Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT).

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Evaluation of vinyl laurate in a battery of in vitro and in vivo tests for genotoxicity.

Vinyl laurate is a potential residual monomer in chewing gum base formulated with polyvinyl acetate vinyl laurate copolymer (PVAcVL). The genotoxic potential of vinyl laurate was examined in a battery of in vitro and in vivo genotoxicity tests. Vinyl laurate was not mutagenic in Ames tests. In addition, it was not mutagenic in the HPRT mutation assay in L5178Y cells. An in vitro mammalian chromosome aberration assay performed in CHO cells was equivocal. Vinyl laurate and/or its metabolites were not clastogenic in the mouse bone marrow micronucleus test. Kinetic data indicate that VL is metabolised to acetaldehyde and lauric acid. Both metabolites are well known and have been studied previously. Model calculations show, that any exposure to acetaldehyde from the consumption of PVAcVL containing chewing gum will remain far below levels of acetaldehyde exposure from food in which acetaldehyde occurs naturally. Direct exposure to VL will primarily be at the site of entry. The lack of toxicity in a 90-day repeated dose toxicity test, performed with VL doses up to approximately 3000 times higher than the maximal VL intake from the consumption of a typical piece of chewing gum, demonstrates a high safety margin.

Toxicity assessment of aggregated/agglomerated cerium oxide nanoparticles in an in vitro 3D airway model: the influence of mucociliary clearance.

We investigated the toxicity of aggregated nanoparticles of cerium oxide (CeO2) using an in vitro 3D human bronchial epithelial model that included a mucociliary apparatus (MucilAir™). CeO2 was dispersed in saline and applied to the apical surface of the model. CeO2 did not induce distinct effects in the model, whereas it did in BEAS-2B and A549 cell cultures. The absence of effects of CeO2 was not because of the model's insensitivity. Nanoparticles of zinc oxide (ZnO) elicited positive responses in the toxicological assays. Respiratory mucus (0.1% and 1%) added to dispersions increased aggregation/agglomeration to such an extent that most CeO2 sedimented within a few minutes. Also, the mucociliary apparatus of the model removed CeO2 from the central part of the apical surface to the borders. This 'clearance' may have prevented the majority of CeO2 from reaching the epithelial cells. Chemical analysis of cerium in the basal tissue culture medium showed only minimal translocation of cerium across the 3D barrier. In conclusion, mucociliary defence appeared to prevent CeO2 reaching the respiratory epithelial cells in this 3D in vitro model. This model and approach can be used to study compounds of specific toxicological concern in airway defence mechanisms in vitro.

A novel safety assessment strategy for non-intentionally added substances (NIAS) in carton food contact materials.

One of the main challenges in food contact materials research is to prove that the presence of non-intentionally added substances (NIAS) is not a safety issue. Migration extracts may contain many unknown substances present at low concentrations. It is difficult and time-consuming to identify all these potential NIAS and concurrently to assess their health risk upon exposure, whereas the health relevance at low exposure levels might not even be an issue. This paper describes a scientifically based, but pragmatic safety assessment approach for unknown substances present at low exposure levels in food contact matrices. This complex mixture safety assessment strategy (CoMSAS) enables one to distinguish toxicologically relevant from toxicologically less relevant substances, when related to their respective levels of exposure, and allows one to focus on the substances of potential health concern. In particular, substances for which exposure will be below certain thresholds may be considered not of health relevance in case specific classes of substances are excluded. This can reduce the amount of work needed for identification, characterisation and evaluation of unknown substances at low concentration. The CoMSAS approach is presented in this paper using a safety assessment of unknown NIAS that may migrate from three carton samples.

Some molecular descriptors for non-specific chromosomal genotoxicity based on hydrophobic interactions.

A concept relating the lipophilicity of chemicals with their genotoxicity on a chromosomal level had been generated by Schultz and Onfelt (Chem Biol Interact 126:97-123, 2000). It was shown that aneuploidy in Chinese hamster V79 cells was elicited by lipophilic chemicals at concentrations related to their hydrophobicity (log P), whereas toxicants with a specific mode of action acted at concentrations consistently lower than predicted based on log P. We have now combined available data sets on aneuploidy/micronucleus formation with procedures used in QSAR modelling, in order to find new molecular descriptors for modelling non-specific chromosomal genotoxicity, and to optimise combinations thereof. Molecular structures of 26 chemicals, including steroids, were converted into single 3D models using Corina (version 3.20), and 11 descriptors of molecular properties were calculated. The data of 16 compounds assigned to a non-specific mode of action were imported into the QSAR module of the software package Cerius(2) (version 4.10). Applying genetic function approximation (GFA), linear equations were set up relating molecular descriptors with experimental concentrations at which doubling of micronuclei occurred in V79 cells (exp -log C). The number of variables (molecular descriptors) was limited to a maximum of three, and linear and quadratic terms were allowed. Based on the descriptions provided by the GFA procedure, log P was the most suitable single property to describe non-specific genotoxicity [r (2 ) = 0.88], confirming the original concept of Schultz and Onfelt. Using more descriptors (up to three in combination) resulted in an optimization of correlations up to r (2 )= 0.97. Such optimal correlation coefficients were obtained by combinations (a) of the numbers of hydrogen bond acceptors, the polar surface and total surface areas of molecules on one hand, and by (b) the dipole moment, polar surface and total surface descriptors on the other hand. In essence, the relation of polar surface to the total molecular surface appears pivotal to determine the non-specific chromosomal genotoxicity of lipophilic compounds.

Proposed criteria for specific and non-specific chromosomal genotoxicity based on hydrophobic interactions.

Tests for chromosomal damage are indispensable in the genotoxicity testing battery. Thus, positive results of clastogenicity or aneugenicity tests are of key relevance in safety assessment and product development. Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.] have studied the chemical induction of bi- and multi-nucleated cells in Chinese hamster V79 cells and compared non-specific agents with inducers acting through a known specific mechanism. They separated compounds with a specific action from those with a non-specific action based on lipophilicity, following a theory of hydrophobic interactions with processes of cytokinesis. It appeared possible to broaden the original database of this concept to include aneugenic as well as clastogenic compounds studied in the micronucleus (MN) test. The datasets used for this purpose were (A) the original dataset of Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.], and two sets (B, C) of our own data from studies in V79 cells in vitro. As the particular endpoints used were different (A: counts of bi- and multi-nucleated cells, B/C: micronucleus counts) the coherence of the experimental data sets was validated by including compounds belonging to both collections. Data set B included compounds with a specific effect on the mitotic spindle (nitrobenzene and benzonitrile) and data set C included the phytoestrogens genistein and daidzein, as well as a number of hormonal steroids with unknown mode of action. Taking all three data sets (A, B, C) together, the 33 compounds investigated covered a total lipophilicity range of logP between -0.51 (diamide) and 5.65 (17alpha-propylmesterolone). In order to separate statistical outliers (with a specific mode of action to be likely) from the large cluster of compounds with non-specific genotoxicity related to hydrophobic interactions, the method of robust regression was applied. It appeared that all compounds with a specific mode of action were in fact outliers of the lipophilicity rule. Genistein, a weak clastogen causing chromosomal aberrations and being discussed to induce topoisomerase-2 mediated DNA breaks, came close to the statistical borderline between compounds with specific and non-specific chromosomal genotoxicity. A general procedure is proposed, applicable in chemical product development, to screen specific and non-specific modes of action.