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The significance of contributions in volumes 11-46 (1983 to 2018) of Alternatives to Laboratory Animals in relation to the reduction, refinement and replacement of animal experimentation in biomedical research and testing is reviewed and discussed by the journal's former editor-in-chief, with particular emphasis on the development and production of the journal itself, FRAME, the European Centre for the Validation of Alternative Methods and other organisations. The role of the journal in promoting the principles of humane research (as spelled out by William Russell and Rex Burch) and highlighting a range of important issues and focus topics is explored. These include: botulinum toxin potency testing; ethical issues; the use of human volunteers, and human cells and tissues; the use of non-human primates (especially chimpanzees) and dogs as laboratory animals; toxicity testing in relation to cosmetics, pharmaceuticals and chemicals; UK and EU politics and legislation; and test validation and invalidation. The review concludes by identifying some of the issues that still need to be discussed and some of the questions that urgently need to be addressed.

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BACKGROUND: Medicinal plants are used by a large proportion of the global population as complementary and alternative medicines. However, little is known about their toxicity. G. africana has been used to treat wounds, coughs and skin diseases and is used in cosmetic formulations such as lotions and shampoos. METHODS: The acute oral and dermal toxicity potential of G. africana was analyzed after a single administration of 300 and 2000 mg/kgbw for acute oral toxicity and 2000 mg/kgbw for acute dermal toxicity. Female Sprague-Dawley rats were used for the acute oral toxicity study whereas both male and female Sprague-Dawley rats were used for the acute dermal toxicity study. In the Episkin skin irritation test, the irritation potential of G. africana (concentrate) and G. africana (in-use dilution) extracts were assessed using the Episkin reconstituted human epidermis. In the dermal sensitization study, female CBA/Ca mice were treated with G. africana concentrations of 50, 100 and 200 mg/ml respectively. The vehicle of choice was dimethylformamide which acted as a control. RESULTS: The results of the acute oral and dermal toxicity studies revealed that the median lethal dosage (LD50) for G. africana extract in Sprague-Dawley rats was considered to exceed 2000 mg/kgbw. In the irritation test, the G. africana (concentrate) and G. africana (in-use dilution) extracts were non-irritant on the Episkin reconstituted human epidermis. In the dermal sensitization study, the stimulation index (SI) values for the mice treated with the G. africana extract at concentrations of 50, 100 and 200 mg/ml/kgbw, when compared to the control group, were 1.3, 0.9 and 1.3 respectively. The open application of the extract at the various concentrations did not result in a SI of ≥ 3 in any group. Hence, it did not elicit a hypersensitivity response. CONCLUSION: These findings demonstrate that the acute toxicity profile for G. africana is acceptable and can subsequently be used for single use in the pharmaceutical and cosmetic industries.

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The main purpose of the present study was to establish a non-animal photosafety assessment approach for cosmetics using in vitro photochemical and photobiochemical screening systems. Fifty-one cosmetics, pharmaceutics and other chemicals were selected as model chemicals on the basis of animal and/or clinical photosafety information. The model chemicals were assessed in terms of photochemical properties by UV/VIS spectral analysis, reactive oxygen species (ROS) assay and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Most phototoxins exhibited potent UV/VIS absorption with molar extinction coefficients of over 1000M(-1)cm(-1), although false-negative prediction occurred for 2 cosmetic phototoxins owing to weak UV/VIS absorption. Among all the cosmetic ingredients, ca. 42% of tested chemicals were non-testable in the ROS assay because of low water solubility; thereby, micellar ROS (mROS) assay using a solubilizing surfactant was employed for follow-up screening. Upon combination use of ROS and mROS assays, the individual specificity was 88.2%, and the positive and negative predictivities were estimated to be 94.4% and 100%, respectively. In the 3T3 NRU PT, 3 cosmetics and 4 drugs were incorrectly predicted not to be phototoxic, although some of them were typical photoallergens. Thus, these in vitro screening systems individually provide false predictions; however, a systematic tiered approach using these assays could provide reliable photosafety assessment without any false-negatives. The combined use of in vitro assays might enable simple and fast non-animal photosafety evaluation of cosmetic ingredients.

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Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.

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INTRODUCTION: Drugs that prolong the QT interval on the electrocardiogram present a major safety concern for pharmaceutical companies and regulatory agencies. Despite a range of assays performed to assess compound effects on the QT interval, QT prolongation remains a major cause of attrition during compound development. In silico assays could alleviate such problems. In this study we evaluated an in silico method of predicting the results of a rabbit left-ventricular wedge assay. METHODS: Concentration-effect data were acquired from either: the high-throughput IonWorks/FLIPR; the medium-throughput PatchXpress ion channel assays; or QSAR, a statistical IC50 value prediction model, for hERG, fast sodium, L-type calcium and KCNQ1/minK channels. Drug block of channels was incorporated into a mathematical differential equation model of rabbit ventricular myocyte electrophysiology through modification of the maximal conductance of each channel by a factor dependent on the IC50 value, Hill coefficient and concentration of each compound tested. Simulations were performed and agreement with experimental results, based upon input data from the different assays, was evaluated. RESULTS: The assay was found to be 78% accurate, 72% sensitive and 81% specific when predicting QT prolongation (>10%) using PatchXpress assay data (77 compounds). Similar levels of predictivity were demonstrated using IonWorks/FLIPR data (121 compounds) with 78% accuracy, 73% sensitivity and 80% specificity. QT shortening (<-10%) was predicted with 77% accuracy, 33% sensitivity and 90% specificity using PatchXpress data and 71% accuracy, 42% sensitivity and 81% specificity using IonWorks/FLIPR data. Strong quantitative agreement between simulation and experimental results was also evident. DISCUSSION: The in silico action potential assay demonstrates good predictive ability, and is suitable for very high-throughput use in early drug development. Adoption of such an assay into cardiovascular safety assessment, integrating ion channel data from routine screens to infer results of animal-based tests, could provide a cost- and time-effective cardiac safety screen.

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Revolutions in thinking and practice are essential in regulatory toxicology if genuine protection of human beings and the environment is truly to be improved. New test development is the key: Tests should have greater relevance than the current animal procedures based on (1) a mechanistic understanding of the basis of the test method itself and of the toxic phenomenon of concern, (2) taking advantage of new developments in cell and molecular biology and computer systems of various kinds, and (3) a clear understanding of the value of good prediction models. In the not-too-distant future, current research in genomics, proteomics, and metabolomics should provide opportunities for the development of valuable new tests. An inescapable requirement of tests intended to be used for regulatory purposes is validation (i.e., an independent assessment of relevance and reliability for stated purposes according to internationally agreed-upon criteria). However, there is no standard validation scheme; a case-by-case approach is essential. It is important to take advantage of experience, which reveals that prevalidation makes formal validation studies faster, less expensive, and more likely to succeed, and that the procedures for independent assessment used by the European Centre for the Validation of Alternative Methods (ECVAM) and the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) are effective in practice.

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The origins of the concept of replacement alternatives in the 1950s, and the impact of societal changes in the 1960s and 1970s, resulting in stricter controls on animal experimentation from the 1980s, based on the Three Rs of Russell and Burch (reduction, refinement and replacement), are reviewed. The range of replacement alternative methods, and some of the ethical issues they raise, and progress toward their incorporation into fundamental and applied research, education, and, in particular, toxicity testing, are discussed. It is concluded that much greater effort should be put into overcoming the barriers to the acceptance of replacement alternatives, which currently limit the contributions they have to make toward greater humanity and better biomedical science. Particular emphasis is place on the need to ensure that the validation of non-animal tests (for their reliability and relevance for specific purposes) is conducted fairly and objectively, and that greater heed is paid to the warning of Russell and Burch about the high fidelity fallacy and the questionable relevance of data provided by animal models for human hazard and risk assessment. Finally, the role of ECVAM in the promotion of valid replacement alternatives, and the opportunities afforded by the Sixth Amendment to the EC Cosmetics Directive, are discussed.

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