Following a request from the European Commission, the EFSA Panel on Plant Protection Products and their Residues (PPR Panel) was asked to deliver a Scientific Opinion on the developmental neurotoxicity (DNT) potential of the neonicotinoid insecticides acetamiprid and imidacloprid.
Acetamiprid and imidacloprid have been evaluated under the criteria of Council Directive 91/414/EEC and approved in Regulation (EU) No 540/2011. In 2012, a paper was published (Kimura-Kuroda et al., 2012) describing in vitro experiments performed with acetamiprid and imidacloprid. It suggested that excitation or desensitisation or both of nicotinic acetylcholine receptors (nAChRs) by these neonicotinoids might affect the developing mammalian nervous system as is known to occur with nicotine.
To evaluate the DNT potential of acetamiprid and imidacloprid, the PPR Panel scrutinised the information available on the two neonicotinoid compounds in the open literature as well as in toxicological assessments for regulatory purposes, including the dossiers submitted for approval within the EU.
The following questions were identified by the European Commission and answered by the PPR Panel:
Q1 Based on the available data, both in the scientific literature and in the toxicological dossier submitted for approval, do acetamiprid and imidacloprid exhibit developmental neurotoxic effects?
Based on the available data, the PPR Panel concludes that both acetamiprid and imidacloprid show some indications of DNT potential. Evidence of effects on offspring from a developmental neurotoxicity study for imidacloprid in rats, submitted within the EU and the US-EPA assessment frameworks, reported decreased pup body weights, reduced motor activity level and changes in dimensions of brain structures (reduction in the thickness of corpus callosum and a decreased width of caudate/putamen). The DNT study for acetamiprid, carried out within the US-EPA assessment framework, showed decreased pup body weights, reduced pup pre-weaning survival and decreased maximum auditory startle response. The Panel therefore concludes that the two neonicotinoid compounds may affect neuronal development and function. However, there are limitations concerning the available evidence due to methodological issues and uncertainties as to whether or not neurotoxic effects in offspring occurred below doses eliciting maternal toxicity.
Q2 Have acetamiprid and imidacloprid been assessed adequately for developmental neurotoxicity and if not, which further information would be required?
Comprehensive toxicological databases are available for these two neonicotinoids. Toxicological in vivo studies include acute, subacute, subchronic, chronic, mutagenicity, carcinogenicity, reproduction and developmental studies carried out for the hazard characterisation of these compounds within the EU regulatory framework. However, available DNT studies show limitations. A DNT study on imidacloprid included data that may not be sufficient for a robust characterisation of dose-response, e.g. for changes in dimensions of brain structures. Regarding the DNT study performed for acetamiprid, important endpoints such as motor activity or learning and memory, which are expected to be the most sensitive DNT endpoints, could not be adequately assessed. Moreover, there are uncertainties with respect to the conclusion of the study report concerning the No Observed Adverse Effect Level (NOAEL) based on reduced pup auditory startle response. Overall, in the opinion of the Panel, the study can only provide supportive evidence, but is inadequate for a robust characterisation of effects and dose-response of acetamiprid-induced DNT. Thus, the PPR Panel concludes that further good quality in vivo studies following OECD TG 426 are required to more properly characterise a DNT potential as well as associated dose-response relationships, particularly for acetamiprid.
Q3 Do the existing health-based guidance values provide adequate protection against any potential developmental neurotoxicity of acetamiprid and imidacloprid and if not what values would be necessary to provide such protection?
Based on the indications provided by the available DNT studies and the associated uncertainties in establishment of the corresponding NOAELs, the Panel considers that the current ARfDs may not be protective enough for the possible developmental neurotoxicity of acetamiprid and imidacloprid. The same uncertainties do not allow to set a reliable ADI for acetamiprid. However, the ADI set for imidacloprid would provide adequate protection against its potential adverse effects on the developing nervous system. Accordingly, more conservative reference values are proposed based on the existing toxicological data.
The PPR Panel recommends that a more conservative NOAEL of 2.5 mg/kg bw per day for acetamiprid should be used as a point of departure for the derivation of ADI, ARfD and AOEL, which should all be set at 0.025 mg/kg bw per day. When new and more reliable DNT data are available, the point of departure can be revised.
As the current ARfD and AOEL for imidacloprid may not be protective enough for potential developmental neurotoxicity of this active substance, the Panel also recommends to conservatively lower these reference values to the same level as the ADI (0.06 mg/kg bw per day).
Q4 Do the approved neonicotinoids need further investigation to clarify the mechanism of action of the nAChRs? Should all neonicotinoids be tested using the in vitro system mentioned in the publication of Kimura-Kuroda et al. (2012)?
The PPR Panel considers that further research is warranted on the role of nAChRs in neuronal differentiation and maturation to fully understand the potential DNT induced by acetamiprid and imidacloprid. The in vitro system used in the study of Kimura-Kuroda et al. (2012) covers only very limited aspects of brain functions and its limitations currently prevent its use as a tool for screening developmental neurotoxicants in the regulatory arena. In particular, the in vitro system as proposed by this study requires considerable further characterisation and should be investigated to assess its relevance to the in vivo situation. To extend confidence in findings, provision of positive and negative controls and scrutiny of data for reliability and reproducibility are required. Only then should a test based on this system be considered as a possible screening tool for neurotoxicity potential. Moreover, the Panel recommends that further studies should be performed using the culture of other brain structures which also express nAChRs (hippocampus, entorhinal cortex, basal ganglia or thalamus) where the key developmental processes such as neuronal and glial proliferation, migration, differentiation, neurite outgrowth, synaptogenesis, networking, myelination and programmed cell death may be evaluated.
Based on the overall appraisal of existing data, revised in the context of the present Scientific Opinion, the PPR Panel considers that in vitro tests currently cannot substitute for in vivo DNT tests. Besides, no in vitro test can be used to date to set health-based reference values. The complexity of neurodevelopmental processes does not allow a proper assessment at the cellular level and behavioural outcomes cannot be assessed by in vitro models. However, in vitro assays may be regarded as complementary to animal testing because they may provide better understanding of the cellular/molecular mechanisms involved in developmental neurotoxicity. As such, in vitro tests could be incorporated into a DNT testing strategy to obtain mechanistic information or for purposes of screening/prioritisation. Following the provision included in section 5.6.2 (“Developmental toxicity studies”) of the Commission Regulation n. 283/2013 on data requirements for active substances, the PPR Panel encourages the definition of clear and consistent criteria at EU level to trigger submission of mandatory DNT studies, which could include development of an integrated and cost-effective, tiered testing strategy composed of robust, reliable and validated in vitro assays and alternative methods complementary to the in vivo test guideline 426 for assessing the developmental neurotoxicity potential of substances.