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This study investigated the response of chick myocardial myocyte reaggregates (MMR) to allylamine, which is specifically toxic to the cardiovascular system, and its putative toxic metabolite, acrolein. Toxicity was assessed by determining cellular ATP content, spontaneous beating and ultrastructural alterations. Both allylamine and acrolein caused a dose-dependent loss of beating and depletion of cellular ATP content. Treatment with either 10 or 100 mum-allylamine produced marked alterations in reaggregate tissue morphology at all times, which was characterized by widespread myocyte necrosis and a reduction in tissue compactness. Exposure of aggregates to acrolein produced generalized tissue destruction following administration of either 10 or 100 mum-acrolein. The concentration of allylamine required to cause loss of MMR beating was greatly increased when incubations were carried out in serum-free medium. It is suggested that the presence of benzylamine oxidase in serum contributes to the toxicity of allylamine to MMR, by metabolizing allylamine to acrolein. This proposal is supported by the finding that the related amines n-methylallylamine and diallylamine had no effect on spontaneous beating, which may be because they are very poor substrates for benzylamine oxidase. The data presented demonstrate that allylamine is highly toxic to MMR, and we believe that further investigations into the role of serum in allylamine toxicity would improve our understanding of the biochemical basis of its action.
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Adverse photoirritant or photoallergic reactions may be caused by the interaction of certain chemicals with light. The behaviour of 20 chemicals (of which 12 were known UV absorbers and photoirritants, four were known UV absorbers and non-photoirritants and four were known non-UV-absorbers and non-photoirritants) have been screened in an in vitro photoirritation assay using cultured cells [3T3 neutral red uptake (NRU) assay]. Treated cells were exposed to UVA (5 J/cm(2) in 50 min) and visible light in Earle's balanced salt solution and compared with dark controls. The 3T3 NRU assay has shown some promising results for screening photoirritants; however, it is likely that a battery of in vitro assays will be required.
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The isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay were evaluated for their ability to predict the eye irritation potential of a range of hair shampoo formulations, some containing a novel non-surfactant ingredient known to be an ocular irritant. The additional endpoints of corneal swelling and histological examination were incorporated into the standard BCOP protocol. Historic Draize data were available for several of the formulations and served as a reference. The standard BCOP assay (without histology) failed to distinguish between shampoos of low and high irritant potential, when exposure times of 10 and 60 min were employed (for undiluted and 10% dilution of the shampoos, respectively) and the in vitro score classified the majority of formulations as mild. The incorporation of the histological endpoint to the BCOP protocol allowed discrimination between formulations of differing irritancy, and should be included to augment the standard BCOP protocol. Corneal swelling values did not, however, correlate with the irritant potential of the shampoos tested. The IRE which includes the endpoints of corneal swelling and histopathological scoring produced classifications of irritancy that were fairly consistent with in vivo data and distinguished between the high and low irritant potential shampoos.
Assuntos
Alternativas aos Testes com Animais/métodos , Córnea/efeitos dos fármacos , Opacidade da Córnea/induzido quimicamente , Olho/efeitos dos fármacos , Preparações para Cabelo/toxicidade , Irritantes/toxicidade , Testes de Toxicidade/métodos , Animais , Bovinos , Córnea/patologia , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Técnicas In Vitro , Permeabilidade/efeitos dos fármacos , Valor Preditivo dos Testes , Coelhos , Fatores de TempoRESUMO
Determining the validity of alternative methods as replacements for the rabbit eye irritation tests is a goal of the scientific, regulatory and political communities and is being evaluated in several studies. The results from the Draize rabbit eye test are used as a standard against which to compare the performance of the in vitro methods. However, the quantitative performance of the modern Draize eye test is unknown. This paper is a review of the findings of a previous study to estimate the historical variability and a comparison with more contemporary data in order to estimate the performance of the modern methods. The question of whether it is practical to obtain an accurate description of in vivo variability of the modern Draize test is considered by calculating the size of the interlaboratory study that would be required to determine whether variability had changed since 1971. The impact that in vivo variability has on the validation of alternative methods is then discussed. The authors conclude that validation studies have a greater chance of success if the alternative methods are soundly based on mechanisms of toxicity operating in vivo, the Draize data are well defined with regard to their variability, the goals of the study are realistic and the customers of the study are in broad agreement with the study design and the in vivo data used as the reference test set.
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The use of MCF-7 cell proliferation assays has been proposed as one of a number of available in vitro assays to determine the oestrogenic potential of chemicals. However, there is concern that differences between strains of MCF-7 cells and protocols used may lead to significant interlaboratory variability, particularly in their response to the natural oestrogen 17beta-oestradiol, and inability to detect weak oestrogens (Villalobos et al., 1995). This investigation adds to the debate by demonstrating that manipulation of assay conditions may modulate the proliferative response of a particular MCF-7 cell strain to 17beta-oestradiol. MCF-7 cells were put in 12-well plates, then exposed for 6 days to 17)3-oestradiol in EMEM without phenol red using serum stripped of endogenous oestrogens (EMEM/H-). A maximum proliferation of 200-250% control (assessed by fluorescein diacetate uptake) was observed, which is lower than some reported values (up to eightfold increase) and would probably reduce the sensitivity of detection of a weak oestrogen. Preincubation of cells in EMEM/H- for 48 hr, prior to the addition of 17beta-estradiol, resulted in an increased maximum proliferation of 400% control or more. Differences were also seen in the shape of the time course of response to 10(-9)m 17beta-oestradiol; preincubated cells exhibited exponential growth after 6 days whereas those exposed directly after plating plateaued after 4 days. The increased response to 17beta-oestradiol may be due to oestrogen receptor induction following transfer of the cells to oestrogen-free medium. This increase in responsiveness of the cells may increase the sensitivity of this strain of MCF-7 cells for the detection of oestrogenic chemicals.
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The cardiotoxicity of allylamine (AA) is considered to be mediated by metabolism to the highly reactive aldehyde, acrolein (ACR). The toxicity of AA to myocardial myocyte reaggregate cultures (MMR) was assessed by measuring percentage beating as a marker of functional viability. The studies demonstrated that AA toxicity could be prevented by inhibitors of benzylamine oxidase (BZO) but not by inhibitors of monoamine oxidase A or B. Addition of exogenous BZO markedly potentiated the toxicity, an effect that could be blocked by semicarbazide, an inhibitor of BZO. The present studies support the view that AA is metabolized by BZO to the proximate toxicant, ACR. In serum-free cultures, high concentrations (0.5-2.5 mm) of AA were required to cause any loss of viability, and as the concentration of serum in the medium was increased so the loss of viability was induced by AA; in MMR maintained in 50% foetal calf serum, all viability was lost after 3 hr of exposure to 100 mum-AA. Mercaptoethanesulphonate (MESNA), a scavenger of reactive species that is known not to penetrate myocytes, prevented the toxicity both of 100 mum-AA and 100 mum-ACR to MMR in serum-supplemented medium. In contrast, when MMR in serum-free medium were exposed to high concentrations of AA, MESNA had no moderating effect. These findings suggest that AA undergoes extracellular metabolism to ACR in serum-supplemented medium because of the presence of BZO in serum. It is clear that extracellular metabolism is of great importance in the pathogenesis of AA-induced toxicity to MMR in serum-supplemented medium. The toxicity of AA was also prevented by the iron chelator desferrioxamine (DF) at a concentration shown not to inhibit significantly the activity of BZO. ACR toxicity too, was inhibited by DF. This suggests a role for free radicals in the toxicity of AA to MMR, as DF chelates iron, thus preventing the catalysis of free radical reactions (Halliwell and Gutteridge, 1986). Addition of alpha-tocopherol succinate, an inhibitor or lipid peroxidation, to the cultures also reduced the toxicity of AA, which provides some evidence of the role of lipid peroxidation in the mechanism of AA toxicity to MMR. That process too, commonly involves free radical mechanisms. The results are discussed with reference to the action of AA in vivo, in order to consider whether extracellular metabolism might be of importance in the mechanism of toxicity to the whole animal.
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Iodide uptake (IU) by thyrocytes from the plasma against chemical and electrical gradients is by a specific iodide transporter or 'pump'. Perchlorate (ClO(4)(-)) and other univalent, symmetrical anions are competitive inhibitors of iodide uptake (IU), and apparent K(i) for individual anions can be correlated with ion size. This study uses cultured thyrocytes and a broad range of anion size, in particular a series of spherical hexafluoride ions, in order to understand more about the parameters governing the activity of competitive inhibitors of IU. (125)I uptake and organification by cultured porcine thyrocytes was combined with biochemical enzyme inhibition studies on thyroid peroxidase in order to identify specific effects on IU. Known inhibitors were used in a validation phase and demonstrated that a combination of the in vitro thyrocyte (125)I assay and thyroid peroxidase inhibition assay could be used to identify selective inhibitors that can be difficult to identify using thyrocytes alone. Anions of less than, or similar, volume to I(-) (35.0 A(3)) were weak inhibitors with potency increasing proportional to ion size up to an apparent maximum for AsF(6)(-) (94.45 A(3)); this correlation was strong (r = 0.96). PF(6)(-), AsF(6)(-) and SbF(6)(-) were identified as novel inhibitors of IU, showing that the size range of anions active in IU inhibition is greater than that previously identified. The biological significance in vivo of the inhibitory action of the hexafluoride ions is not known. Their potency in this study suggests that these anions may have the potential to affect thyroid function in vivo if they were available systemically.
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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 mum) 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 mum. 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.
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Photobinding of drugs to biomolecules constitutes an important early event in the onset of photoallergy. In the present work, UV irradiation of human serum albumin in the presence of either suprofen (SUP) or its major photoproduct, decarboxylated suprofen (DSUP), has been studied as a model system for drug-photosensitised protein binding. Both dark binding and binding in the presence of light were investigated since this will affect the mode, site and mechanism of drug interaction with the protein. In order to determine the binding features of SUP to albumin, competitive binding experiments were carried out using fluorescent probes specific for site I and II. Suprofen was found to selectively dark bind to site II on HSA. Photobinding of DSUP to HSA was more efficient than SUP. Parallel to this, the intrinsic tryptophan fluorescence of HSA decreased when the protein was previously irradiated in the presence of the photoactive compounds, again being DSUP more efficient compared with SUP. As fluorescence quenching involves electron transfer from the excited Trp to the ground state DSUP, it follows that the photoactive compound binding to HSA must be on (or in close proximity to) site I Trp(214) residue. It appears that photobinding of SUP is largely preceded by its photodecomposition to DSUP which, in turn, associates and photobinds to HSA.
Assuntos
Dermatite Fotoalérgica/metabolismo , Albumina Sérica/metabolismo , Suprofeno/metabolismo , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/efeitos da radiação , Descarboxilação , Fluorescência , Humanos , Técnicas In Vitro , Albumina Sérica/efeitos da radiação , Suprofeno/farmacologia , Triptofano/metabolismo , Raios UltravioletaRESUMO
Thirty-three test substances comprising household and personal products and a pure surfactant were used in an independent evaluation of the ability of five selected in vitro assays to predict eye irritation potential. The data were compared with historical archived data from rabbit eye irritation tests conducted on the same subsamples. The data were assessed in three ways. The linear correlation for all 33 test substances with in vivo data was poor for each assay. However, the correlation improved greatly for some assays when a group of similar test substances was considered. Analysis of the data by Cooper's criteria suggested that fluorescein diacetate uptake by Chinese hamster V79 cells (V79) and the Microtox test kit were the best of the assays evaluated in discriminating correctly between the irritants and non-irritants that were identified by rabbit eye irritation testing. Some of the data were selected for further analysis because it was possible to predict differences, based on experience learned from animal data on similar substances, between the test substances without reference to experimental data. Neat or more concentrated test substances were predicted to be more irritant than dilutions and an experimental laundry powder formulation containing 10% sodium metasilicate (MTS) was predicted to be more irritant than two analogous non-MTS containing formulations. The experimental data were then compared with the prediction. The in vivo rabbit eye irritation tests and Microtox distinguished correctly between all the test substances in this analysis. Eytex was unable to distinguish correctly between any of the test substances in this analysis. The other assays were able to distinguish between some of the test substances. No single assay performed well in every type of analysis and it is concluded that a battery of assays is required to obtain reliable predictive data. Overall, Microtox, V79 fluorescein diacetate accumulation and 3T3 neutral red uptake were the most promising assays. Further evaluation is necessary and extreme caution should be used in comparing in vitro results with rabbit eye irritation data to make definitive conclusions.
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Chemicals may interact with the oestrogen receptor (ER) in mammalian cells and thereby exhibit oestrogenic activity which may result in effects on development and reproduction in both wildlife and humans. It has been suggested that in vitro assays provide the best way forward for investigating the oestrogenic potential of large numbers of chemicals. We compared the effects of a series of chemicals in an MCF-7 cell proliferation assay with results from similar assays reported in the literature in order to investigate the practical use of such assays. Alkylphenols and benzylbutylphthalate were found to cause proliferation of MCF-7 cells, which could be antagonized by tamoxifen. However, all the substances tested, including ethanol, progesterone and epidermal growth factor, were found to have some effect on cell proliferation, suggesting that cell proliferation may result from mechanisms other than via the ER. This is in contrast to some reports that MCF-7 cells only respond to oestrogens and highlights MCF-7 cell strain differences. Such differences and other reports in the literature of the effects of growth modulators on MCF-7 cells indicate that interpretation of results from MCF-7 cell proliferation assays needs to be considered carefully. Comparison between the results of in vitro assays and appropriate in vivo assays is also required in order to evaluate the significance of in vitro oestrogenicity assay results and an understanding of the mechanisms responsible for the biological effects is necessary.
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Where substances are intended for use in personal products applied to the skin an assessment of potential phototoxic hazard is required. This report describes a tiered testing strategy involving in vitro assays used for the phototoxic hazard assessment of a personal product ingredient (Ingredient X). The initial assay was measurement of a UV/visible absorption spectrum to identify absorption at relevant wavelengths. This was followed by in vitro assays for phototoxicity (3T3 cell neutral red uptake phototoxicity test) and photoallergy (photobinding to human serum albumin). These in vitro screens gave equivocal results for Ingredient X which appeared to suggest a weak phototoxic reaction. To further evaluate the phototoxic hazard of Ingredient X to human skin, a phototoxicity assay using a 3-D human skin model was conducted. Ingredient X did not cause phototoxicity in this assay. Overall conclusions from these studies were that although Ingredient X showed slight intrinsic potential for photoactivation, it was unlikely to present a hazard to human skin. This report illustrates the value in a step-wise strategy of the use of human skin models to help interpret the results of other in vitro phototoxicity assays.
Assuntos
Cosméticos/toxicidade , Dermatite Fototóxica/patologia , Testes de Toxicidade , Células 3T3 , Animais , Química Farmacêutica , Humanos , Camundongos , Modelos Biológicos , Transtornos de Fotossensibilidade/induzido quimicamente , Transtornos de Fotossensibilidade/patologia , Extratos Vegetais/toxicidade , Testes Cutâneos , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.
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The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.
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Human breast milk is rich in 2-palmitoyl 1,3 unsaturated triacyglycerols and during the neonatal period, when milk is the sole source of nutrients, their role could be particularly important. Betapol is a novel triacylglycerol mix resembling human breast milk in its high palmitic acid content and positional distribution. The total fat absorption from Betapol has been shown to be higher than fat from conventional infant milk formulas and closer to human breast milk in infants. However, the relative fate of purified palmitic acid esterified to glycerol in the 1-, 3- and 2-positions in neonatal and young animals has not previously been established. Therefore, the fate of orally administered 1-[1-14C]palmitoyl, 2,3 dioleoyl glycerol ([14C]POO) and 1,3 dioleoyl,2-[1-14C]palmitoyl glycerol (O[14C]PO) was investigated in suckling and weanling rats using liquid scintillation counting of tissues and expired air and whole-body autoradiography. The results obtained indicate that orally administered [14C]POO and O[14C]PO are extensively absorbed from the gut, probably either as palmitic acid or as a palmitoyl glyceride in both suckling and weanling rats. Radioactivity initially concentrated in brown fat with apparent migration to the white fat of weanling rats by 96 h. Levels of 14C were low in blood, brain and other tissues. Excretion of 14C was mainly by expiration of CO(2) (approximately 72% in 96 h), indicating beta-oxidation as a major route of metabolism. Urine and faeces accounted for only approximately 6% of the excreted radioactivity. The design and size of the experiment did not allow tests of statistical significance between the absorption and excretion of OPO and POO to be conducted. However, the absorption, distribution, beta-oxidation and excretion appeared to be similar.
Assuntos
Triglicerídeos/farmacocinética , Animais , Animais Lactentes , Autorradiografia , Testes Respiratórios , Dióxido de Carbono/análise , Radioisótopos de Carbono , Feminino , Masculino , Ratos , Ratos Wistar , Distribuição Tecidual , Triglicerídeos/síntese química , DesmameRESUMO
The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein. Therefore, decision trees for evaluation of the risk have been developed, the latest being proposed by WHO/FAO early in 2001. Proteins developed using modern biotechnology and derived from fish are being considered for use in food and other applications, and since allergy to fish is well established, a potential risk from such proteins to susceptible human beings exists. The overall aim of the study was to investigate the potential allergenicity of an Ice Structuring Protein (ISP) originating from an arctic fish (the ocean pout, Macrozoarces americanus) using the newly developed decision tree proposed by FAO/WHO. The methods used were those proposed by FAO/WHO including amino acid sequence analysis for sequence similarity to known allergens, methods for assessing degradability under standardised conditions, assays for detection of specific IgE against the protein (Maxisorb RAST) and histamine release from human basophils. In the present paper we describe the serum screening phase of the study and discuss the overall application of the decision tree to the assessment of the potential allergenicity of ISP Type III. In an accompanying paper [Food Chem. Toxicol. 40 (2002) 965], we detail the specific methodology used for the sequence analysis and assessment of resistance to pepsin-catalysed proteolysis of this protein. The ISP showed no sequence similarity to known allergens nor was it stable to proteolytic degradation using standardised methods. Using sera from 20 patients with a well-documented clinical history of fish allergy, positive in skin prick tests to ocean pout, eel pout and eel were used, positive IgE-binding in vitro to extracts of the same fish was confirmed. The sera also elicited histamine release in vitro in the presence of the same extracts. The ISP was negative in all cases in the same experiments. Using the proposed decision tree, we demonstrated the safety of the ISP to patients already sensitised to fish, as well as to individuals potentially susceptible to producing IgE responses to proteins. Furthermore, the practicability of the new decision tree was confirmed.
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Alérgenos/efeitos adversos , Proteínas Anticongelantes Tipo III/efeitos adversos , Árvores de Decisões , Hipersensibilidade Alimentar/prevenção & controle , Imunoglobulina E/imunologia , Adolescente , Adulto , Alérgenos/química , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes Tipo III/química , Criança , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Histamina/metabolismo , Humanos , Hidrólise , Pepsina A/metabolismo , Perciformes , Teste de Radioalergoadsorção , Medição de Risco , Segurança , Homologia de Sequência , Testes Cutâneos , Nações Unidas , Organização Mundial da SaúdeRESUMO
The recently published WHO/FAO guidelines on the assessment of allergenicity of novel food proteins provide a strategy with which to approach the determination of the potential of novel proteins in foods to be allergens. Key to this strategy are the assessment of sequence similarity to known allergens and the assessment of the resistance to pepsin hydrolysis. Ice structuring proteins (also commonly referred to as anti-freeze or thermal hysteresis proteins) are a group of naturally occurring proteins that bind to ice and structure ice crystal formation. The amino acid sequence of the ice structuring protein (ISP) type III HPLC 12 (ISP type III) was compared in silico with the sequences of known allergens. Secondly, the resistance to pepsin hydrolysis of ISP type III and its glycoconjugates (produced in recombinant baker's yeast) was assessed. The results indicate that ISP type III has no sequence similarity with known allergenic proteins. Both ISP type III and ISP type III glycoconjugates contained within the fermentation product were hydrolysed readily by pepsin (50% loss in <10 min at pH 1.5) to give peptide fragments that were too small to be allergenic or to trigger cross-linking to IgE. In an accompanying study, we demonstrated that IgE from fish-allergic individuals did not bind ISP Type III. Therefore, in accordance with the WHO/FAO strategy, the assessment of ISP type III and ISP type III glycoconjugates by sequence analysis together with lack of resistance to pepsin hydrolysis and the absence of IgE binding supports the conclusion that both are unlikely to present a potential sensitisation hazard.
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Alérgenos/efeitos adversos , Pepsina A/metabolismo , Alérgenos/química , Alérgenos/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Hidrólise , Espectrometria de Massas/métodosRESUMO
The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.
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Alternativas aos Testes com Animais , Cosméticos/toxicidade , Preparações para Cabelo/toxicidade , Sabões/toxicidade , Tensoativos/toxicidade , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Estudos de Avaliação como Assunto , Olho/efeitos dos fármacos , Feminino , Hemólise , Humanos , Masculino , Valor Preditivo dos Testes , Coelhos , Distribuição Aleatória , Análise de Regressão , Reprodutibilidade dos Testes , Pele/citologia , Pele/efeitos dos fármacosRESUMO
This report summarises the discussions of a workshop sponsored by the European Cosmetics, Toiletries and Perfumery Association (COLIPA). The workshop discussed the state-of-the-art of eye irritancy testing, and made recommendations as to the best ways in which to validate alternatives to the Draize eye irritation test. The importance of understanding the mechanisms of eye irritation, particularly when attempting to improve in vitro prediction of in vivo eye irritancy, was also emphasised.
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The use of testing strategies which incorporate a range of alternative methods and which use animals only as a last resort is widely considered to provide a reliable way of predicting chemical toxicity while minimising animal testing. The widespread concern over the severity of the Draize rabbit test for assessing skin irritation and corrosion led to the proposal of a stepwise testing strategy at an OECD workshop in January 1996. Subsequently, the proposed testing strategy was adopted, with minor modifications, by the OECD Advisory Group on Harmonization of Classification and Labelling. This article reports an evaluation of the proposed OECD testing strategy as it relates to the classification of skin corrosives. By using a set of 60 chemicals, an assessment was made of the effect of applying three steps in the strategy, taken both individually and in sequence. The results indicate that chemicals can be classified as corrosive (C) or non-corrosive (NC) with sufficient reliability by the sequential application of three alternative methods, i.e., structure-activity relationships (where available), pH measurements, and a single in vitro method (either the rat skin transcutaneous electrical resistance (TER) assay or the EPISKIN™ assay). It is concluded that the proposed OECD strategy for skin corrosion can be simplified without compromising its predictivity. For example, it does not appear necessary to measure acid/alkali reserve (buffering capacity) in addition to pH for the classification of pure chemicals.