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

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.

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

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 of three approaches for regulatory decision making on pesticides with endocrine disrupting properties.

Recent EU legislation has introduced endocrine disrupting properties as a hazard-based "cut-off" criterion for the approval of active substances as pesticides and biocides. Currently, no specific science-based approach for the assessment of substances with endocrine disrupting properties has been agreed upon, although this new legislation provides interim criteria based on classification and labelling. Different proposals for decision making on potential endocrine disrupting properties in human health risk assessment have been developed by the German Federal Institute for Risk Assessment (BfR) and other regulatory bodies. All these frameworks, although differing with regard to hazard characterisation, include a toxicological assessment of adversity of the effects, the evaluation of underlying modes/mechanisms of action in animals and considerations concerning the relevance of effects to humans. Three options for regulatory decision making were tested upon 39 pesticides for their applicability and to analyze their potential impact on the regulatory status of active substances that are currently approved for use in Europe: Option 1, based purely on hazard identification (adversity, mode of action, and the plausibility that both are related); Option 2, based on hazard identification and additional elements of hazard characterisation (severity and potency); Option 3, based on the interim criteria laid down in the recent EU pesticides legislation. Additionally, the data analysed in this study were used to address the questions, which parts of the endocrine system were affected, which studies were the most sensitive and whether no observed adverse effect levels were observed for substance with ED properties. The results of this exercise represent preliminary categorisations and must not be used as a basis for definitive regulatory decisions. They demonstrate that a combination of criteria for hazard identification with additional criteria of hazard characterisation allows prioritising and differentiating between substances with regard to their regulatory concern. It is proposed to integrate these elements into a decision matrix to be used within a weight of evidence approach for the toxicological categorisation of relevant endocrine disruptors and to consider all parts of the endocrine system for regulatory decision making on endocrine disruption.

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.

[Plant protection products and their residues : Aspects of consumer safety in context of the new EU regulations]. / Pflanzenschutzmittel und ihre Rückstände : Aspekte der Verbrauchersicherheit im Kontext neuer EU-Verordnungen.

The law regulating plant protection products (PPP) in the European Union (EU) was fundamentally revised through the introduction of Regulation (EC) No. 1107/2009 which is due to enter into force on 14 June 2011. EU-wide harmonized maximum residue levels (MRLs) for the active substances of PPP in foods are laid down in Regulation (EC) No. 396/2005 and apply since entry into force of the regulation on 1 September 2008. The goal of both regulations is to strengthen the level of consumer protection. PPP are subject to a strict assessment of active substances, which is regulated at the EU level as well as an authorization procedure in the EU Member States. Prior to application for authorization of a PPP, the active substance(s) it contains must be included in a positive list. Tests regarding the toxicity and residue behavior of PPP must be conducted by the applicant, and the respective documents must be submitted to the authorities for evaluation. Following review of the required data, toxicological threshold values are derived, consumer exposure is assessed, and the risk to health is evaluated. The goal of this evaluation is to ensure that the use of PPP according to good plant protection practice does not have any harmful effects on human health.

Harmonisation of rat fetal skeletal terminology and classification. Report of the Third Workshop on the Terminology in Developmental Toxicology. Berlin, 14-16 September 2000.

The initial efforts of the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and the Free University of Berlin to standardise terminology in the field of developmental toxicology began in 1995. Procedures were undertaken to harmonise the terminology used by the International Federation of Teratology Societies (IFTS) and the International Programme on Chemical Safety (IPCS). This article reflects these activities and is a report on the Third Workshop on the Terminology in Developmental Toxicology held in September 2000. This Workshop served as a forum to discuss the results of a survey on the classification of skeletal anomalies that had been previously sent to scientists active in the field. Although high agreement was reached among the evaluators for several terms, the use of a number of terms was rather variable. Therefore, the discussions at the workshop among the experts from research institutions, regulatory agencies, and industry were mainly focussed on those terms for which there was disagreement and/or uncertainties and the possible reasons. Pictures provided by the participants for the illustration of "grey zone" anomalies constituted the basis for detailed discussions. In many of the cases with lower agreement, decisions were facilitated by the provision of the corresponding picture. The main reasons for lower agreement were imprecise terms, insufficient knowledge on postnatal consequences, theoretical terms that are unlikely to occur in isolation, and the possibility of observing a range of severity that might be decisive for the classification of either a malformation or variation. The attendees concluded that "grey-zone" anomalies will never disappear completely and that for the assessment, the grade of severity and/or the frequency of the observation can be decisive for the terminology chosen. A Joint IPCS/IFTS Project was proposed to further consensus of terminology and classification and to link these anomalies to pictures at different skeletal sites. In order to support the harmonisation of regulatory decisions, it was proposed to establish a "Clearinghouse" System under the umbrella of the IPCS. The Clearinghouse could be contacted either by the regulatory authorities or by any company to clarify their queries, particularly with regard to registration or authorisation processes. Finally, it was recommended to also carry out a similar survey on "soft tissue anomalies" and "external findings." The results of this survey will be discussed at a Joint IPCS/IFTS Workshop in Berlin in 2002.

Standard values for reproductive and clinical chemistry parameters of Japanese quail.

Historical control data of Japanese quail collected from reproduction studies conducted at the Federal Institute for Health Protection of Consumers and Veterinary Medicine between 1988 and 1994 are presented in this paper. Reproductive and clinical chemistry data from control animals of 10 ecotoxicological studies are summarized and discussed to develop a normal data base of this species. The data obtained were compared to the control reproductive parameters of bob-white quail and mallard ducks available in the literature. For a long time these two species have been most used in avian reproductive toxicology studies. In summary, the data obtained indicate that Japanese quail appears to be more appropriate to be used for the determination of reproductive effects of pesticides on birds.

Determination of mutational spectrum of the pesticide, captan, with an improved set of Escherichia coli LacZ mutants.

The mutational spectrum of the fungicide, captan, was determined using a set of improved Escherichia coli lacZ mutants. Captan created mutations mostly at dA-dT sites (83%) with only 17% occurring at dG-dC sites. The hydrolysis products of captan do not appear to be mutagenic because samples of captan at different hydrolysis stages showed basically the same mutational spectra: 31% at AT --> CG transversions, 8% of GC --> AT transitions, 2% of GC --> CG transversions, 8% of GC --> TA transversions, 19% of AT --> TA transversions, and 32% of AT --> GC transitions. Prepared solutions of captan lost their mutational activity gradually over time, indicating that the rate of decrease in mutagenicity agreed with the kinetics of captan hydrolysis reported in other studies. Using the change in mutagenicity to predict degradation, the hydrolysis of captan in pH 7.0 buffer was about three times faster than the hydrolysis carried out in pH 4.5 buffer. To our knowledge, this is the first presentation of mutational spectrum of captan.

Relative genotoxic activities of pesticides evaluated by a modified SOS microplate assay.

The genotoxic activities of 47 pesticides were determined using a modified SOS microplate assay in which the induction of beta-galactosidase in E. coli PQ37 was used as a quantitative measure of genotoxic activity. The results were compared with those obtained with anethole, curcumin, and capsaicin, a few examples of naturally occurring compounds present in foods. The assays were conducted with pesticides dissolved either in a suitable solvent, such as 10% DMSO in physiological saline or dispersed in sodium taurocholate micelles, to simulate conditions in the small intestine from where these substances are normally absorbed from the diet. 4-Nitroquinoline oxide (4-NQO) served as the reference standard of a direct acting mutagen. In micellar form, 4-NQO and 25 of the 47 pesticides tested showed significantly higher genotoxic activities than when they were tested in an organic solvent. In micellar form the SOS inducing potency of 4-NQO was almost twice as high as in 10% DMSO in physiological saline. In taurocholate micelles, the five most active compounds had activities in the range of 1,234-3,765 units/mumol and in the order of decreasing activities they were ranked as follows: malathion > dichlorvos > lindane > chlordane > endrin. They were significantly less active than 4-NQO (less than 40%). In micellar solution the naturally occurring compounds, anethole, curcumin, and capsaicin gave activities of 4,594, 928, and 809 units/mumol, respectively. These studies show that genotoxicity may depend upon the environment in which cells are exposed to these potential genotoxins. It appears that testing of the more hydrophobic compounds, both synthetic and naturally occurring, are needed.

Quantitative assessment of groundwater quality using a biological indicator: some preliminary observations.

The genotoxicity of groundwater was evaluated, using a novel application of the SOS microplate assay (SOSMA). Organic residues were extracted from groundwater samples from Maryland, Pennsylvania, and Delaware by using C-18 bonded silica solid phase extraction tubes. Total organic carbon content (TOC) of water samples was also determined. The genotoxicity of the extracts was determined by the SOSMA. Relative activity (RA) as determined by the SOSMA is a quantitative measure of genotoxicity based on a comparison to the activity of the mutagen, 4-nitroquinoline oxide. Low levels of RA (about 2x background) were detected in waters from sites within these states. There was considerable temporal and spatial variation in the observed RA, but no definite patterns were observed in the variation. Between sampling sites there was a positive correlation between RA and TOC; however, this relationship appeared to be reversed occasionally within a sampling site. The extraction and bioassay methods provide an easy and relatively inexpensive means of determining water quality.

Effects of repeated blood samplings on locomotor activity, evasion and wheel-running activity in mice.

The effects of serial blood sampling on nocturnal locomotor activity, evasion, wheel-running activity and body mass were studied in male NMRI mice aged 7-8 weeks. The erythrocyte count, haematocrit and haemoglobin concentration at the beginning and end of the study showed no difference in group 1 (two samples per week, 0.08 ml each) while there was a significant decrease in the group 2 values (three samples per week, 0.08 ml each). The total amount of nocturnal locomotor activity decreased in the animals bled repeatedly while the periods with locomotor activity increased. These alterations appeared particularly after bleeding. In the test-group animals evasion showed a decrease compared with the untreated control animals, but there was no evidence of a relation to the timing of the bleedings.

Identification of new sialic acids derived from glycoprotein of bovine submandibular gland.

Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N-acetyl-8-O-acetylneuraminic acid, N-acetyl-8,9-di-O-acetylneuraminic acid, N-acetyl-7,8,9-tri-O-acetylneuraminic acid, N-glycoloyl-7-O-acetylneuraminic acid, N-glycoloyl-7,9-di-O-acetylneuraminic acid, N-glycoloyl-8,9-di-O-acetylneuraminic acid, and N-glycoloyl-7,8,9-tri-O-acetylneuraminic acid. There are also indications for the presence of N-glycoloyl-8-O-acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7,9-di-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid could be identified. Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anion-exchange column (formate form) by elution with a 0-0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n-butanol/n-propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one-dimensional and two-dimensional thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry.

High-resolution 1H-NMR spectroscopy of free and glycosidically linked O-acetylated sialic acids.

A number of naturally occurring and synthetic, partially O-acetylated derivatives of N-acetylneuraminic and N-glycoloylneuraminic acids have been investigated by 360-MHz 1H-NMR spectroscopy. O-Acetylation causes strong downfield shifts for the resonances of neighbouring sugar-skeleton protons. The chemical shifts of these resonances, together with their characteristic multiplet shapes, can be used for localisation of the position of O-acetyl substituents in the molecule. The number of such substituents in the molecule can be inferred from the number of acetyl-methyl singlets, which also have characteristic resonance positions. This method for determination of the number and position of O-acetyl substituents in sialic acid residues is very powerful for structural analysis of underivatized carbohydrate chains derived from glycoconjugates. This is demonstrated for the oligosaccharide N-acetyl-4-O-acetylneuraminosyl-(alpha 2 leads to 3)-lactose isolated from echidna milk.

Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetyl-neuraminic acid, a new sialic acid from horse submandibular gland.

The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already known equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acid and 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.