The European Food Safety Authority (EFSA) carried out a public consultation to receive input from interested parties on in vitro random mutagenesis techniques. This draft scientific opinion was prepared by the GMO Panel, supported by the Working Group on Molecular Characterization. The draft opinion was endorsed by the EFSA GMO Panel for public consultation on the 5th May 2021. The written public consultation was open from 19 May 2021 until 30 June 2021. EFSA received comments from 16 different interested parties. EFSA and its GMO Panel wish to thank all stakeholders for their contributions to this work. The present report contains the comments received and details how they have been considered for finalisation of the opinion. The final opinion was adopted at the GMO Panel Plenary meeting on the 29th September 2021 and will be published in the EFSA Journal. © European Food Safety Authority,

Dietary exposure assessment is one of the key parts of the risk assessment process. Ideally, it can be assessed by combining data on the concentration in all food products with data on their consumption. Quantitative information on the level of a chemical in food can be obtained through analytic determinations and, in the case of intentionally added substances, from the producers. Dietary surveys are the primary source of food consumption data. The assessment of dietary exposure requires some degree of modeling, and the method chosen usually depends on the degree of accuracy required and the availability of data.

In the most recent risk assessment for Bisphenol A for the first time a multi-route aggregate exposure assessment was conducted by the European Food Safety Authority. This assessment includes exposure via dietary sources, and also contributions of the most important non-dietary sources. Both average and high aggregate exposure were calculated by source-to-dose modeling (forward calculation) for different age groups and compared with estimates based on urinary biomonitoring data (backward calculation). The aggregate exposure estimates obtained by forward and backward modeling are in the same order of magnitude, with forward modeling yielding higher estimates associated with larger uncertainty. Yet, only forward modeling can indicate the relative contribution of different sources. Dietary exposure, especially via canned food, appears to be the most important exposure source and, based on the central aggregate exposure estimates, contributes around 90% to internal exposure to total (conjugated plus unconjugated) BPA. Dermal exposure via thermal paper and to a lesser extent via cosmetic products may contribute around 10% for some age groups. The uncertainty around these estimates is considerable, but since after dermal absorption a first-pass metabolism of BPA by conjugation is lacking, dermal sources may be of equal or even higher toxicological relevance than dietary sources.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91−266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91−266–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91−266–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.

Concern has been raised about the number of juvenile animal studies (JAS) being conducted in the European Union (EU) and the number of species required. The question of whether there has been an increase in the number of JAS requested by European Medicines Agency (EMA) and the question of single or multiple species studies are the focus of this manuscript. All source data on Pediatric Investigation Plans (PIP) were obtained from EMA pediatrics website and evaluated by year as obtained and adjusted to the date of the original PIP agreement rather than the newly assigned date of the PIP modification. The analysis of the data originally suggested an increase in the number of juvenile animal studies per year post 2010. However, a thorough examination of the adjusted PIP data from the EMA from December 2007 through 2013 support that the number of JAS being included in PIPs per year is not increasing but is in fact fairly stable at 15–30% per year. The data also, as expected, support that the JAS are mostly proposed with a single species and that species is predominately the rat and that multi-species JAS for an agreed PIP are infrequent.

In 2009 competent organisations in the European Union provided the European Food Safety Authority (EFSA) with data from the most recent national dietary survey at the level of individuals' consumption. Twenty different Member States provided EFSA with data from 22 different national dietary surveys, with consumption figures for adults and, when available, for children. Member States' dietary data were assembled into the EFSA Comprehensive European Food Consumption Database. In this paper an overview of the methodologies and protocols employed in the different national dietary surveys is provided. Specifically, details about dietary assessment methods, interview administration, sampling design, portion size estimation, dietary software, evaluation of under-reporting and non-dietary information collected are described. This information is crucial to evaluate the level of accuracy of food consumption data and to anticipate and acknowledge the utmost important sources of heterogeneity of national databases included in the Comprehensive Database. The Comprehensive Database constitutes a unique resource for the estimation of consumption figures across the European Union and represents a useful tool to assess dietary exposure to hazardous substances and nutrient intake in Europe. Nevertheless, the many substantial methodological differences that characterise the Comprehensive Database are acknowledged and critically discussed.

BACKGROUND AND AIMS:Italian National Food Consumption Survey, INRAN-SCAI 2005-06, is the third national food consumption survey performed in Italy. This study describes energy and nutrient intakes in Italy. METHODS AND RESULTS:A national cross-sectional food consumption survey was conducted using consecutive 3-day food records between October 2005 and December 2006. A sample of 3323 males and females aged 0.1-97.7 years living in private households was investigated. Individual food records were converted into energy and nutrient intakes with the use of recently updated national food composition databases. For each subject, intakes of energy and of 27 nutrients were calculated, including six minerals (i.e., iron, calcium, phosphorus, magnesium, potassium and zinc) and 10 vitamins (i.e., thiamine, riboflavin, vitamin C, vitamin B₆, retinol, β-carotene, vitamin A as retinol equivalents (REs), vitamin E, vitamin D and vitamin B₁₂. On average, 36% of calories appeared to derive from fat (11% from saturated fatty acids) and 45% from available carbohydrates (15% from soluble carbohydrates). CONCLUSIONS:The results of the INRAN-SCAI 2005-06 survey in terms of nutrient intakes provide an important piece of information for nutrition surveillance of the population and may also be used to identify priorities for further research.

Within the European project called EXPOCHI (Individual Food Consumption Data and Exposure Assessment Studies for Children), 14 different European individual food consumption databases of children were used to conduct harmonised dietary exposure assessments for lead, chromium, selenium and food colours. For this, two food categorisation systems were developed to classify the food consumption data in such a way that these could be linked to occurrence data of the considered compounds. One system served for the exposure calculations of lead, chromium and selenium. The second system was developed for the exposure assessment of food colours. The food categories defined for the lead, chromium and selenium exposure calculations were used as a basis for the food colour categorisation, with adaptations to optimise the linkage with the food colour occurrence data. With this work, an initial impetus was given to make user-friendly food categorisation systems for contaminants and food colours applicable on a pan-European level. However, a set of difficulties were encountered in creating a common food categorisation system for 14 individual food consumption databases that differ in the type and number of foods coded and in level of detail provided about the consumed foods. The work done and the problems encountered in this project can be of interest for future projects in which food consumption data will be collected on a pan-European level and used for common exposure assessments.

Exposure assessment is one of the key parts of the risk assessment process. This task is crucial when evaluating substances for which only intake of toxicologically-important amounts can lead to adverse health effects. Ideally, dietary exposure to hazardous substances can be assessed by combining data on concentration in all food products with data on their consumption. However, it is considered to be neither cost-effective nor necessary to collect detailed data for every substance, and a stepwise procedure is commonly used to focus resources on the most important issues. Screening methods, designed to look for 'worst case' situations, are first used to target chemicals that might be of health concern for the general population or for certain at-risk groups. The quality of the dietary exposure assessments not only depends on the quality of the data collected, but also on the integration tools used for initial screening or for the eventual more precise estimations. A particular challenge is the evaluation of food allergens and components causing other forms of intolerances, since no reliable data seem to be currently available on the type of exposure (amounts and duration) required to induce a food allergy. A different approach from that used for dietary exposure to other hazardous substances has to be adopted. However, the methodologies (such as those used to collect food consumption data) and databases (in particular, information about food labels) developed in such a context could be useful to investigate the exposure conditions leading to the development of food allergies.

The procedure for the safety evaluation of flavourings adopted by the European Commission in order to establish a positive list of these substances is a stepwise approach which was developed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and amended by the Scientific Committee on Food. Within this procedure, a per capita amount based on industrial poundage data of flavourings, is calculated to estimate the dietary intake by means of the maximised survey-derived daily intake (MSDI) method. This paper reviews the MSDI method in order to check if it can provide conservative intake estimates as needed at the first steps of a stepwise procedure. Scientific papers and opinions dealing with the MSDI method were reviewed. Concentration levels reported by the industry were compared with estimates obtained with the MSDI method. It appeared that, in some cases, these estimates could be orders of magnitude (up to 5) lower than those calculated considering concentration levels provided by the industry and regular consumption of flavoured foods and beverages. A critical review of two studies which had been used to support the statement that MSDI is a conservative method for assessing exposure to flavourings among high consumers was performed. Special attention was given to the factors that affect exposure at high percentiles, such as brand loyalty and portion sizes. It is concluded that these studies may not be suitable to validate the MSDI method used to assess intakes of flavours by European consumers due to shortcomings in the assumptions made and in the data used. Exposure assessment is an essential component of risk assessment. The present paper suggests that the MSDI method is not sufficiently conservative. There is therefore a clear need for either using an alternative method to estimate exposure to flavourings in the procedure or for limiting intakes to the levels at which the safety was assessed.

In a previous study, Italian female teenagers regular consumers of sugar free soft drinks and table-top sweeteners were suggested to have a higher intake of intense sweeteners than other teenagers. A food frequency questionnaire designed to identify adolescents who were high consumers of these food products was filled in by a randomly extracted sample of teenagers (n=3982) living in the District of Rome (Italy) in year 2000. A consumer survey was then carried out in a randomly extracted sub-sample of males and females and in all females who reported high consumption of sugar-free soft drinks and/or table-top sweeteners. A total of 362 subjects participated in a detailed food survey by recording, at brand level, all foods and beverages ingested over 12 days. For each sugar-free product, producers provided the concentration of intense sweeteners (saccharin, aspartame, acesulfame K and cyclamate). No intake in excess of the Acceptable Daily Intake (ADI) was observed. Also medicines and supplements were taken into account and these did not result in a large impact on chronic exposure to intense sweeteners. The intake levels did not exceed the ADI even under a worst case scenario which was performed to take into consideration a hypothetical future substitution of all regular food products with their sugar-free version. It can be concluded that, with the observed current consumption patterns and occurrence levels, the risk of an excessive intake of intense sweeteners by Italian teenagers is extremely low.

The present study was designed to estimate the average food and nutrient intake in a sample of Italian students living in the District of Rome. In the school year 1999–2000 all students belonging to the first, second or third year of nine randomly selected secondary public schools were contacted for filling in a food frequency questionnaire (3982 subjects). The next year, 233 of them (125 males and 108 females), randomly extracted, participated in a detailed food survey. Food intake was assessed on the basis of 12 (four consecutive days in three different periods) 24-h dietary records. The average daily intakes of fruit (107±100 g/day), vegetables (186±74 g/day) and legumes (16±29 g/day) were lower than recommended. The percentage of energy intake from fat (39%) and that from saturated fats (12%) were high. On the other hand, the percentage of energy intake from carbohydrates was low (45%) even if that from sugar (14%) was rather high. The students’ diet was characterized by low intakes of fibre (16±6 g/day) and calcium (710±247 mg/day) in both males and females. Iron intakes were lower than 12 mg/day in a very high percentage of females (80%), suggesting that iron deficiency may be present in this population group. Results indicate that the diet of these students is both unbalanced in terms of macronutrients and deficient for some micronutrients, suggesting the need for intervention.

To get a more realistic view of exposure to food chemicals, risk managers are getting more interested in stochastic modelling as an alternative to deterministic approaches based on conservative assumptions. It allows to take into account all the available information in the concentration of the chemical present in foods and in food consumption patterns. Within the EC-funded "Montecarlo" project, a comprehensive set of mathematical algorithms was developed to take into account all the necessary components for stochastic modelling of a variety of food chemicals, nutrients and ingredients. An appropriate computer software is being developed. Since the concentration of food chemicals may vary among different brands of the same product, consumer behaviour with respect to brands may have an impact on exposure assessments. Numeric experiments were carried out on different ways of incorporating indicators of market share and brand loyalty in the mathematical algorithms developed within the stochastic model of exposure to intense sweeteners from sugar-free beverages. The 95th percentiles of intake were shown to vary according to the inclusion/exclusion of these indicators. The market share should be included in the model especially if the market is not equitably distributed between brands. If brand loyalty data are not available, the model may be run under theoretical scenarios.