Proposals for new spray drift exposure values in orchards and vineyards for residents and bystanders.

In the EU, predicted exposure to spray drift for residents and bystanders from applications in orchards and vineyards is based on data from one study published in 1987, where one downwind distance (8 m) was considered. CropLife Europe conducted sixteen new GLP compliant studies in 4 EU countries, 8 in orchards, 8 in vineyards with early and late season applications, using adult and child mannequins located 5, 10 and 15 m downwind from the last row to measure dermal and inhalation exposures. The resulting "Bystander Resident Orchard Vineyard (BROV)" database comprises 288 observations and offers a more comprehensive option for exposure prediction. There were differences between adult and child, crop type, leaf cover and distance from the sprayer, supporting the derivation of mean, median, 75th and 95th percentile exposures for each subset. Exposures did not generally correlate with wind speed, wind direction, sprayer type, spray quality, spray concentration or amount applied. Dermal and inhalation exposure were lower in vineyards than in orchards and further analysis is required to understand why.

Proposals for new transfer coefficient (TC) values for worker re-entry activities in grape vineyards.

New transfer coefficient (TC) values were derived for vineyard workers handling treated grapevines during harvesting and crop maintenance activities. Re-entry exposure and dislodgeable foliar residue (DFR) studies were performed in Europe, covering hand harvesting, pruning/training, pruning/tying and pruning/shoot lifting. Foliar applications of fungicides (iprovalicarb, dimethomorph, dithianon, pyrimethanil and fenbuconazole) were made and 73 workers at 16 sites were monitored over one working day. Exposure was measured on inner and outer dosimeters, face/neck wipes and hand washes. In concurrent DFR studies, leaf punches were taken at each site during the time of worker re-entry. Potential exposure values correlated well with DFR values. TC values were derived for various re-entry activities for potential and actual exposure, with and without gloves. The harvesting task resulted in lower TC values than the other crop maintenance tasks. Additional TC values reflecting the use of protective gloves can be derived from the results. The TC values are much lower than current European Food Safety Authority (EFSA) default values. This project addresses a data gap identified by EFSA for specific EU TC values to permit more realistic and reliable re-entry worker exposure estimates for grapes.

Use of guinea pig data to obtain starting points for skin sensitisation risk assessment - A commentary.

The increasing drive to understand the likelihood of skin sensitisation from plant protection products (PPPs) in workers and the general public has resulted in recent initiatives to establish a quantitative risk assessment (QRA) methodology applicable to these products and their exposure scenarios. The effective evaluation of skin sensitising substances requires not only the identification of that toxicological hazard, but also determination of relative sensitising potency. Typically, this has been achieved by interpretation of local lymph node assay (LLNA) dose response data, delivering what is known as the EC3 value. This permitted regulatory division of skin sensitisers into defined potency sub-categories, but more importantly enabled derivation of a no expected sensitisation induction level (NESIL) as the point of departure for QRA. However, for many existing substances there is no LLNA data, only older guinea pig results exist. To avoid additional (in vivo) testing, an approach has been outlined to employ guinea pig data and existing regulatory guidelines on the determination of potency sub-categorisation to provide a guinea pig based NESIL. The approach adopts a conservative extrapolation from LLNA NESIL benchmarks to deliver points of departure as the basis for the type of QRA process already in successful use by other industries.

Quantitative risk assessment of skin sensitising pesticides: Clinical and toxicological considerations.

Like many other consumer and occupational products, pesticide formulations may contain active ingredients or co-formulants which have the potential to cause skin sensitisation. Currently, there is little evidence they do, but that could just reflect lack of clinical investigation. Consequently, it is necessary to carry out a safety evaluation process, quantifying risks so that they can be properly managed. A workshop on this topic in 2022 discussed how best to undertake quantitative risk assessment (QRA) for pesticide products, including learning from the experience of industries, notably cosmetics, that already undertake such a process routinely. It also addressed ways to remedy the matter of clinical investigation, even if only to demonstrate the absence of a problem. Workshop participants concluded that QRA for skin sensitisers in pesticide formulations was possible, but required careful justification of any safety factors applied, as well as improvements to the estimation of skin exposure. The need for regulations to stay abreast of the science was also noted. Ultimately, the success of any risk assessment/management for skin sensitisers must be judged by the clinical picture. Accordingly, the workshop participants encouraged the development of more active skin health monitoring amongst groups most exposed to the products.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Practical guidance to evaluate in vitro dermal absorption studies for pesticide registration: An industry perspective.

While there are some regulatory assessment criteria available on how to generally evaluate dermal absorption (DA) studies for risk assessment purposes, practical guidance and examples are lacking. The current manuscript highlights the challenges in interpretating data from in vitro assays and proposes holistic data-based assessment strategies from an industry perspective. Inflexible decision criteria may be inadequate for real data and may lead to irrelevant DA estimates. We recommend the use of mean values for reasonably conservative DA estimates from in vitro studies. In cases where additional conservatism is needed, e.g., due to non-robust data and acute exposure scenarios, the upper 95% confidence interval of the mean may be appropriate. It is critical to review the data for potential outliers and we provide some example cases and strategies to identify aberrant responses. Some regional regulatory authorities require the evaluation of stratum corneum (SC) residue, but here, as a very simple pro-rata approach, we propose to review whether the predicted post 24-h absorption flux exceeds the predicted elimination flux by desquamation because otherwise it is not possible for the SC residue to contribute to systemic dose. Overall, the adjustment of DA estimates due to mass balance (normalization) is not recommended.

Assessing the risk of induction of skin sensitization to plant protection products: A quantitative approach.

Exposure to skin sensitizers is common and regulated in many industry sectors. For cosmetics, a risk-based approach has been implemented, focused on preventing the induction of sensitization. First, a No Expected Sensitization Induction Level (NESIL) is derived, then modified by Sensitization Assessment Factors (SAFs) to derive an Acceptable Exposure Level (AEL). The AEL is used in risk assessment, being compared with an estimated exposure dose, specific to the exposure scenario. Since in Europe there is increased concern regarding exposure towards potentially sensitizing pesticides via spray drift, we explore how existing practice can be modified to allow Quantitative Risk Assessment (QRA) of pesticides for bystanders and residents. NESIL derivation by the Local Lymph Node Assay (LLNA), the globally required in vivo assay for this endpoint, is reviewed alongside consideration of appropriate SAFs. Using a case study, the principle that the NESIL in µg/cm2 can be derived by multiplying LLNA EC3% figure by a factor of 250 is adopted. The NESIL is then reduced by an overall SAF of 25 to establish an exposure level below which there is minimal bystander and resident risk. Whilst this paper focuses on European risk assessment and management, the approach is generic and universally applicable.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 µM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.

A new conceptional model for deriving average dermal absorption estimates from studies with multiple tested concentrations for non-dietary risk assessment of pesticides.

Dermal absorption values are used to translate external dermal exposure into potential systemic exposure for non-dietary risk assessment of pesticides. While the Environmental Protection Agency of the United States of America (US EPA) derives a common dermal absorption factor for active substances covering all related products, the European Food Safety Authority (EFSA) requests specific product-based estimates for individual concentrations covering the intended use rates. The latter poses challenges, because it disconnects exposure dose from applied dose in absorption studies, which may not be suitable in scenarios where concentration is not relevant. We analyzed the EFSA dermal absorption database, collected 33 human in vitro studies from CropLife Europe (CLE) companies, where ≥3 in-use dilution concentrations were tested, and 15 dermal absorption triple pack datasets. This shows that absolute dermal absorption correlates with absolute applied dose on a decadic logarithm-scale, which is concordant with the toxicological axiom that risk is driven by exposure dose. This method is radically different from the current European approach focused on concentrations and offers new insights into the relationship of internal and external exposure doses when utilizing data from in vitro studies. A single average dermal absorption value can be simply derived from studies with multiple tested concentrations, by calculating the y-intercept of a linear model on a decadic logarithm scale while assuming a slope of 1. This simplifies risk assessment and frees resources to explore exposure refinements. It also serves as a basis to harmonize dermal absorption estimation globally for use in exposure-driven risk assessments.

An OECD TG 428 study ring trial with 14C-Caffeine demonstrating repeatability and robustness of the dermal absorption in vitro method.

The dermal absorption potential of 14C-Caffeine applied as a 4 mg/mL concentration (10 µL/cm2 finite dose) was investigated in six laboratories under Good Laboratory Practice conditions using an OECD TG 428-compliant in vitro assay with flow-through cells and split-thickness human skin. Potential sources of variation were reduced by a standardized protocol, test item and skin source. Particularly, skin samples from same donors were distributed over two repeats and between labs in a non-random, stratified design. Very similar recovery was achieved in the various assay compartments between laboratories, repeats and donors, demonstrating that the assay can be robustly and reliably performed. The absorption in one laboratory was 5-fold higher than in the others. This did not clearly correlate with skin integrity parameters but might be associated with an accidental COVID-19 pandemic-related interruption in sample shipment. It is possible that other factors may affect dermal absorption variation not routinely assessed or considered in the current method. The mean receptor fluid recovery, potential absorption (recovery in receptor fluid and skin except tape strips 1 and 2) and mass balance of caffeine was 6.99%, 7.14% and 99.13%, respectively, across all and 3.87%, 3.96% and 99.00% in the subset of five laboratories.

Dose Setting for Dermal Absorption Studies on Dried Foliar Residues.

Currently, the standard approach to estimate systemic exposure of workers after contact with dried pesticide residues on crops during re-entry activities relies on using the highest identified dermal absorption value for aqueous spray dilutions. However, recent dermal absorption studies with dried residues and their respective in-use dilutions have shown that this is likely to significantly overestimate their dermal penetration potential and, thus, predicted systemic exposure. The choice of appropriate dose levels for these dermal absorption studies has not been defined. Moreover, actual skin loading during re-entry tasks may differ significantly from that achieved by applying a fixed volume of an aqueous dilution, which is the standard practice in generic dermal absorption studies. To address this, we propose an approach to dose setting for dried residue studies within the current European risk assessment framework. Skin loading for studies can be calculated from the existing exposure algorithms and by taking appropriate body surface areas into account. Thus, skin loading in studies will vary depending on the exact nature and duration of the task and the region of the body exposed, reflecting actual exposure scenarios.

Compounded conservatism in European re-entry worker risk assessment of pesticides.

We review the risk parameters and drivers in the current European Union (EU) worker risk assessment for pesticides, for example considering crop maintenance, crop inspection or harvesting activities, and show that the current approach is very conservative due to multiple worst-case default assumptions. As a case study, we compare generic exposure model estimates with measured worker re-entry exposure values which shows that external cumulative exposure is overpredicted by about 50-fold on average. For this exercise, data from 16 good laboratory practice (GLP)-compliant worker exposure studies in 6 crops were evaluated with a total number of 184 workers. As generic overprediction does not allow efficient risk management or realistic risk communication, we investigate how external exposure can be better predicted within the generic model, and outline options for possible improvements in the current methodology. We show that simply using averages achieves more meaningful exposure estimates, while still being conservative, with an average exposure overprediction of about 9-fold. Overall, EU risk assessment includes several numerically unaccounted "hidden safety factors", which means that workers are well protected; but simultaneously risk assessments are biased towards failing due to compounded conservatism. This should be considered for further global or regional guidance developments and performing more exposure-relevant risk assessment.

Dermal absorption study OECD TG 428 mass balance recommendations based on the EFSA database.

The European Food Safety Authority (EFSA) guidance (EFSA, 2017) for dermal absorption (DA) studies recommends stringent mass balance (MB) limits of 95-105%. EFSA suggested that test material can be lost after penetration and requires that for chemicals with <5% absorption the non-recovered material must be added to the absorbed dose if MB is <95%. This has huge consequences for low absorption pesticides. Indeed, one third of the MBs in the EFSA DA database are outside the refined criteria. This is also true for DA data generated by Cosmetics Europe (Gregoire et al., 2019), indicating that this criterion is often not achieved even when using highly standardized protocols. While EFSA hypothesizes that modern analytical and pipetting techniques would enable to achieve this criterion, no scientific basis was provided. We describe how protocol procedures impact MB and evaluate the EFSA DA database to demonstrate that MB is subject to random variation. Generic application of "the addition rule" skews the measured data and increases the DA estimate, which results in unnecessary risk assessment failure. In conclusion, "missing material" is just a random negative deviation to the nominal dose. We propose a data-driven MB criterion of 90-110%, fully in line with OECD recommendations.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance.

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7-28 days and 8000 ppm metazachlor for 3-28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3-28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5-50 µM) and NaPB (500 µM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5-25 µM metazachlor or 500 µM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

Dermal absorption for pesticide health risk assessment: Harmonization of study design and data reporting for North American Regulatory submissions.

Although an internationally-adopted in vitro dermal absorption test guideline is available (OECD Test Guideline 428), the replacement of the in vivo approach in North America for pesticide formulations has not occurred due to concern over the reliability and consistency of the in vitro results. A 2012 workshop convened a panel of experts in the conduct of in vitro studies used for pesticide risk assessment, together with North American regulators, to examine techniques for in vitro dermal absorption testing. Discussions led to the recommended "best practices" for the conduct of in vitro dermal absorption studies provided herein. The workshop participants also developed recommendations for reporting study results in order to improve the quality and consistency of the data submitted to regulatory agencies in North America. Finally, a case study is presented that illustrates the use of the "triple-pack" approach; the studies, conducted for the registration of sulfoxaflor, follow the standardized recommendations provided at the workshop. In addressing the concerns of these regulators and of the regulated community, and providing harmonized recommendations to facilitate comparative data analyses, it is anticipated that wider acceptance of in vitro dermal absorption studies alone can be achieved for pesticide risk assessment.