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.

Application of in vivo metabolomics to preclinical/toxicological studies: case study on phenytoin-induced systemic toxicity.

BASF and Metanomics have built-up the database MetaMap(®)-Tox containing rat plasma metabolome data for more than 500 reference compounds. Phenytoin was administered to five Wistar rats of both sexes at dietary dose levels of 600 and 2400 ppm over 28 days and metabolome analysis was performed on days 7, 14 and 28. Clinical pathology did not indicate clear evidence for liver toxicity, whereas liver histopathology revealed slight centrilobular hepatocellular hypertrophy. The metabolome analysis of phenytoin shows metabolome changes at both dose levels and the comparison with MetaMap-Tox indicated strong evidence for liver enzyme induction, as well as liver toxicity. Moreover, evidence for kidney and indirect thyroid effects were observed. This assessment was based on the metabolite changes induced, similarities to specific toxicity patterns and the whole metabolome correlation within MetaMap-Tox. As compared with the classical read-out, a more comprehensive picture of phenytoin's effects is obtained from the metabolome analysis, demonstrating the added value of metabolome data in preclinical/ toxicological studies.

Nutritional impact on the plasma metabolome of rats.

Metabolite profiling (metabolomics) elucidates changes in biochemical pathways under various conditions, e.g., different nutrition scenarios or compound administration. BASF and metanomics have obtained plasma metabolic profiles of approximately 500 compounds (agrochemicals, chemicals and pharmaceuticals) from 28-day rat studies. With these profiles the establishment of a database (MetaMap(®)Tox) containing specific metabolic patterns associated with many toxicological modes of action was achieved. To evaluate confounding factors influencing metabolome patterns, the effect of fasting vs. non-fasting prior to blood sampling, the influence of high caloric diet and caloric restriction as well as the administration of corn oil and olive oil was studied for its influence on the metabolome. All mentioned treatments had distinct effects: triacylglycerol, phospholipids and their degradation product levels (fatty acids, glycerol, lysophosphatidylcholine) were often altered depending on the nutritional status. Also some amino acid and related compounds were changed. Some metabolites derived from food (e.g. alpha-tocopherol, ascorbic acid, beta-sitosterol, campesterol) were biomarkers related to food consumption, whereas others indicated a changed energy metabolism (e.g. hydroxybutyrate, pyruvate). Strikingly, there was a profound difference in the metabolite responses to diet restriction in male and female rats. Consequently, when evaluating the metabolic profile of a compound, the effect of nutritional status should be taken into account.

Bioavailability and potency of natural-source and all-racemic alpha-tocopherol in the human: a dispute.

Alpha-tocopherol occurs in nature as a single stereoisomer (RRR) while synthetic vitamin E is a mixture of eight stereoisomers (all-racemic, all-rac). The presently accepted ratio of biopotency (RRR: all-rac) is 1.36, based on the fetal resorption test in rats. This ratio has been disputed for humans. Clinical endpoint studies in humans are lacking, but plasma responses to RRR- and all-rac were measured in bioavailability studies. In nine studies comparing unlabeled forms, the ratio of plasma parameters (AUC, Cmax or steady-state concentration) concurred with the accepted ratio of biopotency within accepted bounds of equivalence. Four recent studies with simultaneous application of trideutero-RRR and hexadeutero-all-rac resulted in ratios of up to 2 for plasma, and of approximately 2.7 and approximately 3.4 for alpha-CEHC (a urinary metabolite) and umbilical cord plasma, respectively. Because these results have been widely assumed to reflect the difference in biopotency, this has prompted a proposal to the Food and Nutrition Board, National Academy of Sciences, USA to change the biopotency factor to 2 : 1. We challenge the validity of bioavailability data in lieu of clinical endpoints. Because RRR and all-rac are not chemically identical and differ in plasma and tissue kinetics and metabolism, the ratio of bioavailability parameters does not reflect the ratio of biopotency. This needs to be determined in adequately designed studies using clinical and biochemical endpoints. Until such studies have been performed it does not appear prudent to exchange the presently accepted ratio based on valid bioassays, albeit in a model animal, for another that is based on erroneous conclusions from human studies.