Acute Systemic Toxicity Test Research Articles

Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance.

Safety assessment of chemicals, pharmaceuticals, food and food ingredients, cosmetics, industrial products is very crucial prior to their approval for human uses. Since the commencement of toxicity testing (about 500 years ago, since 1520), significant advances have been made with respect to the 3Rs (reduction, refinement and replacement) alternative approaches. This review is focused on the update in acute systemic toxicity testing of chemicals. Merits and demerits of these advances were also highlighted. Traditional LD50 test methods are being suspended while new methods are developed and endorsed by the regulatory body. Based on the refinement and reduction approaches, the regulatory body has approved fixed dose procedure (FDP), acute toxic class (ATC) method and up and down procedure (UDP) which involves few numbers of animals. In terms of replacement approach, the regulatory body approved 3T3 neutral red uptake (NRU), the normal human keratinocyte (NHK), and the 3T3 neutral red uptake (NRU) phototoxicity test for acute phototoxicity. However, other promising replacement alternatives such as organ on chip seeded with human cells for acute systemic toxicity and 3T3 neutral red uptake (NRU) cytotoxicity test for identifying substances not requiring classification, as well as the in silico approaches are yet to receive regulatory approval. With this backdrop, a collaborative effort is required from the academia, industries, regulatory agencies, government and scientific organizations to ensure speedily regulatory approval of the prospective alternatives highlighted.

Preclinical safety and efficacy evaluation of ‘BioCaS’ bioactive calcium sulfate bone cement

A new bioactive calcium sulfate-based formulation (named ‘BioCaS’) has been developed for bone filler applications. This is a self-setting injectable cement where the preset form comprises bassanite obtained from the uniform submicron-sized precursor crystals of gypsum, modified with hydrogen orthophosphate ions. The results of the safety and efficacy evaluation of BioCaS cement, done as per the International Standards and guidelines, are presented in this paper. The study plan consisted of in vitro screening tests of cytotoxicity and haemolysis and in vivo biocompatibility evaluation, including an acute systemic toxicity test (in mice), an intracutaneous reactivity test (in rabbits), a pyrogen test (in rabbits) and a maximization sensitization test (in guinea pigs). The efficacy of the material in healing bone defects was investigated by implanting it in artificially created defects in rabbit femora, with clinically established hydroxyapatite porous ceramic as the control, followed by histological analysis at 12, 26 and 52 weeks. Set BioCaS cement consisted of hydrogen orthophosphate incorporating low-dimensional gypsum crystal lattices, the bioactivity of which has been identified by immersion in simulated body fluid. BioCaS was proved to be non-cytotoxic and non-haemolytic in the screening tests. In the live/dead assay, human osteoblast-like human osteosarcoma cells adhered well and spread on the surface of the material, attaining typical morphology and affirming the bone cell compatibility of the material. In the biocompatibility evaluation there were no acute systemic effects and the material proved non-pyrogenic. There was no intracutaneous erythemic or oedematous reactivity and no hypersensitivity observed in the Magnusson–Kligman method. The material satisfied the biocompatibility requirements. Bone implantation study revealed BioCaS to be osteoconductive and its efficacy of healing the experimental bone defects in rabbit femora is on a par with that of hydroxyapatite ceramic. The material resorbed at a pace matching that of new bone formation. This property of osteotransductivity will help the defect to heal and gain strength faster.

GHS additivity formula: A true replacement method for acute systemic toxicity testing of agrochemical formulations

Acute systemic (oral, dermal, inhalation) toxicity testing of agrochemical formulations (end-use products) is mainly needed for Classification and Labelling (C&L) and definition of personal protection equipment (PPE). A retrospective analysis of 225 formulations with available in vivo data showed that: A) LD50/LC50 values were above limit doses in <20.2% via oral route but only in <1% and <2.4% of cases via dermal and inhalation route, respectively; B) for each formulation the acute oral toxicity is always equal or greater than the Acute Toxicity Estimate (ATE) via the other two routes; C) the GHS (Global Harmonised System) computational method based on ATE, currently of limited acceptance, has very high accuracy and specificity for prediction of agrochemical mixture toxicity according to the internationally established classification thresholds.By integrating this evidence, an exposure- and data-based waiving strategy is proposed to determine classification and adequate PPE and to ensure only triggered animal testing is used. Safety characterisation above 2000 mg/kg body weight or 1.0 mg/L air should not be recommended, based on the agrochemical exposure scenarios. The global implementation of these tools would allow a remarkable reduction (up to 95%) in in vivo testing, often inducing lethality and/or severe toxicity, for agrochemical formulations.

Exploring waiving opportunities for mammalian acute systemic toxicity tests.

A survey was carried out to explore opportunities for waiving mammalian acute systemic toxicity tests. We were interested in finding out whether data from a sub-acute toxicity test could be used to predict the outcome of an acute systemic toxicity test. The survey was directed at experts in the field of toxicity testing, and was carried out in the context of the upcoming 2018 final registration deadline for chemicals under the EU REACH Regulation. In addition to the survey, a retrospective data analysis of chemicals that had already been registered with the European Chemicals Agency, and for which both acute and sub-acute toxicity data were available, was carried out. This data analysis was focused on chemicals that were administered via the oral route. The answers to the questionnaire showed a willingness to adopt waiving opportunities. In addition, the responses showed that data from a sub-acute toxicity test or dose-range finding study might be useful for predicting chemicals that do not require classification for acute oral toxicity (LD50 > 2000mg/kg body weight). However, with the exception of substances that fall into the non-classified category, it is difficult to predict current acute oral toxicity categories.

Acute Systemic Toxicity Testing Exploring Waiving Opportunities

Acute Systemic Toxicity Testing Exploring Waiving Opportunities

Standard Biocompatibility Studies Do Not Predict All Effects of PVA/CMC Anti-Adhesive Gel in vivo

Purpose: PVA/CMC (polyvinyl alcohol/carboxymethyl cellulose) hydrogel fulfills various physiochemical properties required for an adhesion barrier and has shown good anti-adhesion properties in previous in vivo studies. In this investigation, we assessed the in vitro and in vivo biocompatibility of PVA/CMC gel and compared this to the functionality and promotion of wound healing for two surgical indications. Methods: Standardized ISO10993 in vitro and in vivo biocompatibility studies, comprising cytotoxicity, genotoxicity, acute systemic toxicity, delayed contact and maximization sensitization test, intracutaneous reactivity and local muscle implantation, were performed on PVA/CMC gel. In the functional studies, PVA/CMC gel was applied - on the one hand - to a rabbit abdominal wall model enforced with a polypropylene mesh for testing the anti-adhesion properties and - on the other hand - to an end- to-end anastomosis model that was selected for surveying potential influences of different dosages of PVA/CMC gel on anastomotic wound healing. Results: The ISO10993 methods indicated generally good biocompatibility properties, such as the absence of cytotoxic and mutagenic effects as well as no signs of systemic toxicity and sensitization potentials. No irritation effects were observed after the intracutaneous injection of lipophilic PVA/CMC sesame oil extract. However, the injection of hydrophilic PVA/CMC physiologic saline extract induced slight irritation. Following rabbit muscle implantation of the PVA membrane for 2, 4, 12, 26 and 52 weeks, a slight irritant effect was observed at 12 weeks due to the peak of phagocytosis. In the functionality tests, PVA/CMC gel showed good anti-adhesive effects in the abdominal wall model enforced with the mesh, with significantly lower and less tense adhesions compared to the untreated control. However, moderate signs of inflammation, especially in the spleen were observed after the intra-abdominal implantation of 3.3 ml PVA/CMC gel per kg body weight. In the end-to-end anastomosis model, PVA/CMC gel had no influence on wound healing. For dosages of 1-6 ml gel per treatment, no signs of intestinal leaks were detected, and tensile strength was equal to that of the untreated control, but again more moderate signs of inflammation in the spleen were observed at a dosage >3 ml. Conclusion: Comparing the standardized ISO10993 methods, anti-adhesive PVA/CMC gel displays good biocompatibility. However, those methods do not seem to be sensitive enough because the rabbit abdominal wall and the end-to-end anastomosis models display more effects with respect to the dosage and routes of the intra-abdominal resorption of PVA/CMC gel - with the recommended <2 ml PVA/CMC gel per kg body weight as a secure dosage.

In vitro and in vivo biocompatibility evaluation of dental zirconia ceramic

Recently, zirconia ceramics have been extensively used as the artificial tooth in modern dentistry on account of their excellent mechanical properties and good esthetics. The purpose of this study was to evaluate biocompatibility of zirconia ceramics using agar diffusion test, acute systemic toxicity, intracutaneous test and local lymph node assay(LLNA). For in vitro test, the agar diffusion assay was used sintered zirconia specimens (In-ceram YZ-55, VITA Co., Bad sackingen, Germany) were performed according to ISO 7405. For in vivo test, three different tests were carried out and samples were prepared according to ISO 10993-12 for all of the test. Acute systemic toxicity test and Intracutaneous test, were performed according to ISO 10993-11 and ISO 10993-10. The murine local lymph node assay test was performed according to OECD guide line 442 B. The overall criterion for the cytotoxicity of zirconia sample was "0". For the acute systemic toxicity test, no symptoms were observed during the visual observation of the animal. For the intracutaneous irritation test, there was no redness and swellings on the reaction sites. The result of LLNA test, the samples extractions in polar solvents showed average OD value of 1.078 and those in non-polar solvents showed average OD value of 1.059. The partially stabilized zirconia materials were proved to be biocompatible. The results of both in vitro and in vivo test concluded that the zirconia materials were biocompatible.

Preparation of laponite bioceramics for potential bone tissue engineering applications.

We report a facile approach to preparing laponite (LAP) bioceramics via sintering LAP powder compacts for bone tissue engineering applications. The sintering behavior and mechanical properties of LAP compacts under different temperatures, heating rates, and soaking times were investigated. We show that LAP bioceramic with a smooth and porous surface can be formed at 800°C with a heating rate of 5°C/h for 6 h under air. The formed LAP bioceramic was systematically characterized via different methods. Our results reveal that the LAP bioceramic possesses an excellent surface hydrophilicity and serum absorption capacity, and good cytocompatibility and hemocompatibility as demonstrated by resazurin reduction assay of rat mesenchymal stem cells (rMSCs) and hemolytic assay of pig red blood cells, respectively. The potential bone tissue engineering applicability of LAP bioceramic was explored by studying the surface mineralization behavior via soaking in simulated body fluid (SBF), as well as the surface cellular response of rMSCs. Our results suggest that LAP bioceramic is able to induce hydroxyapatite deposition on its surface when soaked in SBF and rMSCs can proliferate well on the LAP bioceramic surface. Most strikingly, alkaline phosphatase activity together with alizarin red staining results reveal that the produced LAP bioceramic is able to induce osteoblast differentiation of rMSCs in growth medium without any inducing factors. Finally, in vivo animal implantation, acute systemic toxicity test and hematoxylin and eosin (H&E)-staining data demonstrate that the prepared LAP bioceramic displays an excellent biosafety and is able to heal the bone defect. Findings from this study suggest that the developed LAP bioceramic holds a great promise for treating bone defects in bone tissue engineering.

Degradation behaviour and biological properties of gelatin/hyaluronic acid composite scaffolds

Hydrogels, based on natural polymers, are gaining attention as possible cell scaffolding materials for the regeneration of a variety of tissues. In this work, gelatin (Gel) and hyaluronic acid (HA) were used to fabricate novel scaffold materials using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linker. The degradation behaviours of Gel/HA scaffolds in phosphate buffered saline (PBS) and PBS solution containing lysozyme were investigated respectively. The biological properties including haemolytic activity and acute systemic toxicity were also studied. The results showed that the scaffold had an interconnected pore structure with an average pore size of about 100–500 μm. The degradation of Gel/HA scaffolds in PBS solution containing lysozyme was faster than that in PBS solution. The haemolytic ratio of 0·340–0·781% indicated that the Gel/HA scaffolds have a good blood compatibility. The acute systemic toxicity test showed that Gel/HA extracts have no acute systemic toxicity.

Can acute dermal systemic toxicity tests be replaced with oral tests? A comparison of route-specific systemic toxicity and hazard classifications under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS)

Acute systemic toxicity data (LD50 values) and hazard classifications derived in the rat following oral administration and dermal application have been analysed to examine whether or not orally-derived hazard classification or LD50 values can be used to determine dermal hazard classification. Comparing the oral and dermal classifications for 335 substances derived from oral and dermal LD50 values respectively revealed 17% concordance, and indicated that 7% of substances would be classified less severely while 76% would be classified more severely if oral classifications were applied directly to the dermal route. In contrast, applying the oral LD50 values within the dermal classification criteria to determine the dermal classification reduced the concordance to 15% and the relative ‘under-classification’ to 1%, but increased the relative ‘over-classification’ to 84%. Both under- and over-classification are undesirable, and mitigation strategies are discussed. Finally, no substance with an oral LD50 of >2000mg/kg was classified for acute systemic toxicity by the dermal route, suggesting that dermal testing for acute systemic toxicity of such substances adds nothing to the hazard characterisation and should be removed from routine regulatory data requirements.

Evaluation of Biological Properties In-Vivo of Poly(L-Lactide-Coglycolide) Composites Containing Bioactive Glass

To evaluate the biological safety of composite materials based on poly(L-lactide-co-glycolide) (PLGA) and bioactive glass (BG), which have a potential application in tissue engineering, the in-vivo biological properties were investigated by applying the micronucleus test, acute systemic toxicity test, haemolytic test and pyrogen measurement. The results indicated that the PLGA/BG composite showed no genetic toxicity; there was no toxicosis or death observed in the acute systemic toxicity test. The haemolytic test suggested that the PLGA/BG composite, with a haemolytic index of 0.281%, did not have an obvious haemolytic reaction. In addition, the PLGA/BG composite materials showed no pyrogen reaction. Therefore, PLGA/BG composite materials could be promising candidate biomedical materials for bone tissue engineering.

Biological Assessment of Composite Materials Based on Poly-L-lactide and Bovine Bone

Biodegradable polymers and bioactive fillers are being combined in a variety of composite materials for biomedical application. Biological properties in vivo of poly-L-lactide (PLLA) matrix containing bovine bone were investigated by using acute systemic toxicity in vivo, blood biocompatibility, sensitivity, pyrogen, and genetic toxicity testing methods. The results indicated that there were no toxic and death cases observed in the acute systemic toxicity test. The PLLA/bovine bone composite showed no genetic toxicity, pyrogen, or sensitizing response and the hemolytic index was 0.29%. The results from these studies demonstrated that PLLA/bovine bone has a good biocompatibility and good biological safety. Therefore, PLLA/bovine bone composite materials can be promising biomedical materials for bone tissue engineering.

‘Human-on-a-chip’ Developments: A Translational Cutting-edge Alternative to Systemic Safety Assessment and Efficiency Evaluation of Substances in Laboratory Animals and Man?

Various factors, including the phylogenetic distance between laboratory animals and humans, the discrepancy between current in vitro systems and the human body, and the restrictions of in silico modelling, have generated the need for new solutions to the ever-increasing worldwide dilemma of substance testing. This review provides a historical sketch on the accentuation of this dilemma, and highlights fundamental limitations to the countermeasures taken so far. It describes the potential of recently-introduced microsystems to emulate human organs in 'organ-on-a-chip' devices. Finally, it focuses on an in-depth analysis of the first devices that aimed to mimic human systemic organ interactions in 'human-on-a-chip' systems. Their potential to replace acute systemic toxicity testing in animals, and their inability to provide alternatives to repeated dose long-term testing, are discussed. Inspired by the latest discoveries in human biology, tissue engineering and micro-systems technology, this review proposes a paradigm shift to overcome the apparent challenges. A roadmap is outlined to create a new homeostatic level of biology in 'human-on-a-chip' systems in order to, in the long run, replace systemic repeated dose safety evaluation and disease modelling in animals.

Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests

Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000mg/kgb.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration–response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained.

Evaluation of Physical and Biological Properties of Polyvinyl Alcohol/Chitosan Blend Films

Polyvinyl alcohol/chitosan (PVA/Cs) blend films with various compositions were prepared using glutaraldehyde (GA) as a crosslinker. The hydrophilicity and swelling behavior of the PVA/Cs films were assessed, and the biological properties of the PVA/Cs films, including cell culture in the extract, cell cytotoxicity, and hemolytic and acute systemic toxicity tests were also investigated. The results showed that all composite films were hydrophilic materials with water contact angles changing from 45.3 ± 1.1° to 63.2 ± 1.8° when PVA content was varied from 100 to 0 wt%. The PVA/Cs films showed a significant reduction in swelling behavior as the chitosan content increased. Fibroblasts grown in PVA/Cs extract showed similar appearance with the cells cultured in the negative control medium, and the extract solution of PVA/Cs had no obvious cytotoxic effect. The hemolytic test suggested that the PVA/Cs, with a hemolytic index of 0.91%, did not have an obvious hemolytic reaction. There was no toxicosis or death observed in the acute systemic toxicity test. The results suggest that the complex of PVA and chitosan is a promising candidate biomaterial for tissue engineering.

Glutaraldehyde treatment elicits toxic response compared to decellularization in bovine pericardium

Glutaraldehyde-stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification is reported on account of glutaraldehyde treatment. In this study, commercially available glutaraldehyde cross-linked bovine pericardium was evaluated for its in vitro cytotoxic effect, macrophage activation, and in vivo toxic response in comparison to decellularized bovine pericardium. Glutaraldehyde-treated bovine pericardium and its extract were observed to be cytotoxic and it also caused significant inflammatory cytokine release from activated macrophages. Significant antibody response, calcification response, necrotic, and inflammatory response were noticed in glutaraldehyde-treated bovine pericardium in comparison to decellularized bovine pericardium in a rat subcutaneous implantation model. Glutaraldehyde-treated bovine pericardium also failed in acute systemic toxicity testing and intracutaneous irritation testing as per ISO 10993. With respect to healing and implant remodeling, total lack of host tissue incorporation and angiogenesis was noticed in glutaraldehyde-treated bovine pericardium compared to excellent host fibroblast incorporation and angiogenesis within the implant in decellularized bovine pericardium. In conclusion, using in vitro and in vivo techniques, this study has demonstrated that glutaraldehyde-treated bovine pericardium elicits toxic response compared to decellularized bovine pericardium which is not congenial for long-term implant performance.

Biological safety evaluation of Carbon-Carbon composites

Objective Due to the superior performance,Carbon-Carbon composites,although still at their early stage of development,have gained more and more attention and showed great application potential.Methods According to the National Standard,the biological safety evaluation of carbon-carbon composites were done in the following aspects:cytotoxicity test,acute systemic toxicity test,haemolysis test,pyrogen test,intramuscular implantation test.Results test results showed the biological safety evaluation of carbon-carbon composites well meet the requirement of the national standard with fine biological compatibility.Conclusion The experiment results demonstrate that carbon-carbon composites can be put in clinical application Key words: Carbon-Carbon composites; Biological safety evaluation; Toxicity; Experiment

Study on Biological Safety of Polyvinyl Alcohol/Collagen Hydrogel as a Tissue Substitute (II)

Physically crosslinked polyvinyl alcohol/collagen composite hydrogels were prepared by a cyclic freezing-drying technique. The biological properties of the hydrogels, including hemolysis, anaphylaxis, pyrogen and acute systemic toxicity tests and implantation in-vivo, were investigated. The hemolysis test suggested that the polyvinyl alcohol/collagen, with a hemolysis index of 1.19%, did not have an obvious hemolysis reaction. There was no toxicosis or death cases observed in the acute systemic toxicity test, and the hydrogel showed no anaphylaxis or pyrogen response. The composite hydrogel showed a good histological compatibility in the in-vivo study. The results indicated that the polyvinyl alcohol/collagen composite hydrogels have promising applications for pharmaceutical and biomedical fields.

Synthesis and biological evaluation of PMMA/MMT nanocomposite as denture base material

Inorganic-polymer nanocomposites are of significant interest for emerging materials due to their improved properties and unique combination of properties. Poly (methylmethacrylate) (PMMA)/montmorillonite (MMT) nanocomposites were prepared by in situ suspension polymerization with dodecylamine used as MMT-modifier. X-ray diffraction (XRD) and transmission electron microscopy (TEM) were used to characterize the structures of the nanocomposites. Cytotoxicity test, hemolysis test, acute systemic toxicity test, oral mucous membrane irritation test, guinea-pig maximization test and mouse bone-marrow micronucleus test were used to evaluate the biocompatibility of PMMA/MMT nanocomposites. The results indicated that an exfoliated nanocomposite was achieved, and the resulting nanocomposites exhibited excellent biocompatibility as denture base material and had potential application in dental materials.

Assessment of In-vivo Toxicity of Composite Materials Based on Poly-L-lactic Acid and β-Tricalcium Phosphate

On the investigation of the in-vitro cytotoxicity of composite materials containing poly-L-lactic acid (PLLA) and β-tricalcium phosphate (β-TCP), the second part of the in-vivo cytotoxicity was investigated by acute systemic toxicity test, haemolytic test, skin sensitisation test, micronucleus test and subcutaneous implant test. The results indicated that there was no toxicosis or death cases observed in acute systemic toxicity test. The PLLA/β-TCP composite showed no genetic toxicity and sensitising response, and the haemolytic index was 0.39%. The histological observation of the tissue response showed that the inflammatory response to PLLA/β-TCP continued to decrease, which indicated the composite materials had a good biocompatibility. Therefore, PLLA/β-TCP composite materials can be a promising candidate of biomedical materials for bone tissue engineering.