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We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals.

Mutations in leucine-rich repeat kinase 2 ( LRRK2 ) are the most common genetic risk factors for Parkinson’s disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

As legacy toxicogenomics databases have become available, improved data mining approaches are now key to extracting and visualizing subtle relationships between toxicants and gene expression. In the present study, a novel “aggregating bundles of clusters” (ABC) procedure was applied to separate cholestatic from non-cholestatic drugs and model toxicants in the Johnson & Johnson (Janssen) rat liver toxicogenomics database [3]. Drug-induced cholestasis is an important issue, particularly when a new compound enters the market with this liability, with standard preclinical models often mispredicting this toxicity. Three well-characterized cholestasis-responsive genes (Cyp7a1, Mrp3 and Bsep) were chosen from a previous in-house Janssen gene expression signature; these three genes show differing, non-redundant responses across the 90+ paradigm compounds in our database. Using the ABC procedure, extraneous contributions were minimized in comparisons of compound gene responses. All genes were assigned weights proportional to their correlations with Cyp7a1, Mrp3 and Bsep, and a resampling technique was used to derive a stable measure of compound similarity. The compounds that were known to be associated with rat cholestasis generally had small values of this measure relative to each other but also had large values of this measure relative to non-cholestatic compounds. Visualization of the data with the ABC-derived signature showed a very tight, essentially identically behaving cluster of robust human cholestatic drugs and experimental cholestatic toxicants (ethinyl estradiol, LPS, ANIT and methylene dianiline, disulfiram, naltrexone, methapyrilene, phenacetin, alpha-methyl dopa, flutamide, the NSAIDs–—indomethacin, flurbiprofen, diclofenac, flufenamic acid, sulindac, and nimesulide, butylated hydroxytoluene, piperonyl butoxide, and bromobenzene), some slightly less active compounds (3′-acetamidofluorene, amsacrine, hydralazine, tannic acid), some drugs that behaved very differently, and were distinct from both non-cholestatic and cholestatic drugs (ketoconazole, dipyridamole, cyproheptadine and aniline), and many postulated human cholestatic drugs that in rat showed no evidence of cholestasis (chlorpromazine, erythromycin, niacin, captopril, dapsone, rifampicin, glibenclamide, simvastatin, furosemide, tamoxifen, and sulfamethoxazole). Most of these latter drugs were noted previously by other groups as showing cholestasis only in humans. The results of this work suggest that the ABC procedure and similar statistical approaches can be instrumental in combining data to compare toxicants across toxicogenomics databases, extract similarities among responses and reduce unexplained data varation. Keywords: Cluster analysis, Cholestasis, Gene signature, Microarray, Prediction, Toxicogenomics

Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging.

Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity. The results indicate that alterations in a panel of urinary kidney injury biomarkers can be used to monitor histopathological injury, with Kim-1 and α-GST emerging as the most sensitive biomarkers outperforming clinical standards of care, serum or plasma creatinine and blood urea nitrogen. To enable the prediction of polymyxin-induced nephrotoxicity, an in vitro cytotoxicity assay was employed using human proximal tubule epithelial cells (HK-2). Cytotoxicity data in these HK-2 cells correlated with the renal toxicity detected via safety biomarker data and histopathological evaluation, suggesting that in vitro and in vivo methods can be incorporated within a screening cascade to prioritize polymyxin class analogs with more favorable renal toxicity profiles.

A new class of benzoxazole and benzothiazole amide derivatives exhibiting potent CYP3A4 inhibiting properties was identified. Extensive lead optimization was aimed at improving the CYP3A4 inhibitory properties as well as overall ADME profile of these amide derivatives. This led to the identification of thiazol-5-ylmethyl (2S,3R)-4-(2-(ethyl(methyl)amino)-N-isobutylbenzo[d]oxazole-6-carboxamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (C1) as a lead candidate for this class. This compound together with structurally similar analogues demonstrated excellent ‘boosting’ properties when tested in dogs. These findings warrant further evaluation of their properties in an effort to identify valuable alternatives to Ritonavir as pharmacokinetic enhancers.

BACKGROUND: Testicular toxicity (TT) is a sporadic and challenging issue in pharmaceutical drug development. Efforts to develop TT screening assays or biomarkers have been overshadowed by consortium efforts to predict drug-induced toxicities such as hepatic injury, which are encountered more frequently. METHODS: To gauge the current state of the field and to prioritize future TT activities, the International Life Sciences Institute-Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (DART) Technical Committee sponsored a survey to better understand the incidence and nature of TT findings encountered during drug development. RESULTS: Highlights from the 16 survey respondents include: (1) Although preclinical TT was encountered relatively infrequently, half of the participants observed repeated problems with TT during pharmaceutical development, (2) despite control measures such as use of sexually mature animals to diminish confounding effects of spurious lesions, interpretation of TT remains a challenge, (3) “traditional” evaluation tools such as hormonal monitoring and newer approaches such as -omics are utilized to investigate testicular changes, and (4) an understanding of the risk and relevance of TT findings is achieved through joint consideration of factors such as species specificity, potential mode of action, and safety margins. CONCLUSIONS: TT remains a relatively uncommon but persistent challenge in pharmaceutical development. Although current preclinical TT approaches appear to be effective in limiting the occurrence of pharmaceutical candidate attrition in clinical trials, improved biomarker or screening platforms would allow companies to identify TT at an earlier stage, thus decreasing the time and resources expended on safety evaluation of pharmaceutical candidates. Birth Defects Res (Part B) 92: 511–525, 2011. © 2011 Wiley Periodicals, Inc.

Genomics may be an effective tool in decreasing the lengthy drug development process and reducing compound attrition. It can generate specific gene expression profiles induced by chemicals that can be linked to dose and response. Toxicogenomics can identify sensitive biomarkers of early deleterious effects, distinguish genotoxic from non-genotoxic carcinogens and can provide information on the mechanism of action. It can help bridge in vitro to in vivo findings and provide context for preclinical data and thus address human health risks. Issues and shortcomings that still need to be resolved or improved for efficient incorporation of genomics in drug development and environmental toxicology research include data analysis, data interpretation tools and accessible data repositories. In addition, implementation of toxicogenomics in early screening or drug discovery phases and effective use of this information by project teams remains a challenge.

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 established an Emerging Technologies and New Strategies Workgroup to review the current State of the Art in genetic toxicology testing. The aim of the workgroup was to identify promising technologies that will improve genotoxicity testing and assessment of in vivo hazard and risk, and that have the potential to help meet the objectives of the IVGT. As part of this initiative, HESI convened a workshop in Washington, DC in May 2008 to discuss mature, maturing, and emerging technologies in genetic toxicology. This article collates the abstracts of the New and Emerging Technologies Workshop together with some additional technologies subsequently considered by the workgroup. Each abstract (available in the online version of the article) includes a section addressed specifically to the strengths, weaknesses, opportunities, and threats associated with the respective technology. Importantly, an overview of the technologies and an indication of how their use might be aligned with the objectives of IVGT are presented. In particular, consideration was given with regard to follow-up testing of positive results in the standard IVGT tests (i.e., Salmonella Ames test, chromosome aberration assay, and mouse lymphoma assay) to add weight of evidence and/or provide mechanism of action for improved genetic toxicity risk assessments in humans.

N-oxidation by cytochrome P450 enzymes is an initial step in the metabolic activation of aromatic amine compounds. Once metabolized, these compounds are converted to DNA-reactive species which can exhibit potent mutagenic and/or carcinogenic activity. The precise mechanism of P450 enzyme oxidation is not completely understood, although various theories, involving either one-electron transfer, two-electron transfer or addition–rearrangement, have been debated. In previous studies, selection of the most probable mechanism has been based on consideration of Huckel theory charge distribution calculations and experimentally-derived enzyme metabolism data. This approach can now be improved by incorporating contemporary, ab initio quantum chemical methods to accurately determine the chemical properties of P450 aromatic amine substrates. In this work, we have re-examined the feasibility of three proposed P450 oxidation mechanisms by comparing the experimental oxidation yields and rates of 1-naphthylamine (1-NA), 2-naphthylamine (2-NA) and 2-aminofluorene (2-AF). This data has then been analyzed with respect to ab initio-calculated charge distributions and energies of reactants, oxidation products and proposed intermediates. Our analysis of theoretical and experimental data indicates that the one-electron model is more consistent with oxidation rate data for 1-NA, 2-NA, and 2-AF. We therefore conclude, in contrast to earlier studies, that the one-electron mechanism is more likely to be the pathway for P450-catalyzed aromatic amine oxidation.

2-Nitronaphthalene (2NN) has been identified as a mutagenic atmospheric reaction product of naphthalene in the Ames bacterial reversion assay. Recent experiments have shown this nitroarene to be genotoxic in a human lymphoblastoid cell line (MCL-5) transfected with plasmids encoding epoxide hydrolase and four cytochrome P450 monooxygenase activities. The present study investigated the genotoxicity of 2NN in two related human B-lymphoblastoid cell lines, h1A1v2 containing a single P450 isozyme (cytochrome P450 1A1) and L3 cells which are isogenic with MCL-5 cells and are distinguished only by the absence of transfected plasmids. The results indicate that 2NN-induced mutagenesis at the heterozygous thymidine kinase (tk) locus was dependent on metabolic activities provided by the transfected plasmids in MCL-5; no significant induction of mutants was observed in L3 cells studied in parallel. A similar induction of mutation was observed in h1A1v2 and MCL-5 cell lines at the tk locus and no induction was observed at the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus. The induction of mutations in h1A1v2 cells suggests that cytochrome P450 1A1 alone can activate 2NN to a mutagenic species, however, this interpretation may be confounded by differences between the h1A1v2 and MCL-5 cell lines. The observed genotoxic activity induced by 2NN prompted testing of the amino analogue, beta-naphthylamine (betaNA), to investigate potential similarities in the metabolic activation pathways of the two compounds. The negative response of betaNA in all cell lines suggests that 2NN and betaNA are not activated in these human cells by similar metabolic pathways.

The nitrogen mustard based DNA alkylating agents were the first effective anticancer agents and remain important drugs against many forms of cancer. More than fifty years of research on the nitrogen mustards has yielded a broad range of therapeutically useful compounds and a detailed knowledge of the biochemical mechanism of these drugs. Nevertheless, there is much ongoing research on the phos­ phosphoramidic and other nitrogen mustards to increase their potency and reduce their toxic and mutagenic side effects. To understand the existing nitrogen mustards, and to design the next generation of these drugs, more knowledge is needed about the effects of chemical modifications on their activation and selectivity. Because of the existing knowledge of these drugs, atomic-level chemical modeling can play an important role in the understanding of the phosphoramidic mustard compounds; however, it has not proved straight forward to directly relate the activity of these mustards with simple chemical properties such as bond lengths or atomic charges. Instead, quantum chemical simulations will be required to simulate the activation and alkylation reactions of these compounds, which will require the newest generation of quantum chemical and solvent modeling methods. Additionally, molecular dynamics simulations of the adducted DNA can provide data on the factors favoring crosslinking and its structural consequences. This review summarizes the extensive literature on the metabolism, activation, and action of the phosphoramidic mustards, with an emphasis on the roles that chemical modeling has and will play in the development of this important class of drugs.

Naphthalene is the most abundant polycyclic aromatic hydrocarbon (PAH) in polluted urban areas. Naphthalene is present in the gas phase under typical atmospheric conditions, and its most important atmospheric loss process is reaction with the hydroxyl (OH) radical. The products of the gas-phase reaction of naphthalene with OH radicals and with nitrate (NO3) radicals have been investigated using gas chromatographic techniques with flame ionization, mass spectrometric, and Fourier transform infrared spectroscopic detection and using in situ atmospheric pressure ionization mass spectrometry. The major products identified from the OH radical-initiated reaction of gas-phase naphthalene were ring cleavage species, including 2-formylcinnamaldehyde. The major products observed from the NO3 radical-initiated reaction were 1- and 2-nitronaphthalene.

To address the pressing need for better in vitro testicular toxicity models, a workshop sponsored by the International Life Sciences Institute (ILSI), the Health and Environmental Science Institute (HESI), and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), was held at the Mt. Washington Conference Center in Baltimore, MD, USA on October 26-27, 2011. At this workshop, experts in testis physiology, toxicology, and tissue engineering discussed approaches for creating improved in vitro environments that would be more conducive to maintaining spermatogenesis and steroidogenesis and could provide more predictive models for testicular toxicity testing. This workshop report is intended to provide scientists with a broad overview of relevant testicular toxicity literature and to suggest opportunities where bioengineering principles and techniques could be used to build improved in vitro testicular models for safety evaluation. Tissue engineering techniques could, conceivably, be immediately implemented to improve existing models. However, it is likely that in vitro testis models that use single or multiple cell types will be needed to address such endpoints as accurate prediction of chemically induced testicular toxicity in humans, elucidation of mechanisms of toxicity, and identification of possible biomarkers of testicular toxicity. published

A series of 2-4-ring polycyclic aromatic hydrocarbons (PAH) were reacted under simulated atmospheric conditions in an environmental chamber. The reactant mixtures were adsorbent collected, extracted, fractionated by HPLC, and tested for mutagenic activity in a microsuspension modification of the Ames Salmonella bioassay (strain TA98, -S9). GC/MS analyses were performed on the mutagenic HPLC fractions for identification of the PAH reaction products. In general, the PAH that were studied can be divided into two categories: those which formed mutagenic nitro-PAH products and those which produced more polar mutagenic products, including nitro-PAH lactones. These PAH atmospheric reaction products are estimated to account for a significant portion of the mutagenic activity seen in gas- and particle-phase ambient sample extracts using this bioassay

The detection of drug-induced hepatotoxicity remains an important safety issue in drug development. A liver-specific microRNA species, microRNA-122 (miR-122), has recently shown potential for predicting liver injury in addition to the standard hepatic injury biomarkers. The objective of this study was to measure miR-122 together with several other liver markers in distinct settings of acute liver toxicity in rats to determine the value of miR-122 as a biomarker for liver injury in this species. Rats were exposed to 3 well-established liver toxicants (acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate), a liver-enzyme inducer (phenobarbital), or a cardiotoxicant (doxorubicin). There was a clear increase in plasma miR-122 following administration of acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate. The response of miR-122 paralleled that of other markers and was consistent with liver injury as indicated by histopathological evaluation. Furthermore, the changes in miR-122 were detected earlier than standard liver injury markers and exhibited a wide dynamic range. In contrast, miR-122 responses to phenobarbital and doxorubicin were low. Based on these findings, miR-122 shows significant promise and may provide added value for assessing liver toxicity in drug development.

The nitrogen mustard based DNA alkylating agents were the first effective anticancer agents and remain important drugs against many forms of cancer. More than fifty years of research on the nitrogen mustards has yielded a broad range of therapeutically useful compounds and a detailed knowledge of the biochemical mechanism of these drugs. Nevertheless, there is much ongoing research on the phosphosphoramidic and other nitrogen mustards to increase their potency and reduce their toxic and mutagenic side effects. To understand the existing nitrogen mustards, and to design the next generation of these drugs, more knowledge is needed about the effects of chemical modifications on their activation and selectivity. Because of the existing knowledge of these drugs, atomic-level chemical modeling can play an important role in the understanding of the phosphoramidic mustard compounds; however, it has not proved straight forward to directly relate the activity of these mustards with simple chemical properties such as bond lengths or atomic charges. Instead, quantum chemical simulations will be required to simulate the activation and alkylation reactions of these compounds, which will require the newest generation of quantum chemical and solvent modeling methods. Additionally, molecular dynamics simulations of the adducted DNA can provide data on the factors favoring crosslinking and its structural consequences. This review summarizes the extensive literature on the metabolism, activation, and action of the phosphoramidic mustards, with an emphasis on the roles that chemical modeling has and will play in the development of this important class of drugs.

The genotoxic risks from exposure to polycyclic aromatic hydrocarbons (PAH) have long been recognized. Less well understood are the potential genotoxic risks of the atmospheric reaction products of this class of compounds. In this investigation, we have utilized several human cell genotoxicity assays to evaluate naphthalene, phenanthrene, and their atmospheric reaction products 1-nitronaphthalene, 2-nitronaphthalene, 1-hydroxy-2-nitronaphthalene, 2-hydroxy-1-nitronaphthalene, 1,4-naphthoquinone and 2-nitrodibenzopyranone. In addition, reaction products of naphthalene were generated in a 6700-1 Teflon environmental chamber, collected on a solid adsorbent, extracted and fractionated by normal-phase HPLC. Individual fractions were then analyzed using GC-MS, and tested for genotoxicity. Genotoxicity was determined using the human B-lymphoblastoid cell line, MCL-5, which expresses several transfected P450 and epoxide hydrolase genes. Mutagenicity was evaluated at both the heterozygous tk locus and the hemizygous hprt locus, permitting detection of both intragenic and chromosomal scale mutational events. Test compounds were also screened using the CREST modified micronucleus assay. Genotoxicity results indicate that 2-nitronaphthalene and 2-nitrodibenzopyranone possess greater mutagenic potency than their parent compounds, and interestingly, both compounds induced significant increases in mutation frequency at tk but not hprt. These results suggest a mechanistic difference in human cell response as compared to bacteria, where both compounds were previously shown to induce point mutations in the Salmonella reversion assay. The genotoxicity of 2-nitronaphthalene and 2-nitrodibenzopyranone in human cells, together with their high concentrations in ambient air relative to nitro-PAH directly emitted from combustion sources, emphasizes the need to consider atmospheric reaction products of PAH in genotoxicity assessments.