Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 2: Optimisation of cell line assays.

During the production of clostridial vaccines large numbers of mice are used for various in-process control tests. Replacement in vitro assays had been developed for the testing of the toxins and toxoids of several clostridial species, but none of these assays had been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), a project on clostridial vaccines for veterinary use was started as part of the EDQM-co-ordinated Biological Standardisation Programme (BSP). Within the framework of this project (coded BSP130) a collaborative study was organised to evaluate Vero cell-based alternative methods to the current mouse tests used to measure: i) the toxicity of Clostridium septicum toxin, ii) the absence of toxicity of C. septicum toxoid and iii) the antigenicity of C. septicum toxoid. The principal aims of the study were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the in vitro and current in vivo tests. The study results demonstrated good concordance, but the information gathered through the study (later on called Part 1) and the participants' workshop prompted the extension of the project in order to further optimise the in vitro protocols and improve their repeatability and reproducibility, which were comparable to but not better than those of the in vivo assays in Part 1. The 3 in vitro assays to be optimised in the extension of the BSP130 project were : i) the in vitro toxin neutralisation equivalence plus (TNE+), as a replacement for the in vivo minimum lethal dose (MLD) test for quantification of the toxicity of toxin; ii) the in vitro MLD, as a replacement for the in vivo MLD test for detection of residual toxicity associated with toxoid; iii) the in vitro total combining power (TCP), as a replacement for the in vivo TCP test for quantification of the antigenicity of toxoid. At this point, the Analytical Method Transfer Laboratory of Ceva-Phylaxia (Hungary), supported by the project management team, developed suitable SOPs for the 3 in vitro assays. These optimised methods were further assessed in BSP130 through a second international collaborative study (Part 2) aimed at defining repeatability and reproducibility in different laboratories and determining the levels of improvement compared with the original in vivo tests and the initial in vitro assays used in Part 1 of the project. Fourteen laboratories, comprising 4 public sector and 10 manufacturers' medicines control laboratories, from 11 countries participated in the collaborative Part 2 study, each testing 6 different C. septicum toxins and 6 C. septicum toxoids. Improved repeatability and reproducibility were observed for the optimised assays. The results of this study confirm the suitability of these assays for in-process control of C. septicum vaccines, with better repeatability and reproducibility than their in vivo equivalents. It is expected that, with appropriate minor changes and the use of relevant reagents, these optimised in vitro assays could be used not only for the assessment of C. septicum toxins and toxoids but for all cytotoxin-based clostridial antigens. The development and implementation of such in vitro assays would offer a great opportunity to significantly reduce animal usage, shorten the duration of QC test procedures and increase the precision of toxicity and antigenicity assays in clostridial veterinary vaccine in-process control. This would also provide more accurate and reproducible dosing of antigens in the final vaccine products, help to promote compendial acceptance and to proffer a basis for improved international harmonisation across this area of product testing.

Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Prevalence of mental disorders and deliberate self-harm in Greek male prisoners.

The aim of this survey was to determine the prevalence of current and lifetime mental disorder and deliberate self-harm among male prisoners in Greece. The subjects were 80 randomly selected remanded and sentenced prisoners in a Greek prison. They were assessed for mental disorder including suicidality and substance misuse using the Mini International Neuropsychiatric Interview (MINI). We also collected information regarding contact with psychiatric services, previous deliberate self-harm as well as physical health and conducted a brief assessment of their intellectual functioning. Mental disorder was diagnosed in 63 (78.7%) prisoners. The main diagnoses were: anxiety disorder, 30 (37.5%); major depression, 22 (27.5%); antisocial personality disorder, 30 (37.5%); alcohol dependence, 21 (26.3%) and opiate dependence 22 (27.5%) and schizophrenic or bipolar disorder 9 (11.2%). Deliberate self-harm prior to and during imprisonment was reported by 15% and 2.5% of prisoners, respectively, and 12 prisoners (15%) had IQ below 75. This survey identified a significant level of need for specialist mental health services in prison. Further studies are required to assess the specific needs of those patients who are too unwell to remain in prison, the need for specific treatments for substance misuse and improved assessment/treatment of common psychiatric disorders.

Evaluation of the preservative properties of Thymus vulgaris essential oil in topically applied formulations under a challenge test.

The preservative properties of thyme essential oil (3%) with a known composition were evaluated in two types of final formulations, suitable for use as pharmaceutical or cosmetic vehicles, by means of a standard challenge test proposed by the latest European Pharmacopoeia. The required preservation efficacy criteria were satisfied against the bacterial strains, against the yeast in one of the formulations, but not against the mould strain involved in this study. Interactions between the essential oil compounds and other factors present in the final formulation might have influenced the activity of this essential oil, leading to an incomplete satisfaction of the criteria.

Influence of temperature on irritation in the hand/forearm immersion test.

As indicated by in vitro experiments the penetration of irritants through the skin is significantly influenced by the temperature of the solution. In vivo experiments, demonstrated equally a significant influence of temperature in surfactant-induced skin irritation. In order to evaluate the irritant potential of detergent solutions under normal user conditions, we used the hand/forearm immersion test. We compared 2 detergents with different anionic character in a repetitive immersion protocol (30 min immersion on 4 consecutive days). The solutions were tested at 2 temperatures (37 degrees C and 40 degrees C). The irritation was quantified by assessment of the stratum corneum barrier function (transepidermal water loss), skin redness (a* colour parameter) and skin dryness (capacitance method). Both detergents affected the integrity of the skin in a significant way. The anionic content as well as the temperature of the solutions were found to be determinative for the irritant potential, with a stronger response for higher anionic content and temperature, respectively.

Relationship between anatomical skin site and response to halcinonide and methyl nicotinate studied by bioengineering techniques.

BACKGROUND/AIMS: Regional differences in percutaneous penetration and skin properties are well documented. However, only a few studies have investigated the relationship between substance penetration and specific skin characteristics in function of the body region. It was our aim to evaluate the physiological effect of topically applied substances in function of skin parameters determined at different body regions. METHODS: The characterization of the blanching response to a topically applied corticosteroid and the characterization of the erythema response to topically applied nicotinates were used as two indicators for the percutaneous penetration at selected body regions. Stratum corneum hydration, skin temperature, TEWL, the amount of skin surface lipids and perfusion of the superficial microcirculation were determined at the same body regions. RESULTS: Significant differences in pharmacodynamic response to the corticosteroids and the nicotinates were found in function of the body region. A strong correlation was detected between the parameters characterizing the pharmacodynamic response and the particular skin properties. CONCLUSION: The regional differences in pharmacological response to topically applied halcinonide and nicotinates may point to a body region dependent bioavailability of the vasoactive substances in the upper skin layers. The significant relationship between the parameters characterizing the physiological skin reaction and the determined skin parameters may be an indication of their involvement in the percutaneous penetration process. These regional differences in pharmacological response seem to be independent of the physicochemical properties of the topically applied substances.