A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III.

A prevalidation study sponsored by the European Centre for the Validation of Alternative Methods (ECVAM) on in vitro tests for acute skin irritation is aimed at identifying non-animal tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union and OECD. This paper reports on Phase III for one of the methods, the skin integrity function test (SIFT), assessing the protocol performance of the SIFT, in terms of reproducibility and predictive ability, in three laboratories. The barrier function properties of excised mouse skin were determined using a set of 20 coded chemicals (10 I, 10 NI), using the endpoints of trans-epidermal water loss (TEWL) and electrical resistance (ER). The basis of the SIFT prediction model is if the ratios of the pre- and post-application values for either TEWL or ER are greater than five-fold, then the test chemical is deemed irritant (I). If the ratio of both parameters is less than five-fold then the chemical is deemed non-irritant (NI). Analysis of variance (ANOVA) indicated that the intra-lab reproducibility was acceptable but that the inter-lab reproducibility was not. Overall, the SIFT test under-predicted the irritancy of the test chemicals chosen for Phase III with an overall accuracy of only 55%. The sensitivity value (ability to correctly predict I) was only 30%. The specificity (ability to predict NI) of the test was better at 80%. A retrospective examination of the SIFT results was undertaken using Student's t-test and a significance level of P<0.05 to predict an irritant based on changes in the TEWL ratio values. This improved the predictivity of the SIFT test, giving a specificity of 60%, a sensitivity of 80% and an overall accuracy of 70%. Appropriate modifications to the prediction model have now been made and the SIFT will be re-examined in a new validation exercise to investigate the potential of this non-animal method to predict acute skin irritation potential.

Use of suppression-PCR subtractive hybridisation to identify genes that demonstrate altered expression in male rat and guinea pig livers following exposure to Wy-14,643, a peroxisome proliferator and non-genotoxic hepatocarcinogen.

Understanding the genetic profile of a cell at all stages of normal and carcinogenic development should provide an essential aid to developing new strategies for the prevention, early detection, diagnosis and treatment of cancers. We have attempted to identify some of the genes that may be involved in peroxisome-proliferator (PP)-induced non-genotoxic hepatocarcinogenesis using suppression PCR subtractive hybridisation (SSH). Wistar rats (male) were chosen as a representative susceptible species and Duncan-Hartley guinea pigs (male) as a resistant species to the hepatocarcinogenic effects of the PP, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643). In each case, groups of four test animals were administered a single dose of Wy-14,643 (250 mg/kg per day in corn oil) by gastric intubation for 3 consecutive days. The control animals received corn oil only. On the fourth day the animals were killed and liver mRNA extracted. SSH was carried out using mRNA extracted from the rat and guinea pig livers, and used to isolate genes that were up and downregulated following Wy-14,643 treatment. These genes included some predictable (and hence positive control) species such as CYP4A1 and CYP2C11 (upregulated and downregulated in rat liver, respectively). Several genes that may be implicated in hepatocarcinogenesis have also been identified, as have some unidentified species. This work thus provides a starting point for developing a molecular profile of the early effects of a non-genotoxic carcinogen in sensitive and resistant species that could ultimately lead to a short-term assay for this type of toxicity.

Differential gene expression in drug metabolism and toxicology: practicalities, problems and potential.

1. An important feature of the work of many molecular biologists is identifying which genes are switched on and off in a cell under different environmental conditions or subsequent to xenobiotic challenge. Such information has many uses, including the deciphering of molecular pathways and facilitating the development of new experimental and diagnostic procedures. However, the student of gene hunting should be forgiven for perhaps becoming confused by the mountain of information available as there appears to be almost as many methods of discovering differentially expressed genes as there are research groups using the technique. 2. The aim of this review was to clarify the main methods of differential gene expression analysis and the mechanistic principles underlying them. Also included is a discussion on some of the practical aspects of using this technique. Emphasis is placed on the so-called 'open' systems, which require no prior knowledge of the genes contained within the study model. Whilst these will eventually be replaced by 'closed' systems in the study of human, mouse and other commonly studied laboratory animals, they will remain a powerful tool for those examining less fashionable models. 3. The use of suppression-PCR subtractive hybridization is exemplified in the identification of up- and down-regulated genes in rat liver following exposure to phenobarbital, a well-known inducer of the drug metabolizing enzymes. 4. Differential gene display provides a coherent platform for building libraries and microchip arrays of 'gene fingerprints' characteristic of known enzyme inducers and xenobiotic toxicants, which may be interrogated subsequently for the identification and characterization of xenobiotics of unknown biological properties.

Eye Irritation Testing: The Way Forward. The Report and Recommendations of ECVAM Workshop 34.

This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). The workshop on Eye Irritation Testing: The Way Forward was held in Egham, UK, on 15-17 June 1998, under the chairmanship of Michael Balls (ECVAM, Italy). The workshop had two aims, the first of which was to review some of the previous multi-laboratory validation studies on alternatives to the Draize eye test and assess why many promising alternative methods were not successful in these studies. The second aim was to discuss strategies for making progress toward the short-term reduction, refinement, and eventual replacement, of the Draize test, including: a new approach to the validation of in vitro tests for eye irritancy, based on the use of reference standards, which promises to overcome some of the problems encountered in previous studies; the use of stepwise testing strategies which reduce and refine the use of animals in eye irritation testing; the use of multivariate and other statistical techniques for the further analysis of data generated in previous validation studies; and a programme of research aimed at understanding the underlying mechanisms of eye irritation.

COLIPA validation project on in vitro eye irritation tests for cosmetic ingredients and finished products (phase I): the red blood cell test for the estimation of acute eye irritation potentials. Present status.

The red blood cell test (RBC test) is part of the COLIPA Validation Project on Alternatives to Draize Eye Irritation. It shows good intra- and interlaboratory reproducibility (reliability) and represents one of the promising in vitro alternatives of this project with a good fit to prediction models (relevance) for the assessment of acute ocular irritancy caused by certain classes of chemicals (mainly surfactants) and formulations. Results obtained during the period of test development, prevalidation, and validation are summarized. The method is based on that of Pape et al. (1987), Pape and Hoppe (1990) and Lewis et al. (1993). The protocol has two endpoints: cellular lysis and changes in protein conformation which can be correlated with initial events in tissue injury inducing inflammatory responses as assessed by Draize eye irritation scoring. Both endpoints are detected by spectrophotometric changes in the haemoglobin absorption at 541nm. The protocol also includes a set of prediction models (PM). One PM is designed to predict three classes of irritancy (classification model) based on both endpoints and the three other PMs are designed to predict modified maximum average scores (MMAS) by algorithms based on data from cellular lysis only. These three PMs [with prediction intervals (PIs)] are: (i) for surfactant ingredients, (ii) for surfactant containing finished products, and (iii) for both groups together. The three PMs are based on a common algorithm derived from historic data. It is shown that PMs derived from historic data from several laboratories, by the same procedure, also produce a good fit with the presented data. Therefore, participating laboratories concluded that the protocol as used in this formal validation study can be considered to be validated for the estimation of acute eye irritation potential of surfactant-containing finished products.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team.

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.

An evaluation of the proposed OECD testing strategy for skin corrosion.

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.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

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.

Molecular profiling of non-genotoxic hepatocarcinogenesis using differential display reverse transcription-polymerase chain reaction (ddRT-PCR).

The technique of differential display reverse transcription-polymerase chain reaction (ddRT-PCR) has been used to produce unique profiles of up-regulated and down-regulated gene expression in the liver of male Wistar rats following short term exposure to the non-genotoxic hepatocarcinogens, phenobarbital and WY-14,643. Animals were treated for 3 days, whereupon their livers were extracted and snap frozen. mRNA was prepared from the livers and used for ddRT-PCR. Individual bands from the differential displays were extracted and cloned. False positives were eliminated by dotblot screening and true positives then sequenced and identified.

Feeding studies in rats with mineral hydrocarbon food grade white oils.

This investigation compared the effects of feeding rats diets containing food grade white oil processed by either conventional oleum treatment or the more modern method of catalytic hydrogenation. In two separate experiments, male or female Fischer-344 rats were given free access for 90 days to diets containing 0, 10, 100, 500, 5,000, 10,000, or 20,000 ppm of either oleum-treated white oil (OTWO) or hydrotreated white oil (HTWO). There were no mortalities and no adverse clinical signs associated with feeding either white oil. Treatment-related effects evidenced by hematological, clinical chemical, and pathological changes were generally dose-related and more marked in female than in male rats, and the OTWO caused a greater pathological response than the HTWO. Tissue residues of saturated hydrocarbons were up to 5.2 times higher in female rats than in males. Rats fed 5,000 ppm or more of either white oil showed dose-related alterations in several hematological and clinical chemistry variates associated mainly with hepatic damage or functional alteration. At necropsy, mesenteric lymph nodes were enlarged, and increases in weight of liver, kidney, and spleen were significant. Microscopic changes were characterized by multifocal lipogranulomata in mesenteric lymph node and liver. No changes were observed in rats fed OTWO or HTWO for 90 days at dietary concentrations of 10 or 100 ppm, equivalent to a minimum intake of 0.65 and 6.4 mg/kg/day, respectively. Differences in degree of pathological response associated with each oil may have been due to their differences in specification rather than processing method.

The skin corrosivity test in vitro. Results of an inter-laboratory trial.

The collaborative study reported here was performed to evaluate the reliability of the skin corrosivity test in vitro when performed in independent laboratories. Twenty substances were examined in each of three participating laboratories and the results were compared with existing data from standard assays in vivo. The skin corrosivity test is based on the assumption that corrosive substances destroy the skin's natural outer protective barrier, the stratum corneum. Corrosive action in vitro is measured by a fall in the transcutaneous electrical resistance (TER) below a predetermined threshold. A refined test using a MgSO(4) electrolyte solution for TER measurements has recently been shown to reduce the number of false positive results in the test, while maintaining excellent predictive value for skin corrosive substances. Although in the present study there was some variation between laboratories in terms of the absolute mean TER values obtained, all 6 substances corrosive in vivo were correctly predicted by the three laboratories. The other 14 substances ranged from being non-irritant to severe skin irritants in vivo, but the test was unable to discriminate between these different categories in any of the three laboratories. However, these inter-laboratory comparisons demonstrate that the refined skin corrosivity test is a robust and reliable method in vitro for identifying potential skin corrosive substances.