Re-evaluation of the existing health-based guidance values for copper and exposure assessment from all sources.

Copper is an essential micronutrient and also a regulated product used in organic and in conventional farming pest management. Both deficiency and excessive exposure to copper can have adverse health effects. In this Scientific Opinion, the EFSA 2021 harmonised approach for establishing health-based guidance values (HBGVs) for substances that are regulated products and also nutrients was used to resolve the divergent existing HBGVs for copper. The tightly regulated homeostasis prevents toxicity manifestation in the short term, but the development of chronic copper toxicity is dependent on copper homeostasis and its tissue retention. Evidence from Wilson disease suggests that hepatic retention is indicative of potential future and possibly sudden onset of copper toxicity under conditions of continuous intake. Hence, emphasis was placed on copper retention as an early marker of potential adverse effects. The relationships between (a) chronic copper exposure and its retention in the body, particularly the liver, and (b) hepatic copper concentrations and evidence of toxicity were examined. The Scientific Committee (SC) concludes that no retention of copper is expected to occur with intake of 5 mg/day and established an Acceptable Daily Intake (ADI) of 0.07 mg/kg bw. A refined dietary exposure assessment was performed, assessing contribution from dietary and non-dietary sources. Background copper levels are a significant source of copper. The contribution of copper from its use as plant protection product (PPP), food and feed additives or fertilisers is negligible. The use of copper in fertilisers or PPPs contributes to copper accumulation in soil. Infant formula and follow-on formula are important contributors to dietary exposure of copper in infants and toddlers. Contribution from non-oral sources is negligible. Dietary exposure to total copper does not exceed the HBGV in adolescents, adults, elderly and the very elderly. Neither hepatic copper retention nor adverse effects are expected to occur from the estimated copper exposure in children due to higher nutrient requirements related to growth.

Draft for internal testing Scientific Committee guidance on appraising and integrating evidence from epidemiological studies for use in EFSA's scientific assessments.

EFSA requested its Scientific Committee to prepare a guidance document on appraising and integrating evidence from epidemiological studies for use in EFSA's scientific assessments. The guidance document provides an introduction to epidemiological studies and illustrates the typical biases of the different epidemiological study designs. It describes key epidemiological concepts relevant for evidence appraisal. Regarding study reliability, measures of association, exposure assessment, statistical inferences, systematic error and effect modification are explained. Regarding study relevance, the guidance describes the concept of external validity. The principles of appraising epidemiological studies are illustrated, and an overview of Risk of Bias (RoB) tools is given. A decision tree is developed to assist in the selection of the appropriate Risk of Bias tool, depending on study question, population and design. The customisation of the study appraisal process is explained, detailing the use of RoB tools and assessing the risk of bias in the body of evidence. Several examples of appraising experimental and observational studies using a Risk of Bias tool are annexed to the document to illustrate the application of the approach. This document constitutes a draft that will be applied in EFSA's assessments during a 1-year pilot phase and be revised and complemented as necessary. Before finalisation of the document, a public consultation will be launched.

Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals.

This Guidance document describes harmonised risk assessment methodologies for combined exposure to multiple chemicals for all relevant areas within EFSA's remit, i.e. human health, animal health and ecological areas. First, a short review of the key terms, scientific basis for combined exposure risk assessment and approaches to assessing (eco)toxicology is given, including existing frameworks for these risk assessments. This background was evaluated, resulting in a harmonised framework for risk assessment of combined exposure to multiple chemicals. The framework is based on the risk assessment steps (problem formulation, exposure assessment, hazard identification and characterisation, and risk characterisation including uncertainty analysis), with tiered and stepwise approaches for both whole mixture approaches and component-based approaches. Specific considerations are given to component-based approaches including the grouping of chemicals into common assessment groups, the use of dose addition as a default assumption, approaches to integrate evidence of interactions and the refinement of assessment groups. Case studies are annexed in this guidance document to explore the feasibility and spectrum of applications of the proposed methods and approaches for human and animal health and ecological risk assessment. The Scientific Committee considers that this Guidance is fit for purpose for risk assessments of combined exposure to multiple chemicals and should be applied in all relevant areas of EFSA's work. Future work and research are recommended.

Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment.

The Scientific Committee confirms that the Threshold of Toxicological Concern (TTC) is a pragmatic screening and prioritisation tool for use in food safety assessment. This Guidance provides clear step-by-step instructions for use of the TTC approach. The inclusion and exclusion criteria are defined and the use of the TTC decision tree is explained. The approach can be used when the chemical structure of the substance is known, there are limited chemical-specific toxicity data and the exposure can be estimated. The TTC approach should not be used for substances for which EU food/feed legislation requires the submission of toxicity data or when sufficient data are available for a risk assessment or if the substance under consideration falls into one of the exclusion categories. For substances that have the potential to be DNA-reactive mutagens and/or carcinogens based on the weight of evidence, the relevant TTC value is 0.0025 µg/kg body weight (bw) per day. For organophosphates or carbamates, the relevant TTC value is 0.3 µg/kg bw per day. All other substances are grouped according to the Cramer classification. The TTC values for Cramer Classes I, II and III are 30 µg/kg bw per day, 9 µg/kg bw per day and 1.5 µg/kg bw per day, respectively. For substances with exposures below the TTC values, the probability that they would cause adverse health effects is low. If the estimated exposure to a substance is higher than the relevant TTC value, a non-TTC approach is required to reach a conclusion on potential adverse health effects.

The principles and methods behind EFSA's Guidance on Uncertainty Analysis in Scientific Assessment.

To meet the general requirement for transparency in EFSA's work, all its scientific assessments must consider uncertainty. Assessments must say clearly and unambiguously what sources of uncertainty have been identified and what is their impact on the assessment conclusion. This applies to all EFSA's areas, all types of scientific assessment and all types of uncertainty affecting assessment. This current Opinion describes the principles and methods supporting a concise Guidance Document on Uncertainty in EFSA's Scientific Assessment, published separately. These documents do not prescribe specific methods for uncertainty analysis but rather provide a flexible framework within which different methods may be selected, according to the needs of each assessment. Assessors should systematically identify sources of uncertainty, checking each part of their assessment to minimise the risk of overlooking important uncertainties. Uncertainty may be expressed qualitatively or quantitatively. It is neither necessary nor possible to quantify separately every source of uncertainty affecting an assessment. However, assessors should express in quantitative terms the combined effect of as many as possible of identified sources of uncertainty. The guidance describes practical approaches. Uncertainty analysis should be conducted in a flexible, iterative manner, starting at a level appropriate to the assessment and refining the analysis as far as is needed or possible within the time available. The methods and results of the uncertainty analysis should be reported fully and transparently. Every EFSA Panel and Unit applied the draft Guidance to at least one assessment in their work area during a trial period of one year. Experience gained in this period resulted in improved guidance. The Scientific Committee considers that uncertainty analysis will be unconditional for EFSA Panels and staff and must be embedded into scientific assessment in all areas of EFSA's work.

Guidance on Uncertainty Analysis in Scientific Assessments.

Uncertainty analysis is the process of identifying limitations in scientific knowledge and evaluating their implications for scientific conclusions. It is therefore relevant in all EFSA's scientific assessments and also necessary, to ensure that the assessment conclusions provide reliable information for decision-making. The form and extent of uncertainty analysis, and how the conclusions should be reported, vary widely depending on the nature and context of each assessment and the degree of uncertainty that is present. This document provides concise guidance on how to identify which options for uncertainty analysis are appropriate in each assessment, and how to apply them. It is accompanied by a separate, supporting opinion that explains the key concepts and principles behind this Guidance, and describes the methods in more detail.

Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain: Part 1, human and animal health.

The European Food Safety Authority has produced this Guidance on human and animal health aspects (Part 1) of the risk assessment of nanoscience and nanotechnology applications in the food and feed chain. It covers the application areas within EFSA's remit, e.g. novel foods, food contact materials, food/feed additives and pesticides. The Guidance takes account of the new developments that have taken place since publication of the previous Guidance in 2011. Potential future developments are suggested in the scientific literature for nanoencapsulated delivery systems and nanocomposites in applications such as novel foods, food/feed additives, biocides, pesticides and food contact materials. Therefore, the Guidance has taken account of relevant new scientific studies that provide more insights to physicochemical properties, exposure assessment and hazard characterisation of nanomaterials. It specifically elaborates on physicochemical characterisation of nanomaterials in terms of how to establish whether a material is a nanomaterial, the key parameters that should be measured, the methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. It also details the aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vivo/in vitro toxicological studies are discussed and a tiered framework for toxicological testing is outlined. It describes in vitro degradation, toxicokinetics, genotoxicity as well as general issues relating to testing of nanomaterials. Depending on the initial tier results, studies may be needed to investigate reproductive and developmental toxicity, immunotoxicity, allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read-across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes/mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis, and provides recommendations for further research in this area.

Update: use of the benchmark dose approach in risk assessment.

The Scientific Committee (SC) reconfirms that the benchmark dose (BMD) approach is a scientifically more advanced method compared to the NOAEL approach for deriving a Reference Point (RP). Most of the modifications made to the SC guidance of 2009 concern the section providing guidance on how to apply the BMD approach. Model averaging is recommended as the preferred method for calculating the BMD confidence interval, while acknowledging that the respective tools are still under development and may not be easily accessible to all. Therefore, selecting or rejecting models is still considered as a suboptimal alternative. The set of default models to be used for BMD analysis has been reviewed, and the Akaike information criterion (AIC) has been introduced instead of the log-likelihood to characterise the goodness of fit of different mathematical models to a dose-response data set. A flowchart has also been inserted in this update to guide the reader step-by-step when performing a BMD analysis, as well as a chapter on the distributional part of dose-response models and a template for reporting a BMD analysis in a complete and transparent manner. Finally, it is recommended to always report the BMD confidence interval rather than the value of the BMD. The lower bound (BMDL) is needed as a potential RP, and the upper bound (BMDU) is needed for establishing the BMDU/BMDL per ratio reflecting the uncertainty in the BMD estimate. This updated guidance does not call for a general re-evaluation of previous assessments where the NOAEL approach or the BMD approach as described in the 2009 SC guidance was used, in particular when the exposure is clearly smaller (e.g. more than one order of magnitude) than the health-based guidance value. Finally, the SC firmly reiterates to reconsider test guidelines given the expected wide application of the BMD approach.

Guidance on the risk assessment of substances present in food intended for infants below 16 weeks of age.

Following a request from the European Commission to EFSA, the EFSA Scientific Committee (SC) prepared a guidance for the risk assessment of substances present in food intended for infants below 16 weeks of age. In its approach to develop this guidance, the EFSA SC took into account, among others, (i) an exposure assessment based on infant formula as the only source of nutrition; (ii) knowledge of organ development in human infants, including the development of the gut, metabolic and excretory capacities, the brain and brain barriers, the immune system, the endocrine and reproductive systems; (iii) the overall toxicological profile of the substance identified through the standard toxicological tests, including critical effects; (iv) the relevance for the human infant of the neonatal experimental animal models used. The EFSA SC notes that during the period from birth up to 16 weeks, infants are expected to be exclusively fed on breast milk and/or infant formula. The EFSA SC views this period as the time where health-based guidance values for the general population do not apply without further considerations. High infant formula consumption per body weight is derived from 95th percentile consumption. The first weeks of life is the time of the highest relative consumption on a body weight basis. Therefore, when performing an exposure assessment, the EFSA SC proposes to use the high consumption value of 260 mL/kg bw per day. A decision tree approach is proposed that enables a risk assessment of substances present in food intended for infants below 16 weeks of age. The additional information needed when testing substances present in food for infants below 16 weeks of age and the approach to be taken for the risk assessment are on a case-by-case basis, depending on whether the substance is added intentionally to food and is systemically available.

Guidance on the assessment of the biological relevance of data in scientific assessments.

EFSA requested its Scientific Committee to prepare a guidance document providing generic issues and criteria to consider biological relevance, particularly when deciding on whether an observed effect is of biological relevance, i.e. is adverse (or shows a beneficial health effect) or not. The guidance document provides a general framework for establishing the biological relevance of observations at various stages of the assessment. Biological relevance is considered at three main stages related to the process of dealing with evidence: Development of the assessment strategy. In this context, specification of agents, effects, subjects and conditions in relation to the assessment question(s): Collection and extraction of data; Appraisal and integration of the relevance of the agents, subjects, effects and conditions, i.e. reviewing dimensions of biological relevance for each data set. A decision tree is developed to assist in the collection, identification and appraisal of relevant data for a given specific assessment question to be answered.

Guidance on the use of the weight of evidence approach in scientific assessments.

EFSA requested the Scientific Committee to develop a guidance document on the use of the weight of evidence approach in scientific assessments for use in all areas under EFSA's remit. The guidance document addresses the use of weight of evidence approaches in scientific assessments using both qualitative and quantitative approaches. Several case studies covering the various areas under EFSA's remit are annexed to the guidance document to illustrate the applicability of the proposed approach. Weight of evidence assessment is defined in this guidance as a process in which evidence is integrated to determine the relative support for possible answers to a question. This document considers the weight of evidence assessment as comprising three basic steps: (1) assembling the evidence into lines of evidence of similar type, (2) weighing the evidence, (3) integrating the evidence. The present document identifies reliability, relevance and consistency as three basic considerations for weighing evidence.

Characterization of the dose decrement in regulatory rat pesticide toxicity feeding studies.

In typical rodent pesticide feeding studies of 4 up to 104 weeks, animals are offered the pesticide at constant concentrations in the feed. Throughout the entire study duration of up to 104 weeks, the daily feed consumption per animal remains nearly constant. This results in decreasing doses per kg bodyweight from the first day of treatment onwards as the bodyweight increases. Recently, we have identified this dose decrement as the major cause for lower No Observed Effect Levels (NOAEL, expressed as mg/kg bodyweight) in longer-term studies compared to shorter-term studies, rather than the exposure duration itself. In the current evaluation we investigated the nature of the dose decrement in more detail by using male and female bodyweight and feed consumption data from 118 feeding studies of three rat strains to calculate dose development over time. In male rats, after a steep initial dose decrement, the mean dose at week 7 of treatment is on average half of the initial dose and after 29 weeks one third of the initial dose. In females, 50% of the initial dose is reached at week 18 and in 25% of the studies one third of the initial dose is reached at approximately 75 weeks of treatment. Although bodyweights and feed intakes per animal were different between strains, doses and dose development curves over time are similar. The fact that ingested doses in rats continually decrease, especially in the first 13 weeks, should be taken into account in dietary risk assessments.

Study parameters influencing NOAEL and LOAEL in toxicity feeding studies for pesticides: exposure duration versus dose decrement, dose spacing, group size and chemical class.

The effect of exposure duration on no observed adverse effect levels (NOAEL) and lowest observed adverse effect levels (LOAEL) in rodent pesticide feeding studies was evaluated. Ratios of NOAEL (and LOAEL), expressed as pesticide concentrations in feed, were calculated from subacute to subchronic, subchronic to chronic and subacute to chronic studies. There was no statistical significant effect of exposure duration on ratio distributions. Whereas geometric means of ratios were in a narrow range of 1.1-2.5, the geometric standard deviations and 95th percentiles increased with dose spacing of the involved studies. With the exception of carbamates, the chemical class of pesticides had no influence on the ratio distributions. However, the number of animals in the shorter-term study of ratio couples being ≤ 1 was statistically significantly higher than in ratio couples being >1. Ratios ≤ 1 may be partly explained by the dose decrement over time observed in feeding studies applying the test substances in constant concentrations. The dose decrement possibly converts initially toxic doses to less toxic doses beyond the subacute phase. Ratios >1 seem to be caused predominantly by differences in study design parameters. In dietary risk assessment, the acceptable daily intake (ADI) is compared to pesticide intake estimates based on mean food consumption (i.e. the so called theoretical maximum daily intake, TMDI) being orders of magnitude lower than actual food consumption on eating occasions for certain food commodities. As subacute, subchronic and chronic NOAEL (and LOAEL), expressed as pesticide concentration in feed did not differ statistically significantly, the TMDI as benchmark for the ADI may underestimate the significance of the toxicity of subacute exposure.

The significance of the subchronic toxicity in the dietary risk assessment of pesticides.

Based on 289 public pesticide evaluations, geometric means of subchronic/chronic No Observed Adverse Effect Level (NOAEL) ratios of 2.6, 2.5 and 1.6 in mice, rats and dogs were calculated. The 75th percentiles are 5.5, 5.1 and 3.2. Higher ratios correlate with increased dose spacing in chronic studies and may be mainly explained therewith. In rats fed at constant pesticide concentrations in feed, the mean chronic dose decreases by 1.7- and 2.7-fold compared to the subchronic and subacute phase. These dose decreases match the subchronic/chronic NOAEL ratios. The ratio of predicted rat chronic NOAEL (dose decrement adjusted subchronic NOAEL) to experimental chronic NOAEL is 1.5 and the 75th percentile is 3.0. In dietary risk assessment, the Acute Reference Dose and the Acceptable Daily Intake (derived from acute and chronic NOAEL) are compared to acute (IESTI) or mean (TMDI) exposure estimates. Because IESTI and TMDI base on acute or mean food consumption they differ by orders of magnitude for certain commodities. As subchronic and chronic NOAEL are similar, it remains to be shown whether pesticide intake estimates based on mean food consumption are adequate measures to compare against the ADI if repeated daily exposures considerably higher than mean exposures may occur.

The benchmark dose approach in food risk assessment: is it applicable and worthwhile?

The benchmark dose (BMD) approach is being increasingly used in the area of food risk assessment because it offers several advantages compared to the conventional no-observed-adverse-effect-level approach. The aim of this work was to check the applicability of the BMD approach on toxicity data available from pesticides, mycotoxins and natural toxins. Based on toxicological evaluations, the pivotal study was identified. Detailed data from the original study were retrieved and used for BMD modelling. Twenty-five studies used for BMD modelling were analysed with regard to study design: total number of animals, number of dose levels, and spacing between dose levels. The quality of the modelled endpoints was evaluated according to the following aspects: BMD/BMDL ratio, test for goodness of fit and BMD in the range of dose levels. If one of these aspects was not fulfilled, the BMD derived from this endpoint was considered to be uncertain to some extent and corresponding modelled data sets were examined. The present work demonstrates that the BMD approach is in principle applicable to pesticides, mycotoxins, and natural toxins. Although large differences relating to data availability and data quality were noticed, 69 of 82 modelled endpoints (84%) fulfilled the three quality aspects of BMD modelling.

Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase.

Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.