Development Of Test Guidelines Research Articles

Development of a reference and proficiency chemical list for human steatosis endpoints in vitro.

The most prevalent liver disease in humans is non-alcoholic fatty liver disease, characterised by excessive hepatic fat accumulation, or steatosis. The western diet and a sedentary lifestyle are considered to be major influences, but chemical exposure may also play a role. Suspected environmental chemicals of concern include pesticides, plasticizers, metals, and perfluorinated compounds. Here we present a detailed literature analysis of chemicals that may (or may not) be implicated in lipid accumulation in the liver, to provide a basis for developing and optimizing human steatosis-relevant in vitro test methods. Independently collated and reviewed reference and proficiency chemicals are needed to assist in the test method development where an assay is intended to ultimately be taken forward for OECD Test Guideline development purposes. The selection criteria and considerations required for acceptance of proficiency chemical selection for OECD Test Guideline development. (i.e., structural diversity, range of activity including negatives, relevant chemical sectors, global restrictions, etc.) is described herein. Of 160 chemicals initially screened for inclusion, 36 were prioritized for detailed review. Based on the selection criteria and a weight-of-evidence basis, 18 chemicals (9 steatosis inducers, 9 negatives), including some environmental chemicals of concern, were ranked as high priority chemicals to assist in vitro human steatosis test method optimisation and proficiency testing, and inform potential subsequent test method (pre-)validation.

Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a ‘tier 2’ assay for dermally exposed compounds and was recently accepted into the OECD’s test guideline development program.

Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

First person profile: William C. Faquin, MD, PhD: This Harvard pathologist has helped to standardize how certain tumors are tested and diagnosed.

First person profile: William C. Faquin, MD, PhD: This Harvard pathologist has helped to standardize how certain tumors are tested and diagnosed.

Polypropylene-MWCNT composite degradation, release, detection, and toxicity of MWCNT during accelerated aging.

Nanomaterials (NM) are incorporated into polymers to enhance their properties. However, there are a limited number of studies on the aging of these nanocomposites and the resulting potential release of NM. To characterize NM at critical points in their life cycles, polypropylene (PP) and multiwall carbon nanotube filled PP (PP-MWCNT) plates with different thicknesses (from 0.25 mm to 2 mm) underwent accelerated weathering in a chamber that simulates solar irradiation and rainfall. The physicochemical changes of the plates depended on the radiation exposure, the plate thickness, and the presence of CNT fillers. Photodegradation increased with aging time, making the exposed surface more hydrophilic, decreasing the surface hardness and creating surface stress-cracks. Aged surface and cross-section showed crazing due to the polymer bond scission and the formation of carbonyls. The degradation was higher near the UV-exposed surface as the intensity of the radiation and oxygen diffusion decreased with increasing depth of the plates, resulting in an oxidation layer directly proportional to oxygen diffusion. Thus, sample thickness determines the kinetics of the degradation reaction and the transport of reactive species. Plastic fragments, which are less than 1 mm, and free CNTs were released from weathered MWCNT-PP. The concentrations of released NM that were estimated using ICP-MS, increased with prolonged aging time. Various toxicity tests, including reactive oxygen species generation and cell activity/viability, were performed on the released CNTs. The toxicity of the released fragments and CNTs to A594 adenocarcinomic human alveolar basal epithelial cells was observed. The released polymer fragments and CNTs did not show significant toxicity under the experimental conditions in this study. This study will help manufacturers, users of consumer products with nanocomposites and policymakers in the development of testing guidelines, predictive models, and risk assessments and risk based-formulations of NM exposure.

Comparative analysis of methodological approaches to morphological description of plant varieties in Ukraine and European Union countries

Purpuse. To develop scientific and methodological principles of legal protection of plant varieties in Ukraine in accordance with the requirements of the Community Plant Variety Office (CPVO) and the European Union (EU), to analyze the relevant methodological and legal norms and approa­ches aimed to improve the system of legal protection of plant varieties as intellectual property objects identified during the expert determination the criteria for distinctiveness, uniformity and stability (DUS-test).Results. Determination of the criteria for distinctiveness, uniformity and stability implies the plant varieties identification by the method of phenotype morphological description (the signs of vegetative and generative organs). The morphological code formula of the variety consists of the corresponding codes of the sign and the degree of its expression that is formed according to the technical documents and DUS methods in all countries of the International Union for the Protection of New Varieties of Plants (UPOV) and CPVO. Methodological provision for the protection of breeder’s rights and new plant varieties in the EU and the CPVO protocols were analyzed by the comparative method of the general parts and the tables of signs of DUS techniques for the botanical taxons of 4 plant groups: agricultural, vegetable, fruit and decorative.Conclusions. As a result of comparative assessment of DUS methods for plant varieties (CPVO – Protocols, UPOV – TG, Ukraine – UATG) was found that the morphological des­cription of plant varieties during the field trials (qualitative (QL), quantitative (QN) and pseudo-qualitative (PQ) signs of phenotypes ) is the basic technique for conducting DUS testing according to the international UPOV requirements as well as to CPVO requirements. National Methods for Plant Varieties Examination for Distinctness, Uniformity and Stability are developed under mandatory requirements of the DOCUMENT TGP 7/3 DEVELOPMENT OF TEST GUIDELINES adopted by the Council at its forty-eighth ordinary session on October 16, 2014. The CPVO and EU methodological bases at the 2015 research stage included protocols for 160 botanical taxons, represented by four plant groups: agricultural (18); vegetables (46); fruit (30) and decorative (66). The CPVO protocols for 122 botanical taxons, namely: agricultural (18), vegetable (44); fruit (28) and decorative (32) are of great value for the application’s scientific and technical examination for plant variety registration inUkraine. Field tests of plant phenotypes in accordance with morpho-descriptive method for variety identification by the relevant features and their degree of expression for harmonized description of plant varieties is practically brought to the UPOV international requirements and partly to CPVO protocols of the European Union.

Development of test guidelines and procedures for durian under Plant Varieties Protection Act B.E. 2542 according to ASEAN standards

Development of test guidelines and procedures for durian under Plant Varieties Protection Act B.E. 2542 according to ASEAN standards

Barriers to making recommendations about medical tests: a qualitative study of European guideline developers.

ObjectivesDevelopment of medical test guidelines differs from intervention guideline development. These differences can pose unique challenges in building evidence-based recommendations to guide clinical practice. The aim of our study was to better understand these challenges, explore reasons behind them and identify possible solutions.Setting and participantsIn this qualitative study, we conducted in-depth interviews between February 2012 and April 2013 of a convenience sample of 17 European guideline developers experienced in medical test guideline development.Outcomes measuredWe used framework analysis with deductive and inductive approaches to generate the themes from the interviews. We kept interpretation grounded in the data.ResultsGuideline developers acknowledged that inclusion of patient important outcomes in their guideline development was necessary but lacking. This and other challenges raised fell into 3 broad and overlapping domains: methodological issues, resource limitations and a lack of awareness on the need for evidence that links testing to patient outcomes. Education was mentioned as a key solution to increase awareness and address the resources limitations mentioned.ConclusionsChallenges guideline developers face were interlinked across the domains of methodological issues, resource limitations and a lack of awareness. Solutions that addressed these challenges in parallel are needed. Raising awareness, education and training of relevant stakeholders such as medical doctors, funders and regulators to look beyond test accuracy is key to having a long-term resolution to the issues faced in medical test guideline development.

International regulatory needs for development of an IATA for non-genotoxic carcinogenic chemical substances.

Although regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests. If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, which allows the detection of non-genotoxic carcinogens (NGTxC). However, under most chemical regulations the cancer bioassay is rarely requested, specific requests to obtain information on non-genotoxic mechanisms of carcinogenicity are few, and there are no OECD approved screening methods. When the in vitro genotoxicity battery is negative, usually no further carcinogenicity testing is requested. Consequently NGTxC might remain unidentified and therefore the risks they may pose to human health will not be managed. In contrast to genotoxic carcinogens NGTxCact through a large variety of specific mechanisms, and a panel of tests covering multiple biological traits will be needed. The development of an Integrated Approach to Testing and Assessment (IATA) of NGTxC could assist regulatory decision makers. We examine what NGTxC are and discuss chemical regulatory requirements and limitations. With a strong drive to reduce animal testing and costs in mind, it is essential that proper and robust alternatives for animal testing (3Rs) methods for addressing non-genotoxic modes of action are developed and used. Therefore relevant in vitro mechanisms and assays are described and tentatively organized in levels of information, indicating both a possible structure of the future IATA for NGTxC and associated OECD Test Guideline development priorities.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda)

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol.Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608μgCdL−1). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68<EC50–56d<124μgL−1) and were consistent with literature data. EC50–56d and EC10–56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC50–56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC50–56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin).

Evidence-based toxicology – the toolbox of validation for the 21st century?

Validation has become a primary driver of the evolution of toxicological methods. Agreement at OECD level currently requires validation of new approaches for consideration in test guideline development. Several examples of this exist. However, the toxicology in the 21(st) century movement, prompted by the 2007 NRC/NAS vision document, might lead to a revolutionary change in the toxicological toolbox. The challenge is whether the validation process, as it has been formalized over the last two decades, meets the needs for this paradigm shift.The concept of evidence-based medicine (EBM) has emerged from clinical medicine, which retrospectively assesses the evidence of adequacy of a given approach. This is not typically done in prospective studies - the equivalent of validation studies might be multicenter randomized trials. Evidently, where such unambiguous evidence is available, no other assessment is necessary. EBM, however, has developed procedures, including meta-analysis, to collect and evaluate all the available evidence where no such definitive study is available. The recent successful introduction of retrospective validation, i.e. the collection and evaluation of existing evidence from various sources, represents a step in this direction. Here, we will explore new toxicological approaches via evidence-based toxicology (EBT).

International comparison of criteria for evaluating sensitization of PRTR-designated chemical substances

In this study, we aim to compare the criteria for sensitizers among national organizations in various countries and international organizations, and to specify whether each Pollutant Release and Transfer Register (PRTR)-designated chemical substance is a sensitizer by each organization. The definition of sensitizing chemicals and the designation of respective sensitizers according to the PRTR law, Japan Society for Occupational Health (JSOH), American Conference of Governmental Industrial Hygienists (ACGIH), European Union (EU), and Deutsche Forschungsgemeinshaft (DFG) were studied. Of the 435 PRTR-designated chemical substances, 15 are listed as sensitizers according to the PRTR law, 16 as sensitizers of the airway and 21 as sensitizers of the skin by JSOH, 12 as sensitizers (no discrimination) by ACGIH, 19 (airway) and 85 (skin) by EU, and 15 (airway) and 43 (skin) by DFG. Only 9 substances were designated as sensitizers by all these organizations. The variation in the designation of sensitizers is accounted for by the differences in the classification criteria and grouping of chemical substances. JSOH limits the definition of sensitizers to substances that induce allergic reactions in humans and uses only human data. Other organizations utilize not only human evidence but also appropriate animal tests. In addition, EU designates an isocyanate as a sensitizer except those for which there is evidence showing that they do not cause respiratory sensitivity. The worldwide enforcement of the globally harmonized system (GHS) of classification and labeling of chemicals could promote not only the consistent designation of sensitizers among national and international organizations, but also the development of testing guidelines and classification criteria for mixtures.

Peer Review of Validation Studies: An Assessment of the Role of the OECD by Reference to the Validation of the Uterotrophic Assay for Endocrine Disruptors

The involvement of the OECD in managing the validation of the rat uterotrophic assay for endocrine disruptors, and in organising the peer review of the results of this study, has been assessed and compared with the many conclusions and recommendations in several published reports of international workshops on validation, and information in guidance documents, produced by the European Centre for the Validation of Alternative Methods (ECVAM), the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the OECD itself. It is concluded that the OECD has not followed the recommendations for full transparency and independence of the peer-review process. This is based on the fact that it has published a draft guidance document that differs from the report of a recent OECD workshop on validation, in such a way as to give the OECD the flexibility to fully control the peer-review process and, in so doing, to avoid full transparency. Comparison of the timing of the organisation of workshops by the OECD and the progression of the validation study, together with the fact that a draft test guideline for the assay was written before completion of the peer review, suggest that the OECD has given a higher priority to the expedition of the validation and regulatory acceptance of the uterotrophic assay than it has to good scientific and logistical practice. This severely undermines its credibility in the validation process, so, in order for the OECD to be rightly perceived as an honest broker, it is recommended that the OECD should play no role in the validation of new or revised tests, until after they have been successfully validated, peer reviewed, and endorsed by the appropriate authorities, and are ready for test guideline development. With regard to the on-going OECD validation studies of other in vivo assays for endocrine disruptors, the OECD should take immediate steps to ensure full independence and transparency of their peer review.

Mutual acceptance of data: harmonised test methods and quality assurance of data—the process explained

An essential aspect of the OECD is that it should not be considered a supranational organisation, but rather a center for discussion where governments express their points of view, share their experiences and search for common ground. This implies that decisions are made by consensus instead of majority. Once the Council, which is the highest authority of the OECD, adopts a formal Decision, such a decision is binding on all Member countries. The OECD Guidelines for the Testing of Chemicals, which are considered the leading international standard for safety testing, form an integrated part of such a binding Council Decision. An even more important part of that same Council Decision is that on Mutual Acceptance of Data, where it states that: ‘Data generated in the testing of chemicals in an OECD Member country in accordance with OECD Test Guidelines and OECD Principles of Good Laboratory Practice (GLP) shall be accepted in other Member countries for purposes of assessment and other use relating to the protection of man and the environment.’ In the various steps of the process of Test Guideline development, the National Co-ordinators of the Test Guideline Programme play an important role. The initiative to start the development of a particular guideline can be taken by the OECD Secretariat, by one or more Member countries or, most importantly, by the scientific community itself. Proposals, received by the Secretariat are discussed at the yearly Meeting of the National Co-ordinators. During these meetings, priorities for future activities are set and the approach that should be followed in dealing with the selected activities is discussed. Quite often, so-called Detailed Review Papers (DRP's) form the basis of a new or updated Guideline. These DRP's, which are either prepared by a Member country or by a consultant appointed by the Secretariat, describe the current ‘state of the art’ in scientific progress and technical possibilities of a well-defined area of research. After completion, either an expert meeting or a commenting round will be organised. All Member countries will have sufficient possibilities to express their views. When the DRP is acceptable to the experts of all Member countries, the next step is to actually develop a Test Guideline. Similar to the procedure followed for the DRP, the Test Guideline proposal will be circulated for comment to all Member countries and should reach the desks of relevant experts, nominated by their National Co-ordinator. Frequently, in addition to the commenting rounds, Test Guideline proposals are discussed in special expert meetings. Once the experts reach consensus on a particular Test Guideline, the proposal is put forward to the Meeting of the National Co-ordinators for approval. Since each guideline will form an integrated part of the earlier mentioned Council Decision, each new guideline also needs formal adoption by the Council before it becomes effective.

The role of the UKEMS in the development of testing guidelines.

Twenty years ago UKEMS established a sub-committee to determine the minimal professional criteria that should be achieved to comply with mutagenicity testing requirements in the UK. Recommendations on the conduct of basic and supplementary tests were published in 1983 and 1984, respectively. Despite their local distribution, these recommendations had an impact around the world. Further guidelines for statistical evaluation of mutagenicity test data and revisions to the first two volumes followed. By the early 1990s the mood was for international harmonization rather than national or regional isolation. The processes by which UKEMS had achieved its testing recommendations in the 1980s and early 1990s were successfully employed in the International Workshops for Genotoxicity Testing, of which three have now been held, and made a significant impact on OECD guidelines and ICH guidance. Summary outcomes from the latest meeting (2002 Plymouth Workshop) are given.

HOW TO MEASURE NO EFFECT. PART IV: HOW ACCEPTABLE IS THE ECx FROM AN ENVIRONMENTAL POLICY POINT OF VIEW?

Recently, EC point estimation (ECx, a certain percentile of the concentration–effect curve) has been mentioned in scientific literature as an alternative to the concept of deriving a no observed effect concentration (NOEC) in toxicological and ecotoxicological tests. The question of whether the NOEC approach should be exchanged for the ECx approach concerns not only test guideline development, but has also consequences in the area of risk assessment and environmental quality objectives. Therefore, from an environmental policy point of view it is essential to look critically at the advantages and disadvantages of moving away from an NOEC to ECx estimation; in environmental policy the term ‘no effect’ as well as NOEC values themselves play an important role in legislation and procedures related to risk assessment and environmental quality objectives both at a national and an international level. It seems that much depends on the exact value for the ECx that will be selected, how the resulting data correspond to the NOECs, and whether in risk assessment procedures both NOECs and ECx values can be used side by side. It should be realized however, that even with international acceptance of the ECx concept, it will probably take many years before the necessary changes in legislation and associated documents will have been made. Keeping this in mind as well as the fact that the uncertainty in the whole risk assessment process will usually be much greater than the uncertainty in the NOEC estimation, one might seriously question whether it is worth starting this process. © 1997 John Wiley & Sons, Ltd.

Setting exposure standards: a decision process.

Increased emphasis on routine screening of chemicals for potential neurotoxicity has resulted in the development of testing guidelines and standardized procedures. A multiphased, tiered-testing strategy has been proposed by numerous expert panels to evaluate large numbers of chemicals. In a regulatory context, however, a formal tiered-testing approach is not used, mostly because of the constraints of differing regulatory authorities and the potential cost of such a testing strategy. Instead, current regulatory decision making utilizes all available animal and human data to identify a critical adverse effect which is then used for setting standards. Although the current decision-making process does not use a formal tiered-testing approach, it appears to identify chemicals with neurotoxic effects. An analysis of U.S. Environmental Protection Agency integrated risk information system (IRIS) indicates that about 20% of the chemicals having standards or health advisories are based on neurotoxicity.

Test guideline development and animal welfare: Regulatory acceptance of in vitro studies

Toxicity studies are necessary in order to be able to identify the potential hazards of chemical interference with human reproduction. Until today, most useful contributions to the assessment of possible human reproductive toxicity are considered to be made by animal studies. The OECD Test Guidelines provide the international standards for safety testing and, consequently, have traditionally focused on animal studies. However, the OECD Member countries consider the welfare of laboratory animals also of importance and are of the opinion that animal welfare considerations should significantly influence the work in the OECD Chemical Programme. A constant effort is being made to discover alternative testing systems and to achieve their regulatory acceptance. However, activities predominately focus on finding alternatives to existing animal studies, rather than developing nonanimal tests that could contribute significantly to the hazard identification process. An approach based on the selection of endpoints essential for hazard identification that would focus on demonstrating similarities with the “real life” target events is considered likely to achieve regulatory acceptance much earlier than an approach based on high correlations between the alternative method and the existing animal study it is supposed to replace, simply because it is never better than the existing method, but at most “almost as reliable.”