Species differences and human relevance of the toxicity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and a new approach method in vitro for investigation.

The mode of action (MoA) of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides in mammals is well described and is generally accepted to be due to a build-up of excess systemic tyrosine which is associated with the range of adverse effects reported in laboratory animals. What is less well accepted is the basis for the marked difference in the effects of HPPD inhibitors that has been observed across experimental species and humans, where some species show significant toxicities whereas in other species exposure causes few effects. The activity of the catabolic enzyme tyrosine aminotransferase (TAT) varies across species including humans and it is hypothesized that this primarily accounts for the different levels of tyrosinemia observed between species and leads to the subsequent differences in toxicity. The previously reported activities of TAT in different species showed large variation, were inconsistent, have methodological uncertainties and could lead to a reasonable challenge to the scientific basis for the species difference in response. To provide clarity, a new method was developed for the simultaneous and systematic measurement of TAT in vitro using robust methodologies in a range of mammalian species including human. The results obtained showed general correlation between high TAT activity and low in vivo toxicity when using a model based on hepatic cytosol and a very convincing correlation when using a primary hepatocyte model. These data fully support the role of TAT in explaining the species differences in toxicity. Moreover, this information should give greater confidence in selecting the most appropriate animal model (the mouse) for human health risk assessment and for key classification and labeling decision-making.

Risk assessment of 'endocrine substances': guidance on identifying endocrine disruptors.

The European regulation on plant protection products (1107/2009) and other related legislation only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or wildlife species. This legislation would appear to make the assumption that endocrine active chemicals should be managed differently from other chemicals presumably due to an assumed lack of a threshold for adverse effects. In the absence of agreed scientific criteria and guidance on how to identify and evaluate endocrine activity and disruption within these pieces of legislation, a European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. The first ECETOC technical report and associated workshop, held in 2009, presented a science-based concept on how to identify endocrine activity and disrupting properties of chemicals for both human health and the environment. Specific scientific criteria for the determination of endocrine activity and disrupting properties that integrate information from both regulatory toxicity studies and mechanistic/screening studies were proposed. These criteria combined the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. A key element in the data evaluation is the consideration of all available information in a weight-of-evidence approach. Both sets of data (evidence of the adverse effect in apical studies and conclusive mode of action knowledge) are essential in order to correctly identify endocrine disruption according to accepted definitions. As the legislation seeks to regulate chemicals on a mode of action rather than the more traditional approach of adverse endpoints, then conclusive evidence of the mode of action of concern should be presented. From a human safety perspective and in the absence of any compelling data that endocrine active chemicals exert their adverse effects through anything other than a threshold mechanism there is no scientific justification for not using a margin of exposure approach to risk assessment in order to best protect human health.

A review of the mode of toxicity and relevance to humans of the triketone herbicide 2-(4-methylsulfonyl-2-nitrobenzoyl)-1,3-cyclohexanedione.

The mode of action (MoA) of the herbicide mesotrione has been empirically established in experimental animals. In this review, we evaluate this MoA and the relevance of this MoA to humans against accepted scientific criteria. The key events in the MoA involve inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD), the second enzyme in the tyrosine catabolic pathway, resulting in excess plasma tyrosine (tyrosinemia). When HPPD is completely inhibited, the clearance of excess tyrosine is dependent upon catabolism by the first and rate-limiting enzyme in the catabolic pathway, tyrosine aminotransferase (TAT) and elimination of the products of this catabolism via the urine. The inherent activity of TAT is low in rats and hence they catabolize tyrosine slowly and accumulate tyrosine to very high concentrations in plasma which results in a spectrum of adverse effects that are related to excess tyrosine. There is a large database showing a positive correlation between a range of biological endpoints and elevations in plasma tyrosine. Evidence is presented that clearly establishes a MoA involving tyrosine. Although plausible in humans, the extent and duration of plasma tyrosine elevation in humans is not sufficient to cause adverse effects resulting from the intended use of this herbicide.

The mouse carcinogenicity study is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides.

Regulatory tests investigating pesticide carcinogenicity potential routinely comprise a battery of in vitro and in vivo genotoxicity studies and two cancer bioassays, one in rats and one in mice. The genotoxicity testing strategy essentially ensures that genotoxic compounds are eliminated, and any carcinogens identified in subsequent lifetime studies are probably nongenotoxic in character. Assessment of 202 pesticide evaluations from the European Union review programme under Directive 91/414/EEC indicated that the mouse carcinogenicity study contributed little or nothing to either derivation of an acceptable daily intake (ADI) for assessment of chronic risk to humans, or hazard classification for labelling purposes. From a pesticide approval perspective, the mouse study did not influence a single outcome. From a risk assessment perspective, the ADI for just one pesticide was based on tumours in mice and this would have barely changed if the mouse data had not been available. In total, only 10 (5%) pesticide ADIs were based solely on the mouse carcinogenicity study and even in these few cases, a similar value would have been identified from other studies if the mouse study had not been available. For pesticides with treatment-related tumours only in mice, just three, or 1.5%, were classified as carcinogens and all were in the lowest category, Category 3 (R40). For pesticides with treatment-related tumours in mice and rats, the mouse data were probably the main, if not the only, cause for another three cases of R40 classification. Absence of the mouse studies would not have influenced assignment of the higher, Category 2 (R45), cancer classification for any substance with treatment-related tumours in both species as all decisions for these substances were limited to Category 3 or 'unclassified' outcomes. Over 100,000 mice were used to test these pesticides. This review shows that the mouse carcinogenicity studies did not provide significant information over and above that provided by the rat studies, and underpins the opportunity, from both a scientific and an animal welfare perspective, to remove the mouse carcinogenicity study from regulatory data requirements for the testing of pesticides.

The effects of the phytoestrogen genistein on the postnatal development of the rat.

The present studies report the effects on neonatal rats of oral exposure to genistein during the period from birth to postnatal day (PND) 21 to generate data for use in assessing human risk following oral ingestion of genistein. Failure to demonstrate significant exposure of the newborn pups via the mothers milk led us to subcutaneously inject genistein into the pups over the period PND 1-7, followed by daily gavage dosing to PND 21. The targeted doses throughout were 4 mg/kg/day genistein (equivalent to the average exposure of infants to total isoflavones in soy milk) and a dose 10 times higher than this (40 mg/kg genistein). The dose used during the injection phase of the experiment was based on plasma determinations of genistein and its major metabolites. Diethylstilbestrol (DES) at 10 micro g/kg was used as a positive control agent for assessment of changes in the sexually dimorphic nucleus of the preoptic area (SDN-POA). Administration of 40 mg/kg genistein increased uterus weights at day 22, advanced the mean day of vaginal opening, and induced permanent estrus in the developing female pups. Progesterone concentrations were also decreased in the mature females. There were no effects in females dosed with 4 mg/kg genistein, the predicted exposure level for infants drinking soy-based infant formulas. There were no consistent effects on male offspring at either dose level of genistein. Although genistein is estrogenic at 40 mg/kg/day, as illustrated by the effects described above, this dose does not have the same repercussions as DES in terms of the organizational effects on the SDN-POA.

Recognition of adverse and nonadverse effects in toxicity studies.

One of the most important quantitative outputs from toxicity studies is identification of the highest exposure level (dose or concentration) that does not cause treatment related effects that could be considered relevant to human health risk assessment. A review of regulatory and other scientific literature and of current practices has revealed a lack of consistency in definition and application of frequently used terms such as No Observed Effect Level (NOEL), No Observed Adverse Effect Level (NOAEL), adverse effect, biologically significant effect, or toxicologically significant effect. Moreover, no coherent criteria were found that could be used to guide consistent interpretation of toxicity studies, including the recognition and differentiation between adverse and nonadverse effects. This presentation will address these issues identified first by proposing a standard set of definitions for key terms such as NOEL and NOAEL that are frequently used to describe the overall outcome of a toxicity study. Second, a coherent framework is outlined that can assist the toxicologist in arriving at consistent study interpretation. This structured process involves two main steps. In the first, the toxicologist must decide whether differences from control values are treatment related or if they are chance deviations. In the second step, only those differences judged to be effects are further evaluated in order to discriminate between those that are adverse and those that are not. For each step, criteria are described that can be used to make consistent judgments. In differentiating an effect from a chance finding, consideration is given inter alia to dose response, spurious measurements in individual parameters, the precision of the measurement under evaluation, ranges of natural variation and the overall biological plausibility of the observation. In discriminating between the adverse and the non-adverse effect consideration is given to: whether the effect is an adaptive response, whether it is transient, the magnitude of the effect, its association with effects in other related endpoints, whether it is a precursor to a more significant effect, whether it has an effect on the overall function of the organism. whether it is a specific effect on an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. In interpreting complex studies it is recognised that a weight of the evidence approach, combining the criteria outlined here to reach an overall judgment, is the optimal way of applying the process. It is believed that the use of such a scheme will help to improve the consistency of study interpretation that is the foundation of hazard and risk assessment.