Read-across Hypothesis Research Articles

A Systematic Analysis of Read-Across Adaptations in Testing Proposal Evaluations by the European Chemicals Agency.

An important element of the European Union's "Registration, Evaluation, Authorisation and Restriction of Chemicals" (REACH) regulation is the evaluation by the European Chemicals Agency (ECHA) of testing proposals submitted by the registrants to address data gaps in standard REACH information requirements. The registrants may propose adaptations, and ECHA evaluates the reasoning and issues a written decision. Read-across is a common adaptation type, yet it is widely assumed that ECHA often does not agree that the justifications are adequate to waive standard testing requirements. From 2008 to August 2023, a total of 2,630 Testing Proposals were submitted to ECHA; of these, 1,538 had published decisions that were systematically evaluated in this study. Each document was manually reviewed, and information extracted for further analyses. Read-across hypotheses were standardized into 17 assessment elements (AEs); each submission was classified as to the AEs relied upon by the registrants and by ECHA. Data was analyzed for patterns and associations. Testing Proposal Evaluations (TPEs) with adaptations comprised 23% (353) of the total; analogue (168) or group (136) read-across adaptations were most common. Of 304 read-across-containing TPEs, 49% were accepted; the odds of acceptance were significantly greater for group read-across submissions. The data was analyzed by Annex (i.e., tonnage), test guideline study, read-across hypothesis AEs, as well as target and source substance types and their structural similarity. While most ECHA decisions with both positive and negative decisions on whether the proposed read-across was adequate were context-specific, a number of significant associations were identified that influence the odds of acceptance. Overall, this analysis provides an unbiased overview of 15 years of experience with testing proposal-specific read-across adaptations by both registrants and ECHA. These data will inform future submissions as they identify most critical AEs to increase the odds of read-across acceptance.

The Tissue-Specific Eco-Exposome: Differential Pharmaceutical Bioaccumulation and Disposition in Fish among Trophic Positions.

Though bioaccumulation of pharmaceuticals by aquatic organisms continues to receive scientific attention, the internal disposition of these contaminants among different tissue compartments of fish species has been infrequently investigated, particularly among fish at different trophic positions. We tested a human to fish biological read-across hypothesis for contaminant disposition by examining tissue-specific accumulation in three understudied species, longnose gar (Lepisosteus osseus; piscivore), gizzard shad (Dorosoma cepedianum; planktivore/detritivore), and smallmouth buffalo (Ictiobus bubalus; benthivore), from a river influenced by municipal effluent discharge. In addition to surface water, fish plasma, and brain, gill, gonad, liver, and lateral muscle fillet tissues were analyzed via isotope dilution liquid chromatography tandem mass spectrometry. Caffeine and sucralose, two common effluent tracers, were quantitated at low micrograms per liter levels in surface water, while an anticonvulsant, carbamazepine, was observed at levels up to 37 ng/L. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline and primary metabolites were detected in at least one tissue of all three species at low micrograms per kilogram concentrations. Within each species, brain and liver of select fish contained the highest levels of SSRIs compared to plasma and other tissues, which is generally consistent with human tissue disposition patterns. However, we observed differential accumulation among specific tissue types and species. For example, mean levels of sertraline in brain and liver tissues were 13.4 µg/kg and 1.5 µg/kg in gizzard shad and 1.3 µg/kg and 7.3 µg/kg in longnose gar, respectively. In contrast, smallmouth buffalo did not consistently accumulate SSRIs to detectable levels. Tissue-specific eco-exposome efforts are necessary to understand mechanisms associated with such marked bioaccumulation and internal dispositional differences among freshwater fish species occupying different trophic positions. Environ Toxicol Chem 2024;43:1894-1902. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxicological Assessment of Higher Olefins in OECD TG 422 Repeated Dose and Reproductive /Developmental Toxicity Screening Tests in Han Wistar Rats.

Higher olefins (HO) are used primarily as intermediates in the production of other chemicals, such as polymers, fatty acids, plasticizer alcohols, surfactants, lubricants, amine oxides, and detergent alcohols. The potential toxicity of five HO (i.e., 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene) with carbon ranging from C8 to C18 was examined in a combined repeated dose and reproduction/developmental toxicity screening study (OECD TG 422). These five HO were administered to Han Wistar rats by gavage at 0 (controls), 100, 300, and 1000mg/kg bw/day. As a group of substances, adaptive changes in the liver (liver weight increase without pathological evidence), as well as increased kidney weight in male rats, were observed in HO with carbon numbers from C8 to C10. The overall systemic no observed adverse effect level (NOAEL) for all HO was determined at 1000mg/kg bw/day. In the reproductive/developmental toxicity assessment, offspring viability, size, and weights were reduced in litters from females treated with Nonene at 1000mg/kg bw/day. The overall no observed effects level (NOEL) for reproductive toxicity was considered to be 300mg/kg bw/day for Nonene and 1000mg/kg bw/day for the other four HO, respectively. These data significantly enrich the database on the toxicity of linear and branched HO, allowing comparison with similar data published on a range of linear and branched HO. Comparisons between structural class and study outcome provide further supportive data in order to validate the read-across hypothesis as part of an overall holistic testing strategy.

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-, β-, 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 24h and 72h 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 β-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 β-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.

Assessment of the Intestinal Absorption of Higher Olefins by the Everted Gut Sac Model in Combination with In Silico New Approach Methodologies.

To reduce the number of animals and studies needed to fulfill the information requirements as required by Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (EC no. 1907/2006), a read-across approach was used to support approximately 30 higher olefins. This study aimed to assess the absorption potential of higher olefins through the gut wall as the experimentally determined bioavailability which would strengthen the read-across hypothesis and justification, reducing the need for toxicity studies on all of the higher olefins. The absorption potential of a series of higher olefins (carbon range from 6 to 28, with five configurations of the double bond) was determined in the in vitro everted rat small intestinal sac model and subsequently ranked. In addition, in silico approaches were applied to predict the reactivity, lipophilicity, and permeability of higher olefins. In the in vitro model, everted sacs were incubated in “fed-state simulated small intestinal fluid” saturated with individual higher olefins. The sac contents were then collected, extracted, and analyzed for olefin content using gas chromatography with a flame ionization detector. The C6 to C10 molecules were readily absorbed into the intestinal sacs. Marked inter-compound differences were observed, with the amount of absorption generally decreasing with the increase in carbon number. Higher olefins with ≥C14 carbons were either not absorbed or very poorly absorbed. In the reactivity simulation study, the reactivity is well described by the position of the double bond rather than the number of carbon atoms. In the lipophilicity and permeability analysis, both parameter descriptors depend mainly on the number of carbon atoms and less on the position of the double bond. In conclusion, these new approach methodologies provide supporting information on any trends or breakpoints in intestinal uptake and a hazard matrix based on carbon number and position of the double bond. This matrix will further assist in the selection of substances for inclusion in the mammalian toxicity testing programme.

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.

Assessing metabolic similarity for read-across predictions

Read-across is one of the most frequently used non-animal approaches for filling toxicological data gaps. There is a universal realization that considerations of the metabolic fates of the target substance and proposed source analogue(s) are important in accepting or rejecting a read-across hypothesis. Comparisons of target and source metabolic information most often suffers from the lack of experimental data. These data voids have prompted the development and use of alternative methods. With various limitations, several in silico tools are available to aid an evaluator in appraising metabolic similarity. Metabolic maps, arrays of 2D molecular structures of (a)biotic transformation products, allow for side-by-side visual comparison of stepwise routes of conversion of the target and source chemicals. There are two varieties of metabolic maps – documented and simulated. A document metabolic map is one derived for in vivo or in vitro experimental data. A simulated one is a map derived from metabolic pathway predictions of possible or probable metabolites coupled with expert judgment. Here, through a series of examples, the advantages of using the metabolic information, provided by the OASIS software, are exemplified. This will aid in establishing metabolic consistency between the chemicals used in a read-across argument (i.e., establish similarity in their (a)biotic transformations). Of particular importance are found to be the identification of common metabolic pathway(s), common metabolite(s), and similarity concerning the formation of reactive metabolites.

Application of grouping and read-across for the evaluation of parabens of different chain lengths with a particular focus on endocrine properties

This article presents the outcomes of higher-tier repeated-dose toxicity studies and developmental and reproductive toxicity (DART) studies using Wistar rats requested for methyl paraben and propyl paraben under the European Union chemicals legislation. All studies revealed no-observed adverse effects (NOAELs) at 1000 mg/kg body weight/day. These findings (absence of effects) were then used to interpolate the hazard profile for ethyl paraben, further considering available data for butyl paraben. The underlying read-across hypothesis (all shorter-chained linear n-alkyl parabens are a ‘category’ based on very high structural similarity and are transformed to a common compound) was confirmed by similarity calculations and comparative in vivo toxicokinetics screening studies for methyl paraben, ethyl paraben, propyl paraben and butyl paraben. All four parabens were rapidly taken up systemically following oral gavage administration to rats, metabolised to p-hydroxybenzoic acid, and rapidly eliminated (parabens within one hour; p-hydroxybenzoic acid within 4–8 h). Accordingly, for ethyl paraben, the NOAELs for repeated-dose toxicity and DART were interpolated to be 1000 mg/kg body weight/day. Finally, all evidence was evaluated to address concerns expressed in the literature that parabens might be endocrine disruptors. This evaluation showed that the higher-tier studies do not provide any indication for any endocrine disrupting property. This is the first time that a comprehensive dataset from higher-tier in vivo studies following internationally agreed test protocols has become available for shorter-chained linear n-alkyl parabens. Consistently, the dataset shows that these parabens are devoid of repeated-dose toxicity and do not possess any DART or endocrine disrupting properties.

Incorporating lines of evidence from New Approach Methodologies (NAMs) to reduce uncertainties in a category based read-across: A case study for repeated dose toxicity

A group of triazole compounds was selected to investigate the confidence that may be associated with read-across of a complex data gap: repeated dose toxicity. The read-across was evaluated using Assessment Elements (AEs) from the European Chemicals Agency's (ECHA's) Read-Across Assessment Framework (RAAF), alongside appraisal of associated uncertainties. Following an initial read-across based on chemical structure and properties, uncertainties were reduced by the integration of data streams such as those from New Approach Methodologies (NAM) and other existing data. In addition, addressing the findings of the ECHA RAAF framework, complemented with specific questions concerning uncertainties, increased the confidence that can be placed in read-across. Although a data rich group of compounds with a strong mechanistic basis was analysed, it was clearly demonstrated that NAM data available from publicly available resources could be applied to support read-across. It is acknowledged that most read-across studies will not be so data rich or mechanistically robust, therefore some targeted experimentation may be required to fill the data gaps. In this sense, NAMs should constitute new experimental tests performed with the specific goal of reducing the uncertainties and demonstrating the read-across hypothesis.

Dosimetry considerations for in vivo and in vitro test data and a novel surrogate iTTC approach for read-across based on metabolites

In the context of REACH and the EU ban on animal testing of cosmetic ingredients, development, adoption and acceptance of non-animal methods is urgent. Read-across is the predominate non-animal method being used for higher tier endpoints (repeat dose and developmental and reproductive toxicity). The limitations of read-across include the lack of suitable analogs and analog data for all chemicals of interest, as well as concerns with missing a potential adverse response that results from small differences in chemical structure. In this manuscript we explore two possible enhancements to read-across approaches in the context of a single case study: read-across assessments based on metabolite data and incorporation of in vitro data to support the read-across hypothesis. We use this case study to explore possible paths forward in implementing these enhancements, but more importantly to raise issues that need to be resolved prior to extensive use and hopefully acceptance of these expanded approaches. For the case of read-across based on metabolites, we explore the potential application of an internal dose TTC (iTTC) approach to address uncertainty associated with potential toxicity from exposure to residual parent chemical. This is a relevant scenario for the many cosmetic ingredients containing ester structures. For the use of in vitro data we explore considerations that must be taken before the application of ToxCast™ data to the read-across to ensure positive responses are reliable and relevant. As more cosmetic ingredients are tested in in vitro systems, guidance on the appropriate interpretation of these data will be essential. The concept of dosimetry is a unifying need that must be addressed in both these approaches. In the present paper, we explore how to use and apply internal dose metrics to address some issues associated with expanded approaches in read-across.

Testing the “read-across hypothesis” by investigating the effects of ibuprofen on fish

Human pharmaceuticals present in the environment have the potential to cause adverse effects on non-target organisms. The “read-across hypothesis” stipulates that pharmaceuticals will exhibit similar biological effects across species (e.g. human and fish) if the molecular target has been conserved and the effective drug concentrations are reached (Cmax). We tested this hypothesis by evaluating if ibuprofen, a non-selective inhibitor of prostaglandins and the cyclooxygenase (COX) enzyme, can mimic its primary effect in humans, on fish, at comparable plasma concentrations. The endpoints, prostaglandin E metabolite (PGEM) levels and the mRNA expression of COX (ptgs) gene, were measured in the gills of control and exposed fathead minnows (Pimephales promelas), using enzyme-immunoassay and quantitative real-time PCR (qPCR). Fish were exposed, for 24–72 h, to measured water concentrations of 9 (n = 12), 370 (n = 40) and 470 μg ibuprofen/L (n = 12). Water and blood plasma concentrations were determined using LC-MS/MS. Results showed that PGEM levels in fish exposed to 370 and 470 μg ibuprofen/L were significantly decreased compared to control fish, when mean plasma ibuprofen concentrations were 1.8–5.6-fold below the Cmax. The plasma ibuprofen concentrations and PGEM levels varied greatly between individuals. In fish exposed to 9 μg ibuprofen/L, when the mean plasma ibuprofen concentration was 224-fold below Cmax, no change in PGEM levels was observed. These data provide evidence for the read-across hypothesis, but suggest establishing a direct dose-response between internal plasma and PGEM is difficult, and would require significantly larger numbers of fish to overcome the inter-individual variation.

The effects of antidepressants appear to be rapid and at environmentally relevant concentrations.

The effects of antidepressants on wildlife are currently raising some concern because of an increased number of publications indicating biological effects at environmentally relevant concentrations (<100 ng/L). These results have been met with some scepticism because of the higher concentrations required to detect effects in some species and the perceived slowness to therapeutic effects recorded in humans and other vertebrates. Because their mode of action is thought to be by modulation of the neurotransmitters serotonin, dopamine, and norepinephrine, aquatic invertebrates that possess transporters and receptors sensitive to activation by these pharmaceuticals are potentially affected by them. The authors highlight studies on the effects of antidepressants, particularly on crustacean and molluskan groups, showing that they are susceptible to a wide variety of neuroendocrine disruptions at environmentally relevant concentrations. Interestingly, some effects observed in these species can be observed within minutes to hours of exposure. For example, exposure of amphipod crustaceans to several selective serotonin reuptake inhibitors can invoke changes in swimming behavior within hours. In mollusks, exposure to selective serotonin reuptake inhibitors can induce spawning in male and female mussels and foot detachment in snails within minutes of exposure. In the light of new studies indicating effects on the human brain from selective serotonin reuptake inhibitors using magnetic resonance imaging scans, the authors discuss possible reasons for the discrepancy in former results in relation to the read-across hypothesis, variation in biomarkers used, modes of uptake, phylogenetic distance, and the affinity to different targets and differential sensitivity to receptors.

Quantitative cross-species extrapolation between humans and fish: the case of the anti-depressant fluoxetine.

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.

The Read-Across Hypothesis and Environmental Risk Assessment of Pharmaceuticals

Pharmaceuticals in the environment have received increased attention over the past decade, as they are ubiquitous in rivers and waterways. Concentrations are in sub-ng to low μg/L, well below acute toxic levels, but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern. The read-across hypothesis stipulates that a drug will have an effect in non-target organisms only if the molecular targets such as receptors and enzymes have been conserved, resulting in a (specific) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations. If this holds true for different classes of pharmaceuticals, it should be possible to predict the potential environmental impact from information obtained during the drug development process. This paper critically reviews the evidence for read-across, and finds that few studies include plasma concentrations and mode of action based effects. Thus, despite a large number of apparently relevant papers and a general acceptance of the hypothesis, there is an absence of documented evidence. There is a need for large-scale studies to generate robust data for testing the read-across hypothesis and developing predictive models, the only feasible approach to protecting the environment.