Alternative Test Methods Research Articles

Operator exposure and cabin protection in plant protection product application

AbstractTightly sealed driver's cabs can effectively protect the user from exposure when applying crop protection products. Therefore, under certain conditions, users in such cabins can avoid wearing personal protective equipment for skin and eyes. The protective effect depends on the technical design and handling of the different cabin types and can be divided into categories with different levels of protection. Since the level of protection of the different types of cabins and the associated possibility of not wearing personal protective equipment has not been sufficiently scientifically verified in the past, the present study was carried out to close this gap. Field and laboratory test methods were developed and used to assess exposure levels. External contamination of the cabin was measured using various dosimeters. Exposure inside the cabins was measured on coveralls and gloves to measure dermal exposure and with aerosol sampling pumps to determine inhalation exposure. Tests were conducted on tractors with different cab categories using an air-assisted orchard sprayer as a worst-case scenario for exposure to spray drift. Additionally, an alternative laboratory test method was developed and conducted to evaluate filter efficiency under controlled conditions and compare it with the field tests. The results showed that exposure inside closed cabins was generally low and was not notably correlated with the cabin category. All types of cabins tested were found to provide significant protection from dermal and inhalation exposure.

Factors Affecting Procedural Pain and Discomfort Experienced by Individuals During Nasopharyngeal Swabbing: A Cross-Sectional Study.

This descriptive and cross-sectional study investigated the individual factors that affect procedural pain and discomfort in individuals who had nasopharyngeal swabs by nurses. A total of 193 participants in two COVID-19 test centers located in a city in Southeastern Türkiye between April and May 2022 were included in this study. Personal information form was used to collect data. The visual analog scale was used to assess the pain and discomfort. Risk factors related to pain and discomfort were investigated through a logistic regression model. During the nasopharyngeal swabbing, it was determined that pain was stronger in women and those who considered nasopharyngeal swabbing as a painful procedure (p < .05). Also, discomfort was stronger in women and those who considered nasopharyngeal swabbing as an uncomfortable procedure (p < .05). It was determined that the participants' discomfort scores were higher than their pain scores. Women and individuals with negative opinions about the procedure experienced more pain and discomfort during the nasopharyngeal swabbing. Most of the participants complained more about discomfort than pain. It is recommended to apply alternate test methods and conduct nurse-led information studies to reduce the anticipation of procedural pain/discomfort.

Colistin, Meropenem–Vaborbactam, Imipenem–Relebactam, and Eravacycline Testing in Carbapenem-Resistant Gram-Negative Rods: A Comparative Evaluation of Broth Microdilution, Gradient Test, and VITEK 2

Objectives. This study aimed to evaluate and compare the performance of different assays for antimicrobial susceptibility testing (AST) and minimum inhibitory concentration (MIC) determination for reserve antibiotics in carbapenem-resistant Enterobacterales (CREs), Pseudomonas aeruginosa (CRPAs), and Acinetobacter baumannii (CRABs). Methods. An analysis was conducted on 100 consecutive isolates: 50 CREs, 35 CRPAs, and 15 CRABs. Sensititre broth microdilution was used as a reference standard to evaluate the performance of VITEK 2 card AST-XN24 (bioMérieux), the respective gradient tests (bioMérieux), and UMIC colistin broth microdilution test strips (Bruker Daltonics). Errors, essential agreement (EA), and categorical agreement of MICs for colistin (COL), meropenem–vaborbactam (MVB), imipenem–relebactam (IRL), and eravacycline (ERV) were assessed. Results. The agreement between both of the COL broth microdilution (BMD) methods was perfect (100/100). The gradient test and VITEK 2 analysis yielded comparable EA rates (92/100 and 72/79, respectively), with the latter not registering any very major errors (VMEs). The MVB gradient test achieved EA in 66 of 85 isolates and VITEK 2 in 70/85. For IRL, EA was reached in 69 and 64 of 85 cases by gradient test and VITEK 2 analysis, respectively. The ERV gradient test yielded false results in nearly all (12/15) CRABs but achieved EA in 46 of 50 CREs. The VITEK system recorded EA for ERV in 60 of 65 isolates. Conclusions. We observed substantial variability in the measured MICs between BMD and the alternative methods. In only a few constellations, VITEK 2 or gradient tests could substitute the reference method. BMD is the method of choice for COL analysis, with VITEK 2 representing an alternative method for CRPA testing. Alternative methods for MVB did not provide reliable results, except for Enterobacterales, when tested with the gradient test. However, resistant results need to be confirmed by BMD. Only BMD can be used for IRL MIC determination. VITEK 2 was mostly accurate in measuring ERV MICs, while the corresponding gradient test yielded reliable results exclusively in CREs. It is essential that laboratories are aware of which testing method provides reliable results for each combination of microorganisms and reserve antibiotics.

Analysis of Current Approaches to Monitoring the Content of Depressor Substances

INTRODUCTION. Raw materials of biological origin can contaminate medicines with depressor substances, such as histamine, acetylcholine, bradykinin, serotonin, and prostaglandins. In the Russian Federation, tests for the content of these impurities are conducted in anaesthetised healthy cats in accordance with the General Chapter Test for depressor substances of the State Pharmacopoeia of the Russian Federation. The global trend towards ending animal testing and the search for ways to eliminate animal tests for depressor substances motivate researchers to study international approaches to the determination of histamine-like substances.AIM. This study aimed to analyse Russian and international approaches to the control of biologicals for depressor substances and to consider the applicability of alternative instrument-based physicochemical methods for the determination of histamine-like impurities, with a view to reducing animal testing.DISCUSSION. The rationale for reducing the use of animals as test systems is set forth in Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. In 2023, the European Pharmacopoeia Commission initiated the removal of in vivo tests for histamine-like substances from the European Pharmacopoeia. However, China and India use animal testing as the basic method for assessing the safety of medicines. In the Russian Federation, the control of depressor substances is regulated under General Chapter 1.2.4.0008.18 of the State Pharmacopoeia of the Russian Federation. Since the publication of General Chapter 42-0063-07 Test for histamine in edition XII in 2007, the State Pharmacopoeia of the Russian Federation has included a compendial text providing for a reduction in the number of cats used for testing. At present, Russian scientists are searching for targeted physicochemical and immunochemical methods for the determination of depressor substances. It has been demonstrated that a reasonable approach is to assess the need for testing a particular medicinal product in animals on a case-by-case basis. This approach will help minimise the number of in vivo tests and maintain an adequate quality of medicines at the same time.CONCLUSIONS. Despite the global trend towards ending animal testing, it is still necessary and required to control medicines for the content of depressor substances. Nevertheless, there is no global consensus on the need to use animals for this purpose. The absence of a separate general chapter/monograph, or references to it, will not change the fact that histamine-like substances are controlled worldwide. The decision to exclude in vivo methods from the European Pharmacopoeia necessitates the search for alternative test methods and the implementation of additional manufacturing controls to minimise the contamination risk for finished medicinal products. Studies have shown that high-performance liquid chromatography can determine the content of histamine in a limited amount of a medicinal product.

Comparative analysis of the results of live anthrax vaccine identification by immunofluorescence and immunochromatography

INTRODUCTION. The quality evaluation of live anthrax vaccines will benefit from the implementation of alternative testing methods that are capable of specific identification of the Bacillus anthracis spore antigen using the appropriate diagnostic products that are authorised in the Russian Federation for the detection of B. anthracis spores.AIM. This study aimed to investigate the applicability of immunofluorescence analysis to the identification of live anthrax vaccines and compare this method with immunochromatography.MATERIALS AND METHODS. The study used commercial batches of a live anthrax vaccine and Russian diagnostic products, including diagnostic dry adsorbed fluorescent anti-anthrax spore immunoglobulins and an immunochromatographic assay (ICA) reagent kit for rapid detection and identification of B. anthracis spores (ICA system for B. anthracis). For smears and ICA system reactions, the authors prepared working solutions of bacterial suspensions at spore concentrations typical of the B. anthracis STI-1 vaccine strain. The spore concentrations were achieved using the pharmacopoeial reference standard (RS) for the opacity of bacterial suspensions of 10 international units (IU). Identification reactions involved the registration of immune complex formation.RESULTS. Immunofluorescence tests of the live anthrax vaccine demonstrated bright greenish-yellow envelope fluorescence with an intensity score of 3+ to 4+ for smears stained with diagnostic immunoglobulins at 1:32 and 1:64 dilutions. Immunochromatographic tests of the live anthrax vaccine detected the spore antigen at vaccine concentrations of 109 and 108 spores/mL, with the test strips showing two distinct dark-pink lines indicative of immunoprecipitation. According to the results obtained using the selected methods, the tested microbial culture was confirmed as B. anthracis.CONCLUSIONS. Immunochromatography and immunofluorescence tests with appropriate diagnostic preparations are convenient and reliable tools for the species-specific detection of B. anthracis STI-1 spores in the live anthrax vaccine. The results obtained in the anthrax vaccine identification tests provide a basis for recommending the above methods as supplementary alternatives to Ziehl–Neelsen bacteriological staining, which is currently prescribed by the State Pharmacopoeia of the Russian Federation.

Superhydrophobic Paper Strips with Embedded Agarose-Anthocyanin Mini-Discs for Point-of-Need Quantitative pH Measurements.

Commercial pH paper is a quick and simple tool for measuring a solution's acidity/basicity, but it only provides qualitative or semi-quantitative results, and the synthetic indicator dyes within can be toxic or carcinogenic. Although pH meters enable more accurate and quantitative analysis, they are less convenient to operate and are tedious to calibrate. This presents a need for an alternative pH testing method for applications where it is not easy or possible to use a pH meter, yet quantitative results are desired. We report herein the fabrication of a pH test strip made from superhydrophobic paper and agarose-anthocyanin film discs. In the proposed method, test strips are dipped into samples and then imaged with a portable scanner (or a smartphone). The color of the film is extracted with ImageJ software (or a mobile app), using the RGB color system. By generating a calibration curve relating the film color to the sample pH using standard buffer solutions, we are able to quantify the pH of beverages and other liquids with an accuracy and precision comparable to that of a pH meter. The test strips offer the same convenience as conventional pH paper, with the added capabilities of quantitation and multiplexed testing, which presents a practical tool for point-of-need pH analysis.

Development of a multi-analysis model using an epithelial-fibroblast co-culture system as an alternative to animal testing.

The evaluation of respiratory chemical substances has been mostly performed in animal tests (OECD TG 403, TG 412, TG 413, etc.). However, there have been ongoing discussions about the limited use of these inhalation toxicity tests due to differences in the anatomical structure of the respiratory tract, difficulty in exposure, laborious processes, and ethical reasons. Alternative animal testing methods that mimic in vivo testing are required. Therefore, in this study, we established a co-culture system composed of differentiated epithelial cells under an air-liquid interface (ALI) system in the apical part and fibroblasts in the basal part. This system was designed to mimic the wound-healing mechanism in the respiratory system. In addition, we developed a multi-analysis system that simultaneously performs toxicological and functional evaluations. Several individual assays were used sequentially in a multi-analysis model for pulmonary toxicity. Briefly, cytokine analysis, histology, and cilia motility were measured in the apical part, and cell migration and gel contraction assay were performed by exposing MRC-5 cells to the basal culture. First, human airway epithelial cells from bronchial (hAECB) were cultured under air-liquid interface (ALI) system conditions and validated pseudostratified epithelium by detecting differentiation-related epithelial markers using Transepithelial Electrical Resistance (TEER) measurement, Hematoxylin and Eosin (H&E) staining, and immunocytochemistry (ICC) staining. Afterward, the co-culture cells exposed to Transforming growth factor-beta 1 (TGF-β1), a key mediator of pulmonary fibrosis, induced significant toxicological responses such as cytotoxicity, cell migration, and gel contraction, which are wound-healing markers. In addition, cilia motility in epithelial cells was significantly decreased compared to control. Therefore, the multi-analysis model with a 3D epithelial-fibroblast co-culture system is expected to be useful in predicting pulmonary toxicity as a simple and efficient high-throughput screening method and as an alternative to animal testing.

A new approach for risk assessment of pulmonary toxicants: a preliminary study using didecyldimethylammonium chloride.

Current human risk assessments often rely on animal toxicity data to establish point of departure (POD) values, followed by the application of uncertainty factors. Consequently, there is growing interest in alternative toxicity testing methods that reduce reliance on animal models. In this study, we propose a novel approach for inhalation toxicity risk assessment that integrates in silico and in vitro methods. Human primary alveolar epithelial cells were exposed to aerosolized didecyldimethylammonium chloride (DDAC) to assess cytotoxicity. This was followed by transcriptome analysis and biological pathway investigation, utilizing adverse outcome pathway (AOP), to calculate the POD. Additionally, human DDAC exposure was simulated using a multiple-path particle dosimetry (MPPD) model to predict exposure levels in the human alveolar region via inhalation. The results from in silico and in vitro studies were then compared, and a comprehensive risk assessment was performed. The POD for AOP 452 key events-oxidative stress, inflammation, epithelial-mesenchymal transition (EMT), apoptosis, and autophagy-was found to range between 19.0 and 23.89ng/cm2, according to benchmark dose calculation tools. The estimated human exposure to DDAC in the alveolar region under actual exposure conditions was 0.164ng/cm2/day, resulting in a margin of exposure (MOE) ranging from 121 to 145, suggesting caution regarding DDAC inhalation exposure. This study presents a novel risk assessment method that compares estimated human inhalation exposure values to in vitro results, applying the human equivalent concentration concept. Our findings demonstrate the potential for conducting human risk assessments using both in silico and in vitro methods as alternatives to traditional in vivo studies.

P15-18 Evaluation of transferability and within laboratory reproducibility of an alternative test method with THP-1 cell line for predicting immunotoxicity

P15-18 Evaluation of transferability and within laboratory reproducibility of an alternative test method with THP-1 cell line for predicting immunotoxicity

Rearing conditions (isolated versus group rearing) affect rotenone-induced changes in the behavior of zebrafish (Danio rerio) embryos in the coiling assay

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs’ principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters.

The effect of prior hysterosalpingo-foam sonography or hysterosalpingography on tubal patency: a secondary analysis of a randomized controlled trial.

Does hysterosalpingo-foam sonography (HyFoSy) prior to hysterosalpingography (HSG) or HSG prior to HyFoSy affect visible tubal patency when compared HSG or HyFoSy alone? Undergoing either HyFoSy or HSG prior to tubal patency testing by the alternative method does not demonstrate a significant difference in visible tubal patency when compared to HyFoSy or HSG alone. HyFoSy and HSG are two commonly used visual tubal patency tests with a high and comparable diagnostic accuracy for evaluating tubal patency. These tests may also improve fertility, although the underlying mechanism is still not fully understood. One of the hypotheses points to a dislodgment of mucus plugs that may have disrupted the patency of the Fallopian tubes. This is a secondary analysis of the randomized controlled FOAM study, in which women underwent tubal patency testing by HyFoSy and HSG, randomized for order of the procedure. Participants either had HyFoSy first and then HSG, or vice versa. Here, we evaluate the relative effectiveness of tubal patency testing by HyFoSy or HSG prior to the alternative tubal patency testing method on visible tubal patency, compared to each method alone. Infertile women aged between 18 and 41 years scheduled for tubal patency testing were eligible for participating in the FOAM study. Women with anovulatory cycles, endometriosis, or with a partner with male infertility were excluded. To evaluate the effect HyFoSy on tubal patency, we relied on HSG results by comparing the proportion of women with bilateral tubal patency visible on HSG in those who underwent and who did not undergo HyFoSy prior to their HSG (HyFoSy prior to HSG versus HSG alone). To evaluate the effect of HSG on tubal patency, we relied on HyFoSy results by comparing the proportion of women with bilateral tubal patency visible on HyFoSy in those who underwent and who did not undergo HSG prior to their HyFoSy (HSG prior to HyFoSy versus HyFoSy alone). Between May 2015 and January 2019, we randomized 1160 women (576 underwent HyFoSy first followed by HSG, and 584 underwent HSG first followed by HyFoSy). Among the women randomized to HyFoSy prior to HSG, bilateral tubal patency was visible on HSG in 467/537 (87%) women, compared with 472/544 (87%) women who underwent HSG alone (risk difference 0.2%; 95% CI: -3.8% to 4.2%). Among the women randomized to HSG prior to HyFoSy, bilateral tubal patency was visible on HyFoSy in 394/471 (84%) women, compared with 428/486 (88%) women who underwent HyFoSy alone (risk difference -4.4%; 95% CI: -8.8% to 0.0%). The results of this secondary analysis should be interpreted as exploratory and cannot be regarded as definitive evidence. Furthermore, it has to be noted that pregnancy outcomes were not considered in this analysis. Tubal patency testing by either HyFoSy or HSG, prior to the alternative tubal patency testing method does not significantly affect visible tubal patency, when compared to alternative method alone. This suggests that both methods may have comparable abilities to dislodge mucus plugs in the Fallopian tubes. The FOAM study was an investigator-initiated study, funded by ZonMw, a Dutch organization for Health Research and Development (project number 837001504). IQ Medical Ventures provided the ExEm®-FOAM kits free of charge. The funders had no role in study design, collection, analysis, or interpretation of the data. H.R.V. reports consultancy fees from Ferring. M.v.W. received a travel grant from Oxford University Press in the role of Deputy Editor for Human Reproduction and participates in a Data Safety and Monitoring Board as an independent methodologist in obstetrics studies in which she has no other role. M.v.W. is coordinating editor of Cochrane Fertility and Gynaecology. B.W.J.M. received an investigator grant from NHMRC (GNT1176437) and research funding from Merck KGaA. B.W.J.M. reports consultancy for Organon and Merck KGaA, and travel support from Merck KGaA. B.W.J.M. reports holding stocks of ObsEva. V.M. received research grants from Guerbet, Merck and Ferring and travel and speaker fees from Guerbet. The other authors do not report conflicts of interest. International Clinical Trials Registry Platform No. NTR4746.

Rapid Assessment of Steel Machinability through Spark Analysis and Data-Mining Techniques

The machinability of steel is a crucial factor in manufacturing, influencing tool life, cutting forces, surface finish, and production costs. Traditional machinability assessments are labor-intensive and costly. This study presents a novel methodology to rapidly determine steel machinability using spark testing and convolutional neural networks (CNNs). We evaluated 45 steel samples, including various low-alloy and high-alloy steels, with most samples being calcium steels known for their superior machinability. Grinding experiments were conducted using a CNC machine with a ceramic grinding wheel under controlled conditions to ensure a constant cutting force. Spark images captured during grinding were analyzed using CNN models with the ResNet18 architecture to predict V15 values, which were measured using the standard ISO 3685 test. Our results demonstrate that the created prediction models achieved a mean absolute percentage error (MAPE) of 12.88%. While some samples exhibited high MAPE values, the method overall provided accurate machinability predictions. Compared to the standard ISO test, which takes several hours to complete, our method is significantly faster, taking only a few minutes. This study highlights the potential for a cost-effective and time-efficient alternative testing method, thereby supporting improved manufacturing processes.

Round Robin Study for Evaluation an in vitro skin irritation test for medical device extracts using KeraSkinTM in Korea

With the growing importance of alternative test methods that implement the 3Rs principles (Reduction, Refinement and Replacement) and the global importance of biological safety assessment data for medical devices is increasing. We have developed and optimized the ‘KeraSkin™ Skin Irritation Test (KeraSkin™ SIT) for medical device’ for regulatory application in biological evaluation according to ISO 10993-23. We conducted a round robin study to optimize and evaluate the performance of KeraSkin™ SIT for medical devices using KeraSkin™ Reconstructed Human Epidermis (RhE), which is developed and manufactured in Korea. This round robin study was performed to assess the transferability, reproducibility (within and between laboratories) and predictive capacity in 1 lead laboratory and 3 participating laboratories based on OECD Guidance Document 34. The predictive capacity, the results showed 83.3 % of sensitivity, 100 % of specificity and 91.6 % of accuracy. In conclusion, the results demonstrate that ‘KeraSkin™ SIT for medical device’ provides a robust test method for detecting irritant activity of medical device extracts and can be utilized for identifying low levels of potent irritants in medical device extracts. Therefore, it fulfills the requirements to be included as a ‘me-too’ test method to EpiDerm™ and SkinEthic™ skin irritation test in ISO 10993-23.

Evaluation of gradient diffusion in vitro susceptibility testing of Aerococcus urinae

ObjectiveAerococcus urinae antimicrobial susceptibility testing can be performed via broth microdilution with Mueller-Hinton broth supplemented with lysed horse blood. We sought to compare this with the commonly used gradient diffusion method. MethodsWe compared broth microdilution with Mueller-Hinton broth supplemented with lysed horse blood and gradient diffusion via Mueller-Hinton agar supplemented with sheep blood for 190 A. urinae isolates against 16 antimicrobials. ResultsNo antimicrobials demonstrated more than 90% essential and categorical agreement, and fewer than 3% demonstrated major and very major error rates. Trimethoprim-sulfamethoxazole demonstrated an 81% major error rate and ceftriaxone demonstrated a 76% very major error rate. Agar dilution with lysed horse blood was performed for trimethoprim-sulfamethoxazole against 94 isolates and showed 100% susceptibility, consistent with previous studies. ConclusionsGiven its limitations in detecting resistant strains, our findings cannot support the routine use of gradient diffusion with Mueller-Hinton agar supplemented with sheep blood for A. urinae in lieu of the Clinical and Laboratory Standards Institute method. Our results suggest that A. urinae is usually susceptible to penicillin, linezolid, tetracycline, and vancomycin. Future studies should evaluate alternative testing methods for clinical microbiology laboratories.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method

BackgroundPorcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour.ResultsThis study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method.ConclusionThe developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Raman spectroscopy as an alternative rapid microbial bioburden test method for continuous, automated detection of contamination in biopharmaceutical drug substance manufacturing.

To investigate an in-line Raman method capable of detecting accidental microbial contamination in pharmaceutical vessels, such as bioreactors producing monoclonal antibodies via cell culture. The Raman method consists of a multivariate model built from Raman spectra collected in-line during reduced-scale bioreactor batches producing a monoclonal antibody, as well as a reduced-scale process with intentional spiking of representative compendial method microorganisms (n=4). The orthogonal partial least squares regression discriminant analysis model (OPLS-DA) area under the curve (AUC), specificity and sensitivity were 0.96, 0.99, and 0.95, respectively. Furthermore, the model successfully detected contamination in an accidentally contaminated manufacturing-scale batch. In all cases, the time to detection (TTD) for Raman was superior compared to offline, traditional microbiological culturing. The Raman OPLS-DA method met acceptance criteria for equivalent decision making to be considered a viable alternative to the compendial method for in-process bioburden testing. The in-line method is automated, non-destructive, and provides a continuous assessment of bioburden compared to an offline compendial method, which is manual, results in loss of product, and in practice is only collected once daily and requires 3-5 days for enumeration.

Quantitative assessment of machine learning reliability and resilience.

Advances in machine learning (ML) have led to applications in safety-critical domains, including security, defense, and healthcare. These ML models are confronted with dynamically changing and actively hostile conditions characteristic of real-world applications, requiring systems incorporating ML to be reliable and resilient. Many studies propose techniques to improve the robustness of ML algorithms. However, fewer consider quantitative techniques to assess changes in the reliability and resilience of these systems over time. To address this gap, this study demonstrates how to collect relevant data during the training and testing of ML suitable for the application of software reliability, with and without covariates, and resilience models and the subsequent interpretation of these analyses. The proposed approach promotes quantitative risk assessment of ML technologies, providing the ability to track and predict degradation and improvement in the ML model performance and assisting ML and system engineers with an objective approach to compare the relative effectiveness of alternative training and testing methods. The approach is illustrated in the context of an image recognition model, which is subjected to two generative adversarial attacks and then iteratively retrained to improve the system's performance. Our results indicate that software reliability models incorporating covariates characterized the misclassification discovery process more accurately than models without covariates. Moreover, the resilience model based on multiple linear regression incorporating interactions between covariates tracks and predicts degradation and recovery of performance best. Thus, software reliability and resilience models offer rigorous quantitative assurance methods for ML-enabled systems andprocesses.

A rapid spectrophotometric test for assessing skin sensitization potential of chemicals by using N-acetyl-L-cysteine methyl ester in chemico

ABSTRACT During the key event 1 of skin sensitization defined as covalent binding or haptenization of sensitizer to either thiol or amino group of skin proteins, a sensitizer not only covalently binds with skin proteins but also interacts with nucleophilic small molecules such as glutathione (GSH). Although GSH would not be directly associated with skin sensitization, this interaction may be applied for developing an alternative test method simulating key event 1, haptenization. Thus, the aim of the present study was to examine whether N-acetyl-L-cysteine methyl ester (NACME), a thiol-containing compound, was selected as an electron donor to determine whether NACME reacted with sensitizers. Following a reaction of NACME with a sensitizer in a 96-well plate, the remaining NACME was measured spectrophotometrically using 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions with two different vehicles, such as acetonitrile (ACN) and dimethyl sulfoxide (DMSO), 64 test chemicals were tested to determine the predictive capacity of current NACME test method. The results obtained showed, the predictive capacity of 94.6% sensitivity, 88.9% specificity, and 92.2% accuracy utilizing DMSO as a vehicle with a cutoff NACME depletion of 5.85%. The three parameters were also over 85% in case of ACN. These values were comparable to or better than other OECD-approved test methods. Data demonstrated that a simple thiol-containing compound NACME might constitute as a reliable candidate for identifying reactive skin sensitizers, and that this method be considered as practical method as a screening tool for assessing a chemical’s tendency to initiate skin sensitization.

P-565 Moving towards non – invasive euploid blastocyst prediction. Retrospective euploid blastocyst evaluation using different morphokinetics and Artificial Intelligence (AI) models

Abstract Study question Could Preimplantation Genetic Testing for Aneuploidies be replaced by an Aritificial Intelligence model which evaluates blastocysts and selects the most suitable ones for embryo transfer? Summary answer The use of AI for euploidy prediction has not been validated in a large scale and thus it should not be used to replace PGT-A. What is known already PGT-A is an invasive method that may affects blastocysts implantation potential. Both the biopsy protocol and the experience of the embryologist who performs the biopsy can influence the outcome. Efforts are made to develop non-invasive methods for PGT. Many AI systems have been developed in assisted reproduction.Time- Lapse systems have a specialized AI software, that assist in the evaluation of the embryos and the selection of the most suitable ones based on their morphokinetic characteristics. Other AI systems, such as ERICA, evaluate embryos and provide a measure of their euploidy, serving as an alternative method for non-invasive preimplantation genetic testing. Study design, size, duration The retrospective study was conducted between January 2018 and December 2020. A total of 585 blastocysts, derived from 144 women who had undergone an IVF – PGT-A cycle, were included. The embryos were cultured in a TL incubator and evaluated using KID Score D5 (Vitrolife), iDa Score (Vitrolife) and ERICA (ERICA). Participants/materials, setting, methods The study was conducted in a private IVF clinic. Embryos from women with an average age of 38 years who had a history of recurrent miscarriages and/or &amp;gt;3 failed IVF cycles were included. Biopsy was performed in all blastocysts that were cultured in TL incubators up to day 5-6. Statistical and logistic regression analysis were conducted to assess the comparison between the score of the three AI programs and the result of the biopsy. Main results and the role of chance ERICA seems uncapable to separate euploid from aneuploid embryos. T-test showed that there is no statistically significant difference between the two categories (mean_difference=0,02, p = 0,3&amp;gt;0,001), while the effect size (clinical value) of the difference was very low (0,085). This means that this program cannot replace PGT-A. According to the iDA Score, although there is a difference in scores between euploid and aneuploid embryos, their difference, according to t-test, is not statistically significant [t(581)=2,56, p = 0,01&amp;gt;0,001], while the effect size is low (0,217). According to KID Score D5, there is a statistically significant difference in scores between euploid and aneuploid embryos [t(514)=4,4, p &amp;lt; 0,001], which shows that this program could replace PGT-A in some cases. However, the effect size of the difference (0,37), as well as the determination coefficients are quite low. Finally, the KID Score D5 succeeds in discovering aneuploid embryos in a higher percentage than euploid ones. Limitations, reasons for caution Women under the age of 35 were not included in the study. Permission from the National Greek Authority of Medical Assisted Reproduction was needed from every patient to undergo PGT-A. The size of the sample might limit the conclusions. A larger study would help verify the results of the study. Wider implications of the findings AI models can be used as good indicators for embryo selection/prioritization and predict the euploid status of the embryos. Non-invasive euploid prediction models could be a very useful tool for embryologists especially in countries where law restrictions are strict against the cohort of patients that are interested in PGT-A cycles. Trial registration number Not Applicable

Assessing the setting behavior of ultra-high performance concrete

The setting behavior of ultra-high performance concrete (UHPC) is demonstrably different from that of conventional concrete; thus, tools and guidance extending beyond common test methods such as Vicat and penetration are needed. While UHPC is known for its enhanced mechanical and durability properties, due to the low water and high cementitious contents, UHPC-class materials are prone to early-age autogenous shrinkage. Recognizing that UHPCs are commonly supplied to construction sites as prebagged, proprietary mixes with unknown constituents, and that accurate determination of setting time is crucial in determining the early-age autogenous shrinkage of UHPC-class materials as well as for scheduling construction operations and quality control actions, this study explores alternate test methods such as isothermal calorimetry (ASTM C1679), semi-adiabatic calorimetry (ASTM C1753), autogenous shrinkage (ASTM C1698), chemical shrinkage (ASTM C1608), and dual ring test (American Association of State Highway and Transportation Officials (AASHTO T 363) to evaluate the setting behavior of UHPCs. Setting times obtained using the alternate test methods aligned well with each other and were found to be different than the setting times indicated through standard test methods. Discussion and guidance on the applicability and the use of alternate test methods to determine the setting time of UHPCs for various laboratory and field applications are provided.