With great interest we have read the article from Dr. Paule Benit and colleagues “Evolutionarily conserved susceptibility of the mitochondrial respiratory chain to SDHI pesticides and its consequence on the impact of SDHIs on human cultured cells“ [1] published in PLOS ONE on 7th November 2019.
The purpose of this response is to communicate some concerns regarding the scientific methods used to produce, interpret and report results of work reported by Benit et al. in this journal.

- Assumptions in calculations
The correlations between the in vitro concentrations and the Acceptable Daily Intake (ADI) that are highlighted in Benit et al. seem oversimplified and ignore fundamental pharmacokinetic principles. Therefore, comparisons between the in vitro concentrations and the ADI that are highlighted in Benit et al. are of limited value.

In the discussion, the authors attempted a comparison between SDHI concentrations showing effects under cell culture conditions, with the SDHI exposure of consumers considered to be acceptable by EU authorities ADI. The calculation approach proposed by Benit et al. assumes that the complete daily intake concentrates within the blood vessels. It does not consider that only the free fraction (i.e. not bound to plasma proteins) of any compound (including SDHIs) is biologically active, nor does it account for the fact that SDHI do not remain in the blood vessel compartment indefinitely but are rapidly distributed within the body, degraded to metabolites and are removed from the body by excretion. All these events occur under physiological conditions and are difficult to model in static cell culture experiments. In contrast, the experimental conditions of regulatory studies do consider toxicokinetic aspects and reflect the physiological conditions of mammalian exposure. In addition, exaggerated high-dose conditions are included to maximize the probability of identifying health hazards potentially relevant to humans. The ADI is calculated based on the lowest no observed adverse effect level (NOAEL) for the most sensitive adverse effect, which is then divided by at least an uncertainty factor of 100 to take into account of intra- species and inter- individual variability. In other words, the ADI is a very conservative estimation derived from robust toxicity studies conducted in several species exposed for different durations and at different life stages.

- Methods and Reproducibility
Further methodological information on the experimental design, analysis methodology and interpretation that is not included in the publication or cited references, would be useful to demonstrate the reproducibility of the data presented. In the absence of this information, it is not possible to draw conclusions on the data. Specifically, the reader would benefit from understanding the validation parameters of the enzyme IC50 measurement methodology (for SQDR, SCCR, GQDR, GCCR, QCCR) and having visibility of the individual replicate data to support the IC50 determinations for treated and control mitochondrial preparations. Please could this information be provided?


- Human health relevance and control for variables
The paper presents a set of in vitro data on the effect of selected SDHI fungicides on viability of fibroblast cells derived from healthy human individuals and from a patient suffering from either SDH-deficiency, Friedreich’s ataxia or Alzheimer disease. In experiments using samples from healthy individuals, we would like to understand the author’s rationale for the use of a non-physiological culture system, which itself would provoke toxicity due to the absence of essential energy sources (glucose and pyruvate) critical for normal cellular function, and the relevance of this to the assessment of human health hazards. We note that population variability is not controlled as only cells from a single patient were used in the experiments. The reader would also benefit from understanding the health condition of sampled patients and any relevant confounding health issues. Such information is requested for other (guideline) in vitro studies to be able to judge reliability and reproducibility of the experimental data (see e.g. Eskes et al., 2017 [2]; OECD, 2018 [3]). Please could this information be provided?

- References
[1] P. Benit, A. Kahn, D. Chretien et al. Evolutionarily conserved susceptibility of the mitochondrial respiratory chain to SDHI pesticides and its consequence on the impact of SDHIs on human cultured cells (2019). PLOS ONE 14, e0224132 DOI: 10.1371/journal.pone.0224132
[2] C. Eskes, A.-C. Boström, G. Bowe et al. Good cell culture practices & in vitro toxicology (2017). Toxicology in Vitro 45, 272-277 DOI: 10.1016/j.tiv.2017.04.022
[3] OECD (2018). Guidance Document on Good In Vitro Method Practices (GIVIMP). Series on Testing and Assessment No. 286