Exploring Waiving Opportunities for Mammalian Acute Systemic Toxicity Tests

Graepel EUSurvey

Rabea Graepel, David Asturiol, Pilar Prieto and Andrew P. Worth

A survey was carried out to explore opportunities for waiving mammalian acute systemic toxicity tests. We were interested in finding out whether data from a sub-acute toxicity test could be used to predict the outcome of an acute systemic toxicity test. The survey was directed at experts in the field of toxicity testing, and was carried out in the context of the upcoming 2018 final registration deadline for chemicals under the EU REACH Regulation. In addition to the survey, a retrospective data analysis of chemicals that had already been registered with the European Chemicals Agency, and for which both acute and sub-acute toxicity data were available, was carried out. This data analysis was focused on chemicals that were administered via the oral route. The answers to the questionnaire showed a willingness to adopt waiving opportunities. In addition, the responses showed that data from a sub-acute toxicity test or dose-range finding study might be useful for predicting chemicals that do not require classification for acute oral toxicity (LD50 > 2000mg/kg body weight). However, with the exception of substances that fall into the non-classified category, it is difficult to predict current acute oral toxicity categories.
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Non-animal Replacements for Acute Toxicity Testing

Carol Barker-Treasure, Kevin Coll, Nathalie Belot, Chris Longmore, Karl Bygrave, Suzanne Avey and Richard Clothier

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.
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Determination of the Starting Dose for Acute Oral Toxicity (LD50) Testing in the Up and Down Procedure (UDP) From Cytotoxicity Data

Horst Spielmann, Elke Genschow, Manfred Liebsch and Willi Halle

To reduce the number of animals used in acute oral toxicity testing, cytotoxicity data (IC50) can be used to determine the starting dose for in vivo testing by applying the standard regression between IC50 and acute oral LD50 values in the Register of Cytotoxicity (RC). In the RC, the correlation between cytotoxicity, represented by the mean IC50 (IC50x), and the acute oral LD50 of rats and/or mice has been determined for 347 chemicals by applying the linear regression model for log-transformed pairs of IC50 versus oral LD50. The standard regression line of the two toxicity parameters is characterised by an intercept a = 0.625 and regression coefficient b = 0.435, and 252 of 347 chemicals (72.6 %) are located within a dose-range differing by not more than 0.699 (factor FG ≤ log 5) from the standard regression line. In the present study, we have used the RC and its IC50/LD50 regression model to predict the LD50 values from cytotoxicity data for nine chemicals which were tested in an evaluation study of the Up and Down Procedure (UDP). For seven of the nine chemicals, LD50 values (mg/kg) predicted from the RC were in the same doserange as LD50 values determined in vivo, while the dose-range differed by more than one order of magnitude for the two remaining chemicals. Thus, the prediction of LD50 values from cytotoxicity data was promising in this limited data set. It is proposed that a tiered in vitro/in vivo testing approach will reduce animal use in the UDP method. As the first step, the in vitro cytotoxicity of a new chemical is determined. By applying the RC regression and adapting it to the sensitivity of a specific cell line, the LD50 value (mg/kg) can be predicted from the IC50 value. The predicted LD50 dose is then used as the starting dose in the UDP. In the RC model, the precision of the prediction increases with decreasing toxic potential, and the majority of industrial chemicals (around 90%) are not toxic according to EU classification criteria.
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The High Production Volume Chemical Challenge Program: The Rodent LD50 and its Possible Replacement

Herbert S. Rosenkranz and Albert R. Cunningham

The High Production Volume Chemical Challenge Program provides an opportunity to re-examine the usefulness and informational value of tests currently used to obtain preliminary hazard identification data. With a view to assessing the mechanistic information provided by the rodent LD50 test and to ascertain the possibility of replacing it with other “more acceptable” assays, we used a recently developed approach to determine the relationship of the LD50 assay to other toxicological protocols. Our analyses indicate that, of the assays examined, the LD50 was significantly related to toxicity in cultured cells and to binding at the Ah receptor.
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The Registry of Cytotoxicity: Toxicity Testing in Cell Cultures to Predict Acute Toxicity (LD50) and to Reduce Testing in Animals1

Willi Halle

This is a translation of a report on the Registry of Cytotoxicity (RC), originally published in German in 1998. The report presented an advanced in vitro method, which can significantly reduce the number of animals needed for the toxicity testing of a broad range of compounds/xenobiotics. With the RC method, it was possible to predict the oral or intravenous acute toxicity (LD50) — which is a regulatory requirement for newly developed pharmaceuticals and industrial and household chemicals — from the cytotoxicity data (mean IC50 = IC50X) obtained with mammalian cells. The RC method can be used before the in vivo test, and it does not pose any additional harm or suffering to laboratory animals. The RC method is of broad practical use: it can be applied, for example, in the pharmaceutical industry or the chemical industry in regulatory testing or in research. It is ready for validation, and could then be incorporated into OECD guidelines, thus reducing the total number of animals needed for regulatory toxicity testing. The RC method is based on the comparison of the IC50X values and the LD50 values by using linear regression analysis. With the RC method, it was possible to predict, within a predefined dose range, the acute oral LD50 for 252 of 347 xenobiotics, and the intravenous LD50 for rats and/or mice for 117 of 150 xenobiotics. Comparative studies showed that these results are highly reproducible.
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Growing Old Disgracefully: The Cosmetic Use of Botulinum Toxin

Krys Bottrill

The explosive growth in the use of botulinum toxin for cosmetic purposes has undoubtedly had an impact on the number of animals used in the potency testing of this product. The test used is a classical LD50, a severe procedure during which animals experience increasing paralysis until the occurrence of death. The enthusiastic adoption by the general public of the use of botulinum toxin as an anti-wrinkle treatment has, at least in Europe, paradoxically taken place against a background of moves to stop animal testing of cosmetics and cosmetic ingredients. There appears to be a dearth of information aimed at the public concerning botulinum toxin testing. Botulinum toxin does have important medical applications; however, the question arises whether a blanket licence for the testing can be justified, when a large proportion of the product is being used cosmetically. A further question is why death continues to be the endpoint of the potency test, when a more-humane endpoint has been proposed. In addition, a number of alternative methods have been developed, which could have the potential to replace the lethal potency test altogether. These methods are discussed in this paper, and the importance of establishing a strategy for their validation is emphasised, a need that has become even more urgent in the light of the recently published draft monograph on botulinum toxin by the European Pharmacopoeia Commission.
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Lack of Predictivity of the Rat Lethality (LD50) Test for Ecological and Human Health Effects

Herbert S. Rosenkranz and Albert R. Cunningham

The relationship between acute toxicity in rats (LD50 values) and indicators of potential health hazards in humans was investigated, based on a chemical population-based paradigm (i.e. the “chemical diversity approach”). These structure–activity relationship-based analyses indicate that high toxicity in rats (i.e. a low LD50 value) is not a good predictor of health effects in humans. In fact, it was found that high acute toxicity to minnows, as well as toxicity to cultured cells, showed significantly greater associations with the potential for health effects than rat LD50 values.
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2017-01-09T06:34:11+00:00 Tags: , , , |

The Integrated Acute Systemic Toxicity Project (ACuteTox) for the Optimisation and Validation of Alternative In Vitro Tests

Cecilia Clemedson, Ada Kolman and Anna Forsby

The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.
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The Tubifex tubifex Assay for the Determination of Acute Toxicity

Milonv Tichý, Marián Rucki, Iveta Hanzlíková and Zdenevk Roth

An express (3-minute) test for acute toxicity determination by using the oligochaete annelid, Tubifex tubifex, is described. The EC50(Tubifex tubifex) [EC50(Tt)] for movement inhibition was calculated by using a concentration–response dependence. The reproducibility of the test was checked over several years and by several workers. Its applicability is limited to compounds which are soluble in water. The calculated EC50(Tt) indices correlate with LC50 values determined by using the fish, Pimephales promelas (96- hour assay), and with ICG50 values determined by using the ciliate, Tetrahymena pyriformis (48-hour assay) with high statistical significance (r = 0.822, n = 35, and r = 0.927, n = 80, respectively). The correlation between the EC50(Tt) indices and rat oral LD50 values (48-hour assay) was r = 0.519 (n = 67). The correlation within organic compounds was closer (r = 0.635, n = 60) than with the heterogeneous series of chemicals. A similar trend was noticed for the correlation with mouse oral LD50 values (r = 0.479, n = 56) with the heterogeneous series of chemicals, as compared that with the series without inorganic salts (r = 0.605, n = 42), and similarly with mouse intraperitoneal LD50 values, where r = 0.543 (n = 50) with the heterogeneous series of chemicals and r = 0.893 (n = 33) with the series of organic chemicals.
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