Rudolf Pfeil

A critical review of glyphosate findings in human urine samples and comparison with the exposure of operators and consumers

For active substances in plant protection products (PPP) with well defined urinary elimination, no potential for accumulation and virtually no metabolism, measuring of urine levels could be a powerful tool for human biomonitoring. Such data may provide reliable estimates of actual internal human exposure that can be compared to appropriate reference values, such as the ‘acceptable daily intake (ADI)’ or the ‘acceptable operator exposure level (AOEL)’. Traces of the active compound glyphosate were found in human urine samples, probably resulting either from occupational use for plant protection purposes or from dietary intake of residues. A critical review and comparison of data obtained in a total of seven studies from Europe and the US was performed. The conclusion can be drawn that no health concern was revealed because the resulting exposure estimates were by magnitudes lower than the ADI or the AOEL. The expected internal exposure was clearly below the worst-case predictions made in the evaluation of glyphosate as performed for the renewal of its approval within the European Union. However, differences in the extent of exposure with regard to the predominant occupational and dietary exposure routes and between Europe and North America became apparent.

Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC

Glyphosate is the most widely used herbicide worldwide. It is a broad spectrum herbicide and its agricultural uses increased considerably after the development of glyphosate-resistant genetically modified (GM) varieties. Since glyphosate was introduced in 1974, all regulatory assessments have established that glyphosate has low hazard potential to mammals, however, the International Agency for Research on Cancer (IARC) concluded in March 2015 that it is probably carcinogenic. The IARC conclusion was not confirmed by the EU assessment or the recent joint WHO/FAO evaluation, both using additional evidence. Glyphosate is not the first topic of disagreement between IARC and regulatory evaluations, but has received greater attention. This review presents the scientific basis of the glyphosate health assessment conducted within the European Union (EU) renewal process, and explains the differences in the carcinogenicity assessment with IARC. Use of different data sets, particularly on long-term toxicity/carcinogenicity in rodents, could partially explain the divergent views; but methodological differences in the evaluation of the available evidence have been identified. The EU assessment did not identify a carcinogenicity hazard, revised the toxicological profile proposing new toxicological reference values, and conducted a risk assessment for some representatives uses. Two complementary exposure assessments, human-biomonitoring and food-residues-monitoring, suggests that actual exposure levels are below these reference values and do not represent a public concern.

Combination Effects of (Tri)Azole Fungicides on Hormone Production and Xenobiotic Metabolism in a Human Placental Cell Line

Consumers are exposed to multiple residues of different pesticides via the diet. Therefore, EU legislation for pesticides requires the evaluation of single active substances as well as the consideration of combination effects. Hence the analysis of combined effects of substances in a broad dose range represents a key challenge to current experimental and regulatory toxicology. Here we report evidence for additive effects for (tri)azole fungicides, a widely used group of antifungal agents, in the human placental cell line Jeg-3. In addition to the triazoles cyproconazole, epoxiconazole, flusilazole and tebuconazole and the azole fungicide prochloraz also pesticides from other chemical classes assumed to act via different modes of action (i.e., the organophosphate chlorpyrifos and the triazinylsulfonylurea herbicide triflusulfuron-methyl) were investigated. Endpoints analysed include synthesis of steroid hormone production (progesterone and estradiol) and gene expression of steroidogenic and non-steroidogenic cytochrome-P-450 (CYP) enzymes. For the triazoles and prochloraz, a dose dependent inhibition of progesterone production was observed and additive effects could be confirmed for several combinations of these substances in vitro. The non-triazoles chlorpyrifos and triflusulfuron-methyl did not affect this endpoint and, in line with this finding, no additivity was observed when these substances were applied in mixtures with prochloraz. While prochloraz slightly increased aromatase expression and estradiol production and triflusulfuron-methyl decreased estradiol production, none of the other substances had effects on the expression levels of steroidogenic CYP-enzymes in Jeg-3 cells. For some triazoles, prochloraz and chlorpyrifos a significant induction of CYP1A1 mRNA expression and potential combination effects for this endpoint were observed. Inhibition of CYP1A1 mRNA induction by the AhR inhibitor CH223191 indicated AhR receptor dependence of this effect.

Hepatotoxic effects of (tri)azole fungicides in a broad dose range

Abstract The toxicological relevance of effects observed at molecular stage, which occur at dose levels well below classical no-observed adverse effect levels is currently subject to controversial scientific debate. While the importance of molecular effects for the identification of a mode of action or an adverse outcome pathway is undisputed, their impact for other regulatory purposes remains uncertain. Here, we report the results of a 28-day rat-feeding study including three widely used hepatotoxic (tri)azole fungicides (cyproconazole, epoxiconazole and prochloraz) administered individually at five dose levels, ranging from slightly above the reference values to a clear toxic effect dose. Parameters analysed included pathology, histopathology, clinical chemistry and particularly effects on the molecular level. Since azole fungicides are considered to cause liver toxicity by a mechanism involving the constitutive androstane receptor (CAR), a known CAR activator (phenobarbital, PB) was administered to investigate potential similarities between triazoles and PB-mediated liver toxicity by pathway-focused gene expression analysis. Our results show an increase in liver weights and additionally histopathological changes (hepatocellular hypertrophy) for all substances at the top dose levels. The effects on liver weight were most pronounced for cyproconazole by which also the animals receiving the next lower dose were affected. In addition, vacuolisation of hepatocytes was observed at the top dose level. No such findings were obtained with any substance at lower doses to which consumers and operators might be exposed to. In contrast, the expression of sensitive marker genes (like some cytochrome-P-450 isoforms) was significantly affected also at the lower dose levels. While some of these changes, like the induction of genes related to fatty acid and phospholipid metabolism (e.g. Fasn, Fat/Cd36, Ppargc1a) or xenobiotic metabolism (Cyp1a1, Cyp2b1, Cyp3a2), could be associated with high dose effects like hepatocellular vacuolisation or hypertrophy, a histopathological correlate was lacking for others.

Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Application of omics data in regulatory toxicology: report of an international BfR expert workshop.

Abstract Advances in omics techniques and molecular toxicology are necessary to provide new perspectives for regulatory toxicology. By the application of modern molecular techniques, more mechanistic information should be gained to support standard toxicity studies and to contribute to a reduction and refinement of animal experiments required for certain regulatory purposes. The relevance and applicability of data obtained by omics methods to regulatory purposes such as grouping of chemicals, mode of action analysis or classification and labelling needs further improvement, defined validation and cautious expert judgment. Based on the results of an international expert workshop organized 2014 by the Federal Institute for Risk Assessment in Berlin, this paper is aimed to provide a critical overview of the regulatory relevance and reliability of omics methods, basic requirements on data quality and validation, as well as regulatory criteria to decide which effects observed by omics methods should be considered adverse or non-adverse. As a way forward, it was concluded that the inclusion of omics data can facilitate a more flexible approach for regulatory risk assessment and may help to reduce or refine animal testing.

Human health risk assessment from combined exposure in the framework of plant protection products and biocidal products

Cumulative risk assessment (CRA) is of major importance and one of the biggest challenges for the future as a legal requirement within the EU for active substances used in plant protection products (PPP) and biocidal products (BP). Therefore, it is important to develop a methodology to take into account cumulative and synergistic effects for both active substances and substances of concern (SoC). The implementation of cumulative aspects in regulatory decisions is highly demanded and promoted by EU parliament, EU commission, European Food Safety Authority (EFSA), European Chemicals Agency (ECHA) and national authorities. Based on EFSA’s and ECHA’s work on CRA, the Federal Institute for Risk Assessment (BfR) drafted a concept on how to take cumulative aspects into account in the regulatory context in risk assessments for operators, consumers and other uninvolved third parties. Application of this concept as part of the routine risk assessment for PPP and BP is envisaged as soon as suitable experience has been gained in a testing phase. The BfR draft concept uses dose-addition of individual active substances and SoC as the toxicological standard concept for CRA and proposes a tiered approach. It recommends to start with calculation of a hazard index (HI) for all relevant substances contained in the PPP or BP under consideration. Proceeding to higher tiers is currently foreseen if the HI is larger than 1, i.e., an unacceptable risk cannot be excluded. In higher tiers, the HI should be calculated with respect to common targets and might consider effect-specific NOAEL’s (No Observed Adverse Effect Level) or relative potency factors, if available. Refinements should consider both the toxicity and the exposure part of the CRA and will depend on availability of relevant data. BfR acknowledges the complexity of the refinement work in mixture risk assessment to be done. The exposure assessment for operators, bystanders/residents and workers as well as the acute exposure assessment for consumers rely mainly on the active substances in a PPP or BP under consideration or on combinations of products for which simultaneous use is notified. Chronic consumer exposure assessment needs to take into account all relevant substances contained in the PPP or BP under consideration, but also the residue background of other pesticides in food, which have to be derived from representative food monitoring programmes. A representative food monitoring database is currently being developed. The assessment requires the application of complex probabilistic methods. It is planned that BfR will review the chronic CRA for each active substance and each CAG regularly as soon as all essential monitoring data are available. It is planned to carry out case studies on the impact on regulatory decisions. The paper is intended to promote further discussions of risk assessors, risk managers as well as stakeholders in this area on the applicability of CRA in routine authorisation procedures for PPP and BP and to encourage the flexible use of strategies in CRA.

Die Vorgehensweise des Bundesinstitutes für Risikobewertung bei der Abschätzung der dermalen Absorption von Wirkstoffen in Pflanzenschutzmitteln und Biozidprodukten

ZusammenfassungFür die Bewertung des gesundheitlichen Risikos von Pflanzenschutzmitteln (PSM) und Biozidprodukten (BP) ist die korrekte Abschätzung der Aufnahme der darin enthaltenen Wirkstoffe über die Haut erforderlich. In dieser Veröffentlichung wird dargestellt, wie das Bundesinstitut für Risikobewertung (BfR), in Abhängigkeit von den verfügbaren Daten, die dermale Absorption von Wirkstoffen in PSM und BP ableitet. Dabei finden zwei neue Bewertungsleitfäden der EFSA und der OECD Verwendung. Grundlegende Prinzipien der Studienauswertung werden ebenso beschrieben wie Möglichkeiten zur Abschätzung der Hautabsorption, wenn keine produktspezifischen experimentellen Daten vorliegen. Die Publikation dieser Vorgehensweise stellt einen ersten Schritt für die Harmonisierung der Bewertung in den Zulassungsverfahren von PSM und BP dar.AbstractA correct estimate of dermal absorption of the contained active ingredients is needed for the health risk assessment of plant protection and biocidal products. In this paper, the approach is reported that is currently taken by the Federal Institute for Risk Assessment to derive the dermal absorption rate of active substances in plant protection or biocidal products, depending on the available data. It is explained what use is made of two new guidance documents that were recently released by EFSA and OECD. Basic principles of the assessment of dermal absorption studies are described but also opportunities for estimations in the absence of product-specific experimental data. This publication is considered a first step towards harmonisation of assessment in the authorisation processes for plant protection and biocidal products.

Response to the reply by C. J. Portier and P. Clausing, concerning our review “Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC”

commenting rounds and the public consultation (EFSA 2015b). Although our response will be restricted to the EFSA assessment presented in Tarazona et al. (2017), it is worth noting the similarities in term of complementarity and transparency between the EFSA process under the pesticides regulation and the ECHA process under Regulation (EC) No 1272/2008. Germany as RMS under the Regulation (EC) No 1107/2009 (Germany 2015) and Dossier Submitter under the Regulation (EC) No 1272/2008 (BAuA 2016) expresses the views of the German experts, while EFSA (2015a, b) reports the view of the experts involved in the EFSA peer-review. The RAC Opinion (ECHA 2017) represents the view of the ECHA Committee for Risk Assessment. Portier and Clausing (2017) are suggesting apparent contradictions between the different views regarding the evaluation of study N. But in our opinion, this participates to the strength of the EU complementarity value. Different approaches for the weight of evidence are presented in a transparent way, allowing the scrutiny by the scientific community and interested groups. Even more important, despite different views on how to approach specific issues such as the reliability and relevance of study N, the weights of evidence converge and there is full consensus on the final conclusion, thus confirming that including or excluding study N does not change the overall assessment. EFSA decided to exclude study N due to concerns on the health status of the animal population, identified by US EPA (USEPA 2016), and confirmed by EFSA experts. Viral infections were not investigated by the study authors; therefore, EFSA’s confirmation focused on the health status and was based on indirect indications in the study report, supported by complementary evidence. The GLP status of the study (mandatory for industry-sponsored studies) implies that the test system should be free of any disease or condition that might interfere with the purpose or conduct of We welcome the opportunity for having an evidence-based scientific discussion on glyphosate’s carcinogenicity. We note that all of the findings in Table 1 of the Reply by Portier and Clausing (2017) have been available in the public domain in the supplemental data tables published by Greim et al. (2015; available online since February 2015). In addition, some of these findings were also raised in the report by IARC, but were not considered further. Although not explicitly mentioned by Portier and Clausing, it is relevant to indicate that, following a request to access to documents by four Members of the European Parliament, both authors got access to the findings and raw data of the unpublished industry sponsored studies on the carcinogenicity and genotoxicity of glyphosate submitted to EFSA; and even more important, that this information will be provided, under request, to any other person interested in scrutinizing the EFSA assessment and weight of evidence presented in our review. Two basic values of the EU assessments on pesticides are complementarity and transparency. The first is based on the peer-review principle, with an initial assessment by experts of the rapporteur member state (RMS), which is peer-reviewed by EFSA in close collaboration with experts from other Member States, and resulting in the EFSA Conclusion (EFSA 2015a). Transparency is ensured by publishing all assessments in a peer review report, which includes the comments and responses received during the different

Scientific Basis for Risk Assessment (Reproductive Toxicity) for Pesticides as Practiced by the Bundesgesundheitsamt (BGA)

It is the task of public health control agencies to weigh the risks of a given chemical against its benefits and the need for it. The German pesticides law regulations call for decisions based on safety standards which imply acceptable degrees of risk, without providing any precise definition of permissible degrees of risk. The margin of judgement provided by such legislation confers upon public health agencies the difficult task of making discretionary decisions against a background of conflicting interests.