# Statement on the toxicological properties and maximum residue levels of acetamiprid and its metabolites

Antonio Hernandez-jerez, Tamara Coja, Martin Paparella, Anna Price, Jerome Henri, Andreas Focks, Jochem Louisse, Andrea Terron, Marco Binaglia, Irene Munoz Guajardo, et al.

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## To cite this version:

Antonio Hernandez-jerez, Tamara Coja, Martin Paparella, Anna Price, Jerome Henri, et al. State-ment on the toxicological properties and maximum residue levels of acetamiprid and its metabolites. EFSA Journal, 2024, 22, pp.1-84. 10.2903/j.efsa.2024.8759. anses-04700110

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Submitted on 17 Sep 2024

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STATEMENT

# Statement on the toxicological properties and maximum residue levels of acetamiprid and its metabolites

EFSA (European Food Safety Authority) | Antonio Hernandez-Jerez | Tamara Coja |

Martin Paparella | Anna Price | Jerome Henri | Andreas Focks | Jochem Louisse |

Andrea Terron | Marco Binaglia | Irene Munoz Guajardo | Iris Mangas |

Irene Munoz Guajardo | Lucien Ferreira | Dimitra Kardassi | Chloe De Lentdecker |

Tunde Molnar | Giorgia Vianello

Correspondence:
pesticides Peerreview@efsa.europa.eu

## Abstract

Acetamiprid is a pesticide active substance with insecticidal action whose approval was renewed by Commission Implementing Regulation (EU) 2018/113. In January 2022, the EFSA PPR Panel published a statement following a request from the European Commission to advise on human health or the environment based on new scientific evidence presented by France during the decision-making phase. In July 2022, by means of a further mandate received from the European Commission, EFSA was requested to provide advice if new information and any other scientific evidence that has become available since the assessment conducted for the renewal in 2018 warrant re-evaluation of (i) toxicological parameters used for the risk assessment of acetamiprid during the renewal process, including toxicological endpoints; (ii) the residue definition for acetamiprid in products of plant origin; and (iii) the safety of existing maximum residue levels (MRLs). Meanwhile, the applicant of acetamiprid in the EU submitted new toxicology studies regarding the toxicological profile of the metabolite IM-2-1. Furthermore, the European Commission was made aware that several recent publications in scientific literature were made available after the literature searches conducted by EFSA. As the new data could affect the advice that EFSA was expected to deliver through the 2022 mandate, EFSA was further requested to consider this information by means of a revised mandate received in September 2023. As regards re-evaluation of point (i) in this statement, this was addressed by an EFSA Working Group integrating all the available evidence. The results of the weight of evidence indicated that there are major uncertainties in the body of evidence for the developmental neurotoxicity (DNT) properties of acetamiprid and further data are therefore needed to come to a more robust mechanistic understanding to enable appropriate hazard and risk assessment. In view of these uncertainties, the EFSA WG proposed to lower the acceptable daily intake (ADI) and acute reference dose (ARfD) from 0.025 to 0.005 mg/kg body weight (per day). A revised residue definition for risk assessment was proposed for leafy and fruit crops as sum of acetamiprid and N-desmethyl-acetamiprid (IM-2-1), expressed as acetamiprid. Regarding pulses/oilseeds, root crops and cereals, the new data received did not indicate a need to modify the existing residue definition for risk assessment, which therefore remains as parent acetamiprid. Regarding the residue definition for enforcement, the available data did not indicate a need to modify the existing definition because acetamiprid is still a sufficient marker of the residues in all crop groups. Considering the new health-based guidance values derived in the present statement, a risk for consumer has been identified for 38 MRLs currently in place in the

This is an open access article under the terms of the [Creative Commons Attribution-NoDerivs License](https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made.

© 2024 European Food Safety Authority. *EFSA Journal* published by Wiley-VCH GmbH on behalf of European Food Safety Authority.
EU Regulation. Consequently, EFSA recommended to lower the existing MRLs for 38 commodities based on the assessment of fall-back Good Agricultural Practices received within an ad hoc data call. Some fall-back MRLs proposals require further risk management considerations.

KEYWORDS

acetamiprid, developmental neurotoxicity, insecticides, maximum residue levels, metabolite IM-2-1, monitoring data
CONTENTS

<table><tr><td>Abstract</td><td>1</td></tr><tr><td>Summary</td><td>5</td></tr><tr><td>1. Introduction</td><td>7</td></tr><tr><td>1.1. Background and Terms of Reference as provided by the requestor</td><td>8</td></tr><tr><td>1.2. Interpretation of the Terms of Reference</td><td>9</td></tr><tr><td>1.2.1. Term of Reference 1 (human health)</td><td>9</td></tr><tr><td>1.2.2. Term of Reference 2a (data call, monitoring data)</td><td>10</td></tr><tr><td>1.2.3. Term of Reference 2b (residue definitions)</td><td>11</td></tr><tr><td>1.2.4. Term of Reference 3 (consumer risk assessment)</td><td>11</td></tr><tr><td>2. Human health</td><td>11</td></tr><tr><td>2.1. Hazard assessment acetamiprid</td><td>11</td></tr><tr><td>2.1.1. Data</td><td>12</td></tr><tr><td>2.1.2. Methodologies</td><td>12</td></tr><tr><td>2.1.3. Assessment for non-DNT endpoints</td><td>15</td></tr><tr><td>2.1.3.1. Data collection</td><td>15</td></tr><tr><td>2.1.3.2. Critical appraisal results</td><td>17</td></tr><tr><td>2.1.3.3. Uncertainty analysis and conclusions by the EFSA WG</td><td>21</td></tr><tr><td>2.1.4. Assessment for DNT: Integrated approaches to testing and assessment (IATA) for DNT hazard characterisation of acetamiprid active substance</td><td>21</td></tr><tr><td>2.2. Hazard assessment of acetamiprid metabolites</td><td>37</td></tr><tr><td>2.2.1. Data</td><td>37</td></tr><tr><td>2.2.2. Methodologies</td><td>38</td></tr><tr><td>2.2.2.1. EFSA systematic literature review for potential residue metabolites</td><td>38</td></tr><tr><td>2.2.2.2. IM-2-1 toxicity studies submitted by sole applicant</td><td>38</td></tr><tr><td>2.2.3. Assessment</td><td>38</td></tr><tr><td>2.2.3.1. EFSA systematic literature review for potential residue metabolites</td><td>38</td></tr><tr><td>2.2.3.2. EFSA assessment and conclusion on the toxicological properties of IM-2-1 residue metabolite</td><td>39</td></tr><tr><td>2.3. Human exposure estimation based on human biomonitoring data</td><td>40</td></tr><tr><td>2.3.1. Data</td><td>40</td></tr><tr><td>2.3.1.1. Human biomonitoring data</td><td>40</td></tr><tr><td>2.3.1.2. PBK modelling</td><td>41</td></tr><tr><td>2.3.2. Methods</td><td>41</td></tr><tr><td>2.3.2.1. Human biomonitoring data</td><td>41</td></tr><tr><td>2.3.2.2. PBK modelling</td><td>41</td></tr><tr><td>2.3.3. Assessment</td><td>41</td></tr><tr><td>2.3.3.1. Results appraisal of human studies</td><td>41</td></tr><tr><td>2.3.3.2. Results stepwise approach PBK modelling</td><td>42</td></tr><tr><td>3. Residues</td><td>45</td></tr><tr><td>3.1. Metabolic pattern of acetamiprid and related metabolites identified in pesticide residue monitoring data for unprocessed and processed food of plant origin</td><td>45</td></tr><tr><td>3.1.1. Data collection</td><td>45</td></tr><tr><td>3.1.2. Overview of the results in samples analysed for parent and metabolite IM-2-1</td><td>46</td></tr><tr><td>3.1.3. Relative amount of metabolite IM-2-1 compared to parent compound</td><td>48</td></tr><tr><td>3.2. Metabolic pattern identified in metabolism studies relevant for food products of plant origin</td><td>51</td></tr><tr><td>3.2.1. Primary crop metabolism overview</td><td>51</td></tr><tr><td>3.2.2. Metabolite IM-2-1 in fruit and leafy crops according to metabolism studies</td><td>52</td></tr><tr><td>3.2.3. Other available metabolism studies (rotational crops and hydrolysis studies)</td><td>53</td></tr></table>
<table><tbody><tr><td>3.3. Residue definitions in plant commodities and conversions factors</td><td>54</td></tr><tr><td>3.3.1. Residue definitions</td><td>54</td></tr><tr><td>3.3.2. Conversion factors</td><td>55</td></tr><tr><td>3.4. Consumer risk assessment and identification of MRLs posing risk to consumers</td><td>55</td></tr><tr><td>3.4.1. Consumer risk-assessment using the newly derived HBGVs and existing residue definition for risk assessment (scenario 1)</td><td>56</td></tr><tr><td>3.4.2. Consumer risk-assessment using the new endpoints derived in the present statement (scenario 2)</td><td>56</td></tr><tr><td>3.5. Identification of MRLs that do not pose risk to consumers</td><td>58</td></tr><tr><td>3.5.1. Data call</td><td>58</td></tr><tr><td>3.5.2. Methodology</td><td>59</td></tr><tr><td>3.5.3. Plant commodities with identified fall-back options</td><td>59</td></tr><tr><td>3.5.4. Plant commodities with LOQ as the only fall-back MRL option</td><td>66</td></tr><tr><td>3.5.5. Plant commodities with no fall-back option</td><td>66</td></tr><tr><td>3.5.6. Fall-back options for commodities of animal origin</td><td>67</td></tr><tr><td>3.5.7. Consumer risk assessment using the new endpoints derived in the present statement and considering the identified fall-back MRL options (scenario 3)</td><td>68</td></tr><tr><td>4. Conclusions</td><td>69</td></tr><tr><td>4.1. Human health</td><td>69</td></tr><tr><td>4.1.1. Hazard assessment acetamiprid</td><td>69</td></tr><tr><td>4.1.2. Hazard assessment of acetamiprid metabolites</td><td>70</td></tr><tr><td>4.1.3. Human exposure estimation based on human biomonitoring data</td><td>70</td></tr><tr><td>4.2. Residues</td><td>70</td></tr><tr><td>5. Recommendations</td><td>71</td></tr><tr><td>5.1. Human health</td><td>71</td></tr><tr><td>5.2. Residues</td><td>72</td></tr><tr><td>Abbreviations</td><td>77</td></tr><tr><td>Acknowledgements</td><td>77</td></tr><tr><td>Conflict of interest</td><td>78</td></tr><tr><td>Requestor</td><td>78</td></tr><tr><td>Question number</td><td>78</td></tr><tr><td>Copyright for non-EFSA content</td><td>78</td></tr><tr><td>References</td><td>78</td></tr><tr><td>Appendix A</td><td>82</td></tr><tr><td>Appendices 1-23</td><td>84</td></tr></tbody></table>
## SUMMARY

Acetamiprid is an active substance covered by the third batch of the renewal programme for pesticides ('AIR3') in accordance with Commission Implementing Regulation (EU) No 844/2012. The active substance was first approved by Commission Directive 2004/99/EC and its approval was renewed by Commission Implementing Regulation (EU) 2018/113. A potential next renewal process needs to be initiated by 28 February 2030 at the latest. Maximum residue levels (MRLs) for this substance are set in Annex II to Regulation (EC) No 396/2005.

In January 2022, the European Food Safety Authority (EFSA) Panel on Plant Protection Products and their Residues (PPR Panel) published a statement (EFSA PPR Panel, 2022) following a request from the European Commission to advise on human health based on new scientific evidence presented by France.

For human health, no conclusive evidence of higher hazards of acetamiprid compared to the previous assessment in the context of the renewal was found for genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity. The following recommendations were given by the PPR Panel: (i) for the assessment genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity, the newly submitted evidence did not change the current conclusions from EFSA and ECHA on acetamiprid and no further actions should be taken; (ii) for endocrine disruption, an assessment of endocrine-disrupting properties for acetamiprid should be conducted in line with the EFSA/ECHA guidance document for the identification of endocrine disruptors (ECHA/EFSA, 2018).

Meanwhile, further scientific publications were brought to the attention of the Commission that described methods for detection of pesticide residues in the cerebrospinal fluid (CSF) of children. Using those detection methods, low levels of N-desmethyl-acetamiprid (IM-2-1), a metabolite of acetamiprid, were detected in most samples. In addition, some Member States presented evidence to the Commission that high levels of the metabolite IM-2-1 were found in some food products, especially in spinach. While the provided data showed that the levels of this metabolite in the analysed samples were often higher than the parent compound, IM-2-1 is not included in the existing residue definition for acetamiprid in products of plant origin. Lastly, one Member State informed the Commission that a potential acute consumer risk from exposure to acetamiprid in pears and lettuces was identified if the intake calculation is performed with the most recent version of PRIMO rev. 3.1.

By means of a further mandate received in July 2022 from the European Commission, EFSA was requested to provide advice if the information referred to above and any other scientific evidence that has become available since the assessment conducted for the renewal in 2018 warrants re-evaluation of (i) toxicological parameters used for the risk assessment of acetamiprid during the renewal process, including toxicological endpoints, (ii) the residue definition for acetamiprid in products of plant origin and (iii) the safety of existing MRLs. This technical and scientific assistance addressing the terms of reference (ToRs) included in the Annex of this mandate was requested under Article 31 of Regulation (EC) No 178/2002.

Nisso Chemical Europe GmbH, as the sole applicant of acetamiprid in the EU, on 4 July 2023 submitted to EFSA new toxicology studies claimed to be relevant for the response to the mandate, in particular regarding the toxicological profile of the metabolite IM-2-1 (in vitro micronucleus test with IM-2-1; mouse lymphoma assay with IM-2-1 and a repeated dose 28-day study with IM-2-1).

Furthermore, the Commission received a letter from Pesticide Action Network (PAN) Europe that referred to several recent publications in scientific literature, which might be relevant for the response to the mandate. Following the receipt of the above new data, an extension and revision of the mandate to EFSA was received in September 2023 to consider this information in the ongoing evaluation.

The term of reference 1 (ToR1) concerning the toxicological properties of acetamiprid and its metabolites, including toxicological endpoints, was addressed in this statement by an EFSA working group (WG) by applying the current state of the art methodologies in a transparent and trackable evidence-based AOP-informed IATA approach (Integrated Approaches to Testing and Assessment supported by Adverse Outcome Pathway). This approach relies on the integration of different levels of evidence, i.e. systematic literature review on human epidemiological studies, in vivo studies, and in vitro studies, and on the application of weight of evidence (WoE), including the Developmental Neurotoxicity In vitro Battery (OECD, 2023). The results of the weight of evidence (WoE) indicated that acetamiprid causes activation and rapid desensitisation of the nicotinic acetylcholinesterase receptors (nAChR) at concentrations starting from 1 µM. This is considered per se as a molecular and cellular effect that could lead to an adverse outcome at organism level, and therefore representing a developmental neurotoxicity (DNT) concern. In addition, the WG noted that there are data gaps in the in vivo body of evidence (BoE), including the lack of an acceptable measurement of learning and memory, motor activity and morphometrics evaluation in the available non-guideline DNT study. This represents a regulatory data gap that should be filled to allow to come to a more robust mechanistic understanding, to identify all DNT effects of acetamiprid and obtain concordant dose-response relationships to enable hazard and risk assessment. To account for these uncertainties/limitations on the data set, the WG proposed to include an additional uncertainty factor (UF) of 5 to the current health-based guidance values (HBGVs) to cover the uncertainties in the DNT assessment. As a result of the WG proposal, the acceptable daily intake (ADI) currently set at 0.025 mg/kg body weight (bw) per day would decrease to 0.005 mg/kg bw per day; similarly, the acute reference dose (ARfD) currently set at 0.025 mg/kg bw would be set at 0.005 mg/kg bw. EFSA notes that the same additional UF would be applied for setting the (acute) acceptable operator exposure level ((A)AOEL).

Metabolite IM-2-1 is a major rat metabolite with large structural similarities with the parent compound acetamiprid, unlikely to be genotoxic based on an Ames test, an in vitro micronucleus test and a mouse lymphoma assay. The 28-day study in rats on IM-2-1 does not allow to conclude on a clearly different toxicological profile or potency compared to the parent
compound. Hence, the same HBGVs (ADI of 0.005 mg/kg bw per day and ARfD of 0.005 mg/kg bw) proposed for the parent should also apply to the metabolite.

The **ToR 2a** concerning a data call for monitoring data for plant products was addressed by collecting results of pesticide residue analysis (monitoring data) for acetamiprid and its metabolites in food of plant origin. Data were provided by national competent authorities. Samples with residue analysis for parent acetamiprid and metabolite N-desmethyl acetamiprid (IM-2-1) were reported to EFSA. The **ToR 2b** related to the consideration of the residue definitions for risk assessment and enforcement for plant products was addressed by EFSA with a detailed analysis of the data received under ToR 2a and further analysis of acetamiprid metabolism studies previously submitted and evaluated. The available studies investigating the metabolism of acetamiprid in plants gave an indication that the metabolite IM-2-1 is formed at relatively low levels in edible parts of fruit crops and leafy crops (between 2% and 8% of the TRR; up to 0.3 mg/kg in fruits; up to 1.25 mg/kg in leafy). In inedible leafy matrices, however, this metabolite occurs at higher proportions related to the parent compound, IM-2-1 representing up to 32% of the parent compound (16% of the TRR) in apple leaf at longer preharvest intervals. The monitoring data on the metabolite IM-2-1 confirmed its occurrence in several commodities belonging to the groups of leafy and fruit commodities. In these crop groups, the median proportion of metabolite IM-2-1 compared to the parent compound was found to be significant in fruit and leafy crops (median ratio IM-2-1/acetamiprid accounting for 21%-44%, respectively). It was therefore proposed to include metabolite IM-2-1 in the residue definition of risk assessment for leafy and fruit crops, which is currently limited to the parent acetamiprid. A revised residue definition for risk assessment was proposed for leafy and fruit crops as sum of acetamiprid and N-desmethyl-acetamiprid (IM-2-1), expressed as acetamiprid. Regarding pulses/oilseeds, root crops and cereals, the new data received under ToR 2a did not indicate a need to modify the existing residue definition for risk assessment, which therefore remains as parent acetamiprid. Regarding the residue definition for enforcement, the available data do not indicate a need to modify the existing definition because acetamiprid is still a sufficient marker of the residues in all crop groups.

The **ToR 3** concerning consumer risk assessment related to the existing EU MRLs for acetamiprid in all plant and animal products was addressed by EFSA by means of three consumer risk assessment scenarios performed with the latest PRIMo rev 3.1. Considering the new health-based reference values (HBGVs)<fnref n="1" /> derived in the present statement, a risk for consumer has been identified for 38 MRLs currently in place in the EU Regulation. Furthermore, for granate apples and aubergines, a risk for consumers is unlikely for the existing MRLs, but a risk for consumer has been identified for the higher MRLs proposed in the draft MRL Regulation SANTE/11278/2021. Consequently, EFSA recommends to lower the existing MRLs for 38 commodities and to maintain the existing MRLs for granate apples and aubergines. Data gaps were identified for some of the fall-back MRLs derived in the present statement, for which further risk management considerations are therefore required. For pears, sweet cherries, peaches, strawberries, currants and elderberries, several MRL options are given to risk managers, because of the uncertainty identified with the higher MRL proposal. For five commodities (bananas, lettuces, escaroles/ broad-leaved endives, spinaches, chards/beet leaves), it is recommended to lower the existing MRLs to the enforcement limit of quantification (LOQ) because no safe fall-back MRL options could be identified. For bovine liver and bovine (other edible offals), it is recommended to withdraw the existing Codex MRLs from the EU Regulation. Lower alternative MRL options were derived by EFSA based on an updated EU livestock dietary burden calculation.

<footnote n="1">¹Health-based guidance values (HBGVs) and toxicological reference values (TRVs) are often used interchangeably.</footnote>
# 1 | INTRODUCTION

Acetamiprid is an active substance covered by the third batch of the renewal programme for pesticides ('AIR3') in accordance with Commission Implementing Regulation (EU) No 844/2012.<fnref n="2" /> The active substance was first approved by Commission Directive 2004/99/EC<fnref n="3" /> and its approval was renewed by Commission Implementing Regulation (EU) 2018/113.<fnref n="4" /> A potential next renewal process needs to be initiated by 28 February 2030 at the latest. Maximum residue levels (MRLs) for this substance are set in Annex II to Regulation (EC) No 396/2005.<fnref n="5" />

In January 2022, the European Food Safety Authority (EFSA) Panel on Plant Protection Products and their Residues (PPR Panel) published a statement (EFSA PPR Panel, 2022) following a request from the European Commission to advise on human health or the environment based on new scientific evidence presented by France.

For human health, no conclusive evidence of higher hazards of acetamiprid compared to the previous assessment in the context of the renewal was found for genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity. The following recommendations were given by the PPR Panel: (i) for the assessment of the endpoint categories genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity, the newly submitted evidence did not change the current conclusions from EFSA and ECHA on acetamiprid and no further actions should be taken; (ii) for the endpoint category endocrine disruption, an assessment of endocrine-disrupting properties for acetamiprid should be conducted in line with the EFSA/ECHA guidance document for the identification of endocrine disruptors under Regulations (EU) No 528/2012⁶ and (EC) No 1107/2009⁷ (ECHA/EFSA, 2018; EFSA PPR Panel, 2022).

Recently, further scientific publications were brought to the attention of the Commission that describe methods for detection of pesticide residues in the cerebrospinal fluid (CSF) of children. Using those detection methods, low levels of N-desmethyl-acetamiprid (IM-2-1), a metabolite of acetamiprid, were detected in most samples.

In addition, some Member States presented evidence to the commission that high levels of the metabolite IM-2-1 were found in some food products, especially in spinach. While the provided data showed that the levels of this metabolite in the analysed samples were often higher than the parent compound, IM-2-1 is not included in the existing residue definition for acetamiprid in products of plant origin. Lastly, one Member State informed the commission that a potential acute consumer risk from exposure to acetamiprid in pears and lettuces was identified if the intake calculation is performed with the most recent version of PRIMO (i.e. rev. 3.1).<fnref n="8" />

By means of a further mandate received in July 2022 from the European Commission, EFSA was requested to provide advice if the information referred to above and any other scientific evidence that has become available since the assessment conducted for the renewal in 2018 warrant re-evaluation of (i) toxicological parameters used for the risk assessment of acetamiprid during the renewal process, including toxicological endpoints, (ii) the residue definition for acetamiprid in products of plant origin and (iii) the safety of existing MRLs. This technical and scientific assistance addressing the terms of reference (ToRs) included in the Annex of this mandate was requested under Article 31 of Regulation (EC) No 178/2002⁹ (see Section 1.1).

Meanwhile, Nisso Chemical Europe GmbH, as the sole applicant of acetamiprid in the EU, submitted on 4 July 2023 new toxicology studies to EFSA that the company claimed to be relevant for the response to the mandate, in particular regarding the toxicological profile of the metabolite IM-2-1 (in vitro micronucleus test with IM-2-1; mouse lymphoma assay with IM-2-1 and repeated dose 28-day study with IM-2-1).

Furthermore, the commission received a letter from Pesticide Action Network (PAN) Europe that referred to several recent publications in scientific literature, which might be relevant for the response to the mandate. It was ascertained that these publications became available after the literature searches conducted by EFSA and, therefore, they were not included in the previous evaluation. As the new data from Nisso Chemical Europe and PAN Europe might have affected the advice that EFSA was expected to deliver in response to the mandate, EFSA was requested with an extension and revision of the mandate, to consider also this information in the ongoing evaluation. The references to the studies and publications are included in Annex I to the extension mandate.<fnref n="10" />

<footnote n="2">²Commission Implementing Regulation (EU) No 844/2012 of 18 September 2012 setting out the provisions necessary for the implementation of the renewal procedure for active substances, as provided for in Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market. OJ L 252, 19.9.2012, p. 26–32.</footnote>

<footnote n="3">³Commission Directive 2004/99/EC of 1 October 2004 amending Council Directive 91/414/EEC to include acetamiprid and thiaproprid as active substances. OJ L 309, 6.10.2004, p. 6–8.</footnote>

<footnote n="4">⁴Commission Implementing Regulation (EU) 2018/113 of 24 January 2018 renewing the approval of the active substance acetamiprid in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market, and amending the Annex to Commission Implementing Regulation (EU) No 540/2011. OJ L 20, 25.1.2018, p. 7–10.</footnote>

<footnote n="5">⁵Regulation (EC) No 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC, OJ L 70, 16.3.2005, p. 1.</footnote>

<footnote n="6">⁶Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products. OJ L 167, 27.6.2012, p. 1–123.</footnote>

<footnote n="7">⁷Regulation (EC) No 1107/2009 of 21 October 2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, 24.11.2009, p. 1–50.</footnote>

<footnote n="8">⁸The acute intake concerns for lettuce and pears were also noted in the most recent assessment of EFSA (e.g. EFSA, 2022).</footnote>

<footnote n="9">⁹Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–24.</footnote>

<footnote n="10">¹⁰https://open.efsa.europa.eu/questions/EFSA-Q-2022-00589?search=acetamiprid</footnote>
The above-mentioned new data may altogether impact the evaluation of the safety of existing MRLs. Therefore, as requested in the mandate, fall-back good agricultural practices (GAPs) that could lead to safe scenarios should be investigated in relation to those uses that might lead to intake concerns. Based on a preliminary screening made by EFSA, a list of plant commodities that might lead to intake concerns was identified (full list of products included in Annex II to the extension mandate¹⁰). A call for data for fall-back GAPs and residue trials for the products of plant origin listed in Annex II should be launched with Member States, and the assessment should be performed taking into account the provided fall-back GAPs. In case EFSA considers that the toxicological reference values would need to be modified, the proposed revised values should be used to perform this risk assessment. In case a risk for consumers would be identified for one or more of the existing MRLs, EFSA should recommend new MRLs that ensure safety of consumers, where possible, and advise risk managers on alternative options.

The revised terms of reference with specific points to consider were provided in Annex III of the extension mandate.¹⁰

Considering that the additional analysis requested from EFSA would require additional efforts, and a consultation with experts from Member States in a peer-review meeting may be needed to confirm the reference values of the metabolite, EFSA was requested pursuant to Article 31 of Regulation (EC) No 178/2002, read in conjunction with Regulation (EC) No 1107/2009 and Regulation (EC) No 396/2005, to deliver a scientific output including its advice on the toxicological properties of acetamiprid and its metabolites, and the outcome of the MRL assessment by 31 March 2024.

## 1.1 Background and Terms of Reference as provided by the requestor

Acetamiprid is an α-chloro-N-heteroaromatic compound that belongs to the group of neonicotinoids. The approval of acetamiprid has been renewed until 28 February 2033 by Commission Implementing Regulation (EU) 2018/113 concerning the renewal of approval of acetamiprid as active substance under Regulation (EC) No 1107/2009 that was based on the EFSA Conclusion on the peer review of the pesticide risk assessment of acetamiprid (EFSA, 2016a). This Implementing Regulation sets the conditions for the use of acetamiprid as active substance in plant protection products.

In November 2020, French authorities asked the Commission to prohibit the sale and use of acetamiprid under Article 69 of Regulation (EC) No 1107/2009, in the light of potential concerns that the substance may pose high risks to humans and the environment. The French authorities included in their notification scientific evidence to support this request, including references to published peer-reviewed studies. The Commission mandated the EFSA PPR Panel to advise on the likelihood that the body of evidence would constitute proof of serious risks to humans or the environment, in particular if the new studies indicate new or higher hazards and exposure to humans and the environment compared to previous EU assessments. In its statement published in January 2022 (EFSA PPR Panel, 2022), the EFSA PPR Panel concluded that there is no conclusive evidence of higher hazards from acetamiprid compared to the previous assessment in the context of the renewal with respect to genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity. However, it was recommended that an assessment of endocrine disrupting properties for acetamiprid is conducted in line with the EFSA/ECHA guidance document for the identification of endocrine disruptors under Regulations (EU) No 528/2012 and (EC) No 1107/2009 (ECHA/EFSA, 2018). Those findings were under discussion at the Standing Committee on Plants, Animals, Food and Feed, section Phytopharmaceuticals – Legislation with the view of a possible regulatory action for acetamiprid.

As regards maximum residue levels (MRLs), EFSA published a reasoned opinion on 20 July 2011 on the review of the existing MRLs for the active substance acetamiprid in compliance with Article 12(1) of Regulation (EC) No 396/2005 (EFSA, 2011). In this review, EFSA concluded that, in crops belonging to fruits and fruiting vegetables or to leafy vegetables, only the parent compound was found to be the main component, while for products of animal origin (except muscle of ruminants), the dominant compound was the metabolite N-desmethyl-acetamiprid (IM-2-1). EFSA concluded that the relevant residue definition for enforcement and risk assessment was acetamiprid only for plant products, and sum of acetamiprid and IM-2-1, expressed as acetamiprid, for products of animal origin. The MRLs resulting from this review and the confirmed residue definitions were implemented by Regulation (EU) No 87/2014,<fnref n="11" /> and are now set in Annex II to Regulation (EC) No 396/2005.

In the beginning of 2022, an article was published (Laubscher et al., 2022) that describes methods for detection of pesticide residues in the cerebro-spinal fluid (CSF) of children. Using those detection methods, low levels of metabolite N-desmethyl-acetamiprid (IM-2-1) were detected in most examined samples. The authors concluded that despite of many uncertainties, those findings might indicate potential risks for foetal and children's health and in particular development of the nervous system, due to the mode of action of neonicotinoids. EFSA has already examined the issue of developmental neurotoxicity of acetamiprid in the EFSA PPR Panel (2022). In addition, recently, the OECD published a case study on the developmental neurotoxicity of acetamiprid as part of the OECD Integrated Approaches for Testing and Assessment (IATA) project (OECD Case Study no. 365, 2022c).

At the meeting of the Standing Committee on Plants, Animals, Food and Feed, section Phytopharmaceuticals – Pesticides residues – in April 2022, one Member State informed that it detected high levels of IM-2-1 in spinach, at times higher than

<footnote n="11">¹¹Commission Regulation (EU) No 87/2014 of 31 January 2014 amending Annexes II, III and V to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for acetamiprid, butralin, chlorotoluron, daminozide, isoproturon, picoxystrobin, pyrimethanil and trinexapac in or on certain products Text with EEA relevance. OJ L 35, 5.2.2014, p. 1–48.</footnote>
those of the parent compound. So far, IM-2-1 is not part of the residue definition for acetamiprid in products of plant origin. The Commission had invited Member States to consult with their national laboratories and asked EFSA to investigate if similar findings have occurred in other countries, and to share the information with the Commission. New data were submitted to the Commission from both a Member State and EFSA, confirming that residue levels of IM-2-1 in spinach samples are often higher than the parent compound, and that similar findings occur in other crops.

In addition, another Member State brought to the attention of the Commission that the MRLs for pears and lettuces, which have been previously recommended by EFSA in the focused assessment of certain existing MRLs of concern for acetamiprid, and which have already been established by Regulation (EU) No 2019/88,<fnref n="12" /> may pose acute risks to consumers according to intake calculations performed with the most recent version of PRIMo (rev. 3.1). The EFSA previous assessment from 2018 (EFSA, 2018c) was conducted with a previous version of PRIMo (rev. 2).

The present mandate (Ref. Ares (2023)6354306 of 20 September 2023)¹⁰ is an extension of the mandate received in July 2022 (Ref. Ares (2022)5466053 of 29 of July 2022) by which EFSA was requested to deliver a scientific output including its advice on the toxicological properties of acetamiprid and its metabolites, and the outcome of the MRL assessment. By means of the revised mandate received in September 2023, the Commission requested EFSA to perform the following tasks as detailed in the specific revised terms of reference (ToRs):

1. To provide advice if new scientific evidence that has become available since the assessment conducted for the renewal in 2018 - including data/studies submitted by stakeholders and the additional studies provided by the applicant - warrant a re-evaluation of the toxicological properties of acetamiprid and its metabolites, including toxicological endpoints, in particular those related to developmental neurotoxicity.

2a. To launch a data call for monitoring data for plant products not yet submitted to EFSA.

2b. Based on monitoring data submitted under point 2a and the available metabolism studies in plants, to assess if the residue definitions for risk assessment and for enforcement derived for plant products treated with acetamiprid need to be revised.

3. To perform an assessment of the chronic and acute consumer risk related to the existing EU MRLs for acetamiprid in all plant and animal products,<fnref n="13" /> using the newest version of the PRIMo model. This assessment should be performed considering the Good Agricultural Practices (GAPs) and supporting residue trials already available to EFSA.

In case EFSA considers that toxicological reference values would need to be modified, the proposed revised values should be used to perform this risk assessment. In case EFSA considers that a new residue definition for risk assessment in plant products should be proposed, two separate risk assessment scenarios for the existing and the proposed new residue definitions for risk assessment should be calculated. In parallel, a call for data for possible fall-back GAPs and supporting valid residue trials that could lead to safe scenarios should be launched with Member States for the products of plant origin listed in Annex II of the mandate letter. In case a risk for consumers is identified with the revised toxicological reference values and with the proposed new residue definition(s) for one of more of the existing MRLs, EFSA should recommend new MRLs that do not pose an unacceptable risk to consumers, where possible, and advise risk managers on alternative options. To identify alternative options and MRLs that do not pose an unacceptable risk to consumers, the fall-back GAPs and residue trials collected in the framework of this mandate should be considered. In case EFSA would consider that toxicological reference values and/or the residue definition for risk assessment in plants would need to be modified, the proposed revised values, and/or residue definition(s) should also be used to perform this assessment of the alternative MRL options.

EFSA was requested to deliver a scientific output including its advice on the toxicological properties of acetamiprid and its metabolites and the outcome of the MRL assessment, pursuant to Article 31 of Regulation (EC) No 178/2002, read in conjunction with Regulation (EC) No 1107/2009 and Regulation (EC) No 396/2005, by 31 March 2024.

## 1.2 Interpretation of the Terms of Reference

### 1.2.1 Term of Reference 1 (human health)

In the human health part, an EFSA WG was established in order to address the following ToR: To provide advice if new scientific evidence that has become available since the assessment conducted for the renewal in 2018 – including the elements referred to in Section 1 above – warrant a re-evaluation of the toxicological properties of acetamiprid and its metabolites, including toxicological endpoints, in particular those related to developmental neurotoxicity.

<footnote n="12">¹²Commission Regulation (EU) 2019/88 of 18 January 2019 amending Annex II to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for acetamiprid in certain products). OJ L 22, 24.1.2019, p. 1–12.</footnote>

<footnote n="13">¹³This assessment should also cover those MRLs that were considered safe in recent EFSA outcomes that have not been addressed yet by specific Commission Regulations (e.g. those included in the EFSA, 2021).</footnote>
The EFSA WG interpreted the ToR by considering the following tasks:

1a. To conduct the assessment, including reliability and relevance for risk assessment, of the new biomonitoring data that have become available since 2016 until 2022 (in particular Laubscher et al., 2022 provided in the mandate).

1b. To use the most suitable biomonitoring data to extrapolate the level of acetamiprid and/or its metabolites in children to an external dose of acetamiprid and compare this external dose to the current HBGVs of acetamiprid.

1c. To conduct the assessment of the new evidence that has become available since 2016 until 2022, on toxicological properties that could be used for the risk assessment of acetamiprid and its metabolites.

1d. To conduct the assessment, in particular of the new evidence on DNT that has become available since 2016 until 2022, including reliability and relevance assessment for its use in DNT hazard identification and characterisation of acetamiprid. Evidence from human observational studies (HOS), in vivo studies and new approach methodologies (NAMs) (in vitro, zebrafish) will be used in an adverse outcome pathway (AOP)-informed integrated approach to testing and assessment (IATA).

The EFSA WG made use of the ToR1 to define the assessment questions. The EFSA WG considered two main aspects in the interpretation of the ToR1. The first aspect considers the exposure characterisation, and this is assessed in assessment question 1. The second aspect considers the hazard characterisation of acetamiprid and N-desmethyl-acetamiprid (IM-2-1) metabolite and this is addressed in assessment question 2.

### To address tasks 1a and 1b

1. **Assessment question 1:** Based on the results of the systematic literature review for internal exposure characterisation (and in particular the presence of metabolite N-desmethyl-acetamiprid (IM-2-1) in the cerebrospinal fluid of children), does the estimated external exposure exceed the current HBGVs?

1a. Based on the new biomonitoring evidence of acetamiprid and its metabolites, which information on internal exposure is the most appropriate (reliable, robust) to be used for the exposure pattern characterisation for acetamiprid risk assessment (i.e. derived from biomonitoring studies translated to external exposure by reverse dosimetry, using a physiologically based kinetic (PBK) model for acetamiprid)?

1b. Based on the exposure estimation of acetamiprid from 1a and after the uncertainty analysis (including those uncertainties inherent to the modelling approaches), does the estimated external exposure exceed current HBGVs?

### To address tasks 1c and 1d

2. **Assessment question 2:** Based on the results of the systematic literature review for hazard characterisation, are the current HBGVs for acetamiprid protective for the most sensitive population (fetus and children)?

2a. Based on the newly available evidence for any toxicological endpoint for acetamiprid and its metabolites, are the current HBGVs for acetamiprid protective?

2b. Based on the newly available evidence for DNT hazard characterisation, as outlined in the description of the task, are the current HBGVs for acetamiprid protective for the most sensitive population (fetus and children)?

### 1.2.2 | Term of Reference 2a (data call, monitoring data)

To address ToR 2a, EFSA launched a call for data, inviting Member States to submit to EFSA results of pesticide residue analysis for acetamiprid and its metabolites in food of plant origin derived by national competent authorities. Since the metabolites of acetamiprid are not part of the current residue definition for enforcement, information on these compounds which may be available at national level, might not have been reported in the context of the routine data collections, although such information is available at national level.

Member States were invited to send in particular data that have not been submitted previously to EFSA (i.e. in the context of the routine annual collection of pesticide monitoring results under Art. 31 of Regulation (EC) No 396/2005). The data call was restricted to the period of 2018-2022. The monitoring data should be provided in the format developed for submitting pesticide residue data under Article 31 of Regulation (EC) No 396/2005. Samples with residue analysis for parent acetamiprid and its metabolites such as N-desmethyl-acetamiprid (IM-2-1) were of major relevance for the current mandate.

In order to get a comprehensive database, the new monitoring data were combined with the previously submitted monitoring data: the combined data were assessed in detail to derive conclusions on the metabolic pattern in different crops/crop groups (see Section 3.1).
### 1.2.3 | Term of Reference 2b (residue definitions)

In order to address ToR 2b, EFSA applied the following approach:

1. The pesticide monitoring data (combined data from the data call under ToR 2a and monitoring data previously sent to EFSA in the context of routine data collection under Article 31 of Regulation (EC) No 396/2005) were compared with the results of the metabolism studies, to investigate whether the metabolic patterns are qualitatively and quantitatively comparable (see Section 3.1).

2. Metabolism studies relevant for plant products (i.e. metabolism studies investigating metabolism in primary crops and in rotational crops) submitted by the applicant in the framework of the renewal of the approval of acetamiprid under Regulation (EU) No 844/2012 and in other relevant applications were investigated in view of the metabolic pattern expected in different types of crops/parts of the crops (see Section 3.2).

3. EFSA identified crops/crop groups for which the metabolic pattern found in monitoring data provide evidence that metabolites are likely to be present at levels that would require a re-consideration of the residue definition (risk assessment and/or enforcement). The metabolites relevant for the different crops/crop groups were defined (see Section 3.3).

4. For those commodities where the residue definition for risk assessment was considered for being modified, and therefore became different than the residue definition for monitoring, conversion factors (CF) were derived. A non-standard methodology based on the available monitoring data was used (see Section 3.3).

### 1.2.4 | Term of Reference 3 (consumer risk assessment)

In order to address **ToR 3**, EFSA applied the following approach:

1. A first consumer risk assessment (CRA) was performed using PRIMo rev. 3.1. In this scenario, EFSA applied the newly derived HBGVs for acetamiprid and N-desmethyl-acetamiprid (IM-2-1) and the existing residue definitions for risk assessment, as derived in the framework of the renewal of the approval of acetamiprid under Regulation (EU) No 844/2012. EFSA assessed not only the existing MRLs but also considered those MRLs that were considered safe in the most recent EFSA outcomes (see **scenario 1** in Section 3.4.1).

2. A second consumer risk assessment (CRA) was performed using PRIMo rev. 3.1. In this scenario, EFSA applied the newly derived HBGVs for acetamiprid and N-desmethyl-acetamiprid (IM-2-1) and the newly derived residue definitions for risk assessment (including conversion factors), as proposed in the framework of the present mandate. EFSA assessed not only the existing MRLs but also considered those MRLs that were considered safe in the most recent EFSA outcomes (see **scenario 2** in Section 3.4.2).

3. EFSA launched a call for data for possible fall-back GAPs and supporting valid residue trials that could lead to safe MRL options for the plant commodities for which a risk for consumer has been identified under scenario 2. The data call was addressed to the EU Member States. Specific templates for submitting authorised good agricultural practices (GAPs) and supporting data were made available by EFSA (see Section 3.5.1).

4. EFSA screened and assessed the GAPs and supporting data received from Members States applying a stepwise approach to identify potential fall-back MRL option that would be safe for consumers (see Section 3.5.2).

5. A detailed assessment of the robustness of the identified fall-back MRL options for plant commodities was performed (see Sections 3.5.3 and 3.5.4). Further considerations were also made on the risk characterisation for those plant commodities for which no fall-back MRL could be identified (see Section 3.5.5).

6. For those commodities of animal origin for which a risk for consumer has been identified under scenario 2, further assessment was performed by EFSA to also identify fall-back MRL options.

7. A third consumer risk assessment (CRA) was performed using PRIMo rev. 3.1. EFSA applied the newly derived HBGVs for acetamiprid and N-desmethyl-acetamiprid (IM-2-1) and the newly derived residue definitions for risk assessment (including conversion factors), as proposed in the framework of the present mandate. In this scenario, EFSA used the fall-back MRLs (and risk assessment values) identified under points 4, 5 and 6 (see **scenario 3** in Section 3.5.7).

8. Based on scenario 3, EFSA recommended alternative MRLs for which risk to consumer is unlikely and provided further advice to risk managers where more than one option was identified.

## 2 | HUMAN HEALTH

### 2.1 | Hazard assessment acetamiprid

This section addresses **ToR 1a, ToR 1b, ToR 1c** and **ToR 1d**. To address **ToR 1** a WG of EFSA, including several experts of the PPR Panel was established.
## 2.1.1 | Data

Data related to four sources of evidence were collected and appraised to address the hazard characterisation of acetamiprid and its metabolites:

1. Data related to exposure and hazard characterisation have been retrieved conducting a systematic data collection from 2016 to 2022 and appraisal using critical appraisal tools. In addition, a protocol was developed based on EFSA (2020), which included detailed information on the search strategy, inclusion and exclusion criteria and detailed methods for the systematic review process used (see Appendix 1).

2. The OECD case study on the IATA for DNT of acetamiprid published in 2022 (https://one.oecd.org/document/env/cbc/mono(2022)27/en/pdf) and Annex I (https://one.oecd.org/document/env/cbc/mono(2022)27/ann1/en/pdf) (OECD, 2022c).

3. An in vivo developmental neurotoxicity (DNT) experimental study for regulatory purpose (OPPTS 870.6300) available for acetamiprid active substance. During the appraisal of the literature review retrieved (see source of evidence 1), the experts of the WG noted that in line with the EFSA IATA framework, all the available evidence for DNT should undergo the same reliability and relevance assessment approach (i.e. critical appraisal and uncertainty analysis) in order to conclude on the DNT potential of acetamiprid. For this reason, the DNT regulatory study was also critically appraised.

4. List of publications notified by PAN Europe and published after the period in which EFSA conducted the systematic literature review (up to end of 2022) and included in the extension of the mandate. In particular:

* Li, He, et al. (2022). Neonicotinoid insecticides promote breast cancer progression via G protein-coupled estrogen receptor: In vivo, in vitro and in silico studies. Environment International, 170, 107568. https://doi.org/10.1016/j.envint.2022.107568

* Mishani et al. (2022). The effect of increasing the dose of acetamiprid and dichlorvos pesticides on the reproductive performance of laboratory mice. Advanced Biomedical Research, 11, 114. https://doi.org/10.4103/abr.abr_199_22

* Didenko et al. (2022). Dose dependence of subchronic influencing of acetamiprid on the organism of rats from data of morphological researches. Wiadomosci Lekarskie, 75, 2987-2993. https://doi.org/10.36740/WLek202212116

* Li, Si, et al. (2022). Detection of neonicotinoid insecticides and their metabolites in human cerebrospinal fluid. Environmental Health Perspectives, 130, 127702. https://doi.org/10.1289/EHP11374

* Yang & Liang (2023). Associations between neonicotinoids metabolites and hematologic parameters among US adults in NHANES 2015-2016. Environmental Science and Pollution Research International, 30, 26327-26337. https://doi.org/10.1007/s11356-022-23997-4

* Mendy & Pinney (2022). Exposure to neonicotinoids and serum testosterone in men, women, and children. Environmental Toxicology. https://doi.org/10.1002/tox.23503

* Ma et al. (2022). Long-term exposure to neonicotinoid insecticide acetamiprid at environmentally relevant concentrations impairs endocrine functions in Zebrafish: Bioaccumulation, feminization, and transgenerational effects. Environmental Science Technology, 56, 12494-12505. https://doi.org/10.1021/acs.est.2c04014

It must be noted that it was agreed to evaluate all the studies notified by PAN Europe in detail. Therefore, there was no need for consideration of inclusion/exclusion criteria for these publications as set in the protocol (Appendix 1) was performed. It is also of interest to note that the literature search by EFSA was performed in November 2022 (see Appendix 1), and that some of the studies notified by PAN Europe are from after that period.

## 2.1.2 | Methodologies

To address tasks 1c and 1d, the EFSA WG used the following approaches:

* **An evidence-based approach** for the data retrieval, compilation and integration in a weight of evidence (WoE). The approach to collect, appraise, synthesise/integrate evidence and analyse uncertainty followed EFSA's 'Principles and process for dealing with data and evidence' (EFSA, 2015; EFSA and EBTC, 2018; EFSA Scientific Committee, 2017) and guidance on uncertainty analysis (EFSA Scientific Committee, 2018a, 2018b). This implied planning the methods for conducting the assessment upfront, in a detailed **protocol** based on the draft protocol developed by the EFSA (2020), which included all the detailed information on the methodology of the development of the IATA, systematic literature review, critical appraisal and weight of evidence and uncertainty analysis.

* An **AOP-informed IATA** framework for assessing DNT (OECD, 2016) in line with the framework applied in the EFSA IATA Case Studies for DNT of deltamethrin and flufenacet (EFSA PPR Panel, 2021), where an evidence-based approach for the data compilation and integration in the IATA was used.

Appendix 1 contains the protocol and the details on the methodology. The protocol includes in detail the strategy and methods for the systematic review process, specifically:

* Translation of the mandate into subquestions.
* Search string to retrieve the studies for in vitro (including studies conducted in zebrafish), in vivo and human evidence and a full list of literature databases to be used.

* Studies eligibility criteria and screening for relevance.

* Critical appraisal tools (CATs) and procedures for assessing the risk of bias (RoB), including the rationale used for each line of evidence for DNT.

* Methods for evidence synthesis, integration and uncertainty analysis.

A short summary of the main methodology is reported below for easier reference.

## Systematic literature review

The literature searches were conducted using three electronic bibliographic databases (PubMed, Web of Science, Toxnet). The time period considered was from 2016 until 2022. Search strings are described in the protocol (Appendix 1). A broad search was therefore conducted, and in addition, terms for the exposure were combined with relevant terms for DNT outcomes (human and in vivo studies) or methods (in vitro studies) and the studies were labelled in Distiller SR.

## Critical appraisal of the evidence

The eligible in vivo studies in rodents (experimental toxicological studies) from the literature search, as well as the in vivo rodent studies notified by PAN Europe published after the systematic literature review carried out by EFSA, were appraised for the RoB by the WG using tailored versions of the OHAT-NTP RoB tool (EFSA PPR Panel, 2021; NTP, 2015). For the in vitro studies, a modified tool developed by OHAT-NTP for a specific project (NTP, 2016) was adapted. CATs were defined upfront and are described in the protocol (Appendix 1). Endpoints measured in the studies were classified as being of low (tier 1), moderate (tier 2) or high (tier 3) RoB. These tiers were derived weighing the appraisal from the individual RoB domains where some of the domains were identified as key for the overall appraisal. RoB was appraised endpoint by endpoint in each study as the diverse endpoints measured within the study may have been assessed by different methodology and thus their RoB may differ.

The study of Ma et al. (2022), which was notified by PAN Europe, was appraised in line with previous evaluations of ecotoxicological studies (see EFSA, 2023), i.e. performing a data extraction followed by the appraisal of the measured parameters. For appraising the parameters, the risk of external (relevance) and internal (reliability) bias and precision were assessed. For details about the protocol used, see EFSA (2023) and the related background documents.

## Uncertainty analysis and expert knowledge elicitation

The uncertainty analysis and the expert knowledge elicitation (EKE) methodologies are reported in Appendix 1 and were performed according to EFSA's recommendations (EFSA Scientific Committee, 2018a, 2018b). An uncertainty analysis (Table 1) was therefore performed for each line of evidence and hierarchical level in order to support conclusions on the hazard identification and characterisation questions. The final purpose was to screen the evidence and identify molecular initiating events (MIEs), key events (KEs) and adverse outcomes (AOs) to be included in the putative AOP network. All data were mapped in specific endpoints and endpoint categories (obtained from the systematic review, the DNT in vitro battery (IVB) and the test guideline in vivo study) and this was used with the aim of postulating an AOP network. In the AOP, evidence was classified as MIEs, KEs or AOs based on their categorisation as molecular, cellular, organ, organism or population response. However, this was only possible for the limited available evidence that was considered reliable.

The uncertainty analysis was conducted in two steps (Steps 1 and 2; see Table 1) to identify and characterise the DNT effects in the body of evidence (BoE) by providing answers to specific assessment questions and expressing the uncertainty in a qualitative way using an EKE.

<table><caption>TABLE 1 Assessment questions for the uncertainty analysis for in vivo and in vitro body of evidence (BoE) for acetamiprid DNT hazard identification (see also Appendix 1).</caption><thead><tr><td>Line of evidence</td><td>Question EKE</td><td>Possible outcome options for expression of the uncertainty qualitatively</td></tr></thead><tbody><tr><td>Step 1. In vivo experimental studies</td><td>Based on the in vivo available evidence for DNT hazard characterisation and integrated following an AOP-informed IATA conceptual framework, is the WG of the opinion that the current HBGVs for acetamiprid and IM-2-1 are protective for the most sensitive population (fetus and children)?<br/>The WG must consider EFSA conclusion on acetamiprid (EFSA, 2016a) where the acceptable daily intake (ADI), the acute reference dose (ARfD), the acceptable operator exposure level (AOEL) and the acute acceptable operator exposure level (AAOEL) are set at 0.025 mg/kg bw (per day) on the basis of reduced auditory startle responses in offspring observed from 10 mg/kg bw per day (LOAEL) in the rat DNT study with an uncertainty factor (UF) of 100</td><td>YES, the WG considers that the current HBGVs are adequate and protective for the most sensitive population in view of the new evidence made available since the renewal of approval of acetamiprid<br/>NO, the WG considers that there are additional uncertainties not considered in the previous evaluation. These major limitations in the DNT BoE and data gaps would warrant a re-evaluation of the current HBGV</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><td>Line of evidence</td><td>Question EKE</td><td>Possible outcome options for expression of the uncertainty qualitatively</td></tr></thead><tbody><tr><td>Step 2. In vitro experimental studies</td><td>Once all the mechanistic data will be mapped in an AOP framework, the experts will be asked to answer the following question: Based on the new available evidence and on the AOP-informed IATA, is the result of 1 sufficiently protective for a DNT effect?</td><td>YES, the WG considers that the current HBGVs are adequate and protective for the most sensitive population in view of the new evidence made available since the renewal of approval of acetamiprid<br>NO, the WG considers that there are additional uncertainties not considered in the previous evaluation. These major limitations in the DNT BoE and data gaps would warrant a re-evaluation of the current HBGV</td></tr></tbody></table>

## Adverse outcome pathway (AOP)-informed IATA development and weight of evidence (WoE)

To assess the DNT evidence, an AOP-informed IATA framework was applied. Within an IATA, data from various information sources are evaluated and integrated to draw conclusions on the hazard and/or risk of chemicals (OECD, 2020). IATA is a framework developed by the OECD that allows for the integration of all available hazard and possibly exposure data, including in silico, in chemico, in vitro and in vivo, for use in chemical regulatory assessments (OECD, 2016). Important features of IATAs are the need to explicitly set out the problem formulation and context of use, since these will determine the acceptable level of uncertainty, the choice of methods (building blocks), the approach for evidence integration and the application of the WoE in an iterative process until a conclusion is reached (OECD, 2016). This approach has been previously used by EFSA for DNT hazard characterisation IATAs (EFSA PPR Panel, 2021; OECD, 2022a, 2022b) and represents a step forward in terms of the methodological approach in delivering a sound, transparent and accessible scientific advice in support to decision-making.

IATAs are pragmatic, science-based approaches for chemical evaluations in the context of hazard or risk assessments that rely on an integrated analysis of existing information, with optional use of the AOP framework, coupled with the generation of new information if necessary (OECD, 2016; Sachana & Leinala, 2017; Sakuratani et al., 2018). To fulfil these IATA needs, evidence was structured using the AOP framework. The AOP conceptual framework was applied to integrate information obtained from various lines of evidence by means of a systematic literature review, and the experimental outcome from the DNT IVB in order to provide a structured contextualisation of the molecular initiating events (MIEs) and key events (KEs) leading to the adverse outcomes (Aos) (see Figure 1). Conclusions were drafted for the AO and for the mechanistic pathway (MIE, KEs and key events relationships [KERs]) and in the subsequent step the WoE evaluation of the AOP itself was planned in line with previous EFSA AOP-informed IATAs (OECD Case Study 362 [OECD, 2022a] and OECD Case Study 363 [OECD, 2022b]).

<figure><img src="image_3.png" /><figcaption>FIGURE 1 AOP-informed IATA workflow for acetamiprid assessment.</figcaption></figure>
## 2.1.3 | Assessment for non-DNT endpoints

### 2.1.3.1 | Data collection

A total of 1702 studies were identified for acetamiprid after removing duplicates (see PRISMA flow chart, Figure 2) using an EFSA systematic literature review. Two reviewers screened in two steps the results of the broad search (step 1 title and abstract screening and step 2 full-text reading), i.e. the literature was identified through the searches in DistillerSR® (Evidence Partners, Ottawa, Canada). The evidence was clustered as (1) evidence providing data potentially relevant for any non-DNT toxicological endpoint that could be used for hazard characterisation and risk assessment of acetamiprid active substance and could warrant a re-evaluation of the current toxicological assessment (i.e. studies that could be used for setting HBGVs) and (2) evidence providing indications of a potential DNT effect in line with an AOP-informed IATA framework (i.e. where DNT is defined as any adverse effect on the normal development of nervous system structures and/or function, that is predefined in the protocol by different endpoints measured in in vivo, in vitro and human observational studies. DNT is considered as a molecular/cellular/organ/organism burden that could lead to adversity). The eligibility criteria are detailed in the protocol, Tables 4–8 (Appendix 1).

Since no publication providing evidence relevant for step 1 was retrieved, it was agreed that further assessment for non-DNT endpoints was not needed (Figure 2).

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** PRISMA flow chart of the literature search process for acetamiprid, including the screening for relevance of results.</figcaption></figure>

However, new studies (published in 2022 and 2023) were notified by PAN Europe and included into the appraisal step. A summary of the seven studies is available in the Excel file present in Appendix 2, presenting per study information on the test systems used, the exposure conditions applied, the endpoints/readouts assessed and the results reported by the study authors.

### Description of the studies

Of the seven additional publications notified by PAN Europe, three included data obtained in animal experiments (two studies using rats, one using mice), with one of these studies also including supportive mechanistic data from in vitro studies. Three studies reported on acetamiprid levels in humans, with two of them assessing a possible association between acetamiprid levels and health effects in humans. The remaining study assessed the effects of acetamiprid (and IM-2-1) on zebrafish.

In the following, a short description of the studies is provided, only focusing on acetamiprid and metabolite IM-2-1.

* In the study of Li, He, et al. (2022), the effect of acetamiprid on in situ growth of breast cancer cells in female mice and related mechanisms were studied. Ovariectomised and non-ovariectomised mice were injected with 4T1-luc cells (murine triple-negative breast cancer cell line) and exposed daily by oral gavage for 3 weeks to acetamiprid (ovariectomised mice to 0, 1, 10 or 30 mg/kg bw per day; non-ovariectomised mice to 0 or 10 mg/kg bw per day). Also, a group was included that was exposed to the G protein-coupled oestrogen receptor (GPER) antagonist G15, as well as a group that was exposed to 10 mg acetamiprid/kg bw per day together with the GPER antagonist G15. The authors reported that acetamiprid increases the growth of the in situ tumours and increased metastasis, and that these effects were inhibited by the GPER antagonist G15. In the same publication, experiments are described in which 4T1-luc cells were exposed in vitro to different concentrations of acetamiprid (in the presence or absence of the GPER antagonist G15) and effects on GPER mRNA expression,
GPER activation, as well as on cell migration and cell proliferation were assessed. The authors reported that acetamiprid induced an increase in GPER mRNA expression, GPER activation, as well as increased migration and proliferation of cells exposed to acetamiprid. The authors also reported that effects on GPER activation, cell proliferation and cell migration were inhibited by the GPER antagonist G15.

* In the study of Mishani et al. (2022), male Balb/c mice were exposed to acetamiprid via drinking water (0, 1.5 or 2.5 ppm) for 60 days. After exposure, treated males were mated with untreated females. In males, FSH, LH and testosterone levels were assessed, as well as an analysis was performed to evaluate different cell types in the testes. Pregnant females were sacrificed at gestation day (GD) 15 and the number of embryos was determined. The authors reported a decrease in number of embryos in females mated with males exposed to drinking water with 2.5 ppm acetamiprid. The authors also reported an increase in LH and FSH, a decrease in testosterone and a decrease in spermatogonial stem cells, spermatocytes and spermatogonia in testes of mice exposed to drinking water with 2.5 ppm acetamiprid.

* In the study of Didenko et al. (2022), female rats were daily exposed (5 days per week) for 90 days via oral gavage to 0, 6, 12 or 60 mg acetamiprid/kg bw. The study was performed according to OECD 408 with deviations. The rats were monitored for general effects (behaviour, feed and water consumption, skin and coat condition, mucous membranes, motor activity, tremors, gait/posture disturbances, presence of reaction to care of the animals, presence of clonic or tonic movements, stereotypic movements, chimeric behaviour). Before and during exposure (every 7 days), body weights were determined. At the end of exposure, animals were sacrificed, and absolute and relative organ weights were determined (internal organs: liver, kidneys, spleen, brain). Histological assessment was done for brain, liver, kidneys and spleen. The authors reported a decrease in body weight, absolute weight of spleen and brain and a decrease in relative spleen weight at the top dose (60 mg/kg bw per day) compared to the control group. The authors reported histological changes in various tissues at the mid and top dose levels. Effects at the mid dose were considered by the authors as adaptive changes, whereas these were considered adverse at the top dose in the liver, kidney and brain. The authors set the no observed adverse effect level (NOAEL) at 12 mg/kg bw per day, and the NOEL at 6 mg/kg bw per day.

* In the study of Li, Si, et al. (2022), neonicotinoids and their metabolites were quantified in cerebrospinal fluid (CSF) samples of 314 human donors from 4410 patients available for CSF analysis in First Affiliated Hospital of Shantou University from April 2019 to January 2021, with an age range from 1 month to 89 years. Regarding acetamiprid, the authors reported that the median concentration was below the LOD (0.002 ng/mL), the maximal concentration amounted to 2.07 ng/mL and the detection frequency was 39.5%. Regarding IM-2-1, the authors reported that the median concentration was 0.049 ng/mL, the maximal concentration amounted to 2.38 ng/mL and the detection frequency was 85.4%.

* In the study of Yang and Liang (2023), the relationship between neonicotinoid metabolites (levels measured in urine) and hematologic parameters was assessed using a cross-sectional study design in 1397 adults of the National Health and Nutrition Examination Survey (NHANES) 2015–2016, applying multivariate linear regression models. Regarding acetamiprid/IM-2-1, the authors reported that IM-2-1 levels in urine were associated with reduced white blood cells (WBC) and neutrophils counts (statistically significant for males and females together and for males as separate group). Furthermore, the authors reported that IM-2-1 levels in urine were linked to increased eosinophil counts and percentages (statistically significant only for females as separate group) and to decreased haemoglobin and haematocrit, and reduced platelet count (statistically significant only for males as separate group). The authors concluded that the results from the study provided evidence that exposure to neonicotinoid metabolites can disturb haematologic homeostasis in the general populations and that effects may be sex specific.

* The study of Mendy and Pinney (2022) assessed the association between concentrations of neonicotinoids and their metabolites determined in urine of 2014 participants to the NHANES 2015–2016 (6 years and older) and total serum testosterone concentration and free androgen index (FAI: serum concentration ratio of total testosterone to sex hormone binding globulin [SHBG]). IM-2-1 in urine was associated with reduced testosterone in serum in males (detection rate and log10 concentration). For females, the same association was observed only when adjusted for age, that is, 40 years and older (detection and log10 concentration). Both in males and females, IM-2-1 in urine was reported to be associated with reduced FAI (males only for log10 concentration, females both for detection and log10 concentration). The authors concluded that exposure to neonicotinoids is associated with decreased serum testosterone levels in humans, but that future prospective studies are warranted to confirm these findings.

* The study from Ma et al. (2022) investigated the long-term exposure to acetamiprid on the aquatic test species zebrafish (Danio rerio). The study included three different experiments, (i) a fish full life cycle-type experiment (from larvae to embryos of the second generation [F1]); (ii) an experiment where F1 embryos were monitored but not exposed and (iii) a fish embryo test:

1. Fish full life cycle type test (1_1):

a. Zebrafish were exposed from larval stage (20 days post fertilisation - dpf) up to adulthood (174 dpf) to five nominal concentrations of acetamiprid (0.15, 1.5, 15, 150 and 1500 µg/L). Adult zebrafish were used to examine survival, sex ratio and growth responses. Negative controls and solvent controls containing 0.01% (v/v) DMSO were tested concurrently.

b. From the adult fish, two females and two males (3 replicates per treatment) of the F0 generation were incubated, and the fish embryos (F1) were collected for 3 days. Number of eggs and percentage of fertilisation were recorded. Successfully fertilised and normally developed F1 embryos at 6 h post-fertilisation (hpf) were continuously exposed up to 168 hpf to the same levels of acetamiprid as the F0 in 96-well plates. Hatchability at 72 hpf, malformations and survival at 168 hpf were determined.
2. F1 generation raised in unexposed conditions (1_2): to monitor potential transgenerational effects of parental exposure, successfully fertilised and normally developed F1 embryos at 6 h post-fertilisation (hpf) from the F0 generation exposed to acetamiprid were raised in clean medium in 96-well plates. Hatchability at 72 hpf, malformations and survival at 168 hpf were determined.

3. Fish embryo toxicity experiment (1_3): Studies with acetamiprid and its metabolite IM-2-1 were conducted separately to evaluate their bioaccumulation potential and acute toxicity (LC50/EC50 values) to zebrafish embryos. No specific guideline study was followed/mentioned in the text. Analytically verified treatment levels were 0, 57, 81, 210, 337, 461, 477 and 734 mg/L for acetamiprid, and 0, 69, 104, 287, 366, 434, 520, 628, and 722 mg/L for IM-2-1. After exposure, hatchability at 72 hpf, malformation and survival at 168 hpf were determined.

### 2.1.3.2 | Critical appraisal results

The results of the RoB analysis conducted during the extension of the mandate are presented in **Tables 2–6** by lines of evidence (i.e. in vivo rodent studies [Table 2], in vitro studies [Table 3], human studies [Tables 4 and 5] and zebrafish study [Table 6]). Regarding the human studies, one study reported only internal concentrations (no health effects were measured; Table 4) and two studies evaluated possible associations between internal exposure data and (health) effects (Table 5). These studies were subdivided, because they followed different RoB assessment methods.

All in vivo rodent endpoints were rated as Tier 3 (high RoB). For the Li, He, et al. (2022) and Mishani et al. (2022) studies, risk of bias was considered regarding blinding of research personnel to study group (Q4), possible attrition or exclusion from analysis (Q5), the exposure characterisation (Q6, key questions) and the outcome assessment (Q7, key question). For some of the endpoints of the Li, He, et al. (2022) study, also a risk of bias was considered regarding randomisation of the animals (Q1, key question).

All in vitro endpoints were rated as Tier 3, except for GPER mRNA expression (Tier 2). For all in vitro endpoints, risk of bias was considered regarding the comparability of experimental conditions in control and treatment groups (Q2), outcome reporting (Q7) and internal validity: number of replicates (Q8b). For one endpoint (cell migration: wound healing assay), lack of randomisation of exposure levels was considered to provide a risk of bias (Q1), for two endpoints (cell migration: wound healing assay and cell migration: Boyden chamber assay), lack of blinding of research personnel to study group was considered to provide a risk of bias (Q3), and for two endpoints (GPER activation and cell proliferation), the outcome assessment was considered to provide a risk of bias (Q6, key question).

The human biomonitoring study on acetamiprid/IM-2-1 levels in CSF was rated as Tier 3 (high RoB). There was limited information about the method's validation (Q6) and no reporting about quality assessment/quality control (QA/QC; Q7, key question). Haematologic parameters and testosterone and free androgen index from the human observational studies were rated as Tier 1 (low RoB). More details on the assessments of the rodent, in vitro and human studies can be found in Appendix 3.

<table><caption>**TABLE 2** Heatmap results of the RoB for the endpoints measured in the new in vivo studies notified by PAN Europe in the extension of the mandate for acetamiprid.</caption><thead><tr><th>In vivo endpoints</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>Li, He, et al., (2022): tumour growth<br>ovariectomised mice (bioluminescence)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour growth<br>ovariectomised mice (tumour volume)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour metastasis lung<br>ovariectomised mice (bioluminescence)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour growth non-<br>ovariectomised (bioluminescence)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour growth non-<br>ovariectomised mice (tumour volume)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour metastasis non-<br>ovariectomised mice (bioluminescence)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, He, et al., (2022): tumour metastasis non-<br>ovariectomised mice (tumour nodules lung)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): number of embryos at<br>GD15</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): FSH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): LH</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): testosterone</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): stem cells in testes</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): spermatocytes in testes</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Mishani et al., (2022): spermatogonia in testes</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody><tfoot><tr><td colspan="11">(Continues)</td></tr></tfoot></table>
<table><caption>**TABLE 2** (Continued)</caption><thead><tr><th>In vivo endpoints</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>Didenko et al. (2022): organ weight</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Didenko et al. (2022): organ histopathology</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody></table>

Note: The heatmap includes the endpoint-specific ratings for the RoB for each of the nine questions in the CAT (i.e. domains of randomisation, allocation concealment, experiment conditions, research personnel blinded, attrition, exposure characterisation, outcome assessment, outcome reporting and other aspects, i.e. systemic or maternal toxicity). Each question is scored as either definitely low RoB (dark green), probably low RoB (light green), probably high RoB (light red) or definitely high RoB (dark red). The RoB questions highlighted in yellow correspond to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint (Q1 randomisation domain; Q6 exposure characterisation; Q7 outcome assessment method). For detailed rationale for each rating, see Appendix 3.

<table><caption>**TABLE 3** Heatmap results of the RoB for the endpoints measured in the new in vitro studies notified by PAN Europe in the extension of the mandate for acetamiprid.</caption><thead><tr><th>In vitro endpoints</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8a</th><th>Q8b</th><th>Tier</th></tr></thead><tbody><tr><td>Li, Si, et al. (2022): GPER activation: intracellular calcium</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, Si, et al. (2022): Cell migration: wound healing assay</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, Si, et al. (2022): Cell migration: Boyden chamber assay</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, Si, et al. (2022): Cell proliferation: MTT assay</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Li, Si, et al. (2022): GPER mRNA expression: qPCR</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr></tbody></table>

Notes: The heatmap includes the endpoint-specific ratings for the RoB for each of the nine questions in the CAT (i.e. domains of randomisation, experiment conditions, research personnel blinded, attrition, exposure characterisation, outcome assessment, outcome reporting, other aspects, i.e. cytotoxicity and replicates). Each question is scored as either definitely low RoB (dark green), probably low RoB (light green), probably high RoB (light red) or definitely high RoB (dark red). The RoB questions highlighted in yellow correspond to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint (Q5 exposure characterisation; Q6 outcome assessment method; Q8a cytotoxicity). For detailed rationale for each rating, see Appendix 3.

<table><caption>**TABLE 4** Heatmap results of the RoB for the new human biomonitoring study on reported internal acetamiprid/IM-2-1 concentrations notified by PAN Europe in the extension of the mandate for acetamiprid.</caption><thead><tr><th>Human biomonitoring study</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Tier</th></tr></thead><tbody><tr><td>Li, Si, et al. (2022): acetamiprid/IM-2-1 levels CSF</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody></table>

Note: The heatmap includes the endpoint-specific ratings for the RoB for each of the eight questions in the CAT (i.e. number of participants, analyte measured and biological sample used, potential contamination or non-specific binding to the collection tube, sample storage, method's validation, QA/QC, analytical instrumentation, limited of detection/quantification reported). Each question is scored as either low RoB (green), medium RoB (orange) or high RoB (red). The RoB questions highlighted in yellow correspond to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint (Q2 analyte measured; Q5 method's validation; Q6 QA/QC; Q7 analytical instrumentation). For detailed rationale for each rating, see Appendix 3.

<table><caption>**TABLE 5** Heatmap results of the RoB for the new human observational studies on acetamiprid notified by PAN Europe in the extension of the mandate for acetamiprid.</caption><thead><tr><th>Human observational studies</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Tier</th></tr></thead><tbody><tr><td>Yang and Liang (2023): hematologic parameters</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Mendy and Pinney (2022): testosterone and free androgen index</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr></tbody></table>

Note: The heatmap includes the endpoint-specific ratings for the RoB for each of the seven questions in the CAT (i.e. selection study participants, study design confounding, attrition, exposure characterisation, outcome assessment, reporting, other: statistics). Each question is scored as either definitely low RoB (dark green), probably low RoB (light green), probably high RoB (light red) or definitely high RoB (dark red). The RoB questions highlighted in yellow correspond to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint (Q2 study design confounding; Q4 exposure characterisation; Q5 outcome assessment). For detailed rationale for each rating, see Appendix 3.

**Table 6** presents the assessed parameters and the related RoB for external validity, internal validity and precision of the zebrafish study. The data extracted from the zebrafish study and the appraisal of the measured parameters are presented in Appendix 4.

Although the study is overall well reported, all parameters assessed have high risk of bias for internal validity (Table 6). Two main drawbacks were identified in the full life cycle type test (experiment no. 1_1): the high mortality in control animals (25%) and the high mortality across all treatment levels. Regarding the control mortality, although the study was not performed following an available test guideline, control survival in adult zebrafish should normally be 90% (OECD, 229). Based on this, the validity of the study is overall questionable. For all tested concentrations, clear signs of systemic toxicity (mortality > 10%) were observed, and thus, the tested concentrations were likely inappropriate. Moreover, although some effects were observed, those effects are likely due to excessive toxicity rather than triggered by an ED MoA.

In addition to the parameters reported in Table 6, the bioaccumulation potential of acetamiprid was assessed in the different experiments conducted on both adults of the F0 generation and on the F1 generation. The study authors reported that acetamiprid and its metabolites were observed in a dose-response manner in adult fish tissues, with a bioaccumulation factor of 0.65 ± 0.1 L/kg wet weight. For the F1, acetamiprid and metabolite N-desmethyl-acetamiprid
(IM-2-1) were found in the eggs supporting the initial hypothesis of maternal transfer. Additionally, in the Fish Embryo Acute Toxicity test (FET) the metabolism of acetamiprid was reported to be low based on the ratio of IM-2-1 to acetamiprid. Overall, when observed against the criteria and recommendations of related testing guideline on bioaccumulation in fish (i.e. OECD TG 305), some drawbacks limiting its reliability were identified. For instance, the mortality observed in the F0 generation exceeds 10% (as also mentioned in the previous paragraph). While it could be assumed that a steady state was reached after 154 days of exposure in adult fish, some uncertainties remain since the uptake curve was not reported, together with intermediate analysis of acetamiprid in fish and water over the entirety of the exposure period. Intermediate sampling of fish weight measurement should also have been carried out, to allow correction on growth dilution. In addition, the fish lipid content should have been measured so that the bioconcentration factor (BCF) can be expressed based on a 5% lipid content. It was also noted that a depuration phase was not carried out. It should also be considered that the log k<sub>ow</sub> of acetamiprid is below 3; therefore, the substance is considered having low bioaccumulation potential, which was anyway confirmed by the low BCF obtained by the study authors.

<table><caption>TABLE 6 Heatmap of the parameters assessed in Ma et al. (2022) with zebrafish.</caption><thead><tr><th>Experiment no.</th><th>Assessed endpoint</th><th>External validity</th><th>Internal validity</th><th>Precision</th></tr></thead><tbody><tr><td>1_1</td><td>Survival F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Sex ratio female biased F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Body length F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Body weight F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Condition factor K F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Brain somatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Hepatosomatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_Gonadosomatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_Body length F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_Body weight F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_condition factor K F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_Brain somatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_Hepatosomatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_Gonadosomatic Index F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_pregesterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_17-hydroxyprogesterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_17B-estradiol_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_estriol_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_androstenedione_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_testosterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_pregesterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_17-hydroxyprogesterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_estrone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_17B-estradiol_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_estriol_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_androstenedione_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_testosterone_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_gnrh2 F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_gnrh3_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_fshB_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_lhB_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_cyp19b_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Brain_arB_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females expression_Gonad_fshr_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Gonad_lhr</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Gonad_cyp11a_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Gonad_3Bhsd_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Gonad_cyp17_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Females_gene expression_Gonad_cyp19a_F0</td><td></td><td></td><td></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 6 (Continued)</caption><thead><tr><th>Experiment no.</th><th>Assessed endpoint</th><th>External validity</th><th>Internal validity</th><th>Precision</th></tr></thead><tbody><tr><td>1_1</td><td>Females_gene expression_Liver_vtg1_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain_gnrh2</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain_gnrh3_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain_fshB</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain IhB_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain_cyp19b_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Brain_arB_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_fshr_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_lhr_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_cyp11a_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_3Bhsd_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_cyp17_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Gonad_cyp19a_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>Males_gene expression_Liver_vtg1_F0</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>F1_hatchability_continously exposed</td><td></td><td></td><td></td></tr><tr><td>1_2</td><td>F1_hatchability_unexposed</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>F1_survival_continously exposed</td><td></td><td></td><td></td></tr><tr><td>1_2</td><td>F1_survival_unexposed</td><td></td><td></td><td></td></tr><tr><td>1_1</td><td>F1_malformations_continously exposed</td><td></td><td></td><td></td></tr><tr><td>1_2</td><td>F1_malformations_unexposed</td><td></td><td></td><td></td></tr><tr><td>1_3</td><td>FET survival</td><td></td><td></td><td></td></tr><tr><td>1_3</td><td>FET hatchability</td><td></td><td></td><td></td></tr><tr><td>1_3</td><td>FET abnormalities</td><td></td><td></td><td></td></tr></tbody></table>

Note: Each question is scored as either definitely low RoB (dark green), probably low RoB (light yellow), or definitely high RoB (dark red).

## Relevance assessment

The WG discussed the papers for their possible relevance to the human hazard assessment of acetamiprid. The main points are summarised below per study.

* Li, He, et al. (2022): The WG noted that most lines of evidence were scored as Tier 3 (high RoB), except from the in vitro experiments assessing the effects of acetamiprid on mRNA expression of the GPER (Tier 2). The in vivo investigated endpoints (increase in tumour volume and metastasis of in situ-injected cancer cells) are non-standard endpoints for hazard assessment of active substances and therefore were not considered relevant for the hazard assessment of acetamiprid. The observed in vitro and in vivo effects may be of relevance in the assessment of possible estrogenic activity of acetamiprid mediated via G protein-coupled receptors. However, the lack of an ED assessment for acetamiprid prevents from drawing any conclusion from a single study.

* Mishani et al. (2022): The WG noted that all endpoints were scored as Tier 3 (high RoB); the study cannot be used for hazard characterisation, as exposure levels have not been determined (only concentration in drinking water was reported, but information on consumption of drinking water and related acetamiprid exposure are lacking). Also, in the two acetamiprid exposure groups, reported food consumption was half of the control group, and weight loss was reported within the 60-day exposure period, pointing to general toxicity that biases the study results.

* Didenko et al. (2022): The WG noted that the lines of evidence were scored as Tier 3 (high RoB). Although it was indicated that the study was conducted according to OECD 408 with deviations, providing possible relevant data for the hazard assessment, serious shortcomings in the outcome assessment were identified (scored as high risk of bias), and the study was therefore not further considered. The histopathology procedures are not sufficiently described or not fully adequate, and the histopathological findings (descriptions and figures) are of poor quality and overall indicate artefactual/autolysis findings. No obvious pathological features can be confirmed. The WG also noted that, based on the evaluation of the reported histological findings, the adequateness of organ weight assessment is questioned (e.g. although the brain congestion and oedema reported would contribute to increased brain weight, decreased brain weight was observed). Based on the available pictures of liver histology, it was noted that blood was present in portal and centrilobular veins, as well as in some capillaries, indicating incomplete exsanguination, which may have affected the organ weight findings, especially regarding liver and spleen.

* Li, Si, et al. (2022): The WG noted that the lines of evidence were scored as Tier 3 (high RoB). The relevance of this study is limited as it is a descriptive case series study using a convenience sample. This consisted of patients who experienced neurological symptoms and who underwent a lumbar puncture to obtain CSF. Therefore, results are not generalisable
to the general population. No determinants of exposure other than age and sex are reported. The study only reports on concentrations of acetamiprid and IM-2-1 in CSF, but it did not assess possible associations with health effects. The most important information provided by this study is that neonicotinoids (including acetamiprid and IM-2-1) can be detected in CSF of diseased patients in a hospital setting, but this does not necessarily entail an impairment of the nervous system. As such, the data cannot be used for hazard assessment. As concluded in Section 2.3, an estimation of related external exposure levels based on human biomonitoring data (internal concentrations of acetamiprid and/or IM-2-1) was not deemed feasible, given the lack of sufficient kinetic data to develop a sufficiently robust and reliable PBK model to allow translation of internal exposure data to related estimated external exposure levels.

* Yang and Liang (2023): The WG noted that the lines of evidence were scored as Tier 1 (low RoB). The significant associations found with changes in certain haematological parameters cannot be considered as adverse (i.e. clinically relevant) because there is no indication that these parameters are above or below the normal reference interval. Due to the cross-sectional design, no conclusion about temporality and causality can be drawn between exposure (IM-2-1) and outcome. Furthermore, potential co-exposure to other chemicals affecting haematological parameters was not studied. It is worth noting that only a single spot urine sample was measured, which is not representative of true daily exposure. Furthermore, the detection rate of acetamiprid was < 5.0%, while that for IM-2-1 was 32.6%, which may point to direct exposure to this metabolite, which can be pre-formed in the environment.

* Mendy and Pinney (2022): The WG noted that the lines of evidence were scored as Tier 1 (low RoB). Due to the cross-sectional design of this study, temporality and causality between exposure (IM-2-1) and outcome (reduced testosterone) cannot be established. Only a single spot urine sample was measured, which is not representative of true daily exposure. The accuracy of FAI as a proxy for free testosterone is matter of debate (Vermeulen et al., 1999). The potential contribution of co-exposure to other chemicals affecting serum testosterone levels was not considered. Furthermore, the detection rate of acetamiprid was considered as low, but not reported (between 0.7% and 7.9% for several neonicotinoids), in contrast to the detection rate of IM-2-1 (33.4%), which may suggest possible direct exposure to this metabolite, as IM-2-1 can be pre-formed in the environment. More studies are needed to evaluate the reproducibility of these findings in different populations and using epidemiological designs that allow inferring causality.

* Ma et al. (2022): All parameters assessed have a high risk of bias for internal validity (Table 6). Clear signs of systemic toxicity (mortality > 10%) were observed, and thus, the tested concentrations were likely inappropriate. Moreover, although some effects were observed, those effects are likely due to excessive toxicity rather than triggered by an ED MoA.

### 2.1.3.3 | Uncertainty analysis and conclusions by the EFSA WG

Based on the RoB analysis and relevance assessment, the WG performed an uncertainty analysis to conclude whether the new studies notified by PAN Europe in the mandate provide information that would warrant a re-evaluation of the conclusion achieved for acetamiprid for the in vivo evidence.

Regarding the new evidence from the in vitro and in vivo studies, the WG considered that, based on the RoB analysis and relevance assessment, the studies of Li, He, et al. (2022) and Mishani et al. (2022) cannot be used for the hazard assessment. The lines of evidence in Didenko et al. (2022) were considered of relevance for the hazard assessment, but were scored as high risk of bias (Tier 3), so these were not further considered.

Regarding the evidence from the human studies, the WG considered that the study of Li, Si, et al. (2022) cannot be used for the hazard assessment of acetamiprid, as it solely contains information on internal acetamiprid/IM-2-1 concentrations, but not about possible related (health) effects. Regarding the other human studies (Mendy & Pinney, 2022; Yang & Liang, 2023), the WG noted that given the cross-sectional designs, no conclusion about temporality and causality can be drawn between exposure and outcome, and that potential co-exposure to other chemicals was not controlled for. Therefore, the WG considered that these studies cannot be used for the hazard assessment of acetamiprid.

Regarding the fish study by Ma et al. (2022), overall, all the parameters measured in the study have a high RoB for internal validity (see Table 6). It was noted that, even if data would be considered reliable, a conclusion related to the study by Ma et al. (2022) should not be generalised to draw a conclusion on the ED properties of acetamiprid for non-target organisms, since an ED assessment in line with the ECHA/EFSA (2018) Guidance has not been performed.

In conclusion and answering problem formulation 2a, based on the EFSA systematic data collection and on the studies notified by PAN Europe for any toxicological endpoint but not DNT for acetamiprid and its metabolites, the current HBGVs for acetamiprid are considered protective. It is, however, noted that in line with the PPR Panel recommendation, an assessment of endocrine-disrupting properties for acetamiprid in line with EFSA/ECHA guidance document for the identification of endocrine disruptors is recommended and was outside the scope of this mandate (EFSA PPR Panel, 2022).

### 2.1.4 | Assessment for DNT: Integrated approaches to testing and assessment (IATA) for DNT hazard characterisation of acetamiprid active substance

#### Step 1. Establishment of a protocol with the predefinition of the methods for answering the problem formulation

In line with the summary provided in the methodologies section (see Section 2.1.2), Appendix 1 contains the protocol and the details on the methodology. This was established upfront to reduce data-driven decisions and increase transparency.
## Step 2. Data collection and appraisal results

For step 2, during the title and abstract screening, publications providing potentially relevant evidence for DNT were clustered in in vivo (containing in vivo experimental studies in rodents), in vitro (containing in vitro mechanistic studies and behavioural studies conducted in zebrafish embryos up to 120-h post-fertilisation) or human (containing HOS-biomonitoring) studies. After the title and abstract screening, 44 relevant publications remained that underwent a full-text review (Figure 2). Finally, 14 publications were included and classified in three categories: (a) in vitro (8 studies; 5 in vitro and 3 zebrafish studies), (b) human evidence containing biomonitoring data (3 studies) and (c) in vivo (3 studies). Regarding the human studies, it is noted that these do not describe a link between exposure and (health) effects and were only used for the exposure question (refer to Section 2.3). The full list of relevant references from the systematic literature review is presented in Table 7. DNT was thus the only outcome to be considered in further steps in the AOP-informed IATA in order to assess whether the new available evidence for DNT hazard characterisation, including HOS, experimental data from in vivo animal studies and NAMs (in vitro, zebrafish), integrated following an AOP-informed IATA conceptual framework, could warrant a re-evaluation of the current HBGVs for acetamiprid and/or its metabolites in line with the ToR1 and to answer problem formulation 2b.

Appendix 5 shows a summary of the material and methods and data published in all the selected studies.

<table><caption>TABLE 7 Relevant selected studies from the systematic literature review containing evidence of acetamiprid and measuring DNT effects in in vivo and in vitro studies.</caption><thead><tr><td>RefID*</td><td>Authors</td><td>Year</td><td>Title</td></tr></thead><tbody><tr><td>219</td><td>Loser, D., Hinojosa, M. G., Blum, J., Schaefer, J., Brüll, M., Johansson, Y., Suciu, I., Grillberger, K., Danker, T., Möller, C., Gardner, I., Ecker, G. F., Bennekou, S. H., Forsby, A., Kraushaar, U., Leist, M.</td><td>2021</td><td>Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons</td></tr><tr><td>66</td><td>Christen, V., Rusconi, M., Crettaz, P., Fent, K.</td><td>2017</td><td>Developmental neurotoxicity of different pesticides in PC-12 cells in vitro</td></tr><tr><td>1603</td><td>Lee, J., Escher, B. I., Scholz, S., Schlichting, R.</td><td>2022</td><td>Inhibition of neurite outgrowth and enhanced effects compared to baseline toxicity in SH-SY5Y cells</td></tr><tr><td>173</td><td>Kagawa, N., Nagao, T.</td><td>2018</td><td>Neurodevelopmental toxicity in the mouse neocortex following prenatal exposure to acetamiprid</td></tr><tr><td>261</td><td>Nakayama, A., Yoshida, M., Kagawa, N., Nagao, T.</td><td>2019</td><td>The neonicotinoids acetamiprid and imidacloprid impair neurogenesis and alter the microglial profile in the hippocampal dentate gyrus of mouse neonates</td></tr><tr><td>317</td><td>Sano, K., Isobe, T., Yang, J., Win-Shwe, T. T., Yoshikane, M., Nakayama, S. F., Kawashima, T., Suzuki, G., Hashimoto, S., Nohara, K., Tohyama, C., Maekawa, F.</td><td>2016</td><td>In utero and lactational exposure to acetamiprid induces abnormalities in socio-sexual and anxiety-related behaviors of male mice</td></tr><tr><td>153</td><td>Hussain, A., Audira, G., Malhotra, N., Uapipatanakul, B., Chen, J. R., Lai, Y. H., Huang, J. C., Chen, K. H. C., Lai, H. T., Hsiao, C. D.</td><td>2020</td><td>Multiple screening of pesticides toxicity in zebrafish and daphnia based on locomotor activity alterations</td></tr><tr><td>364</td><td>Von Hellfeld, R., Ovcharova, V., Bevan, S., Lazaridi, M. A., Bauch, C., Walker, P., Hougaard Bennekou, S., Forsby, A., Braunbeck, T.</td><td>2022</td><td>Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays</td></tr><tr><td>225</td><td>Ma, X., Li, H., Xiong, J., Mehler, W. T., You, J.</td><td>2019</td><td>Developmental Toxicity of a Neonicotinoid Insecticide, Acetamiprid to Zebrafish Embryos</td></tr><tr><td>277</td><td>Öztaş, E., Kara, M., Boran, T., Bişirir, E., Karaman, E. F., Kaptan, E., Özhan, G.</td><td>2021</td><td>Cellular Stress Pathways Are Linked to Acetamiprid-Induced Apoptosis in SH-SY5Y Neural Cells</td></tr><tr><td>58</td><td>Cheng, L., Lu, Y., Zhao, Z., Hoogenboom, R. L. A. P., Zhang, Q., Liu, X., Song, W., Guan, S., Song, W., Rao, Q.</td><td>2020</td><td>Assessing the combined toxicity effects of three neonicotinoid pesticide mixtures on human neuroblastoma SK-N-SH and lepidopteran Sf-9 cells</td></tr></tbody></table>

All the selected papers underwent the RoB appraisal step. The outcome of the RoB is presented in Appendix 6 for in vivo (including in vivo mechanistic endpoints), in vitro and zebrafish lines of evidence. For the selected HOS for biomonitoring, see Section 2.3.

Figure 3 presents an overview of the results of the appraisal by specific endpoint for each Tier (Tier 1 (low RoB), Tier 2 (moderate RoB), Tier 3 (high RoB)) and for each line of evidence.

<footnote>*RefID is the identification number in Distiller (see Appendix 1).</footnote>
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Summary of the appraisal of the endpoint categories of the in vivo, in vitro and zebrafish studies selected from the systematic literature review for acetamiprid measuring DNT endpoints.</figcaption></figure>

The body of evidence of in vivo studies

Appendix 5 shows the summary of the materials and methods and results of the selected studies. During the data extraction of the studies selected from the systematic literature review, it was noted that three in vivo studies showed potential DNT effects in rodents. These studies (Kagawa & Nagao, 2018; Nakayama et al., 2019; Sano et al., 2016) underwent a detailed RoB appraisal. Sano et al. (2016) concluded that in utero and lactational exposure to acetamiprid impaired socio-sexual and anxiety-related behaviour in male mice starting from the dose of 1 mg/kg bw per day. Nakayama et al. (2019) concluded that acetamiprid changed the neurogenesis in the hippocampal dentate gyrus and the microglia profile in the hippocampal dentate gyrus at 5 mg/kg bw per day after postnatal exposure; Kagawa and Nagao (2018) concluded that neonatal exposure to 5 mg/kg bw per day resulted in a reduction of hippocampal neurogenesis and increased neuronal apoptosis after prenatal and postnatal exposure. Table 8 shows a summary of the materials and methods and measured endpoints of these studies considered relevant for DNT assessment of acetamiprid.

<table><caption>**TABLE 8** Overview of endpoints and study and exposure characteristics of the studies selected providing evidence for key events (KE) and adverse outcomes (AO) of the postulated AOP and considered in the uncertainty analysis (UA) for in vivo evidence.</caption><thead><tr><th>Publication</th><th>Adverse outcome/key event endpoints measured</th><th>Study characteristics</th><th>Exposure characteristics</th></tr></thead><tbody><tr><td>Sano et al. (2016)</td><td><strong>KE: Number of AVP neurons in hypothalamus</strong><br>Number of arginine vasopressin (AVP) immunoreactive neurons in paraventricular nucleus of the hypothalamus<br><strong>AO: Gross pathology</strong><br>Brain weight (absolute, relative)<br><strong>AO: Behaviour</strong><br>Male sexual behaviour<br>Female sexual behaviour (number of lordosis)<br>Male aggressive behaviour (chasing, boxing, wrestling, biting, tail rattling, lateral attacks)<br>Male anxiety behaviour (light compartment: (a) time spent there, (b) total distance travelled and (c) latency to enter)<br>Female anxiety behaviour<br>Behavioural flexibility test</td><td>C57BL/6J mice<br>Developmental neurotoxicity<br>There were no effects on body weight of male and female mice at birth, PND21 or 23–26 weeks of age</td><td>0, 1, 10 mg/kg bw per day<br>Daily exposure of dams from GD6-LD21<br>Acetamiprid 98% purity</td></tr><tr><td>Kagawa and Nagao (2018)</td><td><strong>KE: Neurogenesis</strong><br>NSC (Ki67 + Cidu + IdU) neocortex E14<br>NSC (Ki67 + Cidu + IdU) neocortex PND14<br>Microglia profile neocortex<br><strong>AO: Gross pathology</strong><br>Brain weight (relative and absolute)</td><td>ICR mice<br>Developmental neurotoxicity study<br>There were no effects on body weight</td><td>0, 5 mg acetamiprid/kg bw per day with oral gavage to the dams from GD6 to GD13 or GD6 to GD18<br>Acetamiprid 99.9% purity</td></tr></tbody></table>

(Continues)
<table><caption>**TABLE 8** (Continued)</caption><thead><tr><th>Publication</th><th>Adverse outcome/key event endpoints measured</th><th>Study characteristics</th><th>Exposure characteristics</th></tr></thead><tbody><tr><td>Nakayama et al. (2019)</td><td><strong>KE: Neurogenesis</strong><br>NSC Proliferation (CidU and Ki67)<br>Neuronal count in DG<br>Cell cycle exit of neural stem cells, stained with anti-Ki67 and anti-IdU 24 h after IdU administration at PND27<br><strong>KE: Change in microglia profile in the hippocampal Dentate Gyrus</strong><br>Microglia profile<br><strong>AO: Gross pathology</strong><br>Brain weight (absolute, relative)</td><td>ICR mice<br>Developmental neurotoxicity study with daily exposure (oral gavage) from PND12-PND26 males and females (12 newborns used from 3 dams)</td><td>0, 5 mg acetamiprid/kg bw per day<br>Acetamiprid 99.9% purity</td></tr></tbody></table>

The results of the systematic literature review indicated that DNT AO and/or KE could occur at doses potentially below the current NOAEL for DNT. It was however noted that all new public literature studies measuring DNT endpoints in vivo that have been made available since the renewal of approval of acetamiprid and considered relevant by the EFSA WG (Kagawa & Nagao, 2018; Nakayama et al., 2019; Sano et al., 2016), were appraised as high RoB and considered not reliable due to major limitations in several key questions of the CAT (see **Table 9** for a summary of the appraisal and **Appendix 6** for the detailed appraisal of each endpoint measured in the papers).

<table><caption>**TABLE 9** Heatmap results of the RoB for in vivo studies from public literature.</caption><thead><tr><th>DNT endpoints measured and appraised in Sano et al. (2016)</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>Brain weight (absolute, relative)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>ACE concentration in brain</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Male sexual behaviour</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Female sexual behaviour (number of lordosis)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Male aggressive behaviour (chasing, boxing, wrestling, biting, tail rattling, lateral attacks)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Male anxiety behaviour (light compartment: (a) time spent there, (b) total distance travelled and (c) latency to enter)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Female anxiety behaviour</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Number of arginin vasopressin (AVP) immunoreactive neurons in paraventricular nucleus of the hypothalamus</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Behavioural flexibility test</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody><thead><tr><th>DNT endpoints measured and appraised in Kagawa and Nagao (2018)</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>NSC (Ki67+CidU+IdU) neocortex E14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>NSC (Ki67+CidU+IdU) neocortex PND14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Microglia profile neocortex</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Brain weight (relative and absolute)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody><thead><tr><th>DNT endpoints measured and appraised in Nakayama et al. (2019)</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>NSC Proliferation (CidU and Ki67)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Neuronal count in DG</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Microglia profile</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Brain weight (absolute, relative)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Cell cycle exit of neural stem cells, stained with anti-Ki67 and anti-IdU 24 h after IdU administration at PND27</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody></table>

Note: The heatmap includes the endpoint-specific ratings for the RoB for each of the nine questions in the CAT (i.e. domains of randomisation, allocation concealment, experiment conditions, research personnel blinded, attrition, exposure characterisation, outcome assessment, outcome reporting and other aspects, i.e. systemic or maternal toxicity). Each question is scored as either definitely low RoB (dark green), probably low RoB (light green), probably high RoB (light red) or definitely high RoB (dark red). The RoB questions highlighted in yellow correspond to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint (Q1 randomisation domain; Q6 exposure characterisation; Q7 outcome assessment method). For detailed rationale for each rating, see Appendices 1 and 6.
Following the IATA framework, which requires all evidence to undergo the same appraisal and uncertainty analysis process for the WoE, an expert uncertainty analysis was conducted for all endpoints measured in the OPPTS 870.6300 regulatory study, as well as in the three in vivo studies selected from public literature. The OPPTS 870.6300 regulatory study was conducted during 1999–2000 and thus not following the current OECD 426 Test Guideline. The study was previously classified as acceptable/non-guideline (Netherlands, 2016; Table 10). It was also noted that the original report lacked methodological details and further amendments were made available providing more methodological details (Netherlands, 2016). Several reporting issues were also noted while evaluating the different reports submitted as part of the revised renewal assessment report (RAR) on acetamiprid (see this section ‘Step 5. Data integration in the AOP framework and final uncertainty analysis’).

Results of the appraisal are presented in **Appendix 6** and a summary by endpoint in **Table 11**.

In addition, the WG conducted a thorough uncertainty analysis in line with the analysis done by EFSA and ECHA for the ECHA report of the Extended One-Generation Reproductive Toxicity Study (EOGRTS) review project (ECHA, 2023). This evaluation included the analysis of the proficiency of the laboratory of the study (e.g. HCD, PCD, dynamic range for each endpoint, statistical analysis), the reporting, including information on the equipment used, the control of extraneous experimental factors, the time of day when testing is performed, as well as recurring methodological inadequacies of these studies. This was considered necessary after the appraisal exercise because of the consolidated experience gained by EFSA on assessing these studies. This detailed assessment is provided in **Appendix 7** and thoroughly discussed in the uncertainty analysis in this section (‘Step 5. Data integration in the AOP framework and final uncertainty analysis’).

<table><caption>**TABLE 10** Overview of the endpoints and study and exposure characteristics of the OPPTS 870.6300 regulatory study included in the revised RAR on acetamiprid (Netherlands, 2016).</caption><thead><tr><th>Adverse outcome/endpoints measured</th><th>Study characteristics</th><th>Exposure characteristics</th></tr></thead><tbody><tr><td><strong>AO: Functional observational battery:</strong><br>Detailed clinical observations in pups (i.e. ease of removal from cage and easy to handle animals; but the measurement of FOB (functional observational battery) manipulative endpoints is lacking)</td><td>Sprague-Dawley Rat Crl:CD*(SD)IGS BR<br>Developmental neurotoxicity study with oral gavage to dams from GD6-LD21<br>Maternal toxicity was observed only at 45 mg/kg bw per day</td><td>0, 2.5, 10, 45 mg/kg bw per day by oral gavage to dams from GD6-LD21<br>Acetamiprid > 99% purity</td></tr><tr><td><strong>AO: Motor activity</strong><br>Motor activity in males and females</td><td rowspan="5">Mean body weight on the day of vaginal patency was statistically significantly reduced in the 45 mg/kg bw per day group</td><td rowspan="5"></td></tr><tr><td><strong>AO: Startle reflex</strong><br>Auditory startle response in males and females</td></tr><tr><td><strong>AO: Learning and memory</strong><br>Learning and memory in males and females</td></tr><tr><td><strong>AO: Gross pathology</strong><br>Brain weight measured at PND11 and PND72</td></tr><tr><td><strong>AO: Morphometrics</strong><br>Morphometrics in males and females and brain size<br>Histopathology in males and females</td></tr></tbody></table>

<table><caption>**TABLE 11** Heatmap results of the RoB for the endpoints measured in the regulatory OPPTS 870.6300 study.</caption><thead><tr><th>DNT endpoints measured:</th><th>Q1</th><th>Q2</th><th>Q3</th><th>Q4</th><th>Q5</th><th>Q6</th><th>Q7</th><th>Q8</th><th>Q9</th><th>Tier</th></tr></thead><tbody><tr><td>Detailed clinical observations in pups (i.e. ease of removal from cage and easy to handle animals; but the measurement of FOB manipulative endpoints is lacking)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Motor activity in males and females (PND 13, PND 17, PND 21, PND 61)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Auditory startle response in males and females (PND 20, PND 60)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Learning and memory in males and females (PND 22 and PND 62)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Brain weight measured at PND11 and PND72</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Morphometrics in males and females and brain size (PND 11 and PND 72)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Histopathology in males and females (PND 11 and PND 72)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Gross pathology in males and females (PND 11 and PND 72)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr></tbody></table>

Note: The heatmap includes the endpoint-specific ratings for the risk of bias for each of the nine questions in the CAT (i.e. domains of randomisation, allocation concealment, experiment conditions, research personnel blinded, attrition, exposure characterisation, outcome assessment, outcome reporting and other aspects – i.e. systemic or maternal toxicity). Each question is scored as either definitely low RoB (dark green), probably low RoB (light green), probably high RoB (light red) or definitely high RoB (dark red). The RoB questions highlighted in yellow (Q1 randomisation domain; Q6 exposure characterisation; Q7 outcome assessment method) corresponds to the key RoB criteria considered in the final Tier (1 [low RoB], 2 [medium RoB] or 3 [high RoB]) of the endpoint. For detailed rationale for each rating, see Appendices 1 and 7.
In the study, a decreased maximum auditory startle response was observed in males from the dose of 10 mg/kg bw per day onwards at PND 20 and PND 60 (see **Table 12** for further details), attaining statistical significance in the 45 mg/kg bw per day group on both days (p < 0.05). The average response amplitudes ($V_{ave}$) in these males were also statistically significantly reduced compared to the control group values at PND 20 and 60, while latencies to maximum response amplitude ($T_{max}$) were similar to those in the control group at PND 20 and 60. On PND 20, $V_{max}$ and $V_{ave}$ were significantly reduced in females in the 45 mg/kg bw per day group in comparison to the control group values, while Tmax was significantly increased in comparison to the control group at this time point. The decreased maximum auditory startle response in males has been the basis for the NOAEL/LOAEL setting of the study by the peer review meeting (EFSA, 2016a; Netherlands, 2016).

<table><caption>**TABLE 12** $V_{max}$ effect as percentage of control in the regulatory OPPTS 870.6300 study.</caption><thead><tr><th colspan="6">V<sub>max</sub> as percent control</th></tr><tr><th>Sex</th><th>Age</th><th>0</th><th>2.5</th><th>10</th><th>45</th></tr></thead><tbody><tr><td rowspan="2">Male</td><td>20</td><td>100</td><td>85</td><td>73</td><td>58</td></tr><tr><td>60</td><td>100</td><td>90</td><td>60</td><td>47</td></tr><tr><td rowspan="2">Female</td><td>20</td><td>100</td><td>101</td><td>89</td><td>71</td></tr><tr><td>60</td><td>100</td><td>100</td><td>102</td><td>102</td></tr></tbody></table>

*Note:* An LOAEL has been set at 10 mg/kg bw per day based on decrease in maximum auditory startle response in males from the dose of 10 mg/kg bw per day onwards at PND20 and PND60.

## The body of evidence of in vitro studies

From the systematic literature review, eight studies were selected as relevant: five were conducted in vitro and three in zebrafish embryos. Two in vitro studies were not evaluated in detail (Cheng et al., 2020; Öztaş et al., 2021), due to the lack of relevance of the test systems as identified during the study appraisal. For more details on the studies, see Appendix 5.

**Table 13** shows information on the methods regarding measured endpoint, test system, exposure and RoB of the studies providing evidence for the MIE, KEs and AO.

<table><caption>**TABLE 13** Summary of endpoints measured, test systems used and exposure characteristics applied in the selected in vitro studies with the related RoB scores.</caption><thead><tr><th>Publication</th><th>Endpoint measured/KE</th><th>Test system</th><th>Exposure characteristics (& significant effect concentrations indicated by*)</th><th>RoB</th></tr></thead><tbody><tr><td>Loser et al. (2021)</td><td><strong>KE: Activation of calcium influx</strong><br>Measurements of intracellular Ca<sup>2+</sup> (preincubation with Cal-520 AM) as the mean of fluorescence signal of each well in 384 well plate (high throughput) or the single cell level</td><td>LUHMES (human DA mesencephalic neurons): 48 h pre-differentiated for Ca<sup>2+</sup>—imaging</td><td>0.1, 1.0, 10* and 100* µM acetamiprid purity > 99% up to 8 min</td><td>1</td></tr><tr><td>Loser et al. (2021)</td><td><strong>KE: Activation of calcium influx</strong><br>Electrophysiology measured using manual patch clamp</td><td>LUHMES (human DA mesencephalic neurons): 48 h pre-differentiated and another 7–8 days for patch clamp recording</td><td>100 µM acetamiprid purity > 99% 5 s</td><td>1</td></tr><tr><td>Loser et al. (2021)</td><td><strong>KE: Activation of calcium influx</strong><br>Measurements of intracellular Ca<sup>2+</sup> using Fura-2AM after 72 h of differentiation</td><td>SH-SY5Y cell line (human neuroblastoma, differentiated during 3DIV in the presence of RA, passage 50–70)</td><td>0.1, 1.0, 10* and 100* µM acetamiprid purity > 99% 150 s</td><td>1</td></tr><tr><td>Christen et al. (2017)</td><td><strong>KE: Neurite outgrowth</strong><br>Neurite outgrowth</td><td>PC12 (rat adrenal cancer) cell line differentiated towards neuronal phenotype with nerve growth factor (NGF) for 5 days</td><td>1, 10 and 100 µM of acetamiprid purity > 99%</td><td>3</td></tr><tr><td>Christen et al. (2017)</td><td><strong>KE: Neurite outgrowth</strong><br>Gene expression linked to altered neurite outgrowth</td><td>PC12 (rat adrenal cancer) cell line differentiated towards neuronal phenotype with NGF for 5 days</td><td>10* and 100* µM of acetamiprid purity > 99%</td><td>3</td></tr></tbody></table>
<table><caption>TABLE 13 (Continued)</caption><thead><tr><td>Publication</td><td>Endpoint measured/KE</td><td>Test system</td><td>Exposure characteristics (&amp; significant effect concentrations indicated by*)</td><td>RoB</td></tr></thead><tbody><tr><td>Lee et al. (2022)</td><td>KE: Neurite outgrowth<br/>Neurite outgrowth<br/>Cytotoxicity</td><td>Human neuroblastoma SH-SY5Y differentiation with RA for 72 h</td><td>620, 1500 and 2600 µM of acetamiprid purity unknown 24 h</td><td>3</td></tr><tr><td>Hussain et al. (2020)</td><td>AO: Locomotor behaviour<br/>Locomotor activity in zebrafish (travelled distance during the light cycle, total travelled distance during the dark cycle, pattern of locomotor activity, burst activity)</td><td>Zebrafish AB strain</td><td>1 ppb acetamiprid in water purity ≥ 98% 24 h</td><td>3</td></tr><tr><td>Ma et al. (2019)</td><td>AO: Locomotor behaviour<br/>Locomotor activity in zebrafish (spontaneous movement, head and tail touch response)</td><td>Zebrafish AB strain</td><td>107, 537, 760 and 974 mg/L acetamiprid purity 98.1% 120 h</td><td>3</td></tr><tr><td>Von Hellfeld et al. (2022)</td><td>AO: Locomotor behaviour<br/>Locomotor activity in zebrafish (coiling assay (21–47 hpf), swimming assay (83–120 hpf))</td><td>Zebrafish West aquarium strain</td><td>6.25; 12.5; 25; 50 and 100 µM acetamiprid purity unknown 120 h</td><td>3</td></tr></tbody></table>

From the in vitro BoE, only the study from Loser et al. (2021) was appraised as low RoB. This study tested whether acetamiprid and other neonicotinoids induced changes of the free intracellular Ca²⁺ concentration ([Ca²⁺]i) in two different test systems, SH-SY5Y human neuroblastoma cells and human dopaminergic post-mitotic neurons generated from LUHMES neuronal precursor cells. Both test systems were characterised for the presence and function of nAChR subunits including a7, a4 and b2 at different days in vitro (DIV). Nicotine, applied as a positive control, triggered typical neuronal signalling responses that were blocked by nAChR antagonists, such as tubocurarine and mecamylamine. Acetamiprid induced Ca²⁺ influx in neuronal cells mediated by the activation of nAChRs subunits (including a7 and a4b2) at 10–100 µM. This effect was strongly potentiated by PTU (an allosteric modulator of a7 nAChR subunit). The presence of functional nAChR subunits was confirmed by using a voltage clamp technique. Similar effects were observed with other neonicotinoids (i.e. imidacloprid, clothianidin and thiacloprid) but not with thiamethoxam and dinotefuran. It was also observed that pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1–10 µM) blunted the signalling response of nicotine causing cross-desensitisation starting from 1 µM. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids resulting in potential toxicity to human brain during development. These results are in line with earlier findings obtained with other mammalian cells, i.e. primary rat brain cultures (Kimura-Kuroda et al., 2012), which provided evidence for potential DNT effects caused by acetamiprid and imidacloprid. However, follow-up studies are required to judge the full toxicological implication for human brain development (i.e. downstream neurofunctional adverse effects).

### Step 3. Data analysis of the developmental neurotoxicity in vitro battery (DNT IVB) results for acetamiprid

Recently, the OECD published the Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In Vitro Testing Battery (DNT IVB; OECD, 2023). The document contains the descriptive standardisation of a battery of 17 assays and recommendations on the use of data in the context of hazard assessment or weight of evidence determination. The assays included are not based on molecular targets, as no comprehensive list of targets is currently known, but instead on fundamental neurodevelopmental processes known to be necessary for proper nervous system development, as reported in Figure 4. The OECD document describes the assays that comprise the battery in terms of neurodevelopmental processes and provides criteria for the evaluation of the relevance of the data to DNT and assists in the determination of the degree of certainty in any positive or negative findings to better inform the use of DNT in vitro data in regulatory hazard assessments (OECD, 2023). It is noted that at the time of publishing this Statement, the OECD project on DNT is still ongoing with further activities to test more chemicals, develop more assays, validate assays already available (e.g. inter-laboratory testing of all assays using defined lists of positive and negative compounds), develop tiered testing strategies as follow-up of the DNT IVB outcomes (e.g. orthogonal assays to confirm positives or negatives or mechanistic testing in rodent assays) and propose more case studies (OECD, 2023).
<figure><img src="image_6.png" /><figcaption>**FIGURE 4** Assays in the current DNT IVB and assays identified as high priority for development (from OECD, 2023). Assays are grouped according to the neurodevelopmental process evaluated (rows) and test system used (columns). Each assay box lists the abbreviated assay name, cell type and assay development laboratory. Assays that need further development, and not included in the current DNT IVB, are identified as data gaps. Each assay is represented as a box that lists the test method name (italics), the test system (cell type used) and the home institution of the developer (IUF, Leibniz Research Institute for Environmental medicine - green; UKON, University of Konstanz - grey; EPA, US Environmental Protection Agency - blue). Other abbreviations can be found in the abbreviations list.</figcaption></figure>

As indicated in the OECD Initial recommendations document Appendix E, acetamiprid has been tested in all assays included in the DNT IVB (i.e. 17 assays as described in Figure 3 of that document) and data are reported in the EFSA report (Masjosthusmann et al., 2020) and can be retrieved from the ToXCast dashboard (https://comptox.epa.gov/dashboard/).

Figures 5, 6 and 7 show the heatmap results for the DNT IVB assays of acetamiprid. The WG extracted the data of the DNT IVB for acetamiprid, nicotine and other neonicotinoids. Results for other neonicotinoids and nicotine are also shown in the figures. A detailed uncertainty analysis was performed for acetamiprid and in part for nicotine, while data for the other neonicotinoids were mainly used for comparison purposes in the uncertainty analysis and the results are presented in this section. A detailed description of the assays is reported in the OECD initial recommendation document (OECD, 2023). Figure 5 shows the heatmap of acetamiprid, nicotine and other neonicotinoids in the US EPA DNT IVB Neural Network Formation (NNF) assay (extracted from invitrodb v3.5 in June, 2023) (Shafer et al., 2019; Harrill et al., 2010; OECD, 2023). Figure 6 shows the heatmap of acetamiprid, nicotine and other neonicotinoids in the US EPA DNT High Content Image (HCI) cellular event assays for Assessing Chemical Effects on Neurodevelopment Processes (i.e. proliferation in neural progenitor cells hNP1; apoptosis in neural progenitor cells hNP1; neurite initiation in human neurons; neurite initiation in cortical rat primary neurons; neurite maturation in rat primary neural culture; synaptogenesis in rat primary neural culture) (extracted from invitrodb v3.5 in June, 2023; OECD, 2023). Figure 7 shows the heatmap of acetamiprid, nicotine and other neonicotinoids in the EU DNT IVB (extracted from Blum et al., 2023). The EU DNT IVB detailed methodological details are fully reported in OECD (2023) and EFSA PPR Panel (2021).
<figure><img src="image_7.png" /><figcaption>**FIGURE 5** Heatmap of acetamiprid, nicotine and other neonicotinoids in the US EPA DNT IVB MEA (NNF) assay, including positive and negative performance controls. Data extracted from ToXCast (invitrodb v3.5 in June, 2023). The following filters applied: ≥ 3 ToxCast flags capturing curve-fitting behaviour such as noisy data; curve-fits with a model top less ≤ 1.2 times the cut-off for a positive and a resultant AC50 lower than the concentration range screened; concentrations series with fewer than four concentrations; excluded MEA NFA '_up' endpoints as not validated by positive performance controls.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**FIGURE 6** Heatmap of acetamiprid, nicotine and other neonicotinoids in the US EPA DNT high content image cellular event assays for assessing chemical effects on neurodevelopment processes. Data extracted from ToXCast (invitrodb v3.5 in June, 2023). Assays included proliferation in neural progenitor cells hNP1; apoptosis in neural progenitor cells hNP1; neurite initiation in human neurons; neurite initiation cortical rat primary neurons; neurite maturation in rat primary neural culture; synaptogenesis in rat primary neural culture.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**FIGURE 7** Heatmap of acetamiprid, nicotine and other neonicotinoids in the EU DNT IVB. Data extracted from Blum et al. (2023) and EFSA PPR Panel (2021).</figcaption></figure>

As indicated in **Figures 5, 6** and **7**, acetamiprid is inactive across the DNT IVB.

The WG, in the context of this IATA case, carefully considered the negative outcome, recognising the limitations of the DNT IVB listed in the OECD initial recommendation document, e.g. lack of coverage for some neurodevelopment processes, and the limited characterisation of some of the test systems for the expression and ontogeny of the nACh receptor subunits (OECD, 2023) (see also **Figure 4**).

Therefore, the WG retrieved gene expression data for the following nAChR subunits, which were analysed in some of the in vitro models (not all) included in the DNT IVB: nAChRa2, nAChRa3, nAChRa4, nAChRa6, nAChRa7, nAChRa8, nAChRa9, nAChRa10, nAChRb2, nAChRb3 and nAChRb4 and classified as present, borderline and absent according to the criteria described in Masjosthusmann et al. (2020). This exercise was part of the uncertainty analysis for the relevance of the DNT IVB for assessing DNT effects of acetamiprid. The WG concluded that:

* Human fetal neural progenitor cells (hNPC) showed borderline expression of nAChRb2 and the remaining subunits was absent. The NPC transcriptomic profile during neural differentiation was analysed using PrimeView Arrays (Affymetrix). In the neural crest cells differentiated from hiPSCs (IMR90), the absence of all nAChR subunits was confirmed, except for
nAChRa8, which was not measured.

* LUHMES cells (post-mitotic mesencephalic DA neurons, CNS) showed the presence of nAChRa3, nAChRb2, nAChRb4, borderline expression of nAChRa4 and nAChRa9. The presence of nAChRa8 subunit has not been measured.

* hiPSC-derived immature dorsal root ganglia (DRG) expressed nAChRa3, nAChRa4, nAChRb2, nAChRb4 and presence of nAChRa8 subunit has not been measured.

* Human iPSC-derived neurons (SynFire, used in the neuronal network formation (hNNF) assay) expressed nAChRa3, nA-ChRa4, nAChRb2, nAChRb4, borderline presence of nAChRa6 and nAChRa7. nAChRa8 subunit expression has not been measured.

The gene expression of the nAChR subunits has not been analysed yet in the following in vitro models of the DNT IVB:

* hNP1 Human Progenitors fully differentiated, derived as adherent cells from hESC WA09 line to evaluate cell viability, apoptosis and high-content imaging assay to screen for changes in neuron proliferation (DNT IVB US EPA).

* hiPSC-derived iCell GlutaNeurons (FujiFilm Cellular Dynamics, USA) for high-content imaging assay to screen for changes in neurite outgrowth.

* Rat primary cortical neurons differentiated for 12-14 days in vitro applied for evaluation of neuronal network formation (rNNF), neurite maturation, synaptogenesis, viability and high-content imaging assay to screen for changes in neurite outgrowth (US EPA). However, published data suggest that primary rat cells from brain cortex, in vitro express α4β2-nAChRs and α7-nAChRs as measured for instance by the spontaneous [Ca²⁺]ᵢ oscillations and nicotine-induced [Ca²⁺]ᵢ oscillations (Wang et al., 2016). Earlier studies also suggest that nAChR-binding sites are present in rat primary neuronal cells in culture (Dávila-García et al., 1999).

It is noted that in vitro models (preferably based on human cells) with an advanced stage of neuronal differentiation and functional nAChR subunits are necessary to produce relevant in vitro data for the assessment of the neurodevelopmental toxicity of acetamiprid. The negative results of the testing of acetamiprid in the DNT IVB are considered uncertain due to the lack of relevant models with advanced stage of neuronal differentiation and functional nAChR subunits. Before chemical testing, each model should be characterised for the presence of different neuronal and glial cell types, expression of critical receptors, neurotransmitters, enzymes, pathways/mechanisms, etc., that are relevant for the specific class of tested chemicals. Such a model characterisation will reduce uncertainty and will facilitate the interpretation of the in vitro data relevant for hazard assessment.

In view of the identified limitations of the DNT IVB, the negative results obtained with acetamiprid are associated with high uncertainty and it cannot be excluded that acetamiprid is a false-negative substance when tested in the DNT IVB.

### Step 4. Data analysis of the EUTOXRisk acetamiprid IATA case study number 365

The EUTOXRisk IATA case study has been published in the OECD Case Study program as Case Study no. 365 (OECD, 2022c) with the following problem formulation: Can new approach methods (NAM) data in an IATA context (integrating existing information) on acetamiprid sufficiently characterise DNT hazard? The EUTOXRisk case study postulated an AOP and concluded as follows:

‘The available in vivo data does not corroborate with each other, and no consistent effects have been established. This is based on the Scientific Opinion on the developmental neurotoxicity potential of acetamiprid and imidacloprid (EFSA PPR Panel, 2013) conclusion that there were uncertainties in the OPPTS 870.6300 regulatory study on acetamiprid that prevented a firm conclusion regarding motor activity and learning and memory, whereas decreased auditory startle response was found at 10 and 45 mg/kg bw per day. The mechanistic data reported by Kimura-Kuroda et al. (2012) did provide some mechanistic understanding, but the study had limitations. Subsequent in vivo DNT studies in mice found that acetamiprid might affect neurogenesis (Kagawa & Nagao, 2018; Nakayama et al., 2019) and reduced anxiety-related behavior in males. Regarding the in vitro evidence, the OECD IATA Cs 355 concluded that acetamiprid induces Ca²⁺ influx via the nAChR as well as attenuated voltage-operated calcium channel (VOCC) function after exposure at concentrations between 1 and 100 µM. Functional endpoints in terms of neurite outgrowth have been investigated but no statically significant effects were seen, yet a reduced dendritic area was observed in rat Purkinje cells. Two of the studies reviewed performed transcriptomics after exposure and observed differentially expressed genes (DEGs) relevant for neuronal development. However, some of the studies were scored as of high RoB because only one concentration was studied, less relevant mammalian models were used, or they did not assess cytotoxicity.

Further testing in the DNT IVB and additional systems/endpoint firmly established that neither acetamiprid nor nicotine affects these endpoints, nor do they exert agonism or antagonism on various nuclear receptors, PPARα and AhR, or activated stress pathways in the CALUX reporter gene assay. However, it is established that acetamiprid has similar, but less potent, activity than nicotine on nAChR-mediated Ca²⁺ influx in neuronal systems. There remain significant uncertainties regarding downstream KEs, including a specific adverse outcome. No effects were observed in the proposed KE3 and KE4 with the assays used after direct exposure with acetamiprid or nicotine, but the possibility cannot be ignored that if the nAChR remains bound by an agonist a conformational change of the receptor leading to a nonfunctional state (termed desensitization) may alter evoked responses, e.g., voltage operated ion channel function, transmitter release or neural network function. This major uncertainty is currently undergoing further investigations. As a first attempt, analysis of DEGs in test systems where acetamiprid and nicotine are active
showed no major effects for any of the systems. Thus, KE3 cannot be confirmed using transcriptomics. Another possibility would be investigating activation/inactivation of signaling pathways as exemplified above.'

Despite some of the data considered are also in the EFSA IATA BoE, a different approach was used by the WG to retrieve, appraise and weight the evidence. More importantly, a different problem formulation and regulatory framework was used. The WG therefore recommends EFSA to update the OECD case study no. 365 or to adapt this statement to an AOP-informed IATA and to submit it to the OECD case study project.

**EFSA has evaluated the data generated and reported in the EUTOX Risk IATA as part of the BoE but notes that an independent analysis of the evidence, including a critical appraisal, would have been necessary for using the data in the regulatory decision-making process of acetamiprid.**

### Step 5. Data integration in the AOP framework and final uncertainty analysis

The evidence was clustered hierarchically by evidence streams first (in vivo, in vitro, zebrafish), then by publication, then by endpoint categories and finally integrated in an AOP framework as potential AOs, KEs and MIEs. Two experts of the WG were independently asked to identify the relevant DNT endpoints in the BoE and classify them as potential MIEs/KEs or AOs, taking into account the previous work in the postulated AOP of the OECD IATA Case Study no. 365. Then, the results of this exercise were discussed in a WG meeting. The postulated AOP from EUTOXRisk was used as a framework to organise the evidence retrieved and appraised independently by EFSA.

The study results are displayed together with the main experiment characteristics and AO/KEs identification in Appendix 5.

Data systematically collected and appraised, as reported in the previous sections, were integrated using the AOP conceptual framework and uncertainty analysis, in two steps. First, synthesis, integration and uncertainty analysis of the evidence on AO were performed from experimental in vivo and zebrafish studies. All endpoints measured in each study were included in the data synthesis, regardless of the Tier allocation in the appraisal. Second, synthesis, integration and uncertainty analysis of mechanistic evidence on MIE and KEs were performed from in vitro and in vivo mechanistic studies. All endpoints measured in the studies were included in the data synthesis, regardless of the Tier of the appraisal. To conduct fit for purpose hazard identification and characterisation, only endpoints falling in Tiers 1 or 2 of internal validity were used in the final uncertainty analysis for drawing conclusions on a DNT effect of acetamiprid and included in the postulated AOP. Tier 3 endpoints were not used in the uncertainty analysis due to the high risk of bias (see Appendix 5 with the data extraction summary data). The uncertainty analysis and the expert knowledge elicitation (EKE) methodologies are reported in Appendix 1 and were performed in line with EFSA Guidance on uncertainty analysis (EFSA and EBTC, 2018; EFSA Scientific Committee, 2018a, 2018b). It is noted that a similar approach and workflow was applied in the AOP-informed IATA for deltamethrin DNT hazard characterisation and this stepwise approach culminated in the development of an evidence-based stressor-based AOP (EFSA PPR Panel, 2021; OECD, case study 362 [OECD, 2022a]).

### Outcome of the EKE for the BoE of in vivo studies

In order to conclude on the AO, in line with the protocol part 2 (see Appendix 1), the evidence retrieved for the AO was assessed for reliability and relevance. An uncertainty analysis was conducted using an EKE exercise in a WG meeting. For AO, seven experts participated in answering the questions (see Table 1) and conducted the exercise in an independent manner. Unanimously, all experts concluded that there are major uncertainties in the in vivo BoE available to identify and characterise the DNT potential of acetamiprid. The following main uncertainties were noted during the EKE discussions.

* Since the renewal of approval of acetamiprid active substance, as indicated in the results of the systematic literature review, new in vivo DNT studies became available (in particular Sano et al., 2016; Kagawa & Nagao, 2018; and Nakayama et al., 2019; see Table 7). The experts appraised these studies as of high RoB (Tier 3), reflecting major limitations in several key questions of the CAT (see Appendix 5 and Section 2.1.4.5). In accordance with the protocol, these endpoints were not included in the postulated AOP due to the high RoB.

* Regarding the DNT regulatory study ( ), evaluated in Netherlands, 2016, all measured endpoints were also appraised using the CAT and were all assigned to Tier 3 (high RoB). This was because at least one critical question was categorised as of high RoB or probably high RoB. Table 14 shows a summary of the uncertainties in the regulatory study, identified by the WG. However, the WG considered the study as acceptable for the auditory startle response and histopathology evaluation, as the only issue identified in the RoB appraisal for these endpoints was the lack of reporting for one aspect of randomisation and the study was conducted following good laboratory practices (GLP). The experts considered the lack of reporting of randomisation as not sufficient to dismiss the positive findings in the acute startle response from 10 mg/kg bw per day onwards, which showed a dose-response pattern and reproducibility over time (decrease was observed in the measurements at PND 20 and PND60 in males, see 'Step 2. Data collection and appraisal results' of this section). In addition, the WG noted that control values are in line with the historical control data (HCD) provided for this endpoint. The WG also noted that the variability in the HCD was limited, which was considered acceptable, indicating a negligible impact of the uncontrolled variables along the different studies. In addition, the positive control data (PCD) submitted for the startle response prove the proficiency of the laboratory to conduct the assay: Propylthiouracil (PTU) showed a clear dose response with up to 50% decrease in auditory startle response in both males
and females and methimazole showed good responses in males and females at PND20 and PND60. In contrast, the RoB assessment for learning and memory, motor activity and morphometric measurements, indicated that the study has major limitations in several key questions in the RoB analysis, particularly for the outcome assessment method. Therefore, no conclusion can be drawn on these measurements. Regarding the outcome assessment for motor activity, and learning and memory, the main constraints were related to HCD (high variability in responses, indicating no confidence in the applied methodology over time and/or lack of control of external variables) and PCD (limited response and high variability). For the morphometric analysis, the methodology and results were poorly reported, making their interpretation difficult. Moreover, the statistically significant effects observed in two measurements in females at high dose should have triggered the measurement of the endpoint in the low and medium doses, which was not done. The experts also noted that only detailed clinical observations were conducted but not a complete FOB is available in the study, which is a shortcoming compared to the current standard.

<table><caption>TABLE 14 Selected uncertainties identified in the in vivo guideline study available.</caption><thead><tr><td>Uncertainty</td><td>Uncertainty number</td><td>Description</td></tr></thead><tbody><tr><td>HCD and PCD for motor activity not acceptable</td><td>U1</td><td>The HCD provided are not acceptable since there is a lack of control of the test methods over time (e.g. PND17 controls range from about 400 to 1800 number of movements in 60 min of session (400–1200 if three inhalation studies are also included)). This was considered as being related to uncontrolled variables in the conduction of the assay by the laboratory. In addition, there are also reporting issues and the studies included were judged inadequate (e.g. use of inhalation studies). Regarding PCD (PTU and methimazole), the selected doses were considered correct (Crofton et al., 2008); however, data were considered as not acceptable because for PTU: (1) the reporting is insufficient (only a figure indicating % of control is provided); (2) the results are not clearly positive (the only statistically significant effect was observed at mid-dose on PND21; (3) habituation was not measured. PCD with methimazole showed an appropriate dynamic range in ontogenetic pattern of habituation, in line with other studies with the test item; however, the variability observed in the control groups is not acceptable</td></tr><tr><td>Learning and Memory assay not acceptable</td><td>U2</td><td>The Biel Water Maze was used for measuring learning and memory. However, several major limitations were noted that makes the data not reliable: (1) In the path A for learning (with 4 trials during 2 days), there is no learning apparent in controls at PND22; learning is not demonstrated in control animals; high-dose adult males made more errors than control but standard deviation (SD) > means; the SD was as large as the means for latencies and for errors at PND60; (2) in path B, the observed variability in the control animals would obscure any treatment related effect; (3) in the path A 2 testing for memory, an inconsistent pattern was observed across sex/age/treatment and high variability was especially observed in the treated groups<br>Overall, the assay was considered not acceptable and of not sufficient quality to determine effects on learning and memory</td></tr><tr><td>HCD and PCD Learning and Memory not acceptable</td><td>U3</td><td>Overall, the WG considered the provided HCD as not acceptable because there is no sufficient evidence in the report that the testing laboratory has control over time of the applied methodology. The data assessment is compromised due to inadequate reporting, as the tables only display mean values and not SD. In addition, means do not seem to be consistent across studies. Therefore, the WG could not properly assess and interpret the data<br>The use of PTU as PCD is considered unacceptable because of inadequate reporting; there is no learning curve and there was no effect of PTU on errors at any dose. There was an increase in latencies, but it was the same at all dose levels despite the doses are considered high. The PCD for methimazole are also considered unacceptable since the observed effects are not in line with the expected positive effects (see Appendix 7 section 5)</td></tr><tr><td>Morphometrics evaluation</td><td>U4</td><td>The WG noted the following limitations: (1) no inclusion of brain size measurements; (2) no samples images of the measured areas are provided in the methodology; (3) the WG considered the statistical analysis inadequate (e.g. sex was not included as a factor); (4) blinding was not reported. More importantly, only control and high-dose animals were investigated. It is noted that a statistically significant effect was observed in the ventral limb of the dentate hilus in the hippocampus in female at PND72. In addition, in females, a statistically significant effect was observed on the height between hippocampal pyramidal neuron layers at PND11. The magnitude of the effect on morphometry was greater than 10% of the controls. The WG noted that these changes should have been further addressed by peer review for the correctness of the sampling, and by conducting further statistical analysis. These changes should have also triggered an investigation of the endpoint in the low and mid dose</td></tr><tr><td>FOB not complete</td><td>U5</td><td>Only detailed clinical observations were measured in the study and the WG noted that the FOB was not available in line with the current standard (i.e. OECD TG 426 and OECD TG 443 Annex 1). See also Sections 1 and 2 of Appendix 7</td></tr><tr><td>Number of animals</td><td>U6</td><td>Only 10 pups per group were included for all DNT endpoints (at least 20/sex per litter/dose are required in the OECD TG 426 for several DNT endpoints)</td></tr></tbody></table>

Additionally, the WG decided to evaluate the ontogeny of the MIE, i.e. the developmental timeline of nAChR expression, as an important step to assess the relevance of the AO. This assessment was not part of the EKE but reported here because
of the relevance of the dynamic changes in the expression and localisation of the nAChR isoforms to better understand the relevance of the in vivo models used in the assessment. The WG also considered the importance of the nAChR ontogeny for the assessment of the relevance of the in vitro models; though, the following discussion will mainly cover ontogeny-related uncertainties for in vivo studies.

nAChRs are pentameric ligand-gated cation channels consisting of various combinations of α and β subunits (e.g. α4β2 heterodimeric receptors) or only α subunits (e.g. α7 homomeric pentamers) (Broide et al., 2019). nAChRs are widely distributed throughout human and rodent brain over all phases of development (Alzu'bi et al., 2020). During ontogeny, nAChRs undergo changes in their distribution, subunit composition and function in the nervous system (Dvorakova et al., 2005). Nine out of 16 genes for human nAChR subunits are selectively expressed in human fetal cerebral cortex between 7.5 and 12 post-conceptional weeks (Alzu'bi et al., 2020). nAChRs are therefore present as early as the first trimester, gradually increasing up to mid-gestation and then declining in the third trimester (Mao et al., 2007).

The mRNA of the α7 subunit is strongly expressed as early as gestational day (GD) 13 in the cortical and hippocampal anlage of mice, and a similar temporal and spatial expression pattern has been observed in rat hippocampus (Broide et al., 2019). At GD 16, epibatidine and α-bungarotoxin binding sites (corresponding to heteromeric and α7 nAChRs, respectively) are widely distributed in the embryonic brain, although with different patterns. They are also abundant in neonate animals, with noticeable changes in some areas during the two to three postnatal weeks. The redistribution of nAChRs in the cerebral cortex, or their transient expression in the brainstem or hippocampus, may be related to the establishment of functional neuronal connections (Tribollet et al., 2004).

In rats, high transcript levels of neuronal nAChRs α4 and β2 subunits are observed at birth in cerebral cortex, medial habenula, CA1/CA3 regions of the hippocampus and several thalamic nuclei. In the latter, the expression of α4 and β2 subunit transcripts shows a biphasic pattern: the lowest levels occurring during the first and second postnatal weeks, respectively, and the highest levels during the second and fourth postnatal weeks, respectively. These findings suggest that the two nAChRs subunits are independently regulated in most of the brain areas (Cimino et al., 1995).

The α7 nAChR subtype is believed to be involved in the regulation of neuronal growth, differentiation and synapse formation during the development of the human brain (Falk et al., 2002). This receptor subtype has been implicated in hippocampal excitatory synapse formation and in the developmental GABAergic switch from excitation to inhibition, suggesting a functional role for these receptors in the developing hippocampus (Broide et al., 2019).

Transiently elevated expression of nAChRs in various brain regions during development contributes to maturation. The nAChR regulation of development occurs during 'critical periods' that are specific to each brain structure (O'Leary & Leslie, 2003).

The WG notes that the DNT OECD TG 426, with dosing during gestation and direct dosing of pups postnatally ensuring adequate exposure during lactation, would have been the appropriate study for acetamiprid to minimise the uncertainties due to the species differences in ontogeny (see Figure 8). However, several uncertainties on the proficiency of the laboratories to conduct the study as well as considerable uncertainties intrinsic to any TG 443 or TG 426 data regarding their reliability and relevance were also noted by the WG (Paparella et al., 2020).

<figure><img src="image_10.png" /><figcaption>**FIGURE 8** Nicotinic acetyl choline receptor (nAChR) ontogeny in rats and humans in the different brain areas. Figure extracted and adapted from OECD IATA Case Study no. 365.</figcaption></figure>
In conclusion, the WG noted that the available evidence is insufficient to robustly characterise a DNT potential and to provide a conclusive pattern of dose-response for acetamiprid. This is mainly due to the lack of adequate measurement of learning and memory, motor activity and brain morphometry during the critical windows of brain development.

## Outcome of the EKE for the BoE of vitro studies

In order to conclude on the mechanistic understanding, i.e. KE and KER, and in line with the pre-established protocol, the evidence retrieved for the identification of KE and their relationship (KER) was assessed for reliability and relevance. All data were extracted, and an uncertainty analysis conducted using an EKE methodology. Seven experts participated in answering the questions used to conduct the exercise (see Table 1) in an independent manner.

During the EKE, unanimously all experts concluded that there is no sufficient data to finalise an AOP-informed IATA that will allow to fully characterise the DNT mechanistic pathway of acetamiprid. The main uncertainties noted during the EKE discussions are presented in Table 15.

<table><caption>TABLE 15 Selected uncertainties identified in the available in vitro BoE.</caption><thead><tr><td>Uncertainty</td><td>Uncertainty number</td><td>Description</td></tr></thead><tbody><tr><td>Limited number of reliable in vitro studies from the literature</td><td>U1</td><td>New public literature studies measuring DNT endpoints in vitro have been made available since the renewal of approval of the active substance. Three have been considered relevant by the WG (Christen et al., 2017; Lee et al., 2022; Loser et al., 2021). According to the studies' authors they show evidence of DNT effects. The WG considered Lee et al. (2022) and Christen et al. (2017) as of high RoB due to limitations in one key question each (purity and cytotoxicity) Regarding Loser et al. (2021), the study was appraised as Tier 1 and a functional effect was observed from the concentration of 1 µM. This effect was considered as an immediate consequence of the nAChR activation and shows that human neuronal cells are functionally affected by low micromolar concentrations of acetamiprid and is therefore relevant</td></tr><tr><td>Expression of (functional) nAChR in DNT IVB test systems and relevant downstream KEs</td><td>U2</td><td>Acetamiprid has been tested in the DNT IVB (OECD, 2023) and no hit was observed. The WG noted that the expression of the nAChR in the DNT IVB test systems was not fully characterised. It is also noted that nicotine and other neonicotinoids showed negative results in the test battery. In addition, the nAChR desensitisation in the experimental conditions of the DNT IVB was considered a remaining uncertainty The WG concluded that there are important knowledge gaps that need to be addressed to identify and/or characterise downstream KEs. This may be done, for example, by the assessment of the neuronal network function (NNF) in the micro electrode array (MEA) using DA neurons derived from LUHMENS cells (Lund human mesencephalic cells representing human embryonic neuronal precursor cells)</td></tr><tr><td>Coverage of neurobiological processes during brain development by in vitro test systems, including those of DNT IVB</td><td>U3</td><td>The relevance of the available in vitro models, in particular the ones included in the DNT IVB, was evaluated by assessing the expression of the relevant targets and their functional activity upon activation. This assessment was not part of the EKE but reported in 'Step 3. Data analysis of the developmental neurotoxicity in vitro battery (DNT IVB) results for acetamiprid' in this section. This was considered as an important exercise to understand the relevance of the available in vitro models and correct in vitro data interpretation The WG noted that the DNT IVB test systems lack some features that are known to be critical in the development of the nervous system. Given the complex nature of brain development and the gaps in our current knowledge on these processes, the DNT IVB assays are not expected to cover the entire array of neurobiological processes during brain development. The applied in vitro models should be well characterised to know what they can or cannot detect</td></tr></tbody></table>

It was noted that limitations and uncertainties in the state of the art must be assessed for in vitro and in vivo data with the same scrutiny, and uncertainties are conceptually similar for in vitro and in vivo methods (Paparella et al., 2020). In vitro methods represent a powerful tool in examining specific mechanisms and have been proved to be fit for purpose when integrated in an AOP-informed IATA. Thus, the incorporation of the DNT IVB in the evaluation of DNT, along with the appropriate uncertainty analysis, would aid the data interpretation, by assembling multiple lines of evidence. Mechanistic in vitro data may represent DNT alerts as such and they may also contextualise negative, uncertain or positive apical endpoints in the in vivo results from guideline studies or public literature, and support a conclusion based on integrated evidence.

It was further noted that additional assays, possibly based on in vitro complex human models, should be developed as part of the DNT IVB. These additional models should be representative of key neuronal and glial subtypes at more advanced stages of morphological and functional differentiation and maturation. Before chemical testing, each model should be characterised for the presence of different neuronal and glial cell types and for the expression of critical molecular targets such as receptors, neurotransmitters, enzymes, pathways/mechanisms, etc.

### Step 6. AOP-informed IATA conclusions for DNT of acetamiprid by the EFSA WG

A critical step in the IATA iterative process is the postulation of an AOP (if not already available) that will inform which KEs are essential to move from the MIE to the AO and how they can be measured using in vitro assays. The WG used the AOP
conceptual framework to organise the evidence and conclude on the mechanistic pathway of acetamiprid. However, the lack of positive outcome in the DNT IVB for acetamiprid and the several uncertainties identified in the test systems and on the DNT IVB outcomes with nicotine and other neonicotinoids, were considered as a critical uncertainty by the WG, such that an acetamiprid-specific postulation of an AOP was considered too uncertain (see also Step 5 EKE results).

**Figure 9** shows the results of integrating the BoE from in vivo and in vitro studies using the AOP conceptual framework. The WG acknowledges the biological relevance of the effect of acetamiprid on the nAChR and the uncertainties within the AOP and the related methods, but nevertheless concluded that more empirical data should be made available to postulate an AOP-informed IATA for acetamiprid.

<figure><img src="image_11.png" /><figcaption>**FIGURE 9** Integration of the acetamiprid DNT BoE into an AOP framework including the related uncertainties. Uncertainties are shaded in grey.</figcaption></figure>

In establishing the AOP postulation, the WG considered the pesticide MoA, i.e. activation of nAChR, as a MIE.

Both neonicotinoid insecticides, as well as nicotine, bind to nAChRs and mimic the action of acetylcholine by opening the ion channels which allow the entry of Na⁺ and Ca²⁺ into cells. These compounds vary in their affinity for different nAChR subtypes, with nicotine showing selective toxicity for vertebrates, whereas neonicotinoids are highly selective for insect nAChRs. The binding of neonicotinoids to insect nAChRs is virtually irreversible (EFSA PPR Panel, 2013).

Recent molecular docking studies using receptor models for the human α7 nAChR and the α4β2 nAChR isoforms showed relevant differences on predominant orientation of the compounds in the binding site using different neonicotinoids and nicotine. This could give a molecular rationale for the observed functional differences (Loser et al., 2021; OECD IATA Case Study no. 365, OECD, 2022c).

Despite the common structural features, neonicotinoids and nicotine have different protonation states at physiological pH. Nicotine tends to be protonated in neutral aqueous solutions. This positive charge gives to nicotine a strong affinity for mammalian nAChRs as it occurs with the ammonium head of ACh; this is a requirement to interact with the mammalian nAChRs (Tomizawa & Casida, 2003). Instead of an easily protonated nitrogen, neonicotinoids have an electronegative pharmacophore, such as a nitro, or a cyano moiety, which is crucial for their insecticide activity. The electronegative pharmacophore is believed to be associated with a cationic subsite (possibly lysine, arginine or histidine) in the insect nAChR (Tomizawa & Casida, 2003, 2005; OECD IATA Case Study no. 365, OECD, 2022c). Acetamiprid shows weak affinity for mammalian nAChRs but strong affinity for insect nAChRs. Indeed, the binding characterisation is considered adequate for neonicotinoids and partially characterised in the IATA Case Study no. 365. Therefore, neonicotinoids are considered adequate stressors to measure the MIE activation.

The immediate downstream KE, following the MIE activation was identified in the activation of calcium influx. This KE was measured in two different neuron types (SH-SY5Y human neuroblastoma cells, an established model of nAChR signaling and dopaminergic neurons generated from LUHMES neuronal precursor cells) at concentrations starting from 10 µM.

Activation of nAChRs (MIE) often results in increased intracellular free Ca²⁺ levels through the direct passage of extracellular Ca²⁺ across the receptor channels in neurons, in addition to Na⁺ and K⁺. Agonist activation of the α7 nAChR can trigger an intracellular transient calcium increase that vary in duration, amplitude and distribution, depending on the subcellular localisation of the nAChR and its proximity to the endoplasmic reticulum (Govind et al., 2009). The raised cytoplasmic calcium levels trigger a series of calcium-dependent intracellular processes. Among the different nAChR subtypes in the brain, the homomeric α7 subtype exhibits higher permeability to Ca²⁺ than the other ligand-gated ion channels permeable to
this divalent cation, such as the NMDA (N-methyl-D-aspartate) receptor (Takarada et al., 2012). This receptor subtype is also known to desensitise rapidly (Wu et al., 2011).

During brain development, the consequences of MIE activation depend on the window of the exposure due to the different stage of cell differentiation/maturation and functional nAChR expression (see Figure 8). Regarding the α7 nAChR subtype, two features of this homomeric receptor must be considered from a toxicological perspective. First, the high levels of expression during brain development as compared to adults in both humans (Falk et al., 2002) and rodents (Tribollet et al., 2004; Zhang et al., 1998) support the important role of nAChRs for the development of the central nervous system (CNS) and are indicative of a potential vulnerability of developing mammals to early neonicotinoid exposures.

Indeed, with agonist binding, receptors become rapidly activated by opening the ion channel through the receptor. This is a metastable event and if the receptor remains bound by agonist, activation is followed by a second conformational change leading to a non-functional state termed desensitisation in which the channel is closed. Normally, when nicotine is removed, receptors rapidly recover from the desensitised state and enter in the resting state. Several lines of evidence indicate that chronic exposure to nicotine causes some of the nAChRs in the brain to undergo long-lasting state changes. These conformational changes are distinguished from activation and desensitisation by much slower kinetics, in the order of hours to days (Govind et al., 2009).

Therefore, the WG considered as a downstream consequence of the KE1, the affected functioning of the nAChR (desensitisation) as KE2. The inhibitory effect of acetamiprid on nAChR activity (i.e. inhibition of agonist-induced Ca²⁺ influx) was tested by pre-exposing LUHMES and SH-SY5Y cells with acetamiprid and acute addition of the receptor agonists nicotine or acetylcholine. Acetamiprid concentrations between 0.01 and 100 µM were tested and effects were observed at concentrations of 1 µM and higher (Loser et al., 2021; OECD IATA Case Study no. 365). The WG noted that the IATA Case Study no. 356 used the same evidence to characterise the MIE, the KE1 and the KE2, but there was no empirical support to the downstream KEs. Overall, the AOP-informed IATA for acetamiprid was considered inconclusive, due to the existing empirical data gaps between KE2 (desensitisation and loss of functionality of nAChRs) and the AO (i.e. impairment of behavioural function).

However, it is worth noting that the biological plausibility of DNT is high when taking into consideration the abundant expression of nAChRs which are functionally important in the developing brain for neurotransmission, neuronal plasticity, neuronal migration and differentiation, neurogenesis and gene expression (e.g. Lauder & Schambra, 1999). Moreover, the AOP Wiki contains three relevant OECD endorsed AOPs (see also Pitzer et al., 2023), i.e. AOPs 12 and 13, which link 'decreased Ca²⁺ influx' (KE52) to reduced levels of brain derived neurotrophic factor (BDNF) (KE381) as well as AOP 48 which links 'increased intracellular Ca²⁺ overload' (KE389) to mitochondrial dysfunction 1 (KE177). Both of these later key events may ultimately lead to 'impairment of learning and memory' (AO341). Thus, the main uncertainty is the critical effect size, exposure duration and exposure window during brain development which are necessary to translate the earlier KEs, for which acetamiprid data are available, into later KEs and finally to an organism level-based AO. Another, relevant uncertainty is that in the rodent in vivo BoE the auditory startle response is affected from 10 mg/kg/bw per day onwards, but it is unknown if other DNT AOs may be affected by acetamiprid at lower doses, since no reliable data are available (i.e. FOB, motor activity, learning and memory, morphometric changes). All these uncertainties need to be contextualised considering the baseline uncertainties resulting from the extrapolation from any experimental system to real-world scenarios. Such uncertainties include variable (epigenetics, multiple stressors and environmental modulators, such that any early molecular or cellular KE triggered may be considered as a burden for an organism reducing its capacity to compensate for additional stress. In addition, there are methodological uncertainties for the assessment of toxicological effects which are conceptually similar between in vivo and in vitro assays (Figure 9 and Paparella et al., 2020). The utility of new data generation can be considered in relation to these baseline uncertainties. Consequently, the amount of evidence needed to regulate pesticides needs to be recognised as a science-policy task.

The WG noted that additional empirical evidence should be provided in terms of experimental data for acetamiprid from assays covering mechanisms between KE2 and the AO. This may be done by, e.g. using LUHMES cell-derived DA neurons in the MEA (neural network formation) assay for the assessment of the neuronal network formation and function. In addition, data and knowledge for other neonicotinoids should be included to minimise the uncertainties. Indeed, integration of mechanistic and AO evidence for the chemical class, by inclusion of several neonicotinoids and nicotine would increase the empirical support for the KERs in terms of critical effect size, effect duration and dose and temporal concordance within the AOP for acetamiprid.

## 2.2 | Hazard assessment of acetamiprid metabolites

### 2.2.1 | Data

Data related to two sources of evidence were obtained and appraised to address hazard characterisation of acetamiprid and its metabolites.

1. Data related to hazard characterisation of acetamiprid metabolites have been retrieved conducting a systematic literature search and data collection from 2016 to 2022 and appraisal using Critical Appraisal Tools (in the context of the same search as for the a.s. [Section 2.1.1]). In addition, a protocol was developed based on EFSA (2020),
which included detailed information on the search strategy, inclusion and exclusion criteria and detailed methods for the systematic review process used (see Appendix 1).

2. New toxicological studies submitted by the sole applicant of acetamiprid on 4 July 2023. These are studies on the toxicological profile of the metabolite IM-2-1 (in vitro micronucleus test with IM-2-1; mouse lymphoma assay with IM-2-1 and repeated dose 28-day toxicity study in rats with IM-2-1)

## 2.2.2 | Methodologies

### 2.2.2.1 | EFSA systematic literature review for potential residue metabolites

The methodologies applied for assessing the data retrieved from the systematic literature search and data collection for potential residue metabolites are the same as the methodologies used for acetamiprid and are described in Section 2.1.2 and in Appendix 1.

### 2.2.2.2 | IM-2-1 toxicity studies submitted by sole applicant

The IM-2-1 toxicity studies submitted by the sole applicant were evaluated by Member State experts in the context of a Peer Review Experts' Meeting. For this purpose, EFSA prepared a background document providing an overview of all the available toxicity data for IM-2-1 for the Member States participating in the Peer Review Meeting in order to facilitate drawing a conclusion on the toxicological properties of the metabolite and assessing its impact on the safety of existing MRLs.

The document takes into account all the available information (regulatory studies submitted by the applicant, public literature studies and in silico predictions) on the metabolite and contains a WoE of all the retrieved available evidence, including studies available in the context of the renewal of the active substance (i.e. included in Netherlands, 2016) and the newly submitted studies under Art 31 of Regulation (EC) No 178/2002, ref. Ref. Ares (2023)6354306 of 20 September 2023, and provides EFSA's considerations for setting toxicological reference values for the consumer risk assessment. This background document can be found in Appendix 8.

In the Peer Review Meeting TC 119 (19 November 2023), the Member State experts discussed the studies and concluded on the toxicological profile and reference values of metabolite IM-2-1 (see the experts' meeting report in Appendix 8a).

## 2.2.3 | Assessment

### 2.2.3.1 | EFSA systematic literature review for potential residue metabolites

A systematic literature review was performed for the list of metabolites included in Table 16. Two reviewers screened the literature identified through the searches in two steps. For potential crops/livestock metabolites, 92 unique references were identified (see PRISMA flow chart, Figure 10). No publication was considered relevant for assessing any toxicological endpoint of any metabolite. Most of the published studies were not conducted with any of the metabolites as listed in Table 16 or were not toxicological studies.

<table><caption>TABLE 16 List of potential crop metabolites for which a systematic literature review was done for toxicological parameters (2016–2022).</caption><thead><tr><td>Name and SMILES code</td></tr></thead><tbody><tr><td>(1E)-N-[(6-chloropyridin-3-yl)methyl]-N'-cyanoethanimidamide (IM-2-1): Clc1ccc(CNC(\C)=N\C#N)cn1</td></tr><tr><td>1-(6-chloropyridin-3-yl)-N-methylmethanamine (IM-1-4): Clc1ccc(CNC)cn1</td></tr><tr><td>(1E)-N'-cyano-N-methylethanimidamide (IS-1-1): C/C(=N\C#N)NC</td></tr><tr><td>(1E)-N'-cyanoethanimidamide (IS-2-1): C/C(N)=N\C#N</td></tr><tr><td>(1E)-N'-carbamoyl-N-[(6-chloropyridin-3-yl)methyl]-N-methylethanimidamide (IM-1-2): Clc1ccc(CN(C)C(\C)=N\C(N)=O)cn1</td></tr><tr><td>N-[(6-chloropyridin-3-yl)methyl]-N-methylacetamide (IM-1-3): Clc1ccc(CN(C)C(C)=O)cn1</td></tr><tr><td>N-[(6-chloropyridin-3-yl)methyl]acetamide (IM-2-3): Clc1ccc(CNC(C)=O)cn1</td></tr><tr><td>6-chloropyridine-3-carboxylic acid (IC-0): OC(=O)c1cnc(CI)cc1</td></tr><tr><td>(6-chloropyridin-3-yl)methanol (IM-0): OCc1cnc(CI)cc1</td></tr></tbody></table>
<figure><img src="image_12.png" /><figcaption>**FIGURE 10** PRISMA flow chart of the literature search process for acetamiprid and its metabolites, including the screening for relevance. *See list of metabolites in Table 16.</figcaption></figure>

2.2.3.2 | EFSA assessment and conclusion on the toxicological properties of IM-2-1 residue metabolite

As IM-2-1 metabolite was considered relevant to include in the consumer risk assessment, the information on IM-2-1 metabolite in the revised RAR on acetamiprid (Netherlands, 2016), (Section B.6.8.1.2 of revised RAR and EFSA conclusion (2016)) was reviewed.

The following data on IM-2-1 were made available for the renewal of the active substance acetamiprid: (1) An acute oral toxicity study with LD50 value for IM-2-1 above 5000 mg/kg bw in both sexes (revised RAR on acetamiprid, Netherlands, 2016); (2) a negative Ames test with IM-2-1 (Netherlands, 2016). In addition, it is noted that, in rats, acetamiprid is metabolised to IM-2-1 by simple demethylation on the chain (Netherlands, 2016). In addition, IM-2-1 metabolite has been quantified as a major rat metabolite in urine (> 10% of absorbed dose).

In rats, acetamiprid is rapidly and extensively absorbed (Cmax was reached at 0.5–7 h) after single (1 or 50 mg/kg bw) (~96%) and repeated (1 mg/kg bw per day) (> 60%) oral administration. It is then distributed reaching the highest concentrations in liver, kidneys, adrenals and thyroid. Excretion is rapid, mainly via urine during the first 24 h after treatment, with more than 90% of the compound being excreted at 96 h. After single oral administration in rats, acetamiprid is extensively metabolised, with 50%–70% of the dose being excreted as metabolites in both urine and faeces. The main metabolic pathway in rats is the demethylation to **IM-2-1** (N-desmethyl-acetamiprid). This metabolite is further transformed to **IC-O** (6-chloronicotinic acid), with the release of **IS-2-1** (N-cyanoacetamidine derivative) after cleavage from the side-chains. In a mouse study from the literature (Ford & Casida, 2006), both acetamiprid and IM-2-1 have been detected in the brain upon intraperitoneal injection of acetamiprid (see detailed RoB assessment for the study in section 2.3). The toxicokinetic data included in the revised RAR on acetamiprid (Netherlands, 2016) also point to exposure of the brain (parent and/or metabolite(s)) upon oral acetamiprid exposure, but since only total radioactivity has been quantified, it cannot be concluded whether the brain exposure includes acetamiprid and/or IM-2-1. In Laubscher et al. (2022), IM-2-1 (but not acetamiprid) was detected in cerebrospinal fluid of 14 children (age range 3–18 years) treated for leukaemia and lymphomas and undergoing therapeutic lumbar punctures. The WG noted that biomonitoring studies may be confounded by preformed pesticide metabolites in the diet due to environmental degradation of parent compounds, as occurs with organophosphate insecticides (Hernández et al., 2019). Since IM-2-1 is an environmental degradation product of acetamiprid (Ospina et al., 2019), it is possible that the quantification of IM-2-1 in plasma, urine and cerebrospinal fluid in all children in Laubscher et al. (2022), with a concurrent lack of quantification of the parent compound acetamiprid in the same specimens may be due to direct exposure to IM-2-1 produced in the environment and not in the body (see Section 4.1.2). In addition, these findings are not generalisable to the general children population, given that the study population consisted of children with lymphohematopoietic tumours (selection bias), and tumours may affect the blood-brain barrier permeability and choroidal plexus barrier function (see Section 2.3.3), thus favouring a greater entry of chemicals into the brain.

Although no quantitative structure-activity relationship analysis is available in the revised RAR on acetamiprid (Netherlands, 2016), the WG noted that IM-2-1 represents a simple demethylated metabolite of acetamiprid, which still contains the N-cyanoamidine group as acetamiprid and thiacloprid. As described in Section 2.1.4.3, the nitro- or cyano- or equivalent electronegative pharmacophore is considered crucial for optimum nAChR binding. It is therefore plausible that IM-2-1 would also bind to mammalian nAChRs, but it is difficult to predict its affinity and potency compared to those of acetamiprid. Further data would be needed to understand the potential concern for DNT of IM-2-1.

Regarding the new toxicity studies submitted by sole applicant on IM-2-1 (see Section 2.2.2.2), details on the assessment of the studies can be found in the background document prepared by EFSA (Appendix 8). The conclusion of the Peer Review Meeting TC 119 (19 November 2023) on the toxicological profile of metabolite IM-2-1 is provided in Appendix 8a and below.

The new in vitro genotoxicity studies (micronucleus (MN) test, mouse lymphoma assays) submitted by the applicant in the context of the present mandate were considered acceptable. In addition, in the revised RAR on acetamiprid (Netherlands, 2016), an acceptable Ames test for IM-2-1 has been included. Based on these data, it was concluded that metabolite IM-2-1 did not induce clastogenicity or aneugenicity in the micronucleus assay in vitro, and it does not induce gene mutations in the mouse lymphoma assay, nor in the Ames test, and was therefore considered unlikely to be genotoxic.
The new 28-day repeated dose toxicity study of IM-2-1 in rats submitted by the applicant in the context of the present mandate was also considered acceptable. The NOAEL of the study was set at 1000 ppm (equivalent to 88.4 mg/kg body weight (bw) per day) based on decreased body weight gain in males (up to 38.0%) and supported by a decrease in body weight in males (10.4%, non-statistically significant), accompanied with a slight decrease in food consumption in male rats at 1600 ppm (138 mg/kg bw per day). The top dose of 1600 ppm (141 mg/kg bw per day) was the NOAEL for females based on the absence of treatment-related adverse effects at all dose levels. An increase in relative liver weight in females at the top dose (of 1600 ppm) was not considered adverse, as no concomitant histopathological changes were observed, and the increase in relative weight was below 15%. It was noted that at the top dose, triglyceride levels increased, and α1-globulin decreased in female rats, pointing to an effect on the liver, although not adverse. It was noted that according to the revised RAR on acetamiprid (Netherlands, 2016), acetamiprid causes liver toxicity in the available short-term study in rats (NOAEL of 90-day rat study amounts to 200 ppm, equivalent to 12.4 and 14.6 mg/kg bw per day for males and females, respectively, based mainly on bw reduction and liver effects from 800 ppm) and that the liver was considered a target organ in the available long-term toxicity study in rats. It was further noted that possible liver toxicity cannot be excluded for the metabolite IM-2-1 based only on this short-term toxicity study. However, the evidence for liver toxicity in females in the current 28-day study is limited and not robust enough to be considered as adverse for the NOAEL setting.

IM-2-1 is found as residue metabolite and the Peer Review Meeting TC 119 (19 November 2023) proposed to set the same toxicological reference values (ADI and ARfD) for IM-2-1 as those for acetamiprid, based on the following considerations:

* Structural similarities: IM-2-1 represents a simple demethylated metabolite of acetamiprid and still contains the active group (i.e. N-cyanoamidine group) as acetamiprid and thiacloprid.

* ADME: IM-2-1 metabolite is a major rat metabolite (18%–24% of dose in urine).

* Genotoxicity: IM-2-1 metabolite does not induce clastogenicity or aneugenicity in the micronucleus assay in vitro, and it does not induce gene mutations in the mouse lymphoma assay, nor in the Ames test. Overall, IM-2-1 metabolite is unlikely to be genotoxic.

* Systemic toxicity: Available 28-day rat study indicated decreased body weight gain, accompanied with bw decrease and decreased food consumption, as the critical toxicological effects in male rats (liver effects observed in females were considered treatment related, but not adverse). In the 90-day rat study with acetamiprid, liver effects were identified as the critical toxicological effects. The critical endpoint used for setting the reference values for acetamiprid, i.e. DNT, is not captured in a short-term repeated dose toxicity study.

* Relative toxicity compared to parent: The available 28-day rat study on IM-2-1 does not allow to conclude on a different qualitative or quantitative toxicological profile of the metabolite compared to the parent. Based on structural similarities to parent and being IM-2-1 a major rat metabolite, the toxicological profile of IM-2-1 is considered as covered by the parent (i.e. IM-2-1 is expected to be at least of equal toxicity as the parent). Therefore, the same toxicological reference values of the parent can apply to the metabolite.

## 2.3 | Human exposure estimation based on human biomonitoring data

To address **ToR1a** and **ToR1b** (assessment question 1) (see Section 1.2.1), the EFSA WG evaluated new human biomonitoring data on acetamiprid and its IM-2-1 metabolite, including the study of Laubscher et al. (2022) as included in the mandate. It was also requested to use these human biomonitoring data to estimate related external (oral) exposure levels using a PBK modelling reverse dosimetry approach (Clewell et al., 2008). These estimated oral exposure levels were then compared with the HBGVs of acetamiprid to evaluate whether exposure, based on the human biomonitoring data, exceeds the HBGVs. It must be noted that it was not intended to perform a full exposure characterisation based on the available human biomonitoring datasets. It was rather aimed to assess whether these datasets would point to possible exceedance of HBGVs of acetamiprid.

Besides application of a PBK model to estimate external oral exposure levels from human biomonitoring data (using a so-called reverse dosimetry approach), a PBK model can be applied to estimate internal exposure levels upon exposure to a given external (e.g. oral) dose (also called forward dosimetry). As such, the PBK model is also intended to estimate internal acetamiprid concentrations upon exposure to levels corresponding to the current HBGVs and to compare these predicted internal concentrations with reported in vitro effect concentrations from relevant and reliable in vitro effect studies identified (Section 2.1).

### 2.3.1 | Data

#### 2.3.1.1 | Human biomonitoring data

The assessment primarily relied on the Laubscher et al. (2022) study, which was included in the mandate. Additionally, two other human biomonitoring studies were identified and considered relevant to estimate acetamiprid (oral) exposure levels for sensitive life stages regarding DNT effects.