The use of dose-response data in a margin of exposure approach to carcinogenic risk assessment for genotoxic chemicals in food.

Genotoxic substances are generally not permitted for deliberate use in food production. However, an appreciable number of known or suspected genotoxic substances occur unavoidably in food, e.g. from natural occurrence, environmental contamination and generation during cooking and processing. Over the past decade a margin of exposure (MOE) approach has increasingly been used in assessing the exposure to substances in food that are genotoxic and carcinogenic. The MOE is defined as a reference point on the dose-response curve (e.g. a benchmark dose lower confidences limit derived from a rodent carcinogenicity study) divided by the estimated human intake. A small MOE indicates a higher concern than a very large MOE. Whilst the MOE cannot be directly equated to risk, it supports prioritisation of substances for further research or for possible regulatory action, and provides a basis for communicating to the public. So far, the MOE approach has been confined to substances for which carcinogenicity data are available. In the absence of carcinogenicity data, evidence of genotoxicity is used only in hazard identification. The challenge to the genetic toxicology community is to develop approaches for characterising risk to human health based on data from genotoxicity studies. In order to achieve wide acceptance, it would be important to further address the issues that have been discussed in the context of dose-response modelling of carcinogenicity data in order to assign levels of concern to particular MOE values, and also whether it is possible to make generic conclusions on how potency in genotoxicity assays relates to carcinogenic potency.

Update of the risk assessment of inorganic arsenic in food.

The European Commission asked EFSA to update its 2009 risk assessment on arsenic in food carrying out a hazard assessment of inorganic arsenic (iAs) and using the revised exposure assessment issued by EFSA in 2021. Epidemiological studies show that the chronic intake of iAs via diet and/or drinking water is associated with increased risk of several adverse outcomes including cancers of the skin, bladder and lung. The CONTAM Panel used the benchmark dose lower confidence limit based on a benchmark response (BMR) of 5% (relative increase of the background incidence after adjustment for confounders, BMDL05) of 0.06 µg iAs/kg bw per day obtained from a study on skin cancer as a Reference Point (RP). Inorganic As is a genotoxic carcinogen with additional epigenetic effects and the CONTAM Panel applied a margin of exposure (MOE) approach for the risk characterisation. In adults, the MOEs are low (range between 2 and 0.4 for mean consumers and between 0.9 and 0.2 at the 95th percentile exposure, respectively) and as such raise a health concern despite the uncertainties.

Update of the risk assessment of polybrominated diphenyl ethers (PBDEs) in food.

The European Commission asked EFSA to update its 2011 risk assessment on polybrominated diphenyl ethers (PBDEs) in food, focusing on 10 congeners: BDE-28, -47, -49, -99, -100, -138, -153, -154, -183 and ­209. The CONTAM Panel concluded that the neurodevelopmental effects on behaviour and reproductive/developmental effects are the critical effects in rodent studies. For four congeners (BDE-47, -99, -153, -209) the Panel derived Reference Points, i.e. benchmark doses and corresponding lower 95% confidence limits (BMDLs), for endpoint-specific benchmark responses. Since repeated exposure to PBDEs results in accumulation of these chemicals in the body, the Panel estimated the body burden at the BMDL in rodents, and the chronic intake that would lead to the same body burden in humans. For the remaining six congeners no studies were available to identify Reference Points. The Panel concluded that there is scientific basis for inclusion of all 10 congeners in a common assessment group and performed a combined risk assessment. The Panel concluded that the combined margin of exposure (MOET) approach was the most appropriate risk metric and applied a tiered approach to the risk characterisation. Over 84,000 analytical results for the 10 congeners in food were used to estimate the exposure across dietary surveys and age groups of the European population. The most important contributors to the chronic dietary Lower Bound exposure to PBDEs were meat and meat products and fish and seafood. Taking into account the uncertainties affecting the assessment, the Panel concluded that it is likely that current dietary exposure to PBDEs in the European population raises a health concern.

Risk assessment of small organoarsenic species in food.

The European Commission asked EFSA for a risk assessment on small organoarsenic species in food. For monomethylarsonic acid MMA(V), decreased body weight resulting from diarrhoea in rats was identified as the critical endpoint and a BMDL10 of 18.2 mg MMA(V)/kg body weight (bw) per day (equivalent to 9.7 mg As/kg bw per day) was calculated as a reference point (RP). For dimethylarsinic acid DMA(V), increased incidence in urinary bladder tumours in rats was identified as the critical endpoint. A BMDL10 of 1.1 mg DMA(V)/kg bw per day (equivalent to 0.6 mg As/kg bw per day) was calculated as an RP. For other small organoarsenic species, the toxicological data are insufficient to identify critical effects and RPs, and they could not be included in the risk assessment. For both MMA(V) and DMA(V), the toxicological database is incomplete and a margin of exposure (MOE) approach was applied for risk characterisation. The highest chronic dietary exposure to DMA(V) was estimated in 'Toddlers', with rice and fish meat as the main contributors across population groups. For MMA(V), the highest chronic dietary exposures were estimated for high consumers of fish meat and processed/preserved fish in 'Infants' and 'Elderly' age class, respectively. For MMA(V), an MOE of ≥ 500 was identified not to raise a health concern. For MMA(V), all MOEs were well above 500 for average and high consumers and thus do not raise a health concern. For DMA(V), an MOE of 10,000 was identified as of low health concern as it is genotoxic and carcinogenic, although the mechanisms of genotoxicity and its role in carcinogenicity of DMA(V) are not fully elucidated. For DMA(V), MOEs were below 10,000 in many cases across dietary surveys and age groups, in particular for some 95th percentile exposures. The Panel considers that this would raise a health concern.

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, which may be present in different epidemiological study designs. It then describes key epidemiological concepts relevant for evidence appraisal. This includes brief explanations for measures of association, exposure assessment, statistical inference, systematic error and effect modification. The guidance then describes the concept of external validity and the principles of appraising epidemiological studies. The customisation of the study appraisal process is explained including tailoring of tools for assessing the risk of bias (RoB). Several examples of appraising experimental and observational studies using a RoB tool are annexed to the document to illustrate the application of the approach. The latter part of this guidance focuses on different steps of evidence integration, first within and then across different streams of evidence. With respect to risk characterisation, the guidance considers how evidence from human epidemiological studies can be used in dose-response modelling with several different options being presented. Finally, the guidance addresses the application of uncertainty factors in risk characterisation when using evidence from human epidemiological studies.

Recent advances in the risk assessment of melamine and cyanuric acid in animal feed.

Melamine can be present at low levels in food and feed mostly from its legal use as a food contact material in laminates and plastics, as a trace contaminant in nitrogen supplements used in animal feeds, and as a metabolite of the pesticide cyromazine. The mechanism of toxicity of melamine involves dose-dependent formation of crystals with either endogenous uric acid or a structural analogue of melamine, cyanuric acid, in renal tubules resulting in potential acute kidney failure. Co-exposure to melamine and cyanuric acid in livestock, fish, pets and laboratory animals shows higher toxicity compared with melamine or cyanuric acid alone. Evidence for crystal formation between melamine and other structural analogs i.e. ammelide and ammeline is limited. Illegal pet food adulterations with melamine and cyanuric acid and adulteration of milk with melamine resulted in melamine-cyanuric acid crystals, kidney damage and deaths of cats and dogs and melamine-uric acid stones, hospitalisation and deaths of children in China respectively. Following these incidents, the tolerable daily intake for melamine was re-evaluated by the U.S. Food and Drug Administration, the World Health Organisation, and the Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA). This review provides an overview of toxicology, the adulteration incidents and risk assessments for melamine and its structural analogues. Particular focus is given to the recent EFSA risk assessment addressing impacts on animal and human health of background levels of melamine and structural analogues in animal feed. Recent research and future directions are discussed.

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.

Guidance on protocol development for EFSA generic scientific assessments.

EFSA Strategy 2027 outlines the need for fit-for-purpose protocols for EFSA generic scientific assessments to aid in delivering trustworthy scientific advice. This EFSA Scientific Committee guidance document helps address this need by providing a harmonised and flexible framework for developing protocols for EFSA generic assessments. The guidance replaces the 'Draft framework for protocol development for EFSA's scientific assessments' published in 2020. The two main steps in protocol development are described. The first is problem formulation, which illustrates the objectives of the assessment. Here a new approach to translating the mandated Terms of Reference into scientifically answerable assessment questions and sub-questions is proposed: the 'APRIO' paradigm (Agent, Pathway, Receptor, Intervention and Output). Owing to its cross-cutting nature, this paradigm is considered adaptable and broadly applicable within and across the various EFSA domains and, if applied using the definitions given in this guidance, is expected to help harmonise the problem formulation process and outputs and foster consistency in protocol development. APRIO may also overcome the difficulty of implementing some existing frameworks across the multiple EFSA disciplines, e.g. the PICO/PECO approach (Population, Intervention/Exposure, Comparator, Outcome). Therefore, although not mandatory, APRIO is recommended. The second step in protocol development is the specification of the evidence needs and the methods that will be applied for answering the assessment questions and sub-questions, including uncertainty analysis. Five possible approaches to answering individual (sub-)questions are outlined: using evidence from scientific literature and study reports; using data from databases other than bibliographic; using expert judgement informally collected or elicited via semi-formal or formal expert knowledge elicitation processes; using mathematical/statistical models; and - not covered in this guidance - generating empirical evidence ex novo. The guidance is complemented by a standalone 'template' for EFSA protocols that guides the users step by step through the process of planning an EFSA scientific assessment.

Assessment of the genotoxicity of acrylamide.

EFSA was requested to deliver a statement on a recent publication revisiting the evidence for genotoxicity of acrylamide (AA). The statement was prepared by a Working Group and was endorsed by the CONTAM Panel before its final approval. In interpreting the Terms of Reference, the statement considered the modes of action underlying the carcinogenicity of AA including genotoxic and non-genotoxic effects. Relevant publications since the 2015 CONTAM Panel Opinion on AA in food were reviewed. Several new studies reported positive results on the clastogenic and mutagenic properties of AA and its active metabolite glycidamide (GA). DNA adducts of GA were induced by AA exposure in experimental animals and have also been observed in humans. In addition to the genotoxicity of AA, there is evidence for both secondary DNA oxidation via generation of reactive oxygen species and for non-genotoxic effects which may contribute to carcinogenesis by AA. These studies extend the information assessed by the CONTAM Panel in its 2015 Opinion, and support its conclusions. That Opinion applied the margin of exposure (MOE) approach, as recommended in the EFSA Guidance for substances that are both genotoxic and carcinogenic, for risk characterisation of the neoplastic effects of AA. Based on the new data evaluated, the MOE approach is still considered appropriate, and an update of the 2015 Opinion is not required at the present time.

Evaluation of existing guidelines for their adequacy for the food and feed risk assessment of microorganisms obtained through synthetic biology.

EFSA was asked by the European Commission to evaluate synthetic biology (SynBio) developments for agri-food use in the near future and to determine whether or not they are expected to constitute potential new hazards/risks. Moreover, EFSA was requested to evaluate the adequacy of existing guidelines for risk assessment of SynBio and if updated guidance is needed. The scope of this Opinion covers food and feed risk assessment, the variety of microorganisms that can be used in the food/feed chain and the whole spectrum of techniques used in SynBio. This Opinion complements a previously adopted Opinion with the evaluation of existing guidelines for the microbial characterisation and environmental risk assessment of microorganisms obtained through SynBio. The present Opinion confirms that microbial SynBio applications for food and feed use, with the exception of xenobionts, could be ready in the European Union in the next decade. New hazards were identified related to the use or production of unusual and/or new-to-nature components. Fifteen cases were selected for evaluating the adequacy of existing guidelines. These were generally adequate for assessing the product, the production process, nutritional and toxicological safety, allergenicity, exposure and post-market monitoring. The comparative approach and a safety assessment per se could be applied depending on the degree of familiarity of the SynBio organism/product with the non-genetically modified counterparts. Updated guidance is recommended for: (i) bacteriophages, protists/microalgae, (ii) exposure to plant protection products and biostimulants, (iii) xenobionts and (iv) feed additives for insects as target species. Development of risk assessment tools is recommended for assessing nutritional value of biomasses, influence of microorganisms on the gut microbiome and the gut function, allergenic potential of new-to-nature proteins, impact of horizontal gene transfer and potential risks of living cell intake. A further development towards a strain-driven risk assessment approach is recommended.

Guidance on the 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 no-observed-adverse-effect-level (NOAEL) approach for deriving a Reference Point (RP). The major change compared to the previous Guidance (EFSA SC, 2017) concerns the Section 2.5, in which a change from the frequentist to the Bayesian paradigm is recommended. In the former, uncertainty about the unknown parameters is measured by confidence and significance levels, interpreted and calibrated under hypothetical repetition, while probability distributions are attached to the unknown parameters in the Bayesian approach, and the notion of probability is extended to reflect uncertainty of knowledge. In addition, the Bayesian approach can mimic a learning process and reflects the accumulation of knowledge over time. Model averaging is again recommended as the preferred method for estimating the BMD and calculating its credible interval. The set of default models to be used for BMD analysis has been reviewed and amended so that there is now a single set of models for quantal and continuous data. The flow chart guiding the reader step-by-step when performing a BMD analysis has also been updated, and a chapter comparing the frequentist to the Bayesian paradigm inserted. Also, when using Bayesian BMD modelling, the lower bound (BMDL) is to be considered as potential RP, and the upper bound (BMDU) is needed for establishing the BMDU/BMDL 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 or 2017 Guidance was used, in particular when the exposure is clearly lower (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 wide application of the BMD approach.

Opinion on the impact of non-monotonic dose responses on EFSA's human health risk assessments.

This Opinion assesses the biological relevance of the non-monotonic dose responses (NMDR) identified in a previous EFSA External Report (Beausoleil et al., 2016) produced under GP/EFSA/SCER/2014/01 and the follow-up probabilistic assessment (Chevillotte et al., 2017a,b), focusing on the in vivo data sets fulfilling most of the checkpoints of the visual/statistical-based analysis identified in Beausoleil et al. (2016). The evaluation was completed with cases discussed in EFSA assessments and the update of the scientific literature. Observations of NMDR were confirmed in certain studies and are particularly relevant for receptor-mediated effects. Based on the results of the evaluation, the Opinion proposes an approach to be applied during the risk assessment process when apparent non-monotonicity is observed, also providing advice on specific elements to be considered to facilitate the assessment of NMDR in EFSA risk assessments. The proposed approach was applied to two case studies, Bisphenol A and bis(2-ethylhexyl phthalate (DEHP) and these evaluations are reported in dedicated annexes. Considering the potential impact of NMDRs in regulatory risk assessment, the Scientific Committee recommends a concerted international effort on developing internationally agreed guidance and harmonised frameworks for identifying and addressing NMDRs in the risk assessment process.

Update of the risk assessment of hexabromocyclododecanes (HBCDDs) in food.

The European Commission asked EFSA to update its 2011 risk assessment on hexabromocyclododecanes (HBCDDs) in food. HBCDDs, predominantly mixtures of the stereoisomers α-, ß- and γ-HBCDD, were widely used additive flame retardants. Concern has been raised because of the occurrence of HBCDDs in the environment, food and in humans. Main targets for toxicity are neurodevelopment, the liver, thyroid hormone homeostasis and the reproductive and immune systems. The CONTAM Panel concluded that the neurodevelopmental effects on behaviour in mice can be considered the critical effects. Based on effects on spontaneous behaviour in mice, the Panel identified a lowest observed adverse effect level (LOAEL) of 0.9 mg/kg body weight (bw) as the Reference Point, corresponding to a body burden of 0.75 mg/kg bw. The chronic intake that would lead to the same body burden in humans was calculated to be 2.35 µg/kg bw per day. The derivation of a health-based guidance value (HBGV) was not considered appropriate. Instead, the margin of exposure (MOE) approach was applied to assess possible health concerns. Over 6,000 analytical results for HBCDDs in food were used to estimate the exposure across dietary surveys and age groups of the European population. The most important contributors to the chronic dietary LB exposure to HBCDDs were fish meat, eggs, livestock meat and poultry. The CONTAM Panel concluded that the resulting MOE values support the conclusion that current dietary exposure to HBCDDs across European countries does not raise a health concern. An exception is breastfed infants with high milk consumption, for which the lowest MOE values may raise a health concern.

Statement on the derivation of Health-Based Guidance Values (HBGVs) for regulated products that are also nutrients.

This Statement presents a proposal for harmonising the establishment of Health-Based Guidance Values (HBGVs) for regulated products that are also nutrients. This is a recurrent issue for food additives and pesticides, and may occasionally occur for other regulated products. The Statement describes the specific considerations that should be followed for establishing the HBGVs during the assessment of a regulated product that is also a nutrient. It also addresses the elements to be considered in the intake assessment; and proposes a decision tree for ensuring a harmonised process for the risk characterisation of regulated products that are also nutrients. The Scientific Committee recommends the involvement of the relevant EFSA Panels and units, in order to ensure an integrated and harmonised approach for the hazard and risk characterisation of regulated products that are also nutrients, considering the intake from all relevant sources.

Guidance Document on Scientific criteria for grouping chemicals into assessment groups for human risk assessment of combined exposure to multiple chemicals.

This guidance document provides harmonised and flexible methodologies to apply scientific criteria and prioritisation methods for grouping chemicals into assessment groups for human risk assessment of combined exposure to multiple chemicals. In the context of EFSA's risk assessments, the problem formulation step defines the chemicals to be assessed in the terms of reference usually through regulatory criteria often set by risk managers based on legislative requirements. Scientific criteria such as hazard-driven criteria can be used to group these chemicals into assessment groups. In this guidance document, a framework is proposed to apply hazard-driven criteria for grouping of chemicals into assessment groups using mechanistic information on toxicity as the gold standard where available (i.e. common mode of action or adverse outcome pathway) through a structured weight of evidence approach. However, when such mechanistic data are not available, grouping may be performed using a common adverse outcome. Toxicokinetic data can also be useful for grouping, particularly when metabolism information is available for a class of compounds and common toxicologically relevant metabolites are shared. In addition, prioritisation methods provide means to identify low-priority chemicals and reduce the number of chemicals in an assessment group. Prioritisation methods include combined risk-based approaches, risk-based approaches for single chemicals and exposure-driven approaches. Case studies have been provided to illustrate the practical application of hazard-driven criteria and the use of prioritisation methods for grouping of chemicals in assessment groups. Recommendations for future work are discussed.

A systems-based approach to the environmental risk assessment of multiple stressors in honey bees.

The European Parliament requested EFSA to develop a holistic risk assessment of multiple stressors in honey bees. To this end, a systems-based approach that is composed of two core components: a monitoring system and a modelling system are put forward with honey bees taken as a showcase. Key developments in the current scientific opinion (including systematic data collection from sentinel beehives and an agent-based simulation) have the potential to substantially contribute to future development of environmental risk assessments of multiple stressors at larger spatial and temporal scales. For the monitoring, sentinel hives would be placed across representative climatic zones and landscapes in the EU and connected to a platform for data storage and analysis. Data on bee health status, chemical residues and the immediate or broader landscape around the hives would be collected in a harmonised and standardised manner, and would be used to inform stakeholders, and the modelling system, ApisRAM, which simulates as accurately as possible a honey bee colony. ApisRAM would be calibrated and continuously updated with incoming monitoring data and emerging scientific knowledge from research. It will be a supportive tool for beekeeping, farming, research, risk assessment and risk management, and it will benefit the wider society. A societal outlook on the proposed approach is included and this was conducted with targeted social science research with 64 beekeepers from eight EU Member States and with members of the EU Bee Partnership. Gaps and opportunities are identified to further implement the approach. Conclusions and recommendations are made on a way forward, both for the application of the approach and its use in a broader context.

Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles.

Following a mandate from the European Commission, EFSA has developed a Guidance on Technical Requirements (Guidance on Particle-TR), defining the criteria for assessing the presence of a fraction of small particles, and setting out information requirements for applications in the regulated food and feed product areas (e.g. novel food, food/feed additives, food contact materials and pesticides). These requirements apply to particles requiring specific assessment at the nanoscale in conventional materials that do not meet the definition of engineered nanomaterial as set out in the Novel Food Regulation (EU) 2015/2283. The guidance outlines appraisal criteria grouped in three sections, to confirm whether or not the conventional risk assessment should be complemented with nanospecific considerations. The first group addresses solubility and dissolution rate as key physicochemical properties to assess whether consumers will be exposed to particles. The second group establishes the information requirements for assessing whether the conventional material contains a fraction or consists of small particles, and its characterisation. The third group describes the information to be presented for existing safety studies to demonstrate that the fraction of small particles, including particles at the nanoscale, has been properly evaluated. In addition, in order to guide the appraisal of existing safety studies, recommendations for closing the data gaps while minimising the need for conducting new animal studies are provided. This Guidance on Particle-TR complements the Guidance on risk assessment of nanomaterials to be applied in the food and feed chain, human and animal health updated by the EFSA Scientific Committee as co-published with this Guidance. Applicants are advised to consult both guidance documents before conducting new studies.

Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health.

The EFSA has updated the Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain, human and animal health. It covers the application areas within EFSA's remit, including novel foods, food contact materials, food/feed additives and pesticides. The updated guidance, now Scientific Committee Guidance on nano risk assessment (SC Guidance on Nano-RA), has taken account of relevant scientific studies that provide insights to physico-chemical properties, exposure assessment and hazard characterisation of nanomaterials and areas of applicability. Together with the accompanying Guidance on Technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (Guidance on Particle-TR), the SC Guidance on Nano-RA specifically elaborates on physico-chemical characterisation, key parameters that should be measured, methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. The SC Guidance on Nano-RA also details aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vitro/in vivo toxicological studies are discussed and a tiered framework for toxicological testing is outlined. Furthermore, in vitro degradation, toxicokinetics, genotoxicity, local and systemic toxicity as well as general issues relating to testing of nanomaterials are described. Depending on the initial tier results, additional studies may be needed to investigate reproductive and developmental toxicity, chronic toxicity and carcinogenicity, immunotoxicity and 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 or mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis.

Guidance on aneugenicity assessment.

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health-based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment.

Safety assessment of titanium dioxide (E171) as a food additive.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO2 nanoparticles (NPs) and data from an extended one-generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO2 NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO2 particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO2 NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO2 NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO2 particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico-chemical properties of TiO2 particles and the outcome of either in vitro or in vivo genotoxicity assays. A concern for genotoxicity of TiO2 particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO2 particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut-off value for TiO2 particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO2 NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.