Red cell haemolysis test as an in vitro approach for the assessment of toxicity of karanja oil

The karanja tree grows in parts of India and Australia. The oil from seed kernels was found to be toxic to animals. The annual potential availability of the oil is around 135,000 tons in India. In order to use it for beneficial purposes, it is necessary to detoxify the oil. In the present study, the oil was assessed for toxicity by the red cell haemolysis test and estimating the LDH in the supernatant. The non-lipid constituents were isolated from raw oil by aqueous methanol extraction. The raw oil and the non-lipid fraction were found to haemolyse the red cells with release of LDH, whereas the extracted oil did not show such a manifestation. There was a good correlation between haemolytic activity and LDH released from cells. These findings were further confirmed with in vivo studies where the raw and extracted karanja oils showed 100% and nil mortality in rats dosed orally at 10 and 20 ml/kg body weight, respectively. This haemolysis test can be used as an in vitro method to predict toxicity and to monitor the detoxification of the oils prior to use in in vivo studies for toxicological evaluation. The fatty acid composition of the raw and extracted karanja oils showed no difference.     

Study of the effects of cardiovascular drugs in heart cell cultures

Compounds that produce myocardial pathology in vivo can be separated into two main classes—those that are directly toxic to the myocardium and those that are considered to act by way of an indirect vascular or neurologically based mechanism. An in vitro model of myocardium without nervous or systemic influences can be used to differentiate between direct myocardial cytotoxic effects and indirect cardiac pathology arising in vivo from exaggerated vascular or neural pharmacological effects of a number of drugs. In this study direct-acting cardiotoxic compounds are distinguished from those causing cardiac pathology by indirect mechanisms by their different pattern of effects in chick embryonic myocardial myocyte reaggregates (MMRs) cultures. The toxicity of the direct-acting cardiotoxic drugs allylamine (positive control, 50 μ ) and doxorubicin were compared with digoxin and isoprenaline, which show both direct and indirect mechanisms in vivo, and the indirectly acting hydralazine and pinacidil. Changes in spontaneous beating activity (SBA), leakage of lactate dehydrogenase (LDH) and cell morphology by light and transmission electron microscopy were used to assess toxicity. The MMRs were cultured for up to 24 hr in a series of concentrations of the five compounds in the range 0.1 to 10,000 μ . Allylamine, doxorubicin, digoxin and, to a lesser extent, isoprenaline were highly toxic to the MMRs, as shown by alterations in SBA, LDH leakage and cellular morphology. In contrast, hydralazine showed a very mild degree of toxicity at the highest concentrations in the absence of LDH leakage; treatment with pinacidil did not show any evidence of morphological degeneration but did cause a dose-related inhibition of SBA. These results are consistent with the view that doxorubicin and digoxin are directly toxic to myocardial cells and also suggests that this is an important mechanism in vivo for isoprenaline. The absence of a significant degree of toxicity with hydralazine and pinacidil is consistent with an indirect toxic mechanism.     

An interlaboratory assessment of the Eytex system

Seventeen raw materials and chemical formulations were evaluated in the Eytex System to determine the ability of this assay in vitro to predict eye irritation potential in vivo. All the test samples, which represented a wide range of chemical types and eye irritancy potential in vivo, were provided by one of the participating laboratories. Historical data from tests in vivo were available for each of the test samples, so testing in vivo specifically for this study was not necessary. Samples were evaluated by both the membrane partition assay (MPA) and the rapid membrane assay (RMA). The sensitivity, specificity, predictivity and equivalence of the Eytex assay were determined by comparison with the rabbit eye irritation data, using each of the different Eytex Draize Equivalent (EDE) classification schemes. Regardless of the classification scheme used, the correlation between the scores in vivo and in vitro was poor. The Eytex System consistently overclassified materials of low irritancy in vivo and underclassified those test materials of moderate irritancy or above. On the basis of the results from the 17 materials tested in this study, the Eytex System appears unsuitable as a replacement in vitro for ocular irritancy testing of all types of chemical. However, Eytex may have a place as a pre-screening method used as part of a test battery.     

The application of in vitro data in the derivation of the Acceptable Daily Intake of food additives

The acceptable daily intake (ADI) for food additives is commonly derived from the NOAEL (no-observed-adverse-effect level) in long-term animal in vivo studies. To derive an ADI a safety or uncertainty factor (commonly 100) is applied to the NOAEL in the most sensitive test species. The 100-fold safety factor is considered to be the product of both species and inter-individual differences in toxicokinetics and toxicodynamics. Although in vitro data have previously been considered during the risk assessment of food additives, they have generally had no direct influence on the calculation of ADI values. In this review 18 food additives are evaluated for the availability of in vitro toxicity data which might be used for the derivation of a specific data-derived uncertainty factor. For the majority of the food additives reviewed, additional in vitro tests have been conducted which supplement and support the short- and long-term in vivo toxicity studies. However, it was recognized that these in vitro studies could not be used in isolation to derive an ADI; only when sufficient in vivo mechanistic data are available can such information be used in a regulatory context. Additional short-term studies are proposed for the food additives which, if conducted, would provide data that could then be used for the calculation of data-derived uncertainty factors.     

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC₅₀ values and in vivo LD₅₀ values. The in vitro data correlate better with rodent parenteral (ip or iv) LD₅₀ values than with oral LD₅₀ values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD₅₀ values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.     

Cytotoxicity testing using neutral red and MTT assays on a three-dimensional human skin substrate

The use of a three-dimensional dermal culture system as a substrate in cytotoxicity assays is described. This substrate consists of several layers of dermal fibroblasts, derived from human foreskin, grown on pretreated nylon mesh. This physiological model of the human dermis has been used in conjunction with the neutral red assay and the MTT assay to assess the in vitro toxicity of a panel of 15 test agents from several different classes. NR₅₀ and MTT₅₀ endpoints (test agent concentrations yielding 50% viability) were obtained for compounds/formulations from the following groups: surfactants, alcohols, antimicrobial preservatives, metal chlorides and pesticides. In addition, the carboxylic ionophore, monensin, was tested in both assays. Limited comparisons of the in vitro neutral red and MTT results, using the three-dimensional culture system, with existing in vivo rabbit ocular irritancy data look promising. This three-dimensional model may afford several advantages over monolayer cultures.     

Development of an in vitro test system with human skin cells for evaluation of phototoxicity

Photosensitizing compounds may cause severe skin reactions after appropriate light exposure. On the cellular level this cutaneous in vivo response can be demonstrated by an in vitro model system with adequate target cells of human skin. Normal keratinocytes and fibroblasts were used to establish a predictive assay for phototoxic compounds. To develop a sensitive, specific and easy-to-handle screening system we determined different viability and proliferation parameters, like neutral red (NR) uptake, MTT-reduction, kenacid blue (KB) test, [³H]thymidine incorporation and a fluorometric assay in response to UVA light after treatment with different classes of compounds. These experiments resulted in the selection of a final test pattern, which we used to screen 26 substances of different phototoxic potential in vivo. We found a qualitative correlation between the phototoxic response in vitro and phototoxicity reported in different test systems in vivo.     

Development of a QSAR for worst case estimates of acute toxicity of chemically reactive compounds

Future EU legislations enforce a fast hazard and risk assessment of thousands of existing chemicals. If conducted by means of present data requirements, this assessment will use a huge number of test animals and will be neither cost nor time effective. The purpose of the current research was to develop methods to increase the acceptability of in vitro data for classification and labelling regarding acute toxicity. For this purpose, a large existing database containing in vitro and in vivo data was analysed. For more than 300 compounds in the database, relations between in vitro cytotoxicity and rat or mouse intravenous and oral in vivo LD50 values were re-evaluated and the possibilities for definition of mechanism based chemical subclasses were investigated.

A high in vitro–in vivo correlation was found for chemicals classified as irritants. This can be explained by a shared unspecific cytotoxicity of these compounds which will act as the predominant mode of action for both endpoints, irritation and acute toxicity. For this subclass, which covered almost 40% of all compounds in the database, the LD50 values after intravenous dosing could be predicted with high accuracy. A somewhat lower accuracy was found for the prediction of oral LD50 values based on in vitro cytotoxicity data.

Based on this successful correlation, a classification and labelling scheme was developed, that includes a hazard based definition of the applicability domain (irritants) and a prediction of the labelling of compounds for their acute iv and oral toxicity. The scheme was tested by an external validation.     

Spermatocyte toxicity of 2-methoxyethanol in vivo and in vitro: Requirement for an intact seminiferous tubule structure for germ cell degeneration

Cultures of isolated testicular cells are widely used for evaluating mechanisms of action of direct-acting testicular toxicants. However, for testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces testicular lesions in rats which are characterized by pachytene spermatocyte degeneration 24 hr after dosing. As part of a study to evaluate mechanisms of male germ cell toxicants, we compared the morphological aspects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposure to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature rats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/kg body weight by gavage and the testes were evaluated 24 hr after dosing. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cultures, and enriched mixed germ cells, all prepared from 24-day-old rats, were evaluated after 24-hr in vitro exposure to 5 m MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats showed the expected pachytene spermatocyte degeneration 24 hr after dosing. Similar changes were observed in cultured seminiferous tubules after 24 hr of exposure to MAA in vitro. However, without the intact seminiferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detachment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was observed in these detached germ cells. Similarly, no increase in degenerating spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture architectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/MAA, and suggests the usefulness of culture seminiferous tubules in future mechanistic studies.     

Whole embryo culture and toxicity testing

The use of post-implantation embryo culture in toxicity testing has been the subject of research as well as debate. Advantages of the method include reduced animal use, and reduced cost and time in comparison with in vivo testing. In addition, the method yields many developmental endpoints: it allows the direct and controlled addition of small amounts of compounds, and the incorporation of metabolizing systems is possible. Disadvantages include the technical skill required, the restricted culture duration, the artificial route of administration of test compounds and the absence of the maternal compartment (and hence the absence of a measure for adult toxicity). Several studies using a variety of compounds have shown promising results with respect to correlations between in vivo effects and the outcome of embryo culture. The question of how a meaningful validation study should be designed is still a matter of dispute. Issues under discussion include: the purpose of validation, culture conditions, endpoint definition, choice of compounds to be tested, the status of in vivo data on test compounds, presentation of results, double-blind and interlaboratory design, and the position of the test within a testing strategy. The validity of whole embryo culture as a toxicity screening test is likely to vary considerably between classes of compounds. Therefore, validation studies with larger sets of related compounds may be more meaningful than those with many unrelated compounds. Whole embryo culture may provide a significant contribution to risk assessment by use in screening, and for mechanistic, structure-activity, and dose-response studies.     

Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) – A new in vitro model for safety assessment of recombinant food compounds

The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.     

Human percutaneous absorption of a direct hair dye comparing in vitro and in vivo results: Implications for safety assessment and animal testing

Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone–aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20 μg/cm² After 0.5 or 48 h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48 h. In vitro, skin was treated with 20 mg/cm² dye for 0.5 h, penetration determined after 24 h. In vivo, at 0.5 h, total recovery (back) was 0.67 μg/cm² (tape strips + CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5 h, scalp tape strips contained 1.80 μg/cm², HFO 0.82 μg/cm². At 48 h, HFO contained 0.21 μg/cm², sebum 0.80 μg/cm². In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50 μg/cm², epidermis/dermis 0.86 μg/cm², receptor fluid < 0.04 μg/cm², a total of 0.90 μg/cm² was considered to be bioavailable. In vitro epidermis/dermis residues were nearly identical to those located in non-living skin in vivo. In conclusion, in vitro percutaneous penetration studies may produce seemingly bioavailable material , which raises the need for a Threshold of Skin Absorption (TSA) addressing a negligible dermal absorption in order to avoid unnecessary in vivo toxicity studies on substances that produce no significant human systemic exposure.     

Inducibility and functionality of rat embryonic/foetal cytochrome P-450: A study of differentiating limb-bud and mid-brain cells in vitro

The presence of constitutive levels of cytochrome P-450 isoenzymes in cultures derived from rat embryo limb-bud (LB) and mid-brain (CNS) cells was demonstrated immunocytochemically by staining with specific monoclonal and polyclonal antibodies of cytochrome P-450. The b and e forms of cytochrome P-450 were found to be non-inducible by either in vitro co-incubation for 5 days or by transplacental maternal induction with phenobarbitone (PB), 3-methylcholanthrene (3MC) or β-naphthoflavone (βNF) in either cell type. Consistent with this lack of response was the observation that both in vitro and in vivo inducer treatment did not alter the toxicity of the teratogens diphenylhydantoin (DPH) or cyclophosphamide (CPA). In contrast, 3MC induction was achieved by both in vitro and transplacental regimens as gauged by the increased intensity of peroxidase staining using a monoclonal antibody to cytochrome t-450 c, in both cell types. There was also a concomitant increase in DPH toxicity (>20% gauged by a decrease in IC₅₀ values) in LB cells by both induction regimens but the CNS cells were refractory. βNF induction of cytochrome P-450 was observed following in vitro and in vivo exposures in both cell types. There was no modulation of DPH or CPA toxicity after in vitro exposure to the inducers, but in vivo induction caused a strong staining reaction in both cell types, commensurate with a 30% increase in DPH toxicity in LB cells and activation of the pro-teratogen CPA. The b and e forms of cytochrome P-450 were non-inducible but it is highly likely that the c form was both inducible (by 3MC and βNF) and functional, the latter being assessed by modulation of DPH toxicity and CPA activation. It may be possible to induce cytochrome P-450 in cells derived from embryos. The system used may be suitable for detailed investigations of the types of metabolizing systems involved in the mechanisms underlying toxicity/teratogenicity.     

Validation—Lessons learned from practical experience

With regard to the problems encountered and the experience gained in validation studies conducted in the past, suggestions have been made concerning criteria for the selection of the tests and laboratories to be included in a validation study, the selection and distribution of test chemicals, and procedures for the handling, analysis and interpretation of the resulting data. In particular, tests should have been developed to the extent that detailed protocols and standard operating procedures have been produced and evaluated. The laboratories should be chosen on the basis of evidence of their appropriate experience, competence and ability to comply with good laboratory practice (GLP) requirements. The choice of test chemicals depends primarily on the goals of the validation study and on the availability of reliable in vivo toxicity data of high quality. A biostatistician should be involved in the initial design of the validation study as well as in the analysis of the resulting data. The quality of the in vivo and in vitro data must be ensured, prior to determining the reproducibility and predictivity of the alternative test.     

Preparation and pharmacokinetics of labeled derivatives of apamin

Apamin, the specific, centrally acting peptide neurotoxin from bee venom, was radiolabeled by different methods in order to study its in vitro and in vivo fate.

Iodination of the histidyl residue resulted in a derivative of diminished toxicity whose label was partially lost within 1 week. Furthermore, it was adsorbed on to Sephadex G-25 gel in a non-specific manner.

Acetylation with ³H-acetic anhydride yielded a slightly less toxic and, according to its behaviour on carboxymethyl cellulose, less basic derivative as compared with the starting material. Acetyl apamin was degraded in vitro by homogenates from liver, kidney, and brain, but not by serum, yielding ³H-acetic acid. In vivo it was degraded to tritiated water.

Reductive alkylation with formaldehyde and sodium borohydride resulted in methylated apamin possessing the basicity and about half the toxicity of the starting peptide. It was also degraded in vitro and in vivo, although to a lesser degree than acetyl apamin. Reductive alkylation appears to be the most favourable approach towards biologically active, labeled apamin.

The radiological or metabolic instability of the different apamin derivatives prevented a quantitative pharmacokinetic approach towards the in vivo fate of intact apamin. However, it is already evident that its acetyl or N-methyl derivatives are enriched not in the pharmacodynamic target organ, i.e. the central nervous system, but in the kidney.     

Nitrofurazone—Genotoxicity studies in mammalian cells in vitro and in vivo

Nitrofurazone has been examined for genetic effects in mammalian cells in vitro and in vivo. In the in vitro studies in Chinese hamster ovary (CHO) cells, metaphase analysis was carried out at different sampling times after nitrofurazone treatment and gene mutation leading to hypoxanthine-guanine phosphoribosyltransferase deficiency was also investigated. Both assays were carried out in the presence and absence of a metabolic activation system (S-9 mix). Metaphase analysis was also carried out on bone-marrow cells at various sampling times after treatment of rats with a single oral dose of nitrofurazone and at one sampling time after administration of five daily oral doses. In the in vitro studies a dose-related increase in aberrant metaphases was observed after nitrofurazone treatment both with and without metabolic activation, but the gene mutation assay was negative. In vivo, despite the use of high doses of nitrofurazone (up to 400 mg/kg body weight) resulting in evidence of toxicity and a reduction in the mitotic index of bone-marrow cells, there was no evidence that nitrofurazone increased chromosomal damage. It is concluded that although the compound has the capacity to cause chromosomal damage in mammalian cells in vitro, it is detoxified by the metabolic system of the animal. These results, together with existing in vivo data, do not suggest that nitrofurazone is likely to give rise to any genotoxic hazard for man.     

Eye irritation: Reference chemicals data bank

A list of 55 chemicals has been developed for which comprehensive in vivo rabbit eye irritation data are available. No new in vivo testing has been carried out to qualify a chemical for inclusion in the list. The 55 chemicals selected are available at high and consistent purity and are expected to be stable on storage. They have been tested undiluted in in vivo studies, except those chemicals where high concentrations of the substance could be expected to cause severe effects. The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the conjunctival sac, and in which observations were made at least at 24, 48 and 72 hr after instillation. The chemicals represent a range of chemical classes (acetates, acids, alcohols, alkalis, aromatics, hydrocarbons, inorganics and surfactants) and different degrees of irritancy. They are ranked for eye irritation potential on the basis of a ‘modified maximum average score’. The reference data bank should be of use in validation tests for promising alternatives to the in vivo rabbit eye irritation test. This is an essential step in the progression to regulatory acceptance of alternative procedures.     

Validation of whole chick embryo cultures, whole rat embryo cultures and aggregating embryonic brain cell cultures using six pairs of coded compounds

A comparative study was performed to assess the effects of six pairs of coded compounds using cultures of whole chick and rat embryos as well as aggregating brain cell cultures. Developed originally for basic studies in developmental biology, these three culture systems have been adapted for the screening of chemicals in the field of prenatal toxicology. Chick and rat embryos were cultured for 2 days during the early stages of organogenesis. Aggregating cell cultures were prepared from early foetal rat telecephalon and grown for 14 days in a chemically defined medium. Concentration-response relationships were established by treating whole embryos in vitro for 2 days, and aggregating brain cell cultures for 9 days. After decoding the compounds, the results showed that, in the three test systems, specific effects were induced at comparable concentration levels. Similar compound-related malformations could be observed in both chick and rat whole embryo cultures. In aggregating brain cell cultures, neuron- and glia-specific effects could be distinguished. Based on the results obtained in the three in vitro systems, the following concentration ranges were determined for the teratogenic/toxic potencies of the test compounds (in mol/litre): <10⁻⁶: retinoids (Ro 13-6307, Ro 1-5488), 6-aminonicotinamide, ketoconazole; 10⁻⁶−10⁻³: 4-hydroxypyridine, sulfadiazine, sulfanilamide, caffeine, theophylline, metronidazole, methoxyacetic acid; >10⁻³: methoxyethanol. In general, the three in vitro test systems were found to provide concordant and complementary data on the toxicity and teratogenicity of a given compound. These data were also comparable with those available from in vivo studies. It is therefore concluded that such a test battery could contribute significantly to risk assessment and to the reduction of in vivo experimentation in reproductive toxicology.     

复方麝香黄芪滴丸的体外及体内血清化学成分分析

目的:应用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap HRMS)对复方麝香黄芪滴丸的体外及体内血清化学成分进行快速分析鉴别.方法:采用Accucore C18(100 mm×2.1 mm,2.6 μm)色谱柱,以0.1％甲酸水溶液-甲醇梯度洗脱,流速0.2mL·min-1,进样量5...