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.
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 ...
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.
Dr. Holger Behrsing, Principal Scientist with IIVS, recently presented "Precision-cut Lung Slices (PCLS)" for the 2018 PETA International Science Consortium webinar series on non-animal inhalation testing methods
Read our latest publication with Bristol-Myers Squibb regarding a tiered testing strategy for occupational hazards of pharmaceutical intermediates now published in the Cutaneous and Ocular Toxicology Journal. Irritation reactions are a frequently reported occupational illness. The potential adverse effects of pharmaceutical compounds (PCs) on employees' eyes and skin can now be assessed using validated in vitro methods. Our overall aim is to reduce animal testing by replacing the historically utilized in vivo test methods with validated in vitro test methods which accurately determine the ocular and dermal irritation/corrosion potential of PCs to inform worker safety within the pharmaceutical space. BMS and IIVS have therefore conceptualized and internally qualified a tiered in vitro testing strategy to inform occupational hazards regarding eye and skin irritation and corrosivity of PCs. which is based on three Organisation for Economic Co-operation and Development (OECD) in vitro methods (OECD TG 435, OECD TG 437, OECD TG 439.) Read the full article.
A framework for the assessment of novel next-generation tobacco and nicotine products with the potential to reduce health risks compared with cigarettes should integrate scientific studies incorporating nonclinical, clinical, and population studies approaches. These products should have lower emissions and exhibit reduced biological effects compared with those of cigarettes, ideally approaching those of smoking cessation. The products should also be acceptable cigarette alternatives for current smokers, while not appealing to nonsmokers or former smokers. Validating harm reduction and promoting regulatory acceptance of the assessment methods require a collaborative approach by industry, independent reviewers, the public health community, and regulatory agencies. This article summarizes the science and approaches applied and considered to substantiate tobacco harm reduction in the light of regulatory requirements. Read the full article.
The U.S. Food and Drug Administration has regulatory authority over tobacco products, including conventional cigarettes and next generation products (NGPs) such as e-cigarettes and tobacco heating products (THPs). There is a desire by the industry, regulators and animal protection organizations to incorporate non-animal test methods for tobacco product and NGP assessment. When assessing respiratory effects in vitro, reliable exposure systems that deliver aerosols to cellular/tissue cultures (such as human reconstructed airways or lung slices) at the air–liquid interface are needed. Using nicotine dosimetry, we report the characterization of a Vitrocell VC1 in our laboratories (IIVS, USA). Nicotine, generated from a 3R4F reference cigarette or NGP (e-cigarette and THP) aerosols at source and the exposure interface (culture media), was assessed using ultra-high-performance liquid chromatography–tandem mass spectrometry. These data were compared to published dosimetry data for the same products, generated at a different laboratory (BAT R&D, Southampton, UK), on different exposure systems (VC10 and Borgwaldt RM20S) to confirm repeatability. Read the full article.