Considerations for the development of guidance on dose level selection for developmental and reproductive toxicity studies.

In 2022, the European Chemicals Agency issued advice on the selection of high dose levels for developmental and reproductive toxicity (DART) studies indicating that the highest dose tested should aim to induce clear evidence of reproductive toxicity without excessive toxicity and severe suffering in parental animals. In addition, a recent publication advocated that a 10% decrease in body weight gain should be replaced with a 10% decrease in bodyweight as a criterion for dose adequacy. Experts from the European Centre for Ecotoxicology and Toxicology of Chemicals evaluated these recent developments and their potential impact on study outcomes and interpretation and identified that the advice was not aligned with OECD test guidelines or with humane endpoints guidance. Furthermore, data analysis from DART studies indicated that a 10% decrease in maternal body weight during gestation equates to a 25% decrease in body weight gain, which differs from the consensus of experts at a 2010 ILSI/HESI workshop. Dose selection should be based on a biological approach that considers a range of other factors. Excessive dose levels that cause frank toxicity and overwhelm homeostasis should be avoided as they can give rise to effects that are not relevant to human health assessments.

Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.

Influence of pregnancy and non-fasting conditions on the plasma metabolome in a rat prenatal toxicity study.

The current parameters for determining maternal toxicity (e.g. clinical signs, food consumption, body weight development) lack specificity and may underestimate the extent of effects of test compounds on the dams. Previous reports have highlighted the use of plasma metabolomics for an improved and mechanism-based identification of maternal toxicity. To establish metabolite profiles of healthy pregnancies and evaluate the influence of food consumption as a confounding factor, metabolite profiling of rat plasma was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. Metabolite changes in response to pregnancy, food consumption prior to blood sampling (non-fasting) as well as the interaction of both conditions were studied. In dams, both conditions, non-fasting and pregnancy, had a marked influence on the plasma metabolome and resulted in distinct individual patterns of changed metabolites. Non-fasting was characterized by increased plasma concentrations of amino acids and diet related compounds and lower levels of ketone bodies. The metabolic profile of pregnant rats was characterized by lower amino acids and glucose levels and higher concentrations of plasma fatty acids, triglycerides and hormones, capturing the normal biochemical changes undergone during pregnancy. The establishment of metabolic profiles of pregnant non-fasted rats serves as a baseline to create metabolic fingerprints for prenatal and maternal toxicity studies.

Analysis of metabolome changes in the bile acid pool in feces and plasma of antibiotic-treated rats.

The bile acid-liver-gut microbiota axis plays an important role in the host's health. The gut microbiota has an impact on the bile acid pool, but also the bile acids themselves can influence the gut microbiota composition. In this study, six antibiotics from five different classes (i.e. lincosamides, glycopeptides, macrolides, fluoroquinolones, aminoglycosides) were used to modulate microbial communities of Wistar rats to elucidate changes in the bile acid metabolism and to identify key metabolites in the bile acid pool related to gut microbial changes. 20 primary and secondary bile acids were analyzed in plasma and feces of control and treated animals. Antibiotics treatment induced significant changes in primary and secondary bile acids in both matrices. Taurine-conjugated primary bile acids significantly increased in plasma and feces. Contrary, cholic acid and most of the analyzed secondary bile acids significantly decreased in plasma, and cholic acid accumulated in the feces after treatment with all antibiotics but roxithromycin. Despite the different activity spectra of the antibiotics applied against gut microbes, the overall effect on the bile acid pool tended to be similar in both matrices except for streptomycin. These results show that changes in the gut microbial community affect the bile acid pool in plasma and feces and that changes in the bile acid profile can be indicative of alterations of the gut microbiome. Due to the important role of bile acids for the host, changes in the bile acid pool can have severe consequences for the host.

Added value of plasma metabolomics to describe maternal effects in rat maternal and prenatal toxicity studies.

For regulatory purposes prenatal developmental toxicity (OECD No. 414) studies are routinely performed in our laboratories. The suitability of metabolomics as technology to identify maternal toxicity in such studies was investigated. Plasma was sampled from pregnant, non-fasted rats on gestation day 20 before cesarean section. Metabolite profiling was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. The sensitivity of routinely examined maternal toxicity parameters (OECD No. 414) was compared to those of metabolome analysis. Evaluating 44 studies, the metabolome-derived NOEL was more sensitive in 45% of the cases in detecting maternal toxicity than the maternal NOAEL. Metabolome patterns indicative for liver effects and 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme-inhibition were established in pregnant rats based on regulated metabolites using reference compounds. The HPPD inhibition and liver toxicity patterns in pregnant rats were reasonably comparable to the ones established in non-pregnant, fasted rats. Metabolomics is a useful tool for an improved and mechanism-based identification of maternal toxicity in maternal and prenatal toxicity studies. The data suggest that the current classical maternal toxicity parameters may underestimate the extent of effects of compounds on the dams.

Impact of lincosamides antibiotics on the composition of the rat gut microbiota and the metabolite profile of plasma and feces.

The importance of the gut microorganisms and their wide range of interactions with the host are well-acknowledged. In this study, lincomycin and clindamycin were used to modulate microbial communities of Wistar rats to gain a comprehensive understanding of the implications of microbiome alterations. A metabolomics approach and taxonomic profiling were applied to characterize the effects of these antibiotics on the functionality of the microbiome and to identify microbiome-related metabolites. After treatment, the diversity of the microbial community was drastically reduced. Bacteroidetes and Verrucomicrobia were drastically reduced, Tenericutes and Deferribacteres completely disappeared, while abundance of Firmicutes and Proteobacteria were highly increased. Changes in plasma and feces metabolites were observed for metabolites belonging mainly to the class of complex lipids, fatty acids and related metabolites as well as amino acids and related compounds. Bile acid metabolism was markedly affected: taurocholic acid, glycochenodeoxycholic acid and cholic acid presented abrupt changes showing a specific metabolite pattern indicating disruption of the microbial community. In both plasma and feces taurocholic acid was highly upregulated upon treatment whereas glycochenodeoxycholic acid was downregulated. Cholic acid was upregulated in feces but downregulated in plasma. These results show that changes in the gut microbial community lead to alterations of the metabolic profile in blood and feces of the host and can be used to identify potentially microbiome-related metabolites. This implies that metabolomics could be a suitable tool to estimate the extent of changes induced in the intestinal microbiome with respect to consequences for the host.

Microbiome-related metabolite changes in gut tissue, cecum content and feces of rats treated with antibiotics.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

The Threshold of Toxicological Concern for prenatal developmental toxicity in rats and rabbits.

The Threshold Toxicological Concern (TTC) is based on the concept that in absence of experimental data reasonable assurance of safety can be given if exposure is sufficiently low. Using the REACH database the low 5th percentile of the NO(A)EL distribution, for prenatal developmental toxicity (OECD guideline 414) was determined. For rats, (434 NO(A)ELs values) for maternal toxicity, this value was 10 mg/kg-bw/day. For developmental toxicity (469 NO(A)ELs): 13 mg/kg-bw/day. For rabbits, (100 NO(A)ELs), the value for maternal toxicity was 4 mg/kg-bw/day, for developmental toxicity, (112 NO(A)EL values): 10 mg/kg-bw/day. The maternal organism may thus be slightly more sensitive than the fetus. Combining REACH- (industrial chemicals) and published BASF-data (mostly agrochemicals), 537 unique compounds with NO(A)EL values for developmental toxicity in rats and 150 in rabbits were evaluated. The low 5th percentile NO(A)EL for developmental toxicity in rats was 10 mg/kg-bw/day and 9.5 mg/kg-bw/day for rabbits. Using an assessment factor of 100, a TTC value for developmental toxicity of 100 µg/kg-bw/day for rats and 95 µg/kg-bw/day for rabbits is calculated. These values could serve as guidance whether or not to perform an animal experiment, if exposure is sufficiently low. In emergency situations this value may be useful for a first tier risk assessment.

Gut microbiome-related metabolic changes in plasma of antibiotic-treated rats.

The intestinal microbiota contributes to the metabolism of its host. Adequate identification of the microbiota's impact on the host plasma metabolites is lacking. As antibiotics have a profound effect on the microbial composition and hence on the mammalian-microbiota co-metabolism, we studied the effects of antibiotics on the "functionality of the microbiome"-defined as the production of metabolites absorbed by the host. This metabolomics study presents insights into the mammalian-microbiome co-metabolism of endogenous metabolites. To identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have applied broad-spectrum antibiotics belonging to the class of aminoglycosides (neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline). These were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. Fluoroquinolones and tetracyclines can be absorbed from the gut; whereas, aminoglycosides are poorly absorbed. Hippuric acid, indole-3-acetic acid and glycerol were identified as key metabolites affected by antibiotic treatment, beside changes mainly concerning amino acids and carbohydrates. Inter alia, effects on indole-3-propionic acid were found to be unique for aminoglycosides, and on 3-indoxylsulfate for tetracyclines. For each class of antibiotics, specific metabolome patterns could be established in the MetaMap®Tox data base, which contains metabolome data for more than 550 reference compounds. The results suggest that plasma-based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight into the mammalian-microbiome co-metabolism.

Metabolomics as read-across tool: A case study with phenoxy herbicides.

New technologies, such as metabolomics, can address chemical grouping and read across from a biological perspective. In a virtual case study, we selected MCPP as target substance and MCPA and 2,4-DP as source substances with the goal to waive a 90-day study with MCPP. In order to develop a convincing case to show how biological data can substantiate read across, we used metabolomics on blood samples from the 28-day studies to show the qualitative and quantitative similarity of the substances. The 28-day metabolome evaluation of source substances and the target substance indicate liver and kidneys as target organs. 2,4-DP was identified as the best source substance. Using the information of the 90-day 2,4-DP study, we predicted MCPP's toxicity profile at 2500 ppm: reduced food consumption and body weight gain, liver and kidney weight increases with clinical-pathology changes and a moderate red blood cell parameter reduction. NOEL prediction for MCPP was below that of 2,4-DP (<500 ppm), and similar to that of MCPA (≥150 ppm). Qualitatively, these predictions are comparable to the results of the real MCPP 90-day study in rats (reduced food consumption and body weight gain, weight increases and clinical-pathology changes in liver and kidneys, reduced red blood cells values). Quantitatively, the predicted NOAEL (150 ppm) is similar to the actual study (NOEL = 75 ppm, NOAEL ≤ 500 ppm). Thus, the 90-day rat toxicity study of MCPP could have been waived and substituted by the 90-day results of 2,4-DP by using metabolome data of 28 day studies.

Metabolite profiles of rats in repeated dose toxicological studies after oral and inhalative exposure.

The MetaMap(®)-Tox database contains plasma-metabolome and toxicity data of rats obtained from oral administration of 550 reference compounds following a standardized adapted OECD 407 protocol. Here, metabolic profiles for aniline (A), chloroform (CL), ethylbenzene (EB), 2-methoxyethanol (ME), N,N-dimethylformamide (DMF) and tetrahydrofurane (THF), dosed inhalatively for six hours/day, five days a week for 4 weeks were compared to oral dosing performed daily for 4 weeks. To investigate if the oral and inhalative metabolome would be comparable statistical analyses were performed. Best correlations for metabolome changes via both routes of exposure were observed for toxicants that induced profound metabolome changes. e.g. CL and ME. Liver and testes were correctly identified as target organs. In contrast, route of exposure dependent differences in metabolic profiles were noted for low profile strength e.g. female rats dosed inhalatively with A or THF. Taken together, the current investigations demonstrate that plasma metabolome changes are generally comparable for systemic effects after oral and inhalation exposure. Differences may result from kinetics and first pass effects. For compounds inducing only weak changes, the differences between both routes of exposure are visible in the metabolome.

Safety assessment of [3S, 3'S]-astaxanthin--Subchronic toxicity study in rats.

Astaxanthin, a naturally occurring xanthophyll, is commercially used as a coloring agent in salmon feed, but also marketed as a dietary supplement. The objective of this study was to investigate the subchronic toxicity of synthetic [3S, 3'S]-Astaxanthin in rats. A powder formulation containing approximately 20% [3S, 3'S]-Astaxanthin was administered via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 5000, 15,000 and 50,000 ppm for a period of 13 weeks. A formulation of comparable composition but without [3S, 3'S]-Astaxanthin served as a placebo control. There were no effects observed on survival, clinical examinations, clinical pathology, estrous cycle as well as on sperm parameters. At terminal necropsy, a macroscopically visible brown-blue discoloration of the gastrointestinal contents was noted which was considered to be secondary to the violet-brown color of the test material. No other significant or dose-related abnormalities were found in the tissues collected at termination. Our observations support that ingestion of [3S, 3'S]-Astaxanthin of up to 700-920 mg/kg bw/day in rats in a gelatin/carbohydrate formulation is without adverse effects.

Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma.

While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential.

In silico models to predict dermal absorption from complex agrochemical formulations.

Dermal absorption is a critical part in the risk assessment of complex mixtures such as agrochemical formulations. To reduce the number of in vivo or in vitro absorption experiments, the present study aimed to develop an in silico prediction model that considers mixture-related effects. Therefore, an experimental 'real-world' dataset derived from regulatory in vitro studies with human and rat skin was processed. Overall, 56 test substances applied in more than 150 mixtures were used. Descriptors for the substances as well as the mixtures were generated and used for multiple linear regression analysis. Considering the heterogeneity of the underlying data set, the final model provides a good fit (r² = 0.75) and is able to estimate the influence of a newly composed formulation on dermal absorption of a well-known substance (predictivity Q²Ext = 0.73). Application of this model would reduce animal and non-animal testings when used for the optimization of formulations in early developmental stages, or would simplify the registration process, if accepted for read-across.

The sensitivity of metabolomics versus classical regulatory toxicology from a NOAEL perspective.

The identification of the no observed adverse effect level (NOAEL) is the key regulatory outcome of toxicity studies. With the introduction of "omics" technologies into toxicological research, the question arises as to how sensitive these technologies are relative to classical regulatory toxicity parameters. BASF SE and metanomics developed the in vivo metabolome database MetaMap®Tox containing metabolome data for more than 500 reference compounds. For several years metabolome analysis has been routinely performed in regulatory toxicity studies (REACH mandated testing or new compound development), mostly in the context of 28 day studies in rats (OECD 407 guideline). For those chemicals for which a toxicological NOAEL level was obtained at either high or mid-dose level, we evaluated the associated metabolome to investigate the sensitivity of metabolomics versus classical toxicology with respect to the NOAEL. For the definition of a metabolomics NOAEL the ECETOC criteria (ECETOC, 2007) were used. In this context we evaluated 104 cases. Comparable sensitivity was noted in 75% of the cases, increased sensitivity of metabolomics in 8%, and decreased sensitivity in 18% of the cases. In conclusion, these data suggest that metabolomics profiling has a similar sensitivity to the classical toxicological study (e.g. OECD 407) design.

Mechanistic analysis of metabolomics patterns in rat plasma during administration of direct thyroid hormone synthesis inhibitors or compounds increasing thyroid hormone clearance.

For identification of toxicological modes of action (MoAs) a database (MetaMap(®)Tox) was established containing plasma metabolome consisting of approximately 300 endogenous metabolites. Each five male and female Wistar rats per groups were treated with >500 reference compounds over a period of 28 days. More than 120 specific toxicity patterns of common metabolite changes associated with unique MoAs were established. To establish patterns predictive effects on the thyroid, animals have been treated with reference compounds directly acting on the thyroid hormone formation (such as methimazole, ethylenethiourea) as well as liver enzyme inducers leading to an increased excretion of thyroid hormones and therewith to a secondary response of the thyroid (such as aroclor 1254 and boscalid). Here we present the plasma metabolite changes which form the patterns for direct and indirect effects on the thyroid. It is possible to identify metabolites which are commonly regulated irrespective of an indirect or direct effect on the thyroid as well as groups of metabolites separating both MoAs. By putting the metabolite regulations in the context of affected pathways helps to identify thyroid hormone inhibiting MoAs even when the hormone levels are not consistently changed. E.g., direct thyroid hormone synthesis inhibitors affect some enzymes in the urea cycle, increase the ω-oxidation of fatty acids and decrease glutamate and oxoproline levels, whereas indirect thyroid hormone inhibiting compounds interact with the lipid mediated and liver metabolism.

Short term inhalation toxicity of a liquid aerosol of glutaraldehyde-coated CdS/Cd(OH)2 core shell quantum dots in rats.

Quantum dots exhibit extraordinary optical and mechanical properties, and the number of their applications is increasing. In order to investigate a possible effect of coating on the inhalation toxicity of previously tested non-coated CdS/Cd(OH)2 quantum dots and translocation of these very small particles from the lungs, rats were exposed to coated quantum dots or CdCl2 aerosol (since Cd(2+) was present as impurity), 6h/d for 5 consecutive days. Cd content was determined in organs and excreta after the end of exposure and three weeks thereafter. Toxicity was determined by examination of broncho-alveolar lavage fluid and microscopic evaluation of the entire respiratory tract. There was no evidence for translocation of particles from the respiratory tract. Evidence of a minimal inflammatory process was observed by examination of broncho-alveolar lavage fluid. Microscopically, minimal to mild epithelial alteration was seen in the larynx. The effects observed with coated quantum dots, non-coated quantum dots and CdCl2 were comparable, indicating that quantum dots elicited no significant effects beyond the toxicity of the Cd(2+) ion itself. Compared to other compounds with larger particle size tested at similarly low concentrations, quantum dots caused much less pronounced toxicological effects. Therefore, the present data show that small particle sizes with corresponding high surfaces are not the only factor triggering the toxic response or translocation.

Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro.

For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273­1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6­60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1­4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5­17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

Prediction of clinically relevant safety signals of nephrotoxicity through plasma metabolite profiling.

Addressing safety concerns such as drug-induced kidney injury (DIKI) early in the drug pharmaceutical development process ensures both patient safety and efficient clinical development. We describe a unique adjunct to standard safety assessment wherein the metabolite profile of treated animals is compared with the MetaMap Tox metabolomics database in order to predict the potential for a wide variety of adverse events, including DIKI. To examine this approach, a study of five compounds (phenytoin, cyclosporin A, doxorubicin, captopril, and lisinopril) was initiated by the Technology Evaluation Consortium under the auspices of the Drug Safety Executive Council (DSEC). The metabolite profiles for rats treated with these compounds matched established reference patterns in the MetaMap Tox metabolomics database indicative of each compound's well-described clinical toxicities. For example, the DIKI associated with cyclosporine A and doxorubicin was correctly predicted by metabolite profiling, while no evidence for DIKI was found for phenytoin, consistent with its clinical picture. In some cases the clinical toxicity (hepatotoxicity), not generally seen in animal studies, was detected with MetaMap Tox. Thus metabolite profiling coupled with the MetaMap Tox metabolomics database offers a unique and powerful approach for augmenting safety assessment and avoiding clinical adverse events such as DIKI.