Practical application of non-whole animal alternatives: summary of IRAG workshop on eye irritation testing. Interagency Regulatory Alternatives Group.

In November 1993, the Interagency Regulatory Alternatives Group (IRAG) sponsored a workshop to examine the current scientific status of alternatives to the Draize eye irritation test by assessing the current practical application of methods used to predict in vivo eye irritation. Laboratories from around the world were invited to submit detailed in vitro and in vivo data in parallel according to a specific set of guidelines in a consistent format. In vitro scores were compared with individual tissue scores. Over 60 data sets from 41 laboratories were received for 29 different test methods. Methods were grouped into five categories: organotypic models, chorioallantoic membrane-based assays, cell function-based assays, cytotoxicity assays and other systems. Data submissions and correlation analyses have been used to demonstrate the application of guidelines in method evaluations. Findings are summarized and future directions are indicated. A significant outcome of the workshop was the co-operation demonstrated among representatives of industry, academia and government in sharing test data on more than 2000 chemicals, products and product formulations for evaluation by their peers. Information obtained from this workshop will add to the weight of scientific evidence and scientific consensus about in vitro test methods and will establish credibility for regulatory acceptance of non-whole animal alternatives for ocular irritation.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.

A primary culture system of adult rat heart cells for the study of toxicologic agents.

Tricyclic antidepressants (TCAs) are currently used in the treatment of mental depression and nocturnal enuresis. Clinically, these drugs are useful; however, cardiotoxicity can occur even with therapeutic dosages. For example, TCAs are known to alter myocardial function, induce arrhythmias, and produce heart block in individuals with a normal cardiovascular history. The present study was undertaken to establish a culture system of spontaneously contracting adult primary myocardial cells for toxicologic testing and to examine their contractility, morphology, and lactate dehydrogenase release (LDH) after treatment with one of the most cardiotoxic TCAs, amitriptyline. Primary myocardial cell cultures were obtained from approximately 60- to 90-day-old Sprague-Dawley rats. After the cells had been grown in culture for 11 days, they were treated with amitriptyline (1 x 10(-3), 1 x 10(-4), and 1 x 10(-5) M) for 2 to 24 h. The highest concentration of amitriptyline (1 x 10(-3) M) completely destroyed the cardiac muscle cells. In addition to moderate and severe vacuole, granule, and pseudopodia formation, all contractile activity was inhibited as early as 2 h after exposure to the intermediate concentration of 1 x 10(-4) M amitriptyline. Significant LDH release did not occur until 8 h after treatment with this intermediate concentration. Even though there was no significant LDH release at all 3 time points tested, there was a 50% decrease in beating activity (154 +/- 9 to 77 +/- 5 beats/min) and initiation of vacuole formation by 2 h with the lowest concentration of amitriptyline (1 x 10(-5) M). This study presents a new apparatus for the isolation of adult cardiac myocytes for the establishment of primary cell cultures for toxicologic testing. Furthermore, these data demonstrate that amitriptyline induces a concentration- and time-dependent cardiotoxic profile in a model of spontaneously contracting adult cardiac muscle cells in culture.

Effects of maternal calorie-restricted diet on development of the foetal heart, as evaluated in primary cultures of rat myocardial cells.

Very-low-calorie diets have been implicated in causing ventricular arrhythmias and sudden cardiac death. Furthermore, studies indicate that maternal carbohydrate-restricted diets consumed during pregnancy and lactation reduce foetal growth, parturition and postnatal survival of rat pups. In this study, Sprague-Dawley rats were maintained on a semi-purified full-calorie or 50% carbohydrate-calorie-restricted diet throughout pregnancy. The function and integrity of myocardial cell cultures obtained from 3-5-day-old offspring from both groups of dams were evaluated after a drug-induced toxic challenge. After the myocytes had been in culture for 4 days, they were exposed to various concentrations of amitriptyline (1 x 10(-3) to 1 x 10(-5) M). Morphology, beating activity, lactate dehydrogenase release, glucose utilization, beta-adrenergic receptor [125I]iodopindolol binding, and cellular adenosine triphosphate content were evaluated for 24 hr after drug exposure. There were no significant differences in morphology, beating activity or glucose utilization between the full-calorie and calorie-restricted groups. When compared with the full-calorie group, lactate dehydrogenase release from the calorie-restricted group was significantly lower at 8 hr for the untreated controls and those cells exposed to 1 x 10(-4) and 1 x 10(-5) M-amitriptyline. Adenosine triphosphate levels were lower in untreated controls from the calorie-restricted group when compared with the full-calorie group at 4 hr. Within the calorie-restricted group, those cultures exposed to 1 x 10(-4) M-amitriptyline had significantly depressed adenosine triphosphate levels after 8 hr of drug treatment when compared with their respective untreated controls. Finally, the calorie-restricted group had significantly increased binding affinities of beta-receptors. Thus, maternal consumption of calorie-restricted diets during pregnancy may affect the myocardial functional capacity and integrity of the offspring.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures.

A protocol based primarily on current laboratory practices in the performance of the unscheduled DNA synthesis (UDS) assay with primary rat hepatocyte cultures has been developed. These guidelines were developed using tabulated responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with the UDS test. This report identifies those modifications to previously described methodologies which are used on a regular basis and also serves to clarify confusing or inconsistent practices. Although this protocol pertains specifically to the use of primary rat hepatocyte cultures, it can be modified to incorporate other types of cells in which certain aspects remain the same.

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations.

Primary myocardial cell cultures and freshly isolated cardiac cells in suspension represent two isolated, whole cell models for investigating cellular transsarcolemmal 45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3- to 5-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol [( 125I]IPIN). The suspensions had a significantly lower Bmax (42 +/- 6 fmol/mg protein) than the membranes and cultures (77 +/- 8 and 95 +/- 10 fmol/mg protein, respectively). The KD of the cultures (218 +/- 2.0 pM) was significantly higher than that for the suspensions (107 +/- 1.3 pM) and membranes (93 +/- 1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0 X 10(-7) M isoproterenol resulted in a significant increase in 45Ca++ exchange as early as 15 s. In contrast, 45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated 45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.

Evaluation of drug and chemical toxicity with cell culture systems.

Approaches to the evaluation of drug and other chemical toxicity with mammalian cell culture systems are designed to enhance the predictability of animal models. Identification of toxic agents by in vitro screening tests and studies of mechanisms through which chemicals induce critical lesions at the cellular and subcellular levels help to make those predictions sooner and perhaps single out those target sites and chemicals of most concern.

Evaluation of purified 4-deoxynivalenol (vomitoxin) for unscheduled DNA synthesis in the primary rat hepatocyte-DNA repair assay.

Primary cultures of rat hepatocytes were used to determine unscheduled DNA synthesis (UDS) and cytotoxicity of purified 4-deoxynivalenol (vomitoxin), a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium. Nontoxic and toxic doses of deoxynivalenol, 0.1 to 1000 micrograms/ml, did not significantly increase UDS as measured by net grains per nucleus, net grains per nuclear area or percentage of cells incorporating greater than or equal to 5, 6, 10 or 20 grains per nucleus. Evidence of cytotoxicity, manifested as a reduction in cell number in autoradiographs, pyknotic nuclei or vacuolated cytoplasm, was observed in hepatocytes treated with deoxynivalenol concentrations of 5 micrograms/ml and above. These findings suggest that the cellular toxicity of deoxynivalenol may not be mediated by a DNA-damaging event in cultured hepatocytes. An increased percentage of large-sized nuclei was also found to be associated with toxic doses of deoxynivalenol as well as 2-acetylaminofluorene used as the positive control.

Studies on the action of nystatin on cultured rat myocardial cells and cell membranes, isolated rat hearts, and intact rats.

The action of nystatin, a polyene antibiotic, was studied in rat myocardial cells, isolated rat hearts, and intact rats. Myocardial cells responded to 10 and 25 micrograms nystatin/ml with arrhythmias that could be minimized by elevated concentrations of K+ and Mg2+ or reversed by washing the cells. Similarly, the isolated heart responded to 100 micrograms nystatin/ml with arrhythmias that could be tempered by addition of elevated concentrations of K+ and Mg2+. The i.v. injection of the drug caused heart failure in intact animals at the 4-mg/kg dose level. At the subcellular level, nystatin made the myocardial cell membranes more rigid, as measured by electron spin resonance spectrometry. These findings indicate a parallel between physiocochemical changes caused by nystatin in the myocardial cell membrane and the biological changes caused by this drug in myocardial cells, isolated heart, and heart of the intact animal.

Screening of fresh water fish extracts for enzyme-inducing substances by an aryl hydrocarbon hydroxylase induction bioassay technique.

A sensitive biological test to detect the presence of certain contaminants, such as highly toxic halogenated dioxins, dibenzofurans, and biphenyls in foods, was applied to extracts of fresh water fish that had been prepared by a food extraction-cleanup procedure developed by the Food and Drug Administration for pesticides and industrial chemicals. Aryl hydrocarbon hydroxylase (AHH) activity in a rat hepatoma cell line was used as the biological detection system for residues that induce enzyme activity. The induction of AHH activity by the extracts was compared with a standard AHH-induction curve for the most active compound known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and results were computed as TCDD equivalents. Several dilutions of fish extracts were used to produce AHH-induction curves from which an optimal dose-response range was determined and used to estimate TCDD equivalents. Cleaned-up extracts of fish obtained from different water bodies in the United States were examined for AHH activity. The samples which had low levels of polyhalogenated contaminants produced low biological activity, while a higher activity was obtained from fish that contained higher levels of polyhalogenated contaminants. The results suggest that the fish extracts can be screened for AHH inducers before chemical analysis.

Influence of Mycoplasma arginini infection on the induction of aryl hydrocarbon hydroxylase by TCDD in rat hepatoma cell cultures.

Mycoplasma arginini was eliminated from a rat hepatoma cell line (H-4-II-E) by plating at low cell density and treatment with chlortetracycline (250 micrograms/ml), kanamycin (250 micrograms/ml), tylosin (100 micrograms/ml), 3% M. arginini antiserum and 5% fresh guinea-pig serum. The induction of AHH activity in the cell culture was measured in response to increasing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ED50 values (estimated doses that produce 50% maximum enzyme induction) were calculated to be 0.256, 0.452 and 0.344 pmol TCDD/plate for original, mycoplasma-free and reinfected cells, respectively. Although the absence of M. arginini in the rat hepatoma cell line makes the cells slightly less responsive to AHH induction by TCDD, this decrease does not detract from the use of the method to screen food extracts and environmental samples for the presence of certain toxic planar organic compounds.

Induction of aryl hydrocarbon hydroxylase activity in cell cultures by aroclors, residues from yusho oil samples, and polychlorinated biphenyl residues from fish samples.

Four Aroclor reference materials, cleaned-up extracts of 2 yusho rice oil samples, and cleaned-up extracts of 3 fish samples containing polychlorinated biphenyl (PCB) residues were tested for their ability to induce aryl hydrocarbon hydroxylase (AHH) activity in a rat hepatoma cell line. Before the AHH bioassay, the samples were fractionated by a Florisil column chromatographic method. All samples contained about 1000 micrograms PCBs before Florisil column chromatography. The first Florisil eluate contains about 95% of the PCBs in a typical Aroclor, and the second contains the more polar or adsorbent PCB congeners. In this study, the first eluate for all samples produced no quantifiable AHH activity. The second Florisil eluates of both Aroclors 1242 and 1248 induced AHH activity, whereas these eluates of both Aroclors 1254 and 1260 did not. This difference may be due to the presence in Aroclors 1242 and 1248 of 3,3',4,4'-tetrachlorobiphenyl, which has not been detected in Aroclors 1254 and 1260. The second Florisil eluates of the fish samples induced somewhat less AHH activity than did Aroclor 1242 or 1248. The second Florisil eluates of the PCB residues from yusho rice oil samples induced significantly greater AHH activity than these eluates of either Aroclor 1242 or 1248, perhaps because yusho rice oil contains a greater amount of polychlorinated dibenzofurans than PCB commercial mixtures on a PCB equivalent basis.

Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic compounds.

Induction of aryl hydrocarbon hydroxylase (AHH) activity in rat hepatoma cell line serves as a simple and rapid method to detect minute (pg) amounts of certain classes of compounds, e.g., dibenzo-p-dioxins, dibenzofurans, and biphenyls. This method may provide a quick screen for such substances in extracts from foods prior to chemical identification. AHH activity is measured by conversion of benzo[a]pyrene (BP) to 3-hydroxy BP in homogenized cell extracts from control and treated cultures and is reported as pmol product formed/mg protein/min. Substances screened by this method include polyhalogenated analogs of dibenzo-p-dioxin (24 compounds), dibenzofuran (11 compounds), biphenyl (7 compounds), and extracts from several food sources. Response of the most reactive compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was used to prepare a standard curve, and the AHH activity induced by mole doses of test substance is reported as an ED50 response (the estimated dose needed to produce 50% maximum enzyme induction). The AHH activity induced by food extracts is equated to the standard curve and reported as TCDD equivalents. A potent ED50 response in cell culture appears to correlate well with known toxic responses in other mammalian and avian systems for certain test substances. This correlation suggests that the cell culture enzyme induction method is a useful model for screening food extracts that are suspected to be contaminated with polychlorinated planar substances. A collaborative study would demonstrate the reproducibility of the method.