Documents & Resources

Promoting the Uptake of Alternatives to Animal Testing Through the Development of eLearning Tools

In order to further promote the implementation of Directive 2010/63/EU, the European Commission issued calls for a number of related projects last year. One of these projects is aimed at facilitating the uptake of non-animal alternatives by developing two e-learning modules. The contract for this project was awarded to a consortium consisting of SYRCLE, the Swiss 3R Competence Centre, lnstitute for ln Vitro Sciences, Pharma Launcher and Ecorys UK. This consortium will develop two modules, i.e., one eLearning module focused on searching for existing non-animal alternatives (including systematic reviews) and one module targeted at researchers who want to develop reliable and relevant non-animal alternatives for regulatory use taking into account Good In Vitro Method Practices (GIVIMP). The quality of the developed modules will be assessed by external review groups. The learning outcomes will be presented to the commission along with the design of the assignments through which these outcomes will be realised.

kDPRA: An in chemico Method to Characterize the Skin Sensitization Potency of Chemicals

While the skin sensitization hazard of substances can readily be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA (OECD TG 442C) wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide is evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times at 25°C. After the respective incubation time, the reaction is stopped by addition of the fluorescent dye monobromobimane (mBBr). The highly reactive and non-fluorescent mBBr rapidly reacts with unbound cysteine moieties of the model peptide to form a fluorescent complex. The remaining non-depleted peptide concentration is determined thereafter by fluorescence measurement at precisely defined time points. Kinetic rates of peptide depletion are then used to distinguish between two levels of skin sensitization potency, i.e. to discriminate between CLP/UN GHS sub-categories 1A and 1B. During an in house validation (Wareing et al., 2017) 35 of 38 substances with LLNA-based sensitizing potency were correctly assigned to the potency sub-categories, and the predictivity for 14 human data was similarly high. These results warranted the kDPRA for further validation. Here we present the results of a ring trial testing 24 blind-coded chemicals in seven labs. In parallel we present the extension of the kDPRA database to further assess the predictive capacity of the assay. Eventually the kDPRA should be used as a part of defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment.

Shipping Study to Evaluate the Performance of the LabCyte EPI-MODEL 24 Tissues for Use in the Skin Irritation Test (OECD TG 439) After Long-Haul Airfreight

It is recommended that an evaluation of the impact of shipping of Reconstructed human Epidermis (RhE) tissues be conducted especially after long-haul airfreight shipments. The OECD Test Guideline 439 (OECD TG 439), In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, recommends that users do so by verifying the barrier properties of the tissues after receipt. In this study, LabCyte EPI-MODEL 24 tissues were received in the USA after an overnight shipment from Japan and were tested to evaluate their performance after shipment using several endpoints. First, the viability of untreated tissues incubated overnight in culture medium was assessed using the vital dye MTT and expressed as Optical Density (OD570-650) values. The calculated OD values were 1.2 (J-TEC) and 0.99 (IIVS) and within the range established by the manufacturer (0.8 - 2.5). The barrier function was further evaluated after the tissues were exposed to the assay negative control (Phosphate Buffered Saline - PBS) and to four concentrations of the positive control, Sodium Lauryl Sulfate (SLS) (1.0, 2.0, 3.0 and 4.0 mg/mL). The calculated IC50 values were 2.7 mg/mL (J-TEC) and 3.4 mg/mL (IIVS), respectively, and within the established historical range (1.4 - 4.0 mg/mL). The analysis of the results generated by the two labs (J-TEC and IIVS) demonstrated that the tissue lot met the acceptance criteria developed by the tissue manufacturer under conditions of shipping stress. Finally, the histological analysis of untreated fixed tissues identified all tissue layers and supported the conclusion that the tissue model was acceptable for use in subsequent studies after long-haul airfreight shipment. Such shipping studies are critical to gaining confidence in the tissues’ performance when used for research, industrial, and regulatory testing purposes.

Regarding the References for Reference Chemicals of Alternative Methods

The selection of reference and proficiency chemicals is an important component of method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate there liability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals, or other chemicals with sufficient supporting data, that are used by naive laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of response (in the validated reference method and in vivo). They shall be commercially available (at non prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency.

Increased Throughput and Cryopreservation of Precision-cut Lung Slices Extend the Utility of Human-relevant, 3-Dimensional Pulmonary Test Systems

Human-relevant, in vitro/ex vivo assays are considered an ethical and economically viable manner by which to screen the thousands of chemicals requiring hazard assessment. Of the 3-dimensional models, human precision-cut lung slices (PCLS) are often considered the most physiologically relevant pulmonary test system, but lower throughput and difficulties in cryopreservation have hampered PCLS use. We have modified a tissue slicer to accommodate 3 tissue cores for simultaneous slicing. Increased slice production was quantified using agarose and tissue cores in the slicer. To evaluate cryopreservation of PCLS, we have tested 5 cryopreservation formulations using PCLS (frozen on the day of slicing, or after overnight culture). Thawed slice viability in each of the groups was assessed with the WST-8 viability assay, prior to fixation and histological evaluation. The slicer modification resulted in 2.8-fold and 2.4-fold more slices from agarose cores, and lung cores, respectively. Cryopreservation efforts indicated freezing after slicing yields better average viability (48-73% of fresh, non-frozen control) than culturing overnight and freezing (13-54% of control) when assessing health over 4 days, post-thaw. Cryopreservation buffers containing University of Wisconsin preservation solution preserved viability the best (54%-90% of non-frozen control). Histological findings concurred with WST-8 viability results and indicated the retention of healthy lung tissue features (>75% of normal), post-thaw. The increased PCLS production indicates larger (or multiple) studies can be initiated from one donor lung. The promising cryopreservation results suggest slices can be banked and utilized at a later date, potentially even allowing the same donor’s tissue to be used repeatedly.

Next Generation Tobacco Products and Skin Staining

Next generation tobacco and nicotine products (NGPs) such as electronic cigarettes (EC) and tobacco heating products (THP) have reduced toxicity and hold great potential for reducing the risks associated with cigarette smoking. NGPs may also have hygiene benefits for consumers that switch to these products. In this study, the level of skin staining was assessed following exposure to a scientific reference cigarette (3R4F) and NGPs across the risk continuum; a prototype EC or a commercial THP (glo™). Test articles (TAs) were prepared by capturing cigarette smoke or EC/THP aerosol on Cambridge filter pads followed by elution with Dimethyl Sulfoxide (DMSO). Abattoir-obtained porcine skin (ø 0.5) samples were incubated at 37°C with each TA or DMSO control for 0, 0.25, 0.5, 1, 4 and 6h. Colour readings (L*, a*, b*) were measured for individual skin samples using a Konica Minolta CM-700d Spectrophotometer and mean colour change (ΔE) for each TA compared. The reference cigarette 3R4F TA showed the greatest colour change, which was significantly higher than the EC and THP TAs, both of which showed relatively little colour change. The mean ΔE values at 6 hrs were: 21.78 ± 2.80, 8.38 ± 2.93, 10.01 ± 2.53 and 9.23 ± 2.87 for 3R4F, a prototype EC, glo™ or DMSO respectively. The cigarette smoke extract significantly increased the level of staining of the skin samples whereas EC or the THP TAs induced little or no staining with values comparable to the DMSO control. For the first time, diverse NGPs across the risk continuum have been assessed in vitro for their impact on skin staining. Further studies are required to assess the long-term impact on skin staining of NGP aerosols.

Video: The 3T3 Neutral Red Uptake Phototoxicity Test

This training video explores a cell-based method for assessing Phototoxicity — or the potential for chemicals to cause damage after being exposed to light.  The method is used widely around the world by many industries, including the cosmetics and pharmaceutical sectors. Disclaimer:  Please note that the procedures shown in this video were for demonstration ...

Non-animal Vaginal Irritation Method Admitted into the US FDA Medical Devices Development Tool (MDDT)

How the Good In Vitro Method Practices Guidance Document Can Help Implement New Toxicological Approaches: A Case Study in China

Webinar: Precision-cut Lung Slices