Mechanisms of Nitrosamine Mutagenicity and Their Relationship to Rodent Carcinogenic Potency.

A thorough literature review was undertaken to understand how the pathways of N-nitrosamine transformation relate to mutagenic potential and carcinogenic potency in rodents. Empirical and computational evidence indicates that a common radical intermediate is created by CYP-mediated hydrogen abstraction at the α-carbon; it is responsible for both activation, leading to the formation of DNA-reactive diazonium species, and deactivation by denitrosation. There are competing sites of CYP metabolism (e.g., ß-carbon), and other reactive species can form following initial bioactivation, although these alternative pathways tend to decrease rather than enhance carcinogenic potency. The activation pathway, oxidative dealkylation, is a common reaction in drug metabolism and evidence indicates that the carbonyl byproduct, e.g., formaldehyde, does not contribute to the toxic properties of N-nitrosamines. Nitric oxide (NO), a side product of denitrosation, can similarly be discounted as an enhancer of N-nitrosamine toxicity based on carcinogenicity data for substances that act as NO-donors. However, not all N-nitrosamines are potent rodent carcinogens. In a significant number of cases, there is a potency overlap with non-N-nitrosamine carcinogens that are not in the Cohort of Concern (CoC; high-potency rodent carcinogens comprising aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds), while other N-nitrosamines are devoid of carcinogenic potential. In this context, mutagenicity is a useful surrogate for carcinogenicity, as proposed in the ICH M7 (R2) (2023) guidance. Thus, in the safety assessment and control of N-nitrosamines in medicines, it is important to understand those complementary attributes of mechanisms of mutagenicity and structure-activity relationships that translate to elevated potency versus those which are associated with a reduction in, or absence of, carcinogenic potency.

Acceptable intakes (AIs) for 11 small molecule N-nitrosamines (NAs).

Low levels of N-nitrosamines (NAs) were detected in pharmaceuticals and, as a result, health authorities (HAs) have published acceptable intakes (AIs) in pharmaceuticals to limit potential carcinogenic risk. The rationales behind the AIs have not been provided to understand the process for selecting a TD50 or read-across analog. In this manuscript we evaluated the toxicity data for eleven common NAs in a comprehensive and transparent process consistent with ICH M7. This evaluation included substances which had datasets that were robust, limited but sufficient, and substances with insufficient experimental animal carcinogenicity data. In the case of robust or limited but sufficient carcinogenicity information, AIs were calculated based on published or derived TD50s from the most sensitive organ site. In the case of insufficient carcinogenicity information, available carcinogenicity data and structure activity relationships (SARs) were applied to categorical-based AIs of 1500 ng/day, 150 ng/day or 18 ng/day; however additional data (such as biological or additional computational modelling) could inform an alternative AI. This approach advances the methodology used to derive AIs for NAs.

Predicting DNA-Reactivity of N-Nitrosamines: A Quantum Chemical Approach.

N-Nitrosamines (NAs) are a class of reactive organic chemicals that humans may be exposed to from environmental sources, food but also impurities in pharmaceutical preparations. Some NAs were identified as DNA-reactive mutagens and many of those have been classified as probable human carcinogens. Beyond high-potency mutagenic carcinogens that need to be strictly controlled, NAs of low potency need to be considered for risk assessment as well. NA impurities and nitrosylated products of active pharmaceutical ingredients (APIs) often arise from production processes or degradation. Most NAs require metabolic activation to ultimately become carcinogens, and their activation can be appropriately described by first-principles computational chemistry approaches. To this end, we treat NA-induced DNA alkylation as a series of subsequent association and dissociation reaction steps that can be calculated stringently by density functional theory (DFT), including α-hydroxylation, proton transfer, hydroxyl elimination, direct SN2/SNAr DNA alkylation, competing hydrolysis and SN1 reactions. Both toxification and detoxification reactions are considered. The activation reactions are modeled by DFT at a high level of theory with an appropriate solvent model to compute Gibbs free energies of the reactions (thermodynamical effects) and activation barriers (kinetic effects). We study congeneric series of aliphatic and cyclic NAs to identify trends. Overall, this work reveals detailed insight into mechanisms of activation for NAs, suggesting that individual steric and electronic factors have directing and rate-determining influence on the formation of carbenium ions as the ultimate pro-mutagens and thus carcinogens. Therefore, an individual risk assessment of NAs is suggested, as exemplified for the complex API-like 4-(N-nitroso-N-methyl)aminoantipyrine which is considered as low-potency NA by in silico prediction.

A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

Use of the bacterial reverse mutation assay to predict carcinogenicity of N-nitrosamines.

Under ICH M7, impurities are assessed using the bacterial reverse mutation assay (i.e., Ames test) when predicted positive using in silico methodologies followed by expert review. N-Nitrosamines (NAs) have been of recent concern as impurities in pharmaceuticals, mainly because of their potential to be highly potent mutagenic carcinogens in rodent bioassays. The purpose of this analysis was to determine the sensitivity of the Ames assay to predict the carcinogenic outcome with curated proprietary Vitic (n = 131) and Leadscope (n = 70) databases. NAs were selected if they had corresponding rodent carcinogenicity assays. Overall, the sensitivity/specificity of the Ames assay was 93-97% and 55-86%, respectively. The sensitivity of the Ames assay was not significantly impacted by plate incorporation (84-89%) versus preincubation (82-89%). Sensitivity was not significantly different between use of rat and hamster liver induced S9 (80-93% versus 77-96%). The sensitivity of the Ames is high when using DMSO as a solvent (87-88%). Based on the analysis of these databases, the Ames assay conducted under OECD 471 guidelines is highly sensitive for detecting the carcinogenic hazards of NAs.

Use of less-than-lifetime (LTL) durational limits for nitrosamines: Case study of N-Nitrosodiethylamine (NDEA).

The ICH M7(R1) guideline describes a framework to assess the carcinogenic risk of mutagenic and carcinogenic pharmaceutical impurities following less-than-lifetime (LTL) exposures. This LTL framework is important as many pharmaceuticals are not administered for a patient's lifetime and as clinical trials typically involve LTL exposures. While there has been regulatory caution about applying LTL concepts to cohort of concern (COC) impurities such as N-nitrosamines, ICH M7 does not preclude this and indeed literature data suggests that the LTL framework will be protective of patient safety for N-nitrosamines. The goal was to investigate if applying the LTL framework in ICH M7 would control exposure to an acceptable excess cancer risk in humans. Using N-nitrosodiethylamine as a case study, empirical data correlating exposure duration (as a percentage of lifespan) and cancer incidence in rodent bioassays indicate that the LTL acceptable intake (AI) as derived using the ICH M7 framework would not exceed a negligible additional risk of cancer. Therefore, controlling N-nitrosamines to an LTL AI based on the ICH M7 framework is thus demonstrated to be protective for potential carcinogenic risk to patients over the exposure durations typical of clinical trials and many prescribed medicines.

Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine Learning.

One of the most appreciated capabilities of computational toxicology is to support the design of pharmaceuticals with reduced toxicological hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compound toxicity assessments and compound optimization methods. To ensure good data quality and aiming at models with global applicability we separately compiled and curated highly chemically diverse data sets from 3T3 NRU phototoxicity reports (450 compounds) and clinical photosensitization alerts (1419 compounds) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extracted from quantum chemical computations at the density functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83-85% could typically be reached for diverse test data sets and other company in-house data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%-90%. Importantly, a computer model-triggered response-map allowed for graphical/chemical interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed molecules.

A critical appraisal of the sensitivity of in vivo genotoxicity assays in detecting human carcinogens.

Genotoxicity testing is an important part of standard safety testing strategies. Animal studies have always been a key component, either as a mandatory part of the regulatory test battery, or to follow-up questionable in vitro findings. The strengths and weaknesses of in vivo assays is a continuous matter of debate, including their capacity to predict (human) carcinogenicity. We have therefore analysed the sensitivity of five routinely used in vivo tests to determine, in addition to other aspects, which tests or combination of tests best identify 73 chemicals classified as IARC Group 1 and 2A carcinogens. The in vivo tests included the micronucleus (MN), unscheduled DNA synthesis (UDS), comet, Pig-a and transgenic rodent assays (TGR). The individual assays detect 74.2% (49/66, MN), 64.3% (9/14, UDS), 92.1% (35/38, comet), 82.4% (14/17, Pig-a) and 90.3% (28/31, TGR) of the probable and confirmed human carcinogens that were tested in these assays. Combining assays that cover different genotoxicity endpoints and multiple tissues, e.g. the bone marrow MN and the liver comet assays, increases the sensitivity further (to 94%). Correlations in terms of organ-specificity for these assays with human cancer target organs revealed only a limited correlation for the hematopoietic system but not for other organs. The data supports the use of the comet and TGR assays for detection of 'site-of-first-contact' genotoxicants, but these chemicals were generally also detected in assays that measure genotoxicity in tissues not directly exposed, e.g. liver and the hematopoietic system. In conclusion, our evaluation confirmed a high sensitivity of the five in vivo genotoxicity assays for prediction of human carcinogens, which can be further increased by combining assays. Moreover, the addition of the comet to the in vivo MN test would identify all DNA reactive human carcinogens. Importantly, integration of some of the study readouts into one experiment is an animal-saving alternative to performing separate experiments.

Computational Models for Human and Animal Hepatotoxicity with a Global Application Scope.

Hepatic toxicity is a key concern for novel pharmaceutical drugs since it is difficult to anticipate in preclinical models, and it can originate from pharmacologically unrelated drug effects, such as pathway interference, metabolism, and drug accumulation. Because liver toxicity still ranks among the top reasons for drug attrition, the reliable prediction of adverse hepatic effects is a substantial challenge in drug discovery and development. To this end, more effort needs to be focused on the development of improved predictive in-vitro and in-silico approaches. Current computational models often lack applicability to novel pharmaceutical candidates, typically due to insufficient coverage of the chemical space of interest, which is either imposed by size or diversity of the training data. Hence, there is an urgent need for better computational models to allow for the identification of safe drug candidates and to support experimental design. In this context, a large data set comprising 3712 compounds with liver related toxicity findings in humans and animals was collected from various sources. The complex pathology was clustered into 21 preclinical and human hepatotoxicity endpoints, which were organized into three levels of detail. Support vector machine models were trained for each endpoint, using optimized descriptor sets from chemometrics software. The optimized global human hepatotoxicity model has high sensitivity (68%) and excellent specificity (95%) in an internal validation set of 221 compounds. Models for preclinical endpoints performed similarly. To allow for reliable prediction of "truly external" novel compounds, all predictions are tagged with confidence parameters. These parameters are derived from a statistical analysis of the predictive probability densities. The whole approach was validated for an external validation set of 269 proprietary compounds. The models are fully integrated into our early safety in-silico workflow.

Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals.

The Ames MPF™ is a miniaturized, microplate fluctuation format of the Ames test. It is a standardized, commercially available product which can be used to assess mutagenicity in Salmonella and E. coli strains in 384-well plates using a color change-based readout. Several peer-reviewed comparisons of the Ames MPF™ to the Ames test in Petri dishes confirmed its suitability to evaluate the mutagenic potential of a variety of test items. An international multicenter study involving seven laboratories tested six coded chemicals with this assay using five bacterial strains, as recommended by the OECD test guideline 471. The data generated by the participating laboratories was in excellent agreement (93%), and the similarity of their dose response curves, as analyzed with sophisticated statistical approaches further confirmed the suitability of the Ames MPF™ assay as an alternative to the Ames test on agar plates, but with advantages with respect to significantly reduced amount of test substance and S9 requirements, speed, hands-on time and, potentially automation.

The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing.

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies.

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.

Optimizing drug discovery by Investigative Toxicology: Current and future trends.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Photochemical genotoxicity: principles and test methods. Report of a GUM task force.

In recent years, assessing the photogenotoxic potential of a compound became an issue for certain drugs and cosmetical products. Therefore, existing methods performed according to international guidelines (e.g. OECD guidelines) were adapted to the use of concurrent UV-visible (UV-Vis) light irradiation for the assessment of photomutagenicity/photogenotoxicity. In this review, photobiological bases of the processes occurring in the cell after irradiation with UV- and/or visible (vis)-light as well as a compilation of testing methods is presented. Methods comprise cell free investigations on naked DNA and in vitro methods, such as the photo-Ames test, the photo-HPRT/photo-mouse lymphoma assay (MLA), the photo-micronucleus test (MNT), the photo-chromosomal aberration test (CA) and the photo-Comet assay. A compilation of the currently available international literature of compounds tested on photogenotoxicity is given for each method. The state of the art of photogenotoxicity testing as well as the rational for testing are outlined in relation to the recommendations reached in expert working groups at different international meetings and to regulatory guidance papers. Finally, photogenotoxicity testing as predictor of photocarcinogenicity and in the light of risk assessment is discussed.

Predictive in silico off-target profiling in drug discovery.

Drug action can be rationalized as interaction of a molecule with proteins in a regulatory network of targets from a specific biological system. Both drug and side effects are often governed by interaction of the drug molecule with many, often unrelated, targets. Accordingly, arrays of protein-ligand interaction data from numerous in vitro profiling assays today provide growing evidence of polypharmacological drug interactions, even for marketed drugs. In vitro off-target profiling has therefore become an important tool in early drug discovery to learn about potential off-target liabilities, which are sometimes beneficial, but more often safety relevant. The rapidly developing field of in silico profiling approaches is complementing in vitro profiling. These approaches capitalize from large amounts of biochemical data from multiple sources to be exploited for optimizing undesirable side effects in pharmaceutical research. Therefore, current in silico profiling models are nowadays perceived as valuable tools in drug discovery, and promise a platform to support optimally informed decisions.

Follow-up actions from positive results of in vitro genetic toxicity testing.

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall risk-based decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing follow-up action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing.