Evaluation of the toxicological properties of Himantura imbricata Venom using a zebrafish model (Danio rerio).

The stingrays of the genus Himantura imbricata are present in all of the world's oceans, but the toxicity of their venoms has not yet been thoroughly characterized. The zebrafish as a toxicology model can be used for general toxicity testing of drugs and the investigation of toxicological mechanisms. The aim of this study was to evaluate the effect of crude venom from the stingray H. imbricata on the zebrafish Danio rerio. Juvenile zebrafish were injected with different concentrations of venom from H. imbricata via subcutaneous injections. The venom's effects were established via histological examination and hemolytic activity in zebrafish. The histopathological analysis revealed significant tissue damage in the organs of the zebrafish injected with venom, including liver necrosis and kidney degeneration. A blood examination revealed echinocytes, hemolysis, and nuclear abnormalities. Bodyweight estimations and histopathological attributes of the gills, heart, muscle, liver, intestine, eye, and brain were determined. The histological staining studies of the gills, liver, and intestine were measurably higher in the venom groups compared with the other two groups. Aggregately, the result shows that zebrafish may act as a valuable biomarker for alterations impelled by H. imbricata venom. The work delivers a useful model with substantial pharmacological potential for new drugs and a better comprehension of research on stingray venom.

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells.

Objective: Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. Methods: In this study, we treated K562 cells with 40 µmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. Results: One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Conclusion: Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

Ensemble multiclassification model for predicting developmental toxicity in zebrafish.

In recent years, with the rapid development of society, organic compounds have been released into aquatic environments in various forms, posing a significant threat to the survival of aquatic organisms. The assessment of developmental toxicity is an important part of environmental safety risk systems, helping to identify the potential impacts of organic compounds on the embryonic development of aquatic organisms and enabling early detection and warning of potential ecological risks. Additionally, binary classification models cannot accurately classify organic compounds. Therefore, it is crucial to construct a multiclassification model for predicting the developmental toxicity of organic compounds. In this study, binary and multiclassification models were developed based on the ToxCast™ Phase I chemical library and literature data. The random forest, support vector machine, extreme gradient boosting, adaptive gradient boosting, and C5.0 decision tree algorithms, as well as 8 types of molecular fingerprint were used to establish a multiclassification base model for predicting developmental toxicity through 5-fold cross-validation and external validation. Ultimately, a multiclassification ensemble model was derived through a voting method. The performance of the binary ensemble model, as measured by the balanced accuracy, was 0.918, while that of the multiclassification model was 0.819. The developmental toxicity voting ensemble model (DT-VEM) achieved accuracies of 0.804, 0.834, and 0.855. Furthermore, by utilizing the XGBoost machine learning algorithm to construct separate models for molecular descriptors and substructure molecular fingerprints, we identified several substructures and physical properties related to developmental toxicity. Our research contributes to a more detailed classification of developmental toxicity, providing a new and valuable tool for predicting the developmental toxicity effects of unknown compounds. This supplement addresses the limitations of previous tools, as it offers an enhanced ability to predict potential developmental toxicity in novel compounds.

Toxicity assessment of animal manure composts containing environmental microplastics by using earthworms Eisenia andrei.

Nowadays, animal manure composting constitutes a sustainable alternative for farmers to enhance the level of nutrients within soils and achieve a good productivity. However, pollutants may be present in manures. This study focuses on the detection of environmental microplastics (EMPs) into composts, as well as on the assessment of their potential toxicity on the earthworm Eisenia andrei. To these aims, animals were exposed to two types of compost, namely bovine (cow) and ovine (sheep) manure, besides to their mixture, for 7 and 14 days. The presence and characterization of EMPs was evaluated in all the tested composts, as well as in tissues of the exposed earthworms. The impact of the tested composts was assessed by a multi-biomarker approach including cytotoxic (lysosomal membrane stability, LMS), genotoxic (micronuclei frequency, MNi), biochemical (activity of catalase, CAT, and glutathione-S-transferase, GST; content of malondialdehyde, MDA), and neurotoxic (activity of acetylcholinesterase, AChE) responses in earthworms. Results indicated the presence of high levels of EMPs in all the tested composts, especially in the sheep manure (2273.14 ± 200.89 items/kg) in comparison to the cow manure (1628.82 ± 175.23 items/kg), with the size <1.22 µm as the most abundant EMPs. A time-dependent decrease in LMS and AChE was noted in exposed earthworms, as well as a concomitant increase in DNA damages (MNi) after 7 and 14 days of exposure. Also, a severe oxidative stress was recorded in animals treated with the different types of compost through an increase in CAT and GST activities, and LPO levels, especially after 14 days of exposure. Therefore, it is necessary to carefully consider these findings for agricultural good practices in terms of plastic mitigation in compost usage, in order to prevent any risk for environment health.

Finding a Direct Method for a Dynamic Process: The DD (Direct and Dynamic) Cell-Tox Method.

The main focus of in vitro toxicity assessment methods is to assess the viability of the cells, which is usually based on metabolism changes. Yet, when exposed to toxic substances, the cell triggers multiple signals in response. With this in mind, we have developed a promising cell-based toxicity method that observes various cell responses when exposed to toxic substances (either death, division, or remain viable). Based on the collective cell response, we observed and predicted the dynamics of the cell population to determine the toxicity of the toxicant. The method was tested with two different conformations: In the first conformation, we exposed a monoculture model of blood macrophages to UV light, hydrogen peroxide, nutrient deprivation, tetrabromobisphenol A, fatty acids, and 5-fluorouracil. In the second, we exposed a coculture liver model consisting of hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells to rifampicin, ibuprofen, and 5-fluorouracil. The method showed good accuracy compared to established toxicity assessment methods. In addition, this approach provided more representative information on the toxic effects of the compounds, as it considers the different cellular responses induced by toxic agents.

Toxic effects of fragmented polyethylene terephthalate particles on the marine rotifer Brachionus koreanus: Based on ingestion and egestion assay, in vivo toxicity test, and multi-omics analysis.

Microplastics (MPs) are a major concern in marine ecosystem because MPs are persistent and ubiquitous in oceans and are easily consumed by marine biota. Although many studies have reported the toxicity of MPs to marine biota, the toxicity of environmentally relevant types of MPs is little understood. We investigated the toxic effects of fragmented polyethylene terephthalate (PET) MP, one of the most abundant MPs in the ocean, on the marine rotifer Brachionus koreanus at the individual and molecular level. No significant rotifer mortality was observed after exposure to PET MPs for 24 and 48 h. The ingestion and egestion assays showed that rotifers readily ingested PET MPs in the absence of food but not when food was supplied; thus, there were also no chronic effects of PET MPs. In contrast, intracellular reactive oxygen species levels and glutathione S-transferase activity in rotifers were significantly increased by PET MPs. Transcriptomic and metabolomic analyses revealed that genes and metabolites related to energy metabolism and immune processes were significantly affected by PET MPs in a concentration-dependent manner. Although acute toxicity of PET MPs was not observed, PET MPs are potentially toxic to the antioxidant system, immune system, and energy metabolism in rotifers.

Toxicity of water-soluble polymers polyethylene glycol and polyvinyl alcohol for fish and frog embryos.

Personal Care Products (PCPs) have been one of the most studied chemicals in the last twenty years since they were identified as pseudo-persistent pollutants by the European Union in the early 2000s. The accumulation of PCPs in the aquatic environment and their effects on non-target species make it necessary to find new, less harmful, substances. Polyethylene glycol (PEGs) and polyvinyl alcohol (PVAs) are two polymers that have increased their presence in the composition of PCPs in recent years, but little is known about the effect of their accumulation in the environment on non-target species. Through embryotoxicity tests on two common models of aquatic organisms (Danio rerio and Xenopus laevis), this work aims to increase the knowledge of PEGs and PVAs' effects on non-target species. Animals were exposed to the pollutant for 96 h. The main embryotoxicity endpoint (mortality, hatching, malformations, heartbeat rate) was recorded every 24 h. The most significant results were hatching delay in Danio rerio exposed to both chemicals, in malformations (oedema, body malformations, changes in pigmentation and deformations of spine and tail) in D. rerio and X. laevis and significant change in the heartbeat rate (decrease or increase in the rate) in both animals for all chemicals tested.

Effects of Chemicals in Reporter Gene Bioassays with Different Metabolic Activities Compared to Baseline Toxicity.

High-throughput cell-based bioassays are used for chemical screening and risk assessment. Chemical transformation processes caused by abiotic degradation or metabolization can reduce the chemical concentration or, in some cases, lead to the formation of more toxic transformation products. Unaccounted loss processes may falsify the bioassay results. Capturing the formation and effects of transformation products is important for relating the in vitro effects to in vivo. Reporter gene cell lines are believed to have low metabolic activity, but inducibility of cytochrome P450 (CYP) enzymes has been reported. Baseline toxicity is the minimal toxicity a chemical can have and is caused by the incorporation of the chemical into cell membranes. In the present study, we improved an existing baseline toxicity model based on a newly defined critical membrane burden derived from freely dissolved effect concentrations, which are directly related to the membrane concentration. Experimental effect concentrations of 94 chemicals in three bioassays (AREc32, ARE-bla and GR-bla) were compared with baseline toxicity by calculating the toxic ratio (TR). CYP activities of all cell lines were determined by using fluorescence-based assays. Only ARE-bla showed a low basal CYP activity and inducibility and AREc32 showed a low inducibility. Overall cytotoxicity was similar in all three assays despite the different metabolic activities indicating that chemical metabolism is not relevant for the cytotoxicity of the tested chemicals in these assays. Up to 28 chemicals showed specific cytotoxicity with TR > 10 in the bioassays, but baseline toxicity could explain the effects of the majority of the remaining chemicals. Seven chemicals showed TR < 0.1 indicating inaccurate physicochemical properties or experimental artifacts like chemical precipitation, volatilization, degradation, or other loss processes during the in vitro bioassay. The new baseline model can be used not only to identify specific cytotoxicity mechanisms but also to identify potential problems in the experimental performance or evaluation of the bioassay and thus improve the quality of the bioassay data.

On Interactions of Sulfamerazine with Cyclodextrins from Coupled Diffusometry and Toxicity Tests.

This scientific study employs the Taylor dispersion technique for diffusion measurements to investigate the interaction between sulfamerazine (NaSMR) and macromolecular cyclodextrins (ß-CD and HP-ß-CD). The results reveal that the presence of ß-CD influences the diffusion of the solution component, NaSMR, indicating a counterflow of this drug due to solute interaction. However, diffusion data indicate no inclusion of NaSMR within the sterically hindered HP-ß-CD cavity. Additionally, toxicity tests were conducted, including pollen germination (Actinidia deliciosa) and growth curve assays in BY-2 cells. The pollen germination tests demonstrate a reduction in sulfamerazine toxicity, suggesting potential applications for this antimicrobial agent with diminished adverse effects. This comprehensive investigation contributes to a deeper understanding of sulfamerazine-cyclodextrin interactions and their implications for pharmaceutical and biological systems.

Safety evaluation of 8 drug degradants present in over-the-counter cough and cold medications.

Although the United States Food & Drug Administration (FDA) has provided guidance on the control of drug degradants for prescription drugs, there is less guidance on how to set degradant specifications for FDA OTC monograph drugs. Given that extensive impurity testing was not part of the safety paradigm in original OTC monographs, a weight of evidence (WOE) approach to qualify OTC degradants is proposed. This approach relies on in silico tools and read-across approaches alongside standard toxicity testing to determine safety. Using several drugs marketed under 21 CFR 341 as case studies, this research demonstrates the utility of a WOE approach across data-rich and data-poor degradants. Based on degradant levels ranging from 1 to 4% of the maximum daily doses of each case study drug and 10th percentile body weight data for each patient group, children were recognized as having the highest potential exposure relative to adults per body mass. Depending on data availability and relationship to the parent API, margins of safety (MOS) or exposure margins were calculated for each degradant. The findings supported safe use, and indicated that this contemporary WOE approach could be utilized to assess OTC degradants. This approach is valuable to establish specifications for degradants in OTCs.

Ultra-high performance concrete alleviates ecotoxicological effects in aquatic organisms.

With the advancement of cementitious material technologies, ultra-high performance concretes incorporating nano- and(or) micro-sized particle materials have been developed; however, their environmental risks are still poorly understood. This study investigates the ecotoxicological effects of ultra-high performance concrete (UC) leachate by comparing with that of the conventional concrete (CC) leachate. For this purpose, a dynamic leaching test and a battery test with algae, water flea, and zebrafish were performed using standardized protocols. The conductivity, concentration of inorganic elements (Al, K, Na, and Fe), and total organic concentration were lower in the UC leachate than in the CC leachate. The EC50 values of the CC and UC leachates were 44.9 % and >100 % in algae, and 8.0 % and 63.1 % in water flea, respectively. All zebrafish exposed to the CC and UC leachates survived. A comprehensive evaluation of the ecotoxicity of the CC and UC leachate based on the toxicity classification system (TCS) showed that their toxicity classification was "highly acute toxicity" and "acute toxicity", respectively. Based on the hazard quotient and principal component analysis, Al and(or) K could be significant factors determining the ecotoxicity of concrete leachate. Furthermore, the ecotoxicity of UC could not be attributed to the use of silica-based materials or multi-wall carbon nanotubes. This study is the first of its kind on the ecotoxicity of UC leachate in aquatic environments, and the results of this study can be used to develop environment-friendly UC.

KoCVAM-led development of phototoxicity alternative test method using reconstructed human epidermis model (KeraSkin™).

Phototoxicity is an acute toxic reaction induced by topical skin exposure to photoreactive chemicals followed by exposure to environmental light and thus chemicals that absorb UV are recommended to be evaluated for phototoxic potential. There are currently three internationally harmonized alternative test methods for phototoxicity. One of them is the in vitro Phototoxicity: RhE Phototoxicity test method (OECD TG498). Korean center for the Validation of Alternative Methods (KoCVAM) developed an in vitro phototoxicity test method using a KeraSkin™ reconstructed human epidermis model (KeraSkin™ Phototoxicity Assay) as a 'me-too' test method of OECD TG498. For the development and optimization of KeraSkin™ Phototoxicity Assay, the following test chemicals were used: 6 proficiency chemicals in OECD TG498 (3 phototoxic and 3 non-phototoxic), 6 reference chemicals in OECD Performance Standard No. 356 (excluding the proficiency test chemicals, 3 phototoxic and 3 non-phototoxic) and 13 additional chemicals (7 phototoxic and 6 non-phototoxic). Based on the test results generated from the test chemicals above, the overall predictive capacity of KeraSkin™ Phototoxicity Assay was calculated. In particular, the assay exhibited 100 % accuracy, 100 % sensitivity, and 100 % specificity. Therefore, it fulfills the requirements to be included as a 'me-too' test method in OECD TG498.

Hybrid non-animal modeling: A mechanistic approach to predict chemical hepatotoxicity.

Developing mechanistic non-animal testing methods based on the adverse outcome pathway (AOP) framework must incorporate molecular and cellular key events associated with target toxicity. Using data from an in vitro assay and chemical structures, we aimed to create a hybrid model to predict hepatotoxicants. We first curated a reference dataset of 869 compounds for hepatotoxicity modeling. Then, we profiled them against PubChem for existing in vitro toxicity data. Of the 2560 resulting assays, we selected the mitochondrial membrane potential (MMP) assay, a high-throughput screening (HTS) tool that can test chemical disruptors for mitochondrial function. Machine learning was applied to develop quantitative structure-activity relationship (QSAR) models with 2536 compounds tested in the MMP assay for screening new compounds. The MMP assay results, including QSAR model outputs, yielded hepatotoxicity predictions for reference set compounds with a Correct Classification Ratio (CCR) of 0.59. The predictivity improved by including 37 structural alerts (CCR = 0.8). We validated our model by testing 37 reference set compounds in human HepG2 hepatoma cells, and reliably predicting them for hepatotoxicity (CCR = 0.79). This study introduces a novel AOP modeling strategy that combines public HTS data, computational modeling, and experimental testing to predict chemical hepatotoxicity.

Toxicity of individual and mixture of organic compounds to P. Phosphoreum and S. Capricornutum using interpretable simple structural parameters.

Human and environmental ecosystem beings are exposed to multicomponent compound mixtures but the toxicity nature of compound mixtures is not alike to the individual chemicals. This work introduces four models for the prediction of the negative logarithm of median effective concentration (pEC50) of individual chemicals to marine bacteria Photobacterium Phosphoreum (P. Phosphoreum) and algal test species Selenastrum Capricornutum (S. Capricornutum) as well as their mixtures to P. Phosphoreum, and S. Capricornutum. These models provide the simplest approaches for the forecast of pEC50 of some classes of organic compounds from their interpretable structural parameters. Due to the lack of adequate toxicity data for chemical mixtures, the largest available experimental data of individual chemicals (55 data) and their mixtures (99 data) are used to derive the new correlations. The models of individual chemicals are based on two simple structural parameters but chemical mixture models require further interaction terms. The new model's results are compared with the outputs of the best accessible quantitative structure-activity relationships (QSARs) models. Various statistical parameters are done on the new and comparative complex QSAR models, which confirm the higher reliability and simplicity of the new correlations.

False positive findings associated with adenoviral vector-based vaccine underscore the regulatory necessity to eliminate abnormal toxicity test.

Accumulating evidence has shown that the abnormal toxicity test (ATT) is not suitable as a quality control batch release test for biologics and vaccines. The purpose of the current study was to explore the optimal ATT experimental design for an adenoviral vector-based vaccine product to avoid false positive results following the standard test conditions stipulated in the Pharmacopoeias. ATT were conducted in both mice and guinea pigs based on methods in Pharmacopeias, with modifications to assess effects of dose volume and amount of virus particles (VPs). The results showed intraperitoneal (IP) dosing at human relevant dose and volume (i.e., VPs), as required by pharmacopeia study design, resulted in false positive findings not associated with extraneous contaminants of a product. Considering many gene therapy products use adeno associated virus as the platform for transgene delivery, data from this study are highly relevant in providing convincing evidence to show the ATT is inappropriate as batch release test for biologics, vaccine and gene therapy products. In conclusion, ATT, which requires unnecessary animal usage and competes for resources which otherwise can be spent on innovative medicine research, should be deleted permanently as batch release test by regulatory authorities around the world.

Revolutionizing developmental neurotoxicity testing - a journey from animal models to advanced in vitro systems.

Developmental neurotoxicity (DNT) testing has seen enormous progress over the last two decades. Preceding even the publication of the animal-based OECD test guideline for DNT testing in 2007, a series of non-animal technology workshops and conferences (starting in 2005) shaped a community that has delivered a comprehensive battery of in vitro test methods (IVB). Its data interpretation is covered by a very recent OECD test guidance (No. 377). Here, we aim to overview the progress in the field, focusing on the evolution of testing strategies, the role of emerging technologies, and the impact of OECD test guidelines on DNT testing. In particular, this is an example of a targeted development of an animal-free testing approach for one of the most complex hazards of chemicals to human health. These developments started literally from a blank slate, with no proposed alternative methods available. Over two decades, cutting-edge science enabled the design of a testing approach that spares animals and enables throughput for this challenging hazard. While it is evident that the field needs guidance and regulation, the massive economic impact of decreased human cognitive capacity caused by chemical exposure should be prioritized more highly. Beyond this, the claim to fame of DNT in vitro testing is the enormous scientific progress it has brought for understanding the human brain, its development, and how it can be perturbed.

Developmental neurotoxicity (DNT) testing predicts the hazard of exposure to chemicals to human brain development. Comprehensive advanced non-animal testing strategies using cutting-edge technology can now replace animal-based approaches to assess this complex hazard. These strategies can assess large numbers of chemicals more accurately and efficiently than the animal-based approach. Recent OECD test guidance has formalized this battery of in vitro test methods for DNT, marking a pivotal achievement in the field. The shift towards non-animal testing reflects both a commitment to animal welfare and a growing recognition of the economic and public health impacts associated with impaired cognitive function caused by chemical exposures. These innovations ultimately contribute to safer chemical management and better protection of human health, especially during the vulnerable stages of brain development.

Terrestrial bioassays for assessing the biochemical and toxicological impact of biosolids application derived from wastewater treatment plants.

Wastewater treatment plants (WWTP) are facilities where municipal wastewater undergoes treatment so that its organic load and its pathogenic potential are minimized. Sewage sludge is a by-product of this process and when properly treated is preferentially called "biosolids". These treatments may include some or most of the following: thickening, dewatering, drying, digestion, composting, liming. Nowadays it is almost impossible to landfill biosolids, which however can well be used as crop fertilizers. Continuous or superfluous biosolids fertilization may negatively affect non-target organisms such as soil macro-organisms or even plants. These effects can be depicted through bioassays on terrestrial animals and plants. It has been shown that earthworms have been affected to various degrees on the following endpoints: pollutants' bioaccumulation, viability, reproduction, avoidance behavior, burrowing behavior. Collembola have been affected on viability, reproduction, avoidance behavior. Other terrestrial organisms such as nematodes and diplopods have also shown adverse health effects. Phytotoxicity have been caused by some biosolids regimes as measured through the following endpoints: seed germination, root length, shoot length, shoot biomass, root biomass, chlorophyll content, antioxidant enzyme activity. Very limited statistical correlations between pollutant concentrations and toxicity endpoints have been established such as between juvenile mortality (earthworms) and As or Ba concentration in the biosolids, between juvenile mortality (collembola) and Cd or S concentration in the biosolids, or between phytotoxicity and some extractable metals in leachates or aquatic extracts from the biosolids; more correlations between physicochemical characteristics and toxicity endpoints have been found such as between phytotoxicity and ammonium N in biosolids or their liquid extracts, or between phytotoxicity and salinity. An inverse correlation between earthworm/collembola mortality and stable organic matter has also been found. Basing the appropriateness of biosolids only on chemical analyses for pollutants is not cost-effective. To enable risk characterization and subsequent risk mitigation it is important to apply a battery of bioassays on soil macro-organisms and on plants, utilizing a combination of endpoints and established protocols. Through combined analytical quantification and toxicity testing, safe use of biosolids in agriculture can be achieved.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.