Use of guinea pig data to obtain starting points for skin sensitisation risk assessment - A commentary.

The increasing drive to understand the likelihood of skin sensitisation from plant protection products (PPPs) in workers and the general public has resulted in recent initiatives to establish a quantitative risk assessment (QRA) methodology applicable to these products and their exposure scenarios. The effective evaluation of skin sensitising substances requires not only the identification of that toxicological hazard, but also determination of relative sensitising potency. Typically, this has been achieved by interpretation of local lymph node assay (LLNA) dose response data, delivering what is known as the EC3 value. This permitted regulatory division of skin sensitisers into defined potency sub-categories, but more importantly enabled derivation of a no expected sensitisation induction level (NESIL) as the point of departure for QRA. However, for many existing substances there is no LLNA data, only older guinea pig results exist. To avoid additional (in vivo) testing, an approach has been outlined to employ guinea pig data and existing regulatory guidelines on the determination of potency sub-categorisation to provide a guinea pig based NESIL. The approach adopts a conservative extrapolation from LLNA NESIL benchmarks to deliver points of departure as the basis for the type of QRA process already in successful use by other industries.

Reference Chemical Potency List (RCPL): A new tool for evaluating the accuracy of skin sensitisation potency measurements by New Approach Methodologies (NAMs).

Considerable progress has been made in the design of New Approach Methodologies (NAMs) for the hazard identification of skin sensitising chemicals. However, effective risk assessment requires accurate measurement of sensitising potency, and this has proven more difficult to achieve without recourse to animal tests. One important requirement for the development and adoption of novel approaches for this purpose is the availability of reliable databases for determining the accuracy with which sensitising potency can be predicted. Some previous approaches have relied on comparisons with potency estimates based on either human or animal (local lymph node assay) data. In contrast, we here describe the development of a carefully curated Reference Chemical Potency List (RCPL) which is based on consideration of the best available human and animal data. The RCPL is comprised of 33 readily available chemicals that span a wide range of chemistry and sensitising potency, and contain examples of both direct and indirect (pre- and pro-) haptens. For each chemical a potency value (PV) was derived, and chemicals ranked according to PV without the use of potency categories. It is proposed that the RCPL provides an effective resource for assessment of the accuracy with which NAMs can measure skin sensitising potency.

Non-animal methods to predict skin sensitization (II): an assessment of defined approaches *.

Skin sensitization is a toxicity endpoint of widespread concern, for which the mechanistic understanding and concurrent necessity for non-animal testing approaches have evolved to a critical juncture, with many available options for predicting sensitization without using animals. Cosmetics Europe and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods collaborated to analyze the performance of multiple non-animal data integration approaches for the skin sensitization safety assessment of cosmetics ingredients. The Cosmetics Europe Skin Tolerance Task Force (STTF) collected and generated data on 128 substances in multiple in vitro and in chemico skin sensitization assays selected based on a systematic assessment by the STTF. These assays, together with certain in silico predictions, are key components of various non-animal testing strategies that have been submitted to the Organization for Economic Cooperation and Development as case studies for skin sensitization. Curated murine local lymph node assay (LLNA) and human skin sensitization data were used to evaluate the performance of six defined approaches, comprising eight non-animal testing strategies, for both hazard and potency characterization. Defined approaches examined included consensus methods, artificial neural networks, support vector machine models, Bayesian networks, and decision trees, most of which were reproduced using open source software tools. Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs demonstrated equivalent or superior performance to the LLNA when compared to both animal and human data for skin sensitization.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays.

Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.

Potency Assessment of CBD Oils by Their Effects on Cell Signaling Pathways.

This study used nanofluidic protein posttranslational modification (PTM) profiling to measure the effects of six cannabidiol (CBD) oils and isolated CBD on the signaling pathways of a cultured SH-SY5Y neuronal cell line. Chemical composition analysis revealed that all CBD oils met the label claims and legal regulatory limit regarding the CBD and tetrahydrocannabinol (THC) contents, respectively. Isolated CBD was cytotoxic, with an effective concentration (EC50) of 40 µM. In contrast, the CBD oils had no effect on cell viability at CBD concentrations exceeding 1.2 mM. Interestingly, only an unadulterated CBD oil had strong and statistically significant suppressive effects on the pI3K/Akt/mTOR signaling pathway with an EC50 value of 143 µM and a slow-acting timescale requiring hours. Systematic profiling of twenty-six proteins, which served as biomarkers for nine signaling pathways, revealed that the unadulterated CBD oil downregulated seven signaling pathways but had no measurable effect on the other two signaling pathways. The remaining CBD oils, which were adulterated, and isolated CBD had weak, variable, or undetectable effects on neuronal signaling pathways. Our data clearly showed that adulteration diminished the biological activities of CBD oils. In addition, nanofluidic protein PTM profiling provided a robust means for potency assessment of CBD oils.