Is read-across for chemicals comparable to medical device equivalence and where to use it for conformity assessment?

Novel medical devices must conform to medical device regulation (MDR) for European market entry. Likewise, chemicals must comply with the Registration, Evaluation, Authorization and Restriction of Chemicals (REACh) regulation. Both pose regulatory challenges for manufacturers, but concordantly provide an approach for transferring data from an already registered device or compound to the one undergoing accreditation. This is called equivalence for medical devices and read-across for chemicals. Although read-across is not explicitly prohibited in the process of medical device accreditation, it is usually not performed due to a lack of guidance and acceptance criteria from the authorities. Nonetheless, a scientifically justified read-across of material-based endpoints, as well as toxicological assessment of chemical aspects, such as extractables and leachables, can prevent failure of MDR device equivalence if data is lacking. Further, read-across, if applied correctly can facilitate the standard MDR conformity assessment. The need for read-across within medical device registration should let authorities to reconsider device accreditation and the formulation of respective guidance documents. Acceptance criteria like in the European Chemicals Agency (ECHA) read-across assessment framework (RAAF) are needed. This can reduce the impact of the MDR and help with keeping high European innovation device rate, beneficial for medical device patients.

Protectiveness of NAM-based hazard assessment - which testing scope is required?

Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.

Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.

Substantiate a read-across hypothesis by using transcriptome data-A case study on volatile diketones.

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, ß-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24 h and 72 h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and ß-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to ß-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.

The effect of radiofrequency electromagnetic fields (RF-EMF) on biomarkers of oxidative stress in vivo and in vitro: A protocol for a systematic review.

BACKGROUND: Oxidative stress is conjectured to be related to many diseases. Furthermore, it is hypothesized that radiofrequency fields may induce oxidative stress in various cell types and thereby compromise human and animal health. This systematic review (SR) aims to summarize and evaluate the literature related to this hypothesis. OBJECTIVES: The main objective of this SR is to evaluate the associations between the exposure to radiofrequency electromagnetic fields and oxidative stress in experimental models (in vivo and in vitro). METHODS: The SR framework has been developed following the guidelines established in the WHO Handbook for Guideline Development and the Handbook for Conducting a Literature-Based Health Assessment). We will include controlled in vivo and in vitro laboratory studies that assess the effects of an exposure to RF-EMF on valid markers for oxidative stress compared to no or sham exposure. The protocol is registered in PROSPERO. We will search the following databases: PubMed, Embase, Web of Science Core Collection, Scopus, and the EMF-Portal. The reference lists of included studies and retrieved review articles will also be manually searched. STUDY APPRAISAL AND SYNTHESIS METHOD: Data will be extracted according to a pre-defined set of forms developed in the DistillerSR online software and synthesized in a meta-analysis when studies are judged sufficiently similar to be combined. If a meta-analysis is not possible, we will describe the effects of the exposure in a narrative way. RISK OF BIAS: The risk of bias will be assessed with the NTP/OHAT risk of bias rating tool for human and animal studies. We will use GRADE to assess the certainty of the conclusions (high, moderate, low, or inadequate) regarding the association between radiofrequency electromagnetic fields and oxidative stress. FUNDING: This work was funded by the World Health Organization (WHO). REGISTRATION: The protocol was registered on the PROSPERO webpage on July 8, 2021.

Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action.

Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity. Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis. To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues. Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds. The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE "lipid accumulation". KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity. Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays. The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.

Comprehensive analysis of chronic rodent inhalation toxicity studies for methyl acrylate with attention to test conditions exceeding a maximum tolerated concentration.

MA is a chemical intermediate, manufactured and processed within closed systems. While so far available subacute to chronic inhalation toxicity studies performed in compliance with OECD TG principles gave no indication of any carcinogenic potential for MA, a recent 2-year inhalation study with F344/DuCrlCrlj rats published in 2017 by the JBRC showed a statistically significant increase of squamous cell carcinoma in the nasal cavity of male rats at the highest tested concentration of 160 ppm. However, the results of the different studies in total indicate that this high concentration exceeded the MTC. As MA has a low potential for genotoxic and mutagenic activity, the increased tumour incidence can be attributed to a non-genotoxic mechanism, namely to a strong inflammatory response observed in this study. Together with mechanistic and epidemiologic data for other compounds related to nasal carcinogenesis via this mode of action, it can be concluded that the relevance of this increased tumour incidence in male rats for humans is questionable. Also, a long-term exposure to higher concentrations of MA is highly unlikely to be reached in the environment or at workplaces. Therefore, a risk for humans including cancer hazard is considered implausible.

Towards grouping concepts based on new approach methodologies in chemical hazard assessment: the read-across approach of the EU-ToxRisk project.

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.

The rat bone marrow micronucleus test: Statistical considerations on historical negative control data.

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

The potential acute and chronic toxicity of cyfluthrin on the soil model organism, Eisenia fetida.

In this study, the acute (72h and 14 d) and chronic (28 d and 8 weeks) effects of cyfluthrin on earthworms were evaluated across different endpoints, which are mortality, growth, reproduction and enzyme activities. Cyfluthrin was rated as moderately toxic in 72-h filter paper test and low toxic in 14-day soil test. The exposure of earthworms to cyfluthrin-polluted soil for 8 weeks showed that growth of earthworms was inhibited by cyfluthrin, cocoon production and hatching were inhibited by 20-60mg/kg cyfluthrin. Moreover, 28-day soil test on the responses of enzymes associated with antioxidation and detoxification showed that the activities of catalase (CAT) and glutathione S- transferase (GST) were initially increased by cyfluthrin at 5-20mg/kg, but reduced at 30-60mg/kg, peroxidase (POD) was increased by 26-102% by cyfluthrin in the early period, except 5mg/kg on day 7, and ethoxyresorufin-O-deethylase (EROD) was increased by 29-335% by cyfluthrin after 3 days. Cyfluthrin degraded with a half-life of 24.8-34.8 d, showing the inconsistency between the continuous toxic responses of earthworms and degradation of cyfluthrin in soil. The variable responses of these indexes indicated that different level endpoints should be jointly considered for better evaluation of the environmental risk of contaminants in soil.

Innovative Strategies to Develop Chemical Categories Using a Combination of Structural and Toxicological Properties.

Interest is increasing in the development of non-animal methods for toxicological evaluations. These methods are however, particularly challenging for complex toxicological endpoints such as repeated dose toxicity. European Legislation, e.g., the European Union's Cosmetic Directive and REACH, demands the use of alternative methods. Frameworks, such as the Read-across Assessment Framework or the Adverse Outcome Pathway Knowledge Base, support the development of these methods. The aim of the project presented in this publication was to develop substance categories for a read-across with complex endpoints of toxicity based on existing databases. The basic conceptual approach was to combine structural similarity with shared mechanisms of action. Substances with similar chemical structure and toxicological profile form candidate categories suitable for read-across. We combined two databases on repeated dose toxicity, RepDose database, and ELINCS database to form a common database for the identification of categories. The resulting database contained physicochemical, structural, and toxicological data, which were refined and curated for cluster analyses. We applied the Predictive Clustering Tree (PCT) approach for clustering chemicals based on structural and on toxicological information to detect groups of chemicals with similar toxic profiles and pathways/mechanisms of toxicity. As many of the experimental toxicity values were not available, this data was imputed by predicting them with a multi-label classification method, prior to clustering. The clustering results were evaluated by assessing chemical and toxicological similarities with the aim of identifying clusters with a concordance between structural information and toxicity profiles/mechanisms. From these chosen clusters, seven were selected for a quantitative read-across, based on a small ratio of NOAEL of the members with the highest and the lowest NOAEL in the cluster (< 5). We discuss the limitations of the approach. Based on this analysis we propose improvements for a follow-up approach, such as incorporation of metabolic information and more detailed mechanistic information. The software enables the user to allocate a substance in a cluster and to use this information for a possible read- across. The clustering tool is provided as a free web service, accessible at http://mlc-reach.informatik.uni-mainz.de.

Emission of biocides from hospitals: comparing current survey results with European Union default values.

Under the European Union (EU) Biocidal Products Directive 98/8/EC, comprehensive evaluations on substances of the Third Priority List were conducted until 31 July 2007. This list includes, among other categories, disinfectants for human hygiene (e.g., skin and surface disinfection). For environmental exposure assessment of biocides, the EU emission scenarios apply. Currently available default values for disinfectants are based on consumption data from not more than 8 hospitals and were originally assembled for other purposes. To revalidate these default values, a survey on annual consumption data was performed in 27 German hospitals. These data were analyzed to provide consumption data per bed and day and per nurse and day for particular categories of active ingredients and were compared with default values from the EU emission scenario documents. Although several deviations were detected, an overall acceptable correspondence between Emission Scenario Documents default values and the current survey data was found.

Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test.

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue(R) mouse), and the lacZ model (commercially available as the Mutatrade mark Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.

Early resistance to cell death and to onset of the mitochondrial permeability transition during hepatocarcinogenesis with 2-acetylaminofluorene.

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 microg of LPS plus 700 mg of d-galactosamine (d-GalN) x 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.