In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

A mechanism for acute aluminium toxicity in fish

Aluminium is acutely toxic to fish in acid waters. The gill is the principal target organ and death is due to a combination of ionoregulatory, osmoregulatory and respiratory dysfunction. The toxic mechanism has hitherto received little direct consideration and is unknown. In this paper the mechanism of acute aluminium toxicity is approached from a chemical perspective. Symptomatic evidence of toxicity is taken from the literature and combined with our own research to elucidate a biochemically sound model to describe a possible mechanism of acute aluminium toxicity in fish. The proposed model delineates the chemical conditions immediately adjacent to the gill surface and emphasizes their importance in aluminium's toxic mode of action. The mechanism is shown to be bipartite. Aluminium binding to functional groups both apically located at the gill surface and intracellularly located within lamellar epithelial cells disrupts the barrier properties of the gill epithelium. The concomitant iono- and osmoregulatory dysfunction results in accelerated cell necrosis, sloughing and death of the fish. The mechanism of epithelial cell death is proposed as a general mechanism of aluminium-induced accelerated cell death.     

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2′,4,4′-tetrabromodiphenylether (BDE-47), 2,2′,4,4′,5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell–based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 μM (5 FRs), 1<10 μM (7 FRs) to the >10 μM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell–based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment.

Graphical abstract

     

In vitro cytotoxicity testing of coladerm membrane

Atpresent, biodegradable and biocompatible membranes based on collagen andglycosaminoglycans play an important role in substitutive medicine. Modernbiomaterials use a chemically modified collagen-based matrix for implants withprogrammable biodegradability as a substitute of buccal mucosa, skin,cartilage,etc. Besides the requirements for biocompatibility and biodegradability, themembranes must be also non-toxic. Therefore, cytotoxicity testing of thesematerials in vitro is an integral part of introducingnewlydeveloped types of membranes into clinical practice. As a biological model forthe tested COLADERM membrane, cell cultures from human embryonic fibroblasts(B-HEF-2) were used for both cytotoxicity testing as well as in tests to assessthe ability of cells to proliferate on this membrane. Along with the ability ofcells to grow on the surface and inside the membrane, immunohistochemicalexamination and scanning electron microscopy (SEM) were performed as well. Theobtained results have shown that the COLADERM membrane is non-toxic withsuitable structural and biological properties for clinical application as asubstitute of buccal mucosa following surgical ablation of malignant tissuesfrom the oral cavity.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.     

Biostatistical methods for the validation of alternative methods for in vitro toxicity testing

Statistical methods for the validation of toxicological in vitro test assays are developed and applied. Validation is performed either in comparison with in vivo assays or in comparison with other in vitro assays of established validity. Biostatistical methods are presented which are of potential use and benefit for the validation of alternative methods for the risk assessment of chemicals, providing at least an equivalent level of protection through in vitro toxicity testing to that obtained through the use of current in vivo methods. Characteristic indices are developed and determined. Qualitative outcomes are characterised by the rates of false-positive and false-negative predictions, sensitivity and specificity, and predictive values. Quantitative outcomes are characterised by regression coefficients derived from predictive models. The receiver operating characteristics (ROC) technique, applicable when a continuum of cut-off values is considered, is discussed in detail, in relation to its use for statistical modelling and statistical inference. The methods presented are examined for their use for the proof of safety and for toxicity detection and testing. We emphasise that the final validation of toxicity testing is human toxicity, and that the in vivo test itself is only a predictor with an inherent uncertainty. Therefore, the validation of the in vitro test has to account for the vagueness and uncertainty of the "gold standard" in vivo test. We address model selection and model validation, and a four-step scheme is proposed for the conduct of validation studies. Gaps and research needs are formulated to improve the validation of alternative methods for in vitro toxicity testing.     

Silica toxicity for macrophages in vitro: a new cytotoxicity test.

Soluble factors released from silica-damaged macrophages inhibit proliferation of various haematopoietic cells in long term cultures. The same holds true for damage induced by heat, non-physiological pH, freezing and thawing. This phenomenon is dose-dependent and correlates with the degree of macrophage viability. Thus, a base for measuring the amount of damage to the macrophage is established.     

In vitro toxicity testing for antibacterials against human keratinocytes

The use of cultured human keratinocytes in an in vitro comparison of topical antibacterial toxicity for epithelial cells was examined. The complement of three assessments allows testing of epithelial migration, growth, and survival. The three assessments included (1) flow cytometry for determination of cell survival, (2) a comparison of confluent cell culture growth after antibacterial exposures, and (3) an evaluation of cell migration using a technique of dermal explants to study radial migration. A comparative ranking of the toxicities of the various topical antibacterials was determined with the three assessments. This has confirmed anecdotal reports that many of the topical antibacterials are cell-toxic and may inhibit wound healing. This information can be directly extrapolated to the clinical setting, unlike many of the animal data for wound healing that currently exist.     

Redox mediated biosensors incorporating cultured fish cells for toxicity assessment

Fish are an important genus within ecosystems, and practical sensing devices, incorporating fish cells, could be used with advantage for environmental monitoring and protection. In this paper, redox mediated biosensors were prepared by immobilizing cultured fish cells at a carbon electrode surface. EPC (from carp) and BF-2 (from bluegill sunfish) cells could be monitored by using a lipophilic mediator (e.g. 2,6-dimethylbenzoquinone) added to the solution bathing the sensor. Currents measured in the external circuit were indicative of the metabolic activity of the immobilized cells and the sensors could be used to determine the presence of chemicals within the bathing medium that perturbed their normal metabolic status.     

Opportunities for reduction in acute toxicity testing via improved design

The conventional method for assessing acute oral toxicity (OECD Test Guideline 401) was designed to identify the median lethal dose (LD50), using the death of animals as an endpoint. Introduced as an alternative method (OECD Test Guideline 420), the Fixed Dose Procedure (FDP) relies on the observation of clear signs of toxicity, uses fewer animals and causes less suffering. More recently, the Acute Toxic Class method and the Up-and-Down Procedure have also been adopted as OECD test guidelines. Both of these methods also use fewer animals than the conventional method, although they still use death as an endpoint. Each of the three new methods incorporates a sequential dosing procedure, which results in increased efficiency. In 1999, with a view to replacing OECD Test Guideline 401, the OECD requested that the three new test guidelines be updated. This was to bring them in line with the regulatory needs of all OECD Member Countries, provide further reductions in the number of animals used, and introduce refinements to reduce the pain and distress experienced by the animals. This paper describes a statistical modelling approach for the evaluation of acute oral toxicity tests, by using the revised FDP for illustration. Opportunities for further design improvements are discussed.     

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.     

In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

In vitro assessment of the toxicity of metal compounds

A review has been compiled illustrating the directions taken in examining the genotoxic effects of metals and their compounds centering only on those studies pertaining to effects of metals and their compounds on DNA structure and function, such as the induction of DNA strand breaks, production of DNA-protein crosslinks, induction of chromosomal aberrations, and sister chromatid exchanges. Although it is premature to declare a cause and effect relationship between the carcinogenic activity of metals and their ability to induce one or more lesions in DNA, strong evidence is emerging to suggest such a relationship. Low concentrations of metals induce the appearance of DNA lesions, such as strand breaks and crosslinks, or induce sister chromatid exchanges or DNA repair synthesis. Assays based upon these events constitute extremely sensitive probes for genotoxic effects of metals and their compounds. These effects of metals on DNA are consistent with the currently accepted mechanism of chemical carcinogenesis, allowing the acquisition and propagation of altered DNA function. The lack of complete information on the activity of metals in producing DNA lesions allow only preliminary conclusions to be drawn. Certain compounds containing potentially or actually carcinogenic elements, such as Ni, Be, As, Cr, Cd, and to a minor extent Pb, have yielded positive responses in one or more DNA lesion assays. At relatively nontoxic levels of Ni and Cr, considerable evidence suggests that multiple types of DNA lesions are induced.     

In vitro assessment of the toxicity of metal compounds

A review of the activity of metal compounds in mammalian cell transformation assays has been completed. Results from these assays appear to correlate well with the known carcinogenic activity displayed by specific metal compounds in vivo. Studies of cell transformation in vitro may provide information pertaining to the mechanism of the induction of carcinogenesis by certain metals.     

In vitro assessment of the toxicity of metal compounds

This review has focused on several parameters related to the delivery of carcinogenic metal compounds to the cell nucleus as a basis for understanding the intermediates formed between metals and cellular components and the effect of these intermediates on DNA structure and function. Emphasis has been placed on metal interactions at the cellular membrane, including lipid peroxidation, metal interactions with glutathione and their relation to membrane injury, and metal effects on the membrane bound enzyme, Na⁺/K⁺ ATPase. Metal binding to metallothionein is also considered, particularly as related to transport and utilization of metal ions and to genetic defects in these processes exemplified in Menkes disease. The ability of cadmium to induce the synthesis of metallothionein more strongly than zinc is also discussed in relation to other toxic and carcinogenic metals. The effects of metal ions on purified DNA and RNA polymerase systems are presented with some of the recent studies using biological ligand-metal complexes. This review points out the importance of considering how metals affect in vitro systems when presented as ionic forms or complexed to relevant biological ligands.     

In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity

BackgroundThimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 μg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains.MethodsThe safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations.ResultsThe viability of all examined cell lines was suppressed entirely in the presence of 4.6 μg/ml (12.5 μM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 μg/ml (5.5, 4.3, 2.7 and 0.8 μM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 μg/ml (7.1, 8.5, 3.5 and 2.4 μM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 μM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 μM) for Aspergillus brasiliensis.ConclusionAccording to our results Thimerosal should be present in culture media at 100 μg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 μg/ml (12.5 μM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.     

Functional assessment in vitro of human-complement-dependent antibody-induced cytotoxicity of neoplastic cells

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GM1. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors, such as C4, should be further validated in cancer patients at various disease stages and phases of treatment. Received: 25 November 1999 / Accepted: 13 January 2000