The objective of this study was to develop and test a procedure for the identification of chemicals registered under the REACH Regulation that are of potential health concern and are likely to occur in the food chain. For this purpose, 100 data-rich substances registered under REACH together with four positive controls were evaluated. The evaluation of the 104 substances took into account parameters related to exposure (tonnage, release, biodegradation and potential bioaccumulation) and toxicity (repeated dose toxicity, genotoxicity and reproductive toxicity) organised in six blocks. All substances were scored for each block. ACC-HUMANsteady software was used to evaluate the potential for bioaccumulation in eleven different food items using input data derived from QSAR predictions. The extraction of the relevant experimental data generated under REACH was successful, but encountered several problems in relation to the data extraction process and subsequent evaluation steps. Several weighting scenarios were tested to aggregate scores for the six blocks into a total score to enable a ranking of the 104 substances. Scenarios that assigned high total scores to chemicals that combined high scores in the exposure blocks with high scores in the toxicity blocks identified a set of substances of potential concern (including the positive controls). In addition, a Pivot table selection was implemented that can be used without weighting. Further analyses compared the scores derived from experimental data with those derived from predicted data. These analyses found a good agreement of scores for biodegradability, but considerable disagreement of scores for toxicity endpoints. In conclusion, a scoring and ranking procedure was developed for the identification of chemicals of potential concern in the food chain (potential emerging risks) that showed a good level of differentiation. The focus on (semi-)automated processes ensures that this procedure can be applied to all chemicals registered under the REACH Regulation. © European Food Safety Authority

Read-across is one of the most frequently used alternative tools for hazard assessment, in particular for complex endpoints such as repeated dose or developmental and reproductive toxicity. Read-across extrapolates the outcome of a specific toxicological in vivo endpoint from tested (source) compounds to "similar" (target) compound(s). If appropriately applied, a read-across approach can be used instead of de novo animal testing. The read-across approach starts with structural/ physicochemical similarity between target and source compounds, assuming that similar structural characteristics lead to similar human hazards. In addition, similarity also has to be shown for the toxicokinetic and toxicodynamic properties of the grouped compounds. To date, many read-across cases fail to demonstrate toxicokinetic and toxicodynamic similarities. New concepts, in vitro and in silico tools are needed to better characterise these properties, collectively called new approach methodologies (NAMs). This white paper outlines a general read-across assessment concept using NAMs to support hazard characterization of the grouped compounds by generating data on their dynamic and kinetic properties. Based on the overarching read-across hypothesis, the read-across workflow suggests targeted or untargeted NAM testing also outlining how mechanistic knowledge such as adverse outcome pathways (AOPs) can be utilized. Toxicokinetic models (biokinetic and PBPK), enriched by in vitro parameters such as plasma protein binding and hepatocellular clearance, are proposed to show (dis)similarity of target and source compound toxicokinetics. Furthermore, in vitro to in vivo extrapolation is proposed to predict a human equivalent dose, as potential point of departure for risk assessment. Finally, the generated NAM data are anchored to the existing in vivo data of source compounds to predict the hazard of the target compound in a qualitative and/ or quantitative manner. To build this EU-ToxRisk read-across concept, case studies have been conducted and discussed with the regulatory community. These case studies are briefly outlined.

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A hallmark of tumorigenesis is resistance to apoptosis. To explore whether resistance to cell death precedes tumor formation, we have studied the short-term effects of the hepatocarcinogen 2-acetylaminofluorene (AAF) on liver mitochondria, on hepatocytes, and on the response to bacterial endotoxin lipopolysaccharide (LPS) in albino Wistar rats. We show that after as early as two weeks of AAF feeding liver mitochondria developed an increased resistance to opening of the permeability transition pore (PTP), an inner membrane channel that is involved in various forms of cell death. Consistent with a mitochondrial adaptive response in vivo, (i) AAF feeding increased the expression of BCL-2 in mitochondria, and (ii) hepatocytes isolated from AAF-fed rats became resistant to PTP-dependent depolarization, cytochrome c release, and cell death, which were instead observed in hepatocytes from rats fed a control diet. AAF-fed rats were fully protected from the hepatotoxic effects of the injection of 20 -30 g of LPS plus 700 mg of D-galactosamine (D-GalN) ؋ kg ؊1 of body weight, a treatment that in control rats readily caused a large increase of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in liver cryosections and release of alanine and aspartate aminotransferase into the bloodstream. Treatment with LPS and D-GalN triggered cleavage of BID, a BCL-2 family member, in the livers of both control-and AAF-fed animals, whereas caspase 3 was cleaved only in control-fed animals, indicating that the mitochondrial proapoptotic pathway had been selectively suppressed during AAF feeding. Phenotypic reversion was observed after stopping the carcinogenic diet. These results underscore a key role of mitochondria in apoptosis and demonstrate that regulation of the mitochondrial PTP is altered early during AAF carcinogenesis, which matches, and possibly causes, the increased resistance of hepatocytes to death stimuli in vivo. Both events precede tumor formation, suggesting that suppression of apoptosis may contribute to the selection of a resistant phenotype, eventually increasing the probability of cell progression to the transformed state.