells, an immune component of 3D reconstructed skin, improves the functionality and predictive capability of these models. Information about the MOA of a substance can also be gleaned from these models. These models have the advantages of directly visualizable and quantifiable outcomes that are comparable to traditional histopathology. The systems are versatile, being manipulable in many ways [14].

Another distinct advantage of 3D cultures is that they may detect changes in cells leading to cancer that are not normally detectable using other types of genetic toxicity assays. Either direct or indirect (i.e., epigenetic) changes, such as those associated with phototoxicity, wound healing, fibrosis, and inflammation, leading to cancer, can be detected and visualized.

These systems have the disadvantage of not being a high-throughput process in any respect, and are time-consuming, labor-intensive, and technologically demanding.

Recently accepted for the OECD TG development program, this assay was validated by a Cosmetics Europe-led ‘round robin’ laboratory validation project intended to address the lack of alternatives to traditional in vivo genotoxicity testing. This is because, under EU rules for cosmetics, an in vitro positive test result would rule out the commercial use of a substance without further confirmatory in vivo testing being permitted. This effort also supports the development of dermal genotoxicity assays [1416]. It aims to evaluate the performance of the test using the Phenion^® Full-Thickness skin model in various regulatory, academic, and industry laboratory settings. The researchers applied chemicals three times over a 48-hour period, then isolated keratinocytes and fibroblasts, which were subjected to electrophoresis using the standard Comet assay, with the percent tail DNA as the recorded outcome. The experiment was conducted on 32 substances in a blinded manner. Results were evaluated by a statistician and then decoded [14].

The assay was highly predictive (sensitivity 80%), specific (97%), and accurate (92%). Intra- and inter-laboratory reproducibility were 93% and 88%, respectively. It was asserted that the method is useful for confirming the results of standard genotoxicity assays, such as the Ames test, and can fulfill EU Cosmetics Regulation EC No. 1223/2009 requirements that ban animal testing. It can also confirm in vivo results under REACH.

This assay combines the micronucleus (MN) assay with the EpiDerm™ three-dimensional in vitro reconstructed skin (RS) model. RSMN is intended for dermally applied products, not as a stand-alone assay, but rather as a follow-up to verify the results of a standard genotoxicity assay, and it is accepted by European regulatory agencies [1719].

Validation studies have demonstrated good transferability, inter- and intra-laboratory reproducibility, specificity (87%), and sensitivity (65%). However, sensitivity was further increased to 80% by the addition of a 72-hour treatment to resolve equivocal results. In combination with the 3D skin comet assay, the assay sensitivity increased to 92%. Fluorescently labelled cells are visually scored for the presence of micronuclei in binucleated cells; automation may speed the process.

Some of the advantages include topical application of the test substances, the relative rapidity of the test (total treatment time of 48 hours), and the fact that it has been thoroughly validated. Another advantage is that compounds testing negative after 48 hours can be easily retested up to 72 hours, which was found to increase test sensitivity. These qualities are likely to result in significantly lowered resource requirements when measured against traditional animal skin testing. This method is human-based and does not utilize cells of animal origin, but it complements other methods that may employ animal-based components.

Also in the OECD TG pipeline, the Bhas 42 CTA is a short-term, sensitive assay for the detection of chemical carcinogenicity. It is not a genetic toxicity assay per se, but it can assess the potential of a substance to cause changes to cells that might signal potential nongenotoxic carcinogenesis [20]. As a modification of the NIH 3T3 method, it was developed through the efforts of several labs [2123] and later validated by an inter-laboratory study [22] and an international consortium [2426].

Sasaki et al. [26] described the method of using v-Ha-ras gene-transfected mouse BALB/c 3T3 A31-1-1 cells to determine whether a chemical is an initiating or promoting (non-genotoxic) carcinogen. However, the method is not used to distinguish between genotoxic and non-genotoxic chemicals, but to detect carcinogenicity regardless of genotoxicity. The Bhas 42 cells were developed from BALB/c 3T3 cells through transfection with plasmid pBR322 containing Ha-MuSV-DNA, clone H1 (v-Ha-ras) [2628], and transformed using 12-O-tetradecanoylphorbol-13-acetate (TPA).

The two components of the assay initially were termed the initiation activity assay and the promotion activity assay but are now termed the ‘proliferation phase’ test to address the late initiation stage that the test assesses, and the ‘stationary phase’ test to define the proliferative stage where cells are treated at the stationary phase, and this provides a growth advantage for anomalous cells.

These two phases vary in terms of time and treatment conditions. In the first component, cells are seeded at 4 x 10³ cells/well (day 0) and treated early in the assay period (days 1-4) only. This allows target cells to undergo several rounds of division before contact inhibition occurs, allowing fixation of DNA mutations. In the second component, cells are seeded at 1.4 x 10³ cells/well and treated at sub-confluence (days 4-14), then continued without further treatment for a total of 21 days.

The use of the stationary phase test is intended to detect chemical compounds that can act as tumor promoters. However, they are considered negative or equivocal in the Ames assay. For those compounds that are positive in the first or proliferation phase, the Bhas 42 CTA can serve as a confirmatory assay. Compounds positive in both components are considered ‘complete carcinogens’. Currently, this assay has been commercialized and is available from multiple sources as a service or in kit form [29], and has been undergoing OECD TG acceptance for some time.

Ohmori et al. have since measured gene expression over time during the cellular transformation of Bhas 42 cells by TPA [30] and described the pathways and specific gene changes observed. Guichard et al. [31] then evaluated whether a 12-gene panel could predict the cell transformation potential of tumor-promoting agents, using the Bhas 42 CTA. They tested 12 genes that had previously been shown to be altered during transformation using either silica nanoparticles or TPA. Four soluble transforming agents (mezerein, methylarsonic acid, cholic acid, quercetin) were tested, and it was found that one (mezerein) modified all 12 genes, two (methylarsonic acid and cholic acid) gave an incomplete signature, sharing some gene changes but not all, and one (quercetin) induced no change in the 12 genes but induced cytotoxicity. Thus, at least for these four agents, they were unable to predict the signature of a transforming agent using the 12-gene panel. They hypothesized that these agents used different cellular pathways or molecular initiating events and thus could not be classed together using a single gene expression pattern.

Masumoto et al. [32] developed a trained convolutional neural network (CNN) for the automated determination of transformed foci in Bhas 42 cells, which exhibited an AUC of 0.95 and significantly outperformed conventional classification methods, as learned using the OECD guidance document. This was true even using untrained images. An important advantage is that CNN does not require feature extraction and can learn feature extraction from the data, thus reducing the time taken to classify transformed or non-transformed foci and the error rate in classification.

For an in vitro method, the assay length is somewhat lengthy (21 days), meaning that repeated studies could become quite time-consuming. As with all cell culture methods, any significant deviations that occur can require a complete restart of the procedure. Bhas 42 CTA is not a standalone assay for the detection of genetic toxicity; it is used as a confirmatory assay only for compounds negative or equivocal in the Ames assay. It can differentiate tumor promoters (both genotoxic and non-genotoxic) from non-tumor promoters, which is a useful approach but has limited application.

Advantages include its sensitivity and the ability to determine the transforming potential of a substance without an initiator, as the cell line already contains v-Ha ras. It reduces the time to correctly classify a transformed versus a non-transformed focus.

Originally developed by Hendriks et al. [3337], ToxTracker is a fluorescence-based assay that measures the activation of six reporter systems. The assay uses mouse embryonic stem cells (mESC) and detection by flow cytometry in a 96-well plate format [38].

The first step is to determine the appropriate dose range by exposing the cells to multiple concentrations in a serial dilution, up to a maximum concentration that produces 50-75% cytotoxicity, or if not reached, 1 mg/mL or the maximal soluble concentration. In a 96-well plate, five concentrations plus positive and negative or vehicle controls are applied for 24 hours, followed by measurement of relative mean fluorescence in the treated vs. (vehicle) control wells, corrected for relative cell count.

Like the Ames and in vitro MN assays, the ToxTracker assay relies on metabolic activation using rat S9 liver homogenate. The Hendriks protocol specified co-treatment of cells with compounds and S9 mix for 3 to 4 hours, followed by recovery for 17 to 24 hours, and then detection. However, this procedure required a significant recovery period due to S9 toxicity. Subsequently, others [38, 39] modified the procedure to increase sensitivity. Their modification reduced the concentration of S9, increased incubation to 24 hours, and specified no recovery period, which apparently produces less interference with assay results.

ToxTracker can detect several different forms of cellular damage. The two major reporter constructspredicting genotoxicity in the ToxTracker assay are Bscl2-GFP (activated upon the formation of bulky DNA adducts, which subsequently inhibits DNA replication) and Rtkn-GFP (activated upon the formation of DNA double-strand breaks). Other types of damage that are detectable include oxidative stress (Srxn1, Blvrb reporters) and protein damage (Ddit3 reporter), which constitute non-genotoxic mechanisms. Btg2 reporter induction may signal cell cycle arrest or general genotoxic stress. Together, the responses can differentiate between direct and indirect DNA damage and provide information about the specific pathways involved [40].

In a recent interlaboratory validation study, seven labs tested 64 chemicals (both genotoxic and non-genotoxic) using OECD TG 34 and achieved intralaboratory reproducibility of 73 to 98% and interlaboratory reproducibility of 83%. The sensitivity of the assay was 84.4%, and the specificity was 91.2% [39].

The assay requires metabolic activation and utilizes mouse embryo donors. The maximum soluble concentration is 1 mg/mL for some compounds, which may make it challenging to find a concentration that does not cause cytotoxicity, is soluble, and yet is sufficiently concentrated to produce a significantly measurable effect in the assay.

Bryce et al. [4146] developed a miniaturized flow cytometry-based assay that automates MN scoring (included in OECD TG 487) and a multiplexed flow cytometric-based assay that measures phosphorylation of histone H3 (p-H3; mitosis marker), phosphorylation of H2AX at serine 139 (γH2AX; double strand DNA breaks), nuclear p53 content (p53 translocation marker, response to DNA damage), frequency of 8n cells (marker of polyploidization), and nuclei counts (cell enumeration) for evaluation of cellular genotoxicity.

A sophisticated data analysis strategy is employed, utilizing multinomial logistic regression (MLR to generate probability scores, which are then used to classify chemicals by mechanism of action (MOA), including clastogen, aneugen, and non-genotoxic. The same authors later extended these results to new chemicals with known genotoxic properties and tested the applicability of LR algorithms (and others) to data generated from TK6 cells exposed to 103 chemicals not previously evaluated, tested, or used in training. Multinomial logistic regression (LR), artificial neural network (ANN), and random forest (RF) models were built using 4-hour and 24-hour MultiFlow data to predict whether a chemical is genotoxic and to determine its predicted mechanism of action (MOA) as clastogenic, aneugenic, or non-genotoxic. These were fed through the models after a set of 83 previously studied chemicals was applied to train the models. Both the individual model performance and a ‘majority vote ensemble’ approach were determined. Specific criteria for the number of positive scores from successive concentrations were applied, and compounds were ranked based on a probability score. The authors aimed to enhance the throughput, predictivity, and overall generalizability of genotoxicity testing by employing this strategy. The ANN model performed particularly well, and the ensemble majority vote approach added validity to the conclusions.

This test aims to determine only directly genotoxic-active substances, and no metabolic activation is applied. Therefore, any substance known or predicted to require metabolic activation would, by definition, be classified as non-genotoxic. The method was cross-validated in a 7-laboratory multi-center study of 60 chemicals. The majority vote ensemble score (2 of the 3 model approaches in agreement) was able to produce high accuracy, specificity, and sensitivity values of between 90 and 95%. The assay could not test 49 of 103 chemicals based on inability to reach the 1 mM limit, failure to meetthe assay’s cytotoxicity threshold, or precipitate formation.

Advantages of the MultiFlow™ assay are its ability to screen compounds and classify them by MOA as clastogen, aneugen, or non-genotoxic, which can support de-risking of an adverse finding. It would be a suitable choice as a pre-screen or a mechanistic follow-up for cosmetics under EU rules, or for marketed chemicals under REACH. For non-genotoxic carcinogens, it is useful to study the MOA, especially for data-poor substances. It is a multiplex, high-throughput assay with high sensitivity and specificity, providing mechanistic insights.

The TGx-DDI assay, developed by Li et al. [47], is designed to identify potential genotoxic substances and discriminate between DNA- and other types of damage [48]. It includes gene expression data for 64 individual genes, identified as relevant to DNA-damage-inducible substances and known non-DNA damage-inducible genes. Originally, TK6 cultured mammalian cells were exposed to 28 chemical substances (one of which is a validated biomarker for aneugenicity, or a change in chromosome number), and the resulting gene expression changes were measured. The results were then generalized to newly tested substances that produce the same changes in vitro.

Gene expression analysis is used to assess genotoxicity after cells in culture are exposed to the test substance for four hours. Cell collection, lysis, RNA extraction, and transcriptomic analysis are performed.

Buick et al. [49] employed this combinatorial approach to assess the potential genotoxicity of ten data-poor compounds. Six of the ten were identified as genotoxins by all three assays in the multiplex, despite being data-poor, and the mechanism of action (MOA) was defined as clastogenic. In four other compounds, the results of the three assays did not align, and the MultiFlow^® assay results indicating non-genotoxicity were used to conclude that these two compounds were likely false positives in the MicroFlow^® test. The last two compounds were weakly DNA-damage inducing in the presence of S9 and MN-inducing by MicroFlow^®, but were identified as non-genotoxic by MultiFlow^®. Therefore, they were deemed equivocal and recommended for further definitive testing. The authors then potency-ranked each of the test substances using benchmark concentration (BMC) modeling.

TGX DDI is an effective screening and confirmatory assay as part of a battery of tests to identify potential genotoxins, DNA damage, other cellular damage, and mechanisms of action (MOAs). It is particularly useful for data-poor substances.

Prototypical substances have been used to confirm the assay performance [4951]. Multiplexing the TGx-DDI together with MicroFlow^® and MultiFlow^® assays (above) is particularly useful because classifiers from the two approaches can then be compared and the results corroborated. The information that can be derived from this multiplex of assays is clearly much more useful than a simple test of positive or negative genotoxicity alone.

It was noted that the resulting BMCs could be converted to administered equivalent doses (AEDs, as referred to in qIVIVE in the Discussion) using HTTK models. Since qIVIVE can be used to determine a human MOE (known as a bioactivity exposure ratio, BER), it may be practically employed for risk assessment if toxicokinetic parameters, such as plasma protein binding and metabolic clearance, are known for the compound(s). This makes it an extraordinarily valuable technique for human risk assessment.

This assay is amenable to high-throughput analysis and can be completed in as little as one to two days with experienced hands and automated facilities.

Disadvantages include that it is an indirect measure of damage and has not yet been fully validated (although it has been cross-tested in experiments).

The FE1 in vitro version of MutaMouse™ Transgenic Rodent Gene Mutation Assay [5254] is an in vitro transgene mutation assay that uses the FE1 epithelial cell line derived from MutaMouse™ lung. The cells contain a shuttle vector with a lacZ mutation target that is amenable to positive selection of mutants using an E. coli galE-lacZ host and the PGal (phenyl-β-galactosidase) selection system.

Maertens et al. [51] demonstrated that for nine compounds that previously produced false positive in vitro test results, none of them showed positive results in the FE1 in vitro MutaMouse™ transgenic assay. Furthermore, when compared with the results of Fowler et al. [54] for ability to induce micronuclei in three p53-deficient rodent cell lines (V79, CHO, and CHL) or three p53-competent human cell lines (primary human lymphocyte HuLy cells, human lymphoblastoid TK6 cells, and human hepatocellular carcinoma HepG2 cells), the FE1 MutaMouse cells outperformed the V79, CHO, and CHL cells in identifying the false positive chemicals, and were equal in performance to the human p53-competent cell lines.

Some positive attributes of FE1 include its cytogenetic stability, normal p53 functionality, endogenous metabolic capability (constitutive CYP1A1 and GST enzymes), and the presence of a retrievable transgene for mutational scoring.

As the in vivo MutaMouse™ transgenic assay is accepted by the OECD (OECD TG 488) [55], the in vitro FEI MutaMouse™ assay serves as a complementary test and should be considered an appropriate screen for compounds that previously produced false positive results in conventional assays, or prior to conducting the in vivo MutaMouse™ assay. It has reportedly been submitted to the OECD multistep evaluation process for validation [56].

Cox et al. characterized and developed a second MutaMouse™ transgenic in vitro assay, based on primary hepatocytes [57, 58]. This assay was intended to overcome problems with in vitro genotoxicity assays, including a need for metabolically competent cells (and the attendant problems with using rodent liver S9), and karyotype instability issues (deletions, duplications, translocations, impaired p53 function, genomic drift, and changing cell growth characteristics).

After thorough characterization, it was determined that cells exhibited a normal phenotype, were metabolically competent, and contained the lacZ shuttle vector on chromosome 3, demonstrating that the cells could be used to measure mutational events after treatment with candidate compounds in vitro. Cytochrome P450 induction by a canonical Cyp1a1 and 1a2 gene inducer, β-naphthoflavone, was also observed.

Later, the same authors tested 13 mutagenic and non-mutagenic compounds, including a range of compounds (direct acting, requiring metabolic activation) and detected a concentration-dependent increase in mutant frequency of up to 14.4-fold vs. control in all but one of the mutagens, and in none of the four non-mutagens (two of which had previously elicited false positive results). They concluded that for either chemicals that require metabolic activation or direct-acting mutagens, the MutaMouse™ primary hepatocyte (PH) assay can be used as an in vitro gene mutation assay.

The PH assay has the same advantages and disadvantages as the FE1 test, except that it uses primary hepatocytes and does not require metabolic activation.