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.
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.
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 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.
FDA’s recently launched Predictive Toxicology Roadmap calls for the optimization of non-animal methods for the safety evaluation of drugs, consumer products and medical devices. We have created an Industry Consortium comprised of manufacturers of personal lubricants/vaginal moisturizers and companies interested in the advancement of animal alternatives working collaboratively with stakeholders and the US FDA to develop an in vitro testing approach that could be used in place of the rabbit vaginal irritation (RVI) in pre-market submissions.
Currently China is striving to adopt and implement non-animal, including in vitro, testing approaches for the safety assessment of cosmetics and ingredients. Collaborative efforts between industry and the Institute for In Vitro Sciences (IIVS, Gaithersburg, USA) have focused on the transfer of several OECD Test Guideline methods to government laboratories in China and have supported the creation of an in vitro toxicology testing laboratory within the Zhejiang Institute for Food and Drug Control (Hangzhou, China). Recently BASF SE (Ludwigshafen, Germany) and IIVS have partnered to introduce a cell based in vitro skin sensitization test, LuSens, into China using the principles of GIVIMP as a standard. This case study exemplifies the practical way in which the GIVIMP guidance can assist interested parties in the development, transfer and establishment of in vitro approaches.
To address ocular device induced phototoxicity, an in vitro 3T3 Neutral Red Uptake Phototoxicity test (OECD 432) for chemicals has been modified. Soft contact lenses formulated with three different photo-absorbing compounds and chemical solutions of these compounds were evaluated.
Alternative methods, including the validated 3T3 Neutral Red Uptake (NRU) Phototoxicity assay (OECD TG 432) may be used as a pre-clinical test to address phototoxicity. Currently, there are no validated alternative test methods to identify photoallergens; however, there are several validated alternative test methods to address skin sensitization, including the Direct Peptide Reactivity Assay (DPRA) (OECD 442C). To address photoallergy, we utilized the 3T3 Phototoxicity assay in combination with a modified photo-DPRA assay to determine if these assays were able to 1) identify compounds with phototoxicity potential and 2) discriminate between photoirritants and photoallergens.
Robust non-animal models and assays for pulmonary toxicology are required to make competent product development and risk assessments for new materials requiring toxicity testing. Three in vitro assays (goblet cell hyperplasia , ciliary beat frequency , and MUC5AC quantitation) were evaluated for performance and reproducibility. To assess these assays, 6 laboratories contributed data using a common protocol utilizing IL-13 as an inducer of adverse mucociliary-relevant tissue changes. MatTek EpiAirway™ and Epithelix MucilAir™ 3D tissue models were used to evaluate endpoints using histology for GCH, software-based applications, Cilia FA and SAVA, for CBF, and ELISA assay for MUC5AC.
E-vapor product liquids contain a variety of ingredient combinations that should be assessed for human risk. One human lung-relevant testing platform with reasonable throughput, is human precision-cut lung slices (HuPCLS). HuPCLS are arguably the most complex non-animal model of the lung, retaining native architecture and immune-competent cells over multi-week culture periods. HuPCLS were exposed to three concentrations (0.1%, 0.5%, and 1.2%) of propylene glycol (PG; an E-vapor product constituent) continuously for 16-days. Exposure-effects were evaluated biochemically (WST-8 assay) and histologically (viability assessment of H&E stained slides). Positive control treatments consisted of 10 µM Phortress and 13 µM bleomycin.