The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene ...modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.
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This guidance document is intended to assist the applicant in the preparation and the presentation of an application, as foreseen in Article 7.6 of Regulation (EC) No 1831/2003, for the authorisation ...of additives used in animal nutrition. It specifically covers the assessment of the safety for the environment.
Draft endorsed by the FEEDAP Panel
2 October 2018
Submitted for public consultation
8 October 2018
End of public consultation
19 November 2018
Adoption by the FEEDAP Panel
27 February 2019
Implementation date
1 September 2019
This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2019.EN-1595/full
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The likelihood of oral exposure to nanoparticles (NPs) is increasing, and it is necessary to evaluate the oral bioavailability of NPs. In vitro approaches could help reducing animal studies, but ...validation against in vivo studies is essential. Previously, we assessed the translocation of 50 nm polystyrene NPs of different charges (neutral, positive and negative) using a Caco-2/HT29-MTX in vitro intestinal translocation model. The NPs translocated in a surface charge-dependent manner. The present study aimed to validate this in vitro intestinal model by an in vivo study. For this, rats were orally exposed to a single dose of these polystyrene NPs and the uptake in organs was determined. A negatively charged NP was taken up more than other NPs, with the highest amounts in kidney (37.4 µg/g tissue), heart (52.8 µg/g tissue), stomach wall (98.3 µg/g tissue) and small intestinal wall (94.4 µg/g tissue). This partly confirms our in vitro findings, where the same NPs translocated to the highest extent. The estimated bioavailability of different types of NPs ranged from 0.2 to 1.7 % in vivo, which was much lower than in vitro (1.6–12.3 %). Therefore, the integrated in vitro model cannot be used for a direct prediction of the bioavailability of orally administered NPs. However, the model can be used for prioritizing NPs before further in vivo testing for risk assessment.
Chemical substances are subjected to assessment of genotoxic and carcinogenic effects before being marketed to protect man and the environment from health risks. For agrochemicals, the long-term ...rodent carcinogenicity study is currently required from a regulatory perspective. Although it is the current mainstay for the detection of nongenotoxic carcinogens, carcinogenicity studies are shown to have prominent weaknesses and are subject to ethical and scientific debate. A transition toward a mechanism-based weight-of-evidence approach is considered a requirement to enhance the prediction of carcinogenic potential for environmental (agro)chemicals. The resulting approach should make optimal use of innovative (computational) tools and be less animal demanding. To identify the various mode of actions (MOAs) underlying the nongenotoxic carcinogenic potential of agrochemicals, we conducted an extensive analysis of 411 unique agrochemicals that have been evaluated for carcinogenicity by the United States Environmental Protection Agency (US EPA) and the European Chemicals Agency (ECHA). About one-third of these substances could be categorized as nongenotoxic carcinogens with an average of approximately two tumor types per substance, observed in a variety of organs. For two-third of the tumor cases, an underlying MOA (network) could be identified. This analysis demonstrates that a limited set of MOA (networks) is underlying nongenotoxic carcinogenicity of agrochemicals, illustrating that the transition toward a MOA-driven approach appears manageable. Ultimately the approach should cover relevant MOAs and its associated key events; this will also facilitate the evaluation of the human relevance. This manuscript describes the results of the analysis while identifying knowledge gaps and necessities to achieve a mechanism-based weight-of-evidence approach.
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With our recently developed in vitro physiologically based kinetic (PBK) modelling approach, we could extrapolate in vitro toxicity data to human toxicity values applying PBK-based reverse dosimetry. ...Ideally information on kinetic differences among human individuals within a population should be considered. In the present study, we demonstrated a modelling approach that integrated in vitro toxicity data, PBK modelling and Monte Carlo simulations to obtain insight in interindividual human kinetic variation and derive chemical specific adjustment factors (CSAFs) for phenol-induced developmental toxicity. The present study revealed that UGT1A6 is the primary enzyme responsible for the glucuronidation of phenol in humans followed by UGT1A9. Monte Carlo simulations were performed taking into account interindividual variation in glucuronidation by these specific UGTs and in the oral absorption coefficient. Linking Monte Carlo simulations with PBK modelling, population variability in the maximum plasma concentration of phenol for the human population could be predicted. This approach provided a CSAF for interindividual variation of 2.0 which covers the 99th percentile of the population, which is lower than the default safety factor of 3.16 for interindividual human kinetic differences. Dividing the dose-response curve data obtained with in vitro PBK-based reverse dosimetry, with the CSAF provided a dose-response curve that reflects the consequences of the interindividual variability in phenol kinetics for the developmental toxicity of phenol. The strength of the presented approach is that it provides insight in the effect of interindividual variation in kinetics for phenol-induced developmental toxicity, based on only in vitro and in silico testing.
At present, regulatory assessment of systemic toxicity is almost solely carried out using animal models. The European Commission’s REACH legislation stimulates the use of animal-free approaches to ...obtain information on the toxicity of chemicals. In vitro toxicity tests provide in vitro concentration-response curves for specific target cells, whereas in vivo dose-response curves are regularly used for human risk assessment. The present study shows an approach to predict in vivo dose-response curves for developmental toxicity by combining in vitro toxicity data and in silico kinetic modeling. A physiologically based kinetic (PBK) model was developed, describing the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites in rat and man. In vitro toxicity data of these metabolites derived in the embryonic stem cell test were used as input in the PBK model to extrapolate in vitro concentration-response curves to predicted in vivo dose-response curves for developmental toxicity of the parent glycol ethers in rat and man. The predicted dose-response curves for rat were found to be in concordance with the embryotoxic dose levels measured in reported in vivo rat studies. Therefore, predicted dose-response curves for rat could be used to set a point of departure for deriving safe exposure limits in human risk assessment. Combining the in vitro toxicity data with a human PBK model allows the prediction of dose-response curves for human developmental toxicity. This approach could therefore provide a means to reduce the need for animal testing in human risk assessment practices.
Assessment of genotoxic and carcinogenic potential is considered one of the basic requirements when evaluating possible human health risks associated with exposure to chemicals. Test strategies ...currently in place focus primarily on identifying genotoxic potential due to the strong association between the accumulation of genetic damage and cancer. Using genotoxicity assays to predict carcinogenic potential has the significant drawback that risks from non-genotoxic carcinogens remain largely undetected unless carcinogenicity studies are performed. Furthermore, test systems already developed to reduce animal use are not easily accepted and implemented by either industries or regulators. This manuscript reviews the test methods for cancer hazard identification that have been adopted by the regulatory authorities, and discusses the most promising alternative methods that have been developed to date. Based on these findings, a generally applicable tiered test strategy is proposed that can be considered capable of detecting both genotoxic as well as non-genotoxic carcinogens and will improve understanding of the underlying mode of action. Finally, strengths and weaknesses of this new integrative test strategy for cancer hazard identification are presented.
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Toxicity outcomes derived in vitro do not always reflect in vivo toxicity values, which was previously observed for a series of phenols tested in the embryonic stem cell test (EST). Translation of in ...vitro data to the in vivo situation is therefore an important, but still limiting step for the use of in vitro toxicity outcomes in the safety assessment of chemicals. The aim of the present study was to translate in vitro embryotoxicity data for a series of phenols to in vivo developmental toxic potency values for the rat by physiologically based kinetic (PBK) modelling-based reverse dosimetry. To this purpose, PBK models were developed for each of the phenols. The models were parameterised with in vitro-derived values defining metabolism and transport of the compounds across the intestinal and placental barrier and with in silico predictions and data from the literature. Using PBK-based reverse dosimetry, in vitro concentration–response curves from the EST were translated into in vivo dose–response curves from which points of departure (PoDs) were derived. The predicted PoDs differed less than 3.6-fold from PoDs derived from in vivo toxicity data for the phenols available in the literature. Moreover, the in vitro PBK-based reverse dosimetry approach could overcome the large disparity that was observed previously between the in vitro and the in vivo relative potency of the series of phenols. In conclusion, this study shows another proof-of-principle that the in vitro PBK approach is a promising strategy for non-animal-based safety assessment of chemicals.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic ...Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.
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Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large ...numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.
•EPAA workshop: mechanism-based approach for cancer risk assessment of agrochemicals•Combining non-genotoxic carcinogenicity MOA data with subchronic toxicity studies•Weight-of-evidence approach to identify carcinogenic potential of a test substance•Novel realistic approach that could satisfy different regulatory needs
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