Guidance

Genotoxicity testing using 3D models

Published 18 July 2024

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Background

The Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) has a remit to provide UK Government Departments and Agencies with advice on the most suitable approaches to testing chemical substances for genotoxicity. The COM views regarding the most appropriate strategy for genotoxicity testing are outlined in full in the COM (2021) ‘Guidance On A Strategy For Genotoxicity Testing Of Chemical Substances’ [footnote 1].

In brief, the COM recommend a staged approach to genotoxicity testing.

Stage 0, in the absence of test data from adequately designed and conducted genotoxicity tests, consists of preliminary considerations of the test chemical substance, including, physicochemical properties, structure-activity relationships (SAR), and information from screening tests.

Stage 1 consists of in vitro genotoxicity tests that provide information on 3 types of genetic damage (namely, gene mutation, chromosomal damage and aneuploidy) and gives appropriate sensitivity to detect chemical genotoxins.

Stage 2 consists of in vivo genotoxicity tests which are chosen on a case-by-case basis to address any genotoxic endpoints identified in Stage 1.

  1. Investigate genotoxicity in tumour target tissue(s) and/or site of contact tissues.
  2. Investigate the potential for germ cell genotoxicity.
  3. Investigate potential genotoxicity for chemicals where high/moderate and prolonged exposure is anticipated, even if negative in Stage 1.

The use of 3-dimensional (3D) models for genotoxicity testing has not previously been discussed in the full COM guidance document [footnote 1]. However, as the development of 3D models is a rapidly evolving field, members considered it appropriate to prepare guidance in this area, that can be updated at regular intervals. As such, a brief summary of this area is provided in the full guidance document, while this document outlines in more detail the 3D models currently used for genotoxicity testing and those under development and/or validation.

Application of 3D models for genotoxicity testing

The main drivers for the development/use of 3D tissue models (artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all 3 dimensions) were the Cosmetics Directive, which prevented the use of in vivo testing for cosmetics, and the 3Rs principle that requires the reduction, replacement and refinement of the use of animals in toxicity testing. 3D tissue models have also been developed to undertake testing for which there is no robust in vivo system, such as site of contact studies, and have shown a utility that is now being assessed for genotoxicity testing. Human 3D tissue models may also be more scientifically valid when predicting human genotoxicity hazards.

Although currently used in vitro genotoxicity testing batteries can reliably identify in vivo genotoxicants, there are a number of positives which, when tested in vivo, are non-genotoxic, that is, these are misleading positive findings, commonly referred to as ‘false positives’. As a consequence, animal usage, testing time and costs can be unnecessarily increased which go against current initiatives that attempt to reduce the number of misleading positives from in vitro testing.

Such misleading positive findings are considered to occur for a number of reasons, including the use of cell lines of rodent origin (V79, CHO or CL) that partially lack normal cell cycle control, have limited metabolic capacity (even with the addition of rat liver S9) and do not mimic site-specific metabolic capacity [footnote 2]. However, the impact of these factors has become increasingly recognised and has led to the development of cell models which more closely reflect tissue structure and tissue metabolic activity.

A number of types of 3D tissue model, ranging from single cell microtissues to multicellular types grown within scaffolds have been developed. It is hoped the use of such models will improve the accuracy of predictions due to their improved metabolic capacity and closer correlation with in vivo human gene and protein expression profiles [footnote 3], [footnote 4].

The International Workshop on Genotoxicity Testing (IWGT) concluded that ‘3D tissue models offer a more ‘in-vivo-like’ behaviour for key parameters like cell viability, proliferation, differentiation, morphology, gene and protein expression, and function and therefore provide a valuable complement to the classical ‘2-dimensional (2D)’ cell culture-based assays’ [footnote 5].

3D models of skin

3D tissue models have, to date, mainly been developed for the skin. These models mimic the architectural features and behaviour of normal human skin and the changes that occur during early skin cancer progression and wound re-epithelialisation. Reconstructed 3D human epidermal skin models are used in Organisation for Economic Co-operation and Development (OECD) TG 431 (in vitro skin corrosion: reconstituted human epidermis (RHE) test) [footnote 6], [footnote 7], [footnote 8], which can be used in addition to the acute dermal irritation/corrosion test in rats (OECD TG 404). OECD TG439 (in vitro skin irritation: reconstituted human epidermis test) also utilises reconstructed 3D epidermal skin models [footnote 9], [footnote 10], [footnote 11]. Assessment of phototoxic properties [footnote 12], [footnote 13] and sensitisation potential [footnote 14], [footnote 15] are also being explored using reconstructed 3D skin models and are considered to have a high potential to be accepted as OECD TGs [footnote 2], [footnote 5], [footnote 16], [footnote 17], [footnote 18].

For genotoxicity testing purposes, 3D skin models have been linked to the standard genotoxicity endpoints of the micronucleus (MN) test and Comet assay. Two endpoints are utilised to reflect different types of genetic damage, namely clastogenicity/aneugenicity and DNA strand breaks/incomplete excision repair sites/alkali labile sites, respectively. The 3D Skin Comet assay and reconstituted skin micronucleus (RSMN) test are described below. These assays allow the in vitro assessment of DNA damage following dermal exposure, which has only previously been possible using in vivo assays. This is despite dermal exposure being a common route for a number of compounds found in household products, cosmetics, and industrial chemicals [footnote 19].

3D Skin Comet assay

The comet assay has been adapted for use with 2 reconstructed full thickness human skin models: the EpiDerm™ and Phenion® Full Thickness Skin Models. Both skin models are comprised of primary (p53 competent) cells of human origin. These models have a number of advantages over current monolayer-type assays including:

  • species specificity, with a phenotype close to native human skin
  • normal cell cycle control
  • DNA-repair competence
  • similar gene and protein expression patterns
  • and the mimicking of conditions of use and barrier function for dermally applied substances/products [footnote 19]

As the comet assay does not rely on proliferating cells and can be used with a wide range of cell types, it is particularly suitable for application to skin tissue models. The assay also detects a wide range of DNA damage including double-stranded and single-strand breaks from direct interaction of the test chemical or related to incomplete excision repair sites as well as alkali labile sites [footnote 20]. This ensures that both clastogenic DNA damage and lesions that may give rise to gene mutation are detected.

The 3D Skin Comet assay has undergone inter-laboratory validation using the Phenion® Full-Thickness Skin Model to assess its potential use as a new in vitro tool for following up positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals. The authors reported that the skin model has similar metabolic competency to natural human skin. Further, for the 32 substances tested, there was a high predictive capacity with a sensitivity of 80%, a specificity of 97% and an overall accuracy of 92% when compared to in vivo animal genotoxicity test outcomes. Improved predictability of the assay was seen when combined with the RSMN assay in a testing battery, with the sensitivity increasing to 90% and specificity remaining high [footnote 18], [footnote 19].

The RSMN assay has been developed to assess the genotoxicity of dermally applied compounds incorporated into cosmetics utilising a highly differentiated in vitro model of the human epidermis (EpiDerm™). Automated MN detection using the standard cytokinesis block MN assay is being developed [footnote 21]. The RSMN offers a close approximation of natural human skin due to the origin of the cells used and its physiological properties for cosmetic testing. The model also allows topical administration which ensures that all parts of the model are exposed, regardless of the lipophilic nature of the test substance. The assay has been successfully expanded to the Episkin LM™ model [footnote 4],[footnote 18], [footnote 22], [footnote 23], [footnote 24], [footnote 25], [footnote 26], which has previously been shown to have a similar metabolic capacity to that of native human skin [footnote 27] allowing the assessment of genotoxic potential by metabolic activation as an intrinsic feature.

A global validation of the assay has been carried out with the blinded testing of over 40 coded chemicals using the EpiDerm™ model. Findings showed an overall accuracy of 80%, a sensitivity of 75% and specificity of 84% when compared to in vivo genotoxicity outcomes [footnote 18]. It was noted that the sensitivity of the 72-hour protocol was superior to that of the 48-hour protocol and that the assay was now suitable for OECD TG development [footnote 25]. A submission has now been made to the OECD to include this assay into the test guideline programme. Further the working group (WG) concluded that the ‘RSMN assay was an acceptable alternative to the in vivo test for cosmetic testing and that the high predictivity also demonstrates that the test complies with all requirements to be accepted as a second-tier test’ [footnote 5].

Other 3D tissue models

3D human airway model

A number of 3D human airway models (also called lung models) have been established that closely resemble the lining of the human airway (discussed below). As such, their utility for the genotoxicity testing of inhaled chemicals is being evaluated. The IWGT reported that ‘initial data show that the comet assay can be applied to the 3D airway models and the WG encourages further development of this assay’. It was emphasised that ‘the lack of 3D airway assays that can detect aneugenicity is considered a gap and the development of such an assay is strongly encouraged’ [footnote 5].

The IWGT also suggested that use of the MN assay with the current 3D airway models may be restricted by the limited proliferation rate of the cells in the models [footnote 5]. However, developments are being made in this area and a recent publication has described the use of the cytokinesis-block micronucleus assay (CBMN) to detect secondary toxicity of nanomaterials in a dual cell co-culture model of the bronchial cell line, 16HBE14o- and differentiated THP-1 (dTHP-1) macrophages [footnote 28].

3D tissue models of the airway epithelium 18. In conventional monolayer (2D) cultures of basal cells, only maintenance and expansion of cells is possible. However, in 3D airway tissue models, basal cells can differentiate into a mucociliary pseudostratified epithelium containing ciliated, goblet and basal cells. Other properties similar to the native human airway epithelium include beating cilia, mucus secretion, barrier properties and remodelling and restoration properties [footnote 29].

The 2 most widely used models of the airway epithelium are 3D microtissue models and co-cultures of multiple cell types, both of which can be grown at the air-liquid interface (ALI) [footnote 28], [footnote 30], [footnote 31], [footnote 5].

ALI cultures reflect physiological conditions in vivo, with the respiratory epithelium being exposed to the air. These cultures are currently used to study cell biology and infection, culture patient-derived cells to model diseases, and test the effects of aerosolised particles (including drug formulations and cigarette smoke) on the respiratory epithelium (for example [footnote 32]. IWGT considered that these models may enable a more realistic (geno)toxicity assessment of inhaled compounds, and subsequent developments have been reported using the CBMN assay to detect secondary toxicity in a dual cell co-culture of 16HBE14o- bronchial cells and dTHP-1 macrophages. In addition, as the models can be kept in culture for months, IGWT considered that this presented the possibility of assessing subchronic exposures, and this has subsequently been demonstrated for 3D HepG2 (Immortalised cell line consisting of human liver carcinoma cells) model (see below).

3D liver microtissue model

In vitro liver models are valuable for hazard identification strategies due to the liver being the site of genotoxic metabolism. Conventional in vitro monolayer assays using hepatic cell lines may not be the most relevant assays to carry out functional and metabolic studies as the cells can lose key liver-specific functions, in particular cytochrome P450 activity [footnote 33], [footnote 34], [footnote 35], [footnote 5]. In addition, non-parenchymal cells (for example, immune cells) are absent which play an important role in clearance and in the initiation of an immune response. Due to the limited lifespan of the conventional 2D assays, repeated exposures are not possible [footnote 36].

A 3D liver microtissue model has been described for toxicological studies [footnote 36], [footnote 37], [footnote 38] which has a number of advantages over conventional 2D hepatic assays. These include:

  • the use of primary human hepatic cells
  • viability of cells for long periods which allows multiple exposures to be assessed
  • maintenance of a high level of metabolic activity across the lifespan of cells

A 3D liver spheroid model utilising HepG2 cells grown using a ‘hanging-drop’ technique has also been assessed for genotoxicity testing, with MN detection in the 3D spheroid models. MN induction by test agents (for example, B[a]P) was seen to be greater in the 3D structures than in the 2D format [footnote 39], likely as a result of the enhanced P450 expression in the 3D model compared to 2D HepG2 cells. The IWGT concluded that for 3D liver spheroids ‘initial data show that the MN assay can be applied to 3D liver spheroids and the WG encourages further development of this assay’. It is also recognised by the WG that this technique is being investigated within the EU Horizon 2020 project PATROLS (Physiologically anchored tools for realistic nanomaterial hazard assessment) which includes characterisation of their metabolic competence[footnote 5], [footnote 40]. A recent study has shown adaptation of the 3D HepG2 model for the assessment of genotoxicity following longer-term low dose exposure [footnote 41]. Currently, a gene mutation endpoint has not been developed for use in the 3D liver models. However, 3D liver spheroids have been used to provide metabolic competence in genotoxicity assays as a more human-relevant alternative to rat liver S9 [footnote 42].

Regulatory challenges

There is a requirement within the genotoxicity OECD TGs to show proliferative index and viability when undertaking in vitro assays for genotoxicity. This is challenging for some slow-growing 3D models and requires further consideration.

The cosmetics industry is more accepting of the findings of 3D models as no in vivo testing can be carried out. However, the application and acceptance of data to other areas of chemical genotoxicity testing is currently not known. In both cases though, data from such models would be considered as part of an overall weight of evidence (WoE). For example, the Scientific Committee on Consumer Safety (SCCS) guidance recommends the comet assay with 3D-reconstructed human skin as a tool to support a WoE approach in the case of a positive or equivocal gene mutation test in bacteria or mammalian gene mutation test. To evaluate a positive or equivocal result, the in vitro MN test on 3D- reconstructed human skin could be considered for dermally applied compounds [footnote 43].

Liver 3D models have advanced but there is a need for the development of gene mutation assays. Both airway and liver gene mutation models require validation.

Conclusion

3D human tissue models may offer an alternative testing strategy to in vivo assays for substances that are found to be positive using the traditional in vitro genotoxicity battery of tests. Extensive progress has been made on the development and validation of 3D genotoxicity models and models are available for the major routes of exposure in humans.

The most advanced of such models, the 3D skin models, have undergone inter-laboratory validation and been shown to comply with all requirements to be accepted as a second-tier test for cosmetic ingredients testing.

The 3D RSMN assay is currently moving into OECD TG development. For the 3D airway model, measurement of clastogenicity and gene mutation are possible, and detection of aneuploidy has been demonstrated.

Using historic data, chemicals that are positive for genotoxic activity in vivo have been shown to be positive in either the 3D-micronucleus or 3D-Comet Assay skin models. In the main, chemicals that are negative for genotoxic activity in vivo are also negative in the 2 3D models [footnote 44].

COM Secretariat
UK Health Security Agency
Radiation, Chemical and Environmental Hazards
Chilton, Didcot, Oxfordshire OX11 0RQ

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