Abstract Following the submission of dossier GMFF‐2023‐21253 under Regulation (EC) No 1829/2003 from Bayer CropScience LP, the Panel on Genetically Modified Organisms of the European Food Safety Authority was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the stearidonic acid producing genetically modified soybean MON 87769, for food and feed uses, excluding cultivation within the European Union. The data received in the context of this renewal application contained post‐market environmental monitoring reports, post‐market monitoring reports, an evaluation of the literature retrieved by a scoping review, a search for additional studies performed by or on behalf of the applicant and updated bioinformatics analyses. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in soybean MON 87769 considered for renewal is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in renewal dossier GMFF‐2023‐21253 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean MON 87769.

After adoption of the Novel Foods Regulation in the European Union, insects are gaining recognition as sustainable protein sources and potential novel allergens. We developed and partially validated a targeted immunoaffinity tandem mass spectrometric assay originally optimized for allergen-focused detection of insect proteins. Its high analytical sensitivity also supports applications in species authentication and quantitative assessment. The assay targets five edible insect species Alphitobius diaperinus, Tenebrio molitor, Locusta migratoria, Gryllodes sigillatus, Hermetia illucens, and a pan-insect marker based on a conserved tropomyosin peptide. The method demonstrated high accuracy (80% - 120%), precision (coefficient of variation <20%), selectivity, and sensitivity (limits of quantification: 6.17 fmol to 1500 fmol (>30-4500 ppm insect). Matrix and carryover effects observed for some analytes were mitigated by introduction of an additional wash step. Intra- and inter-assay reproducibility and calibration parallelism were confirmed for all analytes. The method was tested on commercial insectbased products and spiked model foods at allergen-relevant levels. Quantitative performance of the assays to determine the insect content in food varied by matrix. The insect allergens were detectable by the pan-insect marker tropomyosin down to 30 ppm and two species-specific markers down to 5 ppm but not consistently across all species-specific analytes. These results demonstrate the capability to detect insects at reasonable levels that might raise allergenic reactions. This assay offers a sensitive, specific approach for detecting and quantifying insect proteins in food.

Pregnane X receptor (PXR) is a nuclear receptor acting as a master xenobiotic receptor for many exogenous chemicals. Activation of PXR has been linked to liver growth in mice, usually via interaction with Yes-associated protein (YAP), but this adaptive response has not been observed in humans. We investigated the effect of human PXR agonist, rifampicin, on liver size in a controlled clinical trial in healthy volunteers. Moreover, we developed a novel protocol to reveal mechanisms of PXR-mediated liver growth in mice. One-week rifampicin treatment caused a 2.7

Genetically modified soybean MON 94313 was developed to confer tolerance to dicamba, glufosinate, 2,4-D and mesotrione-based herbicides. These properties were achieved by introducing the dmo, pat, ft_t.1 and tdo expression cassettes. The molecular characterisation data and bioinformatics analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between soybean MON 94313 and its conventional counterpart need further assessment, except for methionine and Gly m Bd 28K, which underwent additional evaluation and were found to not raise any safety or nutritional concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the DMO, PAT, FT_T.1 and TDO proteins as expressed in soybean MON 94313 and finds no evidence that the genetic modification would change the overall safety of soybean MON 94313. In the context of this application, the consumption of food and feed from soybean MON 94313 does not represent a nutritional concern in humans and animals, and no post-market monitoring of food/feed is considered necessary. In the case of release of processed soybean MON 94313 material, or accidental spillage of viable GM soybean seeds into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean MON 94313. The GMO Panel concludes that soybean MON 94313 is as safe as its conventional counterpart and the tested non-GM soybean varieties with respect to potential effects on human and animal health and the environment.

Following a mandate from EFSA, the EFSA Panel on Genetically Modified Organisms (GMO Panel) addressed the food and feed risk assessment of genetically modified plants containing stacked transformation events ('stacks'). The GMO Panel examined the experience gained in the assessment of stacks in the last 20 years. Based on this, the GMO Panel concluded that under specific conditions, field trials for comparative analysis may not always be required for the assessment of stacks and a derogation may be considered. The conditions are: all the events combined in the stack should express traits through newly expressed proteins (NEPs) and these should not be metabolic modifiers. Under the same conditions and in the absence of interactions affecting NEP expression levels, a derogation from current requirements for dietary exposure assessment may also be considered. The GMO Panel also proposes improvements for applicants regarding the presentation of molecular characterisation data for stacks.

Standardized and reproducible cell models are key to replace animal testing in toxicology. The composition of culture medium is a major, yet frequently undercontrolled, determinant of cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic metabolism and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings show that medium effects must be interpreted in the context of cell line identity and indicate that HepaRG cells cultured in CDM provide a more PHH-like transcriptomic background for in vitro studies of xenobiotic metabolism and hepatotoxicity-related readouts than HuH7 cells.

Abstract The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro . For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

Abstract Genetically modified soybean GMB151 × DAS‐44406‐6 was developed by crossing to combine two single events: GMB151 and DAS‐44406‐6. The two‐event stack soybean expresses the Cry14Ab‐1, HPPD‐4, PAT, AAD‐12 and 2mEPSPS proteins to confer herbicide tolerance and resistance to plant parasitic nematodes. The GMO Panel previously assessed the two single soybean events and did not identify safety concerns. No new data on the single soybean events were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single soybean events and of the newly expressed proteins in the two‐event stack soybean does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that two‐event stack soybean, as described in this application, is as safe as the comparator and non‐GM reference soybean varieties tested and no post‐market monitoring of food/feed is considered necessary. In the case of accidental release of GM two‐event stack soybean material into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean GMB151 × DAS‐44406‐6. Post‐market monitoring of food/feed is not considered necessary. The GMO Panel concludes that two‐event stack soybean is as safe as its comparator and the tested non‐GM reference varieties with respect to potential effects on human and animal health and the environment.

Enzymes from the cytochrome P450 (CYP) superfamily, especially from families CYP1, CYP2, and CYP3, play a decisive role in phase I of drug and xenobiotic metabolism in mammalian organisms. The enzymes are responsible for metabolic conversion and detoxification of a plethora of foreign molecules. Metabolic conversion of pro-carcinogenic compounds links CYP enzyme activities to cancer development, while in addition oncogenic pathways have been shown to regulate the expression of CYP genes, together with the well-known regulation by nuclear receptors acting as ligand-activated transcription factors triggered by exposure to xenobiotics. Specifically, the Wnt/β-catenin signaling pathway is among the recently established transcriptional regulators of CYP enzymes. β-Catenin is well-known as a key player in organism development and, when aberrantly activated, a major oncogenic driver of carcinogenesis. While the latter phenomena are rather well-described, new evidence suggests that CYP enzymes themselves may, under certain conditions, also affect the activity of the β-catenin pathway and thereby could impact on carcinogenesis in a way different from toxifying or detoxifying foreign compounds. This review focuses on the currently available knowledge about the regulation of β-catenin-dependent signaling by CYP enzymes. The synopsis of data reveals the possibility of a previously undervalued role of CYPs in the regulation of Wnt/β-catenin signaling, and possible molecular mechanisms are highlighted.

Following the submission of dossier GMFF-2025-33580 under Regulation (EC) No 1829/2003 from Syngenta Crop Protection AG, the Panel on Genetically Modified Organisms of the EFSA was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide-tolerant genetically modified soybean FG72, for food and feed uses, excluding cultivation within the European Union. The data received in the context of this renewal application contained post-market environmental monitoring reports, an evaluation of the literature retrieved by a scoping review, additional studies performed by or on behalf of the applicant and updated bioinformatics analyses. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Assuming that the DNA sequence of the event in soybean FG72 considered for renewal is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in renewal dossier GMFF-2025-33580 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean FG72.

Abstract Genetically modified RF3 Canola Quality (CQ) Brassica juncea was developed to confer tolerance to glufosinate‐based herbicides and to restore fertility in Barnase‐expressing plants. The RF3 event was originally developed in Brassica napus and has previously been assessed by EFSA in application EFSA‐GMO‐RX‐MS8‐RF3. The GMO Panel assessment of the molecular characterisation data and bioinformatics analyses did not raise issues that would require specific food and feed safety assessments. None of the identified differences in the agronomic, phenotypic and compositional characteristics tested between RF3 CQ B. juncea and its comparator need further assessment, except for yield, crude fat, aspartic acid and behenic acid (C22:0), which underwent additional evaluation and were found to not raise any environmental impact and safety or nutritional concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the PAT/bar and Barstar proteins as expressed in RF3 CQ B. juncea and finds no evidence that the genetic modification would change the overall allergenicity of RF3 CQ B. juncea. In the context of this application, the consumption of food and feed from RF3 CQ B. juncea does not represent a nutritional concern in humans and animals. The GMO Panel concludes that RF3 CQ B. juncea is as safe as the comparator and CQ non‐GM B. juncea varieties tested, and no post‐market monitoring of food and feed is considered necessary. In the case of release of RF3 CQ B. juncea material, including viable seeds, into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of RF3 CQ B. juncea. The GMO Panel concludes that RF3 CQ B. juncea is as safe as its comparator and the tested CQ non‐GM B. juncea varieties with respect to potential effects on human and animal health and the environment.

Due to their high persistence and adverse health effects in humans the use of a number of per- and polyfluoroalkyl substances (PFAS) has been restricted. As a consequence, novel PFAS are increasingly being introduced for industrial applications, although toxicological data are still limited or lacking for many of these compounds. This study examined the molecular mechanisms of action of novel PFAS with a focus on mono- and polyether PFAS with linear or branched structures and either carboxylic acid or sulfonic acid functional groups. Differentiated HepaRG cells, a model of human hepatocytes, were exposed for 24 h to different PFAS congeners at three non-cytotoxic concentrations each. Total RNA was isolated and subjected to whole transcriptome analysis. The study provides transcriptomic data for in total 33 PFAS congeners, for 13 of them for the first time. For most PFAS, the number of differentially expressed genes (DEG) increased in a concentration-dependent manner, whereas five PFAS induced only minor transcriptional changes even at the highest test concentration. Ingenuity Pathway Analysis (IPA) revealed broadly comparable transcriptional responses across all 33 PFAS, indicating convergent molecular effects in HepaRG cells despite marked structural differences among the PFAS congeners. The tested PFAS consistently activated canonical pathways related to fatty acid and lipid metabolism, mainly regulated by the nuclear receptor PPARα, and also affected pathways related to xenobiotic metabolism, partially linked to PXR and CAR signaling. In addition, several PFAS inhibited cholesterol and bile acid biosynthesis pathways. IPA further predicted effects on hepatocyte-relevant upstream regulators such as HNF4A, HNF1A, and FOXA2. Finally, IPA tox-function analysis indicated associations between PFAS-induced transcriptional changes and liver diseases related to cholestasis.

Zinc (Zn) deficiency affects approximately 1 billion people worldwide with severe consequences for their health, including increased intestinal infections, inflammation, and diarrhea. Accordingly, the intestinal defense barrier is compromised, leading to epithelial destruction and alteration of mucus. However, the processes and the extent to which Zn deficiency affects mucin synthesis in intestinal goblet cells (GCs) remain poorly understood. To this end, we investigated the impact of Zn deficiency on mucin expression and glycosylation in the human GC model HT-29-MTX. Zn deprivation altered the GC transcriptome, affecting genes involved in Zn transport, mucin synthesis and glycosylation. Accordingly, mucus composition was changed in Zn-deficient GCs, significantly increasing MUC2 and MUC17 on the mRNA and protein level. Several Zn transporters, mostly those associated with the early secretory pathway (ESP), were dysregulated, indicating an adaptive response of cellular Zn homeostasis. Additionally, free Zn was markedly reduced in the ESP, a critical location for glycosylation. Zn deficit substantially changed mucin glycosylation, characterized by an increase in sialylation and a strong decrease in complex N-glycans. All these changes involved widespread dysregulation of glycosyltransferase expression, including an increase in COSMC, a Zn-binding chaperone essential for the core 1 O-glycan formation. Collectively, our in vitro findings demonstrate that Zn is a critical regulator of mucin production and glycosylation in GCs. Zn deficiency might weaken the protective and functional qualities of intestinal mucus, increasing the risk of infections and potentially disrupting host-microbiome interactions.

Per- and polyfluoroalkyl substances (PFAS) are a large family of persistent environmental contaminants. Some PFAS are known to bioaccumulate, are frequently detected in human serum, and are associated with several adverse effects on the immune system, the endocrine system, and the liver. PFAS-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα), which plays a key role in lipid and cholesterol homeostasis, is suggested to be an important molecular initiating event triggering PFAS toxicity. The aim of this study was to evaluate the PPARα activation potential of a diverse panel of 34 PFAS congeners, consisting of both legacy and novel compounds, using a PPARα-dependent transactivation assay in transfected HEK293T cells. The resulting concentration-response data were analyzed using benchmark dose (BMD) modelling to quantify PPARα activation potency. A key finding was that PFAS with a sulfonic acid group showed a lower potency compared to those with a carboxylic group. The most potent activators belonged to the perfluoroalkylether carboxylic acid (PFECA) subgroup. Computational descriptors were generated to characterize each congener, and quantitative structure-activity relationship (QSAR) modelling was applied to relate molecular features to in vitro PPARα activation potency, as expressed by BMD estimates. For prioritization purposes in the context of PFAS hazard characterization, the QSAR model was used to screen about 10,000 PFAS congeners. Of these, roughly 10

The Adverse Outcome Pathway (AOP) framework is a pivotal tool for organizing mechanistic knowledge and linking it to adverse outcomes of regulatory significance. However, the integration of test method information, particularly New Approach Methods (NAMs), within the central repository for AOP knowledge, (the AOP-Wiki), has been suboptimal, limiting the framework's utility for regulatory decision-making. The Methods2AOP collaboration, comprised of various international stakeholders, was established to address this gap and enhance the role of test methods within the AOP framework. This paper reviews their work emphasizing the importance of linking detailed test method information and conceptually proposes how it may be included in the AOP knowledgebase in alignment with existing assay documentation standards and governance frameworks. The Methods2AOP collaboration proposes using ontologies to standardize and structure information, thereby facilitating interoperability, enabling reusability, and establishing clear connections between test methods and Key Events (KEs). A conceptual model is presented to demonstrate qualitative similarities between concepts in key event components and structured methods information. The implementation of Methods2AOP recommendations would increase the clarity and transparency of method descriptions, which could support regulatory acceptance and a wider adoption of NAMs. The broad community of stakeholders impacted by this work stands to benefit from the Methods2AOP recommendations through enhanced regulatory decisions, increased visibility and scientific impact, new market opportunities, and the accelerated adoption of NAMs in regulatory affairs. In summary, the Methods2AOP collaboration presents a comprehensive effort to formally standardize the integration of test methods into the AOP framework, thereby fostering a more robust, and transparent system that aligns with the goals of the scientific and regulatory communities.

Abstract In a previous scientific opinion on application EFSA‐GMO‐BE‐2016‐138, the EFSA Panel on Genetically Modified Organisms (GMO Panel) could not conclude on the comparative analysis and on the food and feed safety assessment of genetically modified (GM) oilseed rape MS11 because of the lack of an appropriate compositional data set. Following a request from the European Commission, the GMO Panel assessed additional information on the composition of oilseed rape MS11. The GMO Panel concluded that the information can be used to complement the original assessment. None of the differences identified in seed composition between oilseed rape MS11 and the conventional counterpart needed further assessment regarding food and feed safety. The GMO Panel found no evidence that the genetic modification impacts the overall safety of oilseed rape MS11. Τhe consumption of oilseed rape MS11 does not represent any nutritional concern. Νo post‐market monitoring of food and feed is considered necessary. Considering the assessment reported in the original scientific opinion and in this statement, the GMO Panel concludes that oilseed rape MS11 is as safe as its conventional counterpart and the tested non‐GM oilseed rape reference varieties with respect to potential effects on human and animal health and the environment.

Abstract Genetically modified soybean DAS‐44406‐6 × FG72 was developed by conventional crossing to combine two single events: DAS‐44406‐6 and FG72. The soybean DAS‐44406‐6 × FG72 expresses the AAD‐12, 2mEPSPS, PAT and HPPD W336 proteins to confer herbicide tolerance to 2,4‐D‐, glyphosate‐, glufosinate‐containing and HPPD‐inhibiting herbicide products, respectively. The GMO Panel previously assessed the two single soybean events and did not identify safety concerns. No new data on the single soybean events were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single soybean events and of the newly expressed proteins in the soybean DAS‐44406‐6 × FG72 does not give rise to food and feed safety or nutritional concerns. The GMO Panel concludes that the DAS‐44406‐6 × FG72 stack soybean, as described in this application, is as safe as the non‐GM comparator and non‐GM reference varieties tested, and no post‐market monitoring of food and feed is considered necessary. In the case of release of processed soybean DAS‐44406‐6 × FG72 or accidental spillage of viable GM soybean seeds into the environment, this would not raise environmental safety concerns. The post market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DAS‐44406‐6 × FG72. The GMO Panel concludes that soybean DAS‐44406‐6 × FG72 is as safe as its non‐GM comparator and the tested non‐GM reference varieties with respect to potential effects on human and animal health and the environment.

Following the submission of application GMFF-2023-21236 under Regulation (EC) No 1829/2003 from Bayer CropScience LP, the Panel on Genetically Modified Organisms of the European Food Safety Authority was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide-tolerant, increased oleic acid genetically modified soybean MON 87705, for food and feed uses, excluding cultivation within the European Union. The data received in the context of this renewal application contained post-market environmental monitoring reports, post-market monitoring reports, an evaluation of the literature retrieved by a scoping review, a search for additional studies performed by or on behalf of the applicant and updated bioinformatics analyses. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in soybean MON 87705 considered for renewal is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in renewal application GMFF-2023-21236 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on soybean MON 87705.

The Adverse Outcome Pathway (AOP) framework is a pivotal tool for organizing mechanistic knowledge and linking it to adverse outcomes of regulatory significance. However, the integration of test method information, particularly New Approach Methods (NAMs), within the central repository for AOP knowledge, (the AOP-Wiki), has been suboptimal, limiting the framework’s utility for regulatory decision-making. The Methods2AOP collaboration, comprised of various international stakeholders, was established to address this gap and enhance the role of test methods within the AOP framework. This paper reviews their work emphasizing the importance of linking detailed test method information and conceptually proposes how it may be included in the AOP knowledgebase in alignment with existing assay documentation standards and governance frameworks. The Methods2AOP collaboration proposes using ontologies to standardize and structure information, thereby facilitating interoperability, enabling reusability, and establishing clear connections between test methods and Key Events (KEs). A conceptual model is presented to demonstrate qualitative similarities between concepts in key event components and structured methods information. The implementation of Methods2AOP recommendations would increase the clarity and transparency of method descriptions, which could support regulatory acceptance and a wider adoption of NAMs. The broad community of stakeholders impacted by this work stands to benefit from the Methods2AOP recommendations through enhanced regulatory decisions, increased visibility and scientific impact, new market opportunities, and the accelerated adoption of NAMs in regulatory affairs. In summary, the Methods2AOP collaboration presents a comprehensive effort to formally standardize the integration of test methods into the AOP framework, thereby fostering a more robust, and transparent system that aligns with the goals of the scientific and regulatory communities.

The risk assessment (RA) requirements for genetically modified plants (GMPs) are defined in Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA GMO Panel, 2011). When a GMP is developed to silence transcripts by RNA interference (RNAi), some specific additional analysis needs to be provided by the applicant. This guidance describes the requirements and recommendations for the GMP applications submitted to EFSA. It covers the molecular characterisation, focusing on bioinformatic analysis and confirmation of the trait, as well as the food and feed safety and dietary exposure assessment of RNAi-based GMPs. This document replaces the GMO panel strategy for the risk assessment of RNAi off targets in plants, described in Annex II to the minutes of the 118th Plenary meeting of the Scientific Panel on GMO and takes into account the current knowledge on the mechanisms of RNAi in plants.