Alternative Test Systems Research Articles

Alternative testing systems for evaluating noncarcinogenic, hematologic toxicity.

Hematopoietic tissues are the targets of numerous xenobiotics. Clinical hematotoxicity is either a decrease or an increase in peripheral blood cell counts in one or more cell lineages--a cytopenia or a cytosis, respectively--that carries a risk of an adverse clinical event. The purpose of in vitro hematotoxicology is the prediction of these adverse hematologic effects from the effects of the toxicants on human hematopoietic targets under controlled experimental conditions in the laboratory. Building on its important foundations in experimental hematology and the wealth of hematotoxicology data found in experimental oncology, this field of alternative toxicology has developed rapidly during the past decade. Although the colony-forming unit-granulocyte/monocyte neutrophil progenitor is most frequently evaluated, other defined progenitors and stem cells as well as cell types found in the marrow stroma can be evaluated in vitro. End points have been proposed for predicting toxicant exposure levels at the maximum tolerated dose and the no observable adverse effect level for the neutrophil lineage, and several clinical prediction models for neutropenia have developed to the point that they are ready for prospective evaluation and validation in both preclinical species and humans. Known predictive end points are the key to successful comparisons across species or across chemical structures when in vitro dose-response curves are nonparallel. Analytical chemistry support is critical for accurate interpretation of in vitro data and for relating the in vitro pharmacodynamics to the in vivo pharmacokinetics. In contrast to acute neutropenia, anemia and acute thrombocytopenia, as well as adverse effects from chronic toxicant exposure, are much more difficult to predict from in vitro data. Pharmacologic principles critical for clinical predictions from in vitro data very likely will apply to toxicities to other proliferative tissues, such as mucositis.

Neonatal Mouse Assay for Tumorigenicity: Alternative to the Chronic Rodent Bioassay

The chronic rodent bioassay for tumors has been utilized systematically for 25 years to identify chemicals with carcinogenic potential in man. In general, those chemicals exhibiting tumorigenicity at multiple sites in both mice and rats have been regarded as possessing strong carcinogenic potential in humans. In comparison, the value of data collected for those test chemicals exhibiting more sporadic tumorigenicity results (e.g., single species/single sex or dose-independent) has been questioned. As knowledge of the carcinogenic process has increased, several alternative test systems, usually faster and less expensive than the 2-year bioassay, have been suggested for identification of the strongly acting, transspecies carcinogens. The International Conference on Harmonization for Technical Requirements for the Registration of Pharmaceuticals for Human Use has proposed an international standard that allows for the use of one long-term rodent carcinogenicity study, plus one supplementary study to identify potential human pharmaceutical carcinogens. The neonatal mouse assay for tumorigenicity has been used since 1959; however, relative to other alternate tests, little has been written about this system. It is clear that this assay system successfully identifies transspecies carcinogens from numerous chemical classes, thus recommending itself as a strong candidate for a supplementary study to identify potential human carcinogens. In contrast, there are decidedly less data available from this assay in response to pharmaceuticals shown to exhibit weak and/or conflicting results in the 2-year bioassay, knowledge invaluable to the regulatory process. This paper reviews the historical development and our experience with the neonatal mouse assay and includes suggestions for a standardized protocol and strategies to document its response to “weak” and/or “nongenotoxic” carcinogens.

Medium-term Rat Liver Bioassay for Rapid Detection of Hepatocarcinogenic Substances.

We have developed a medium-term liver bioassay system for rapid detection of carcinogenic agents using male F344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system is fundamen-tally based on the two-stage hypothesis with regard to tumor production, employing initiation by diethylnitrosamine (200mg/kg, ip) in the first stage and test chemical administration during the second, in combination with 2/3 partial hepatectomy. It requires only 8 weeks for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically-demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 277 chemicals/substances have already been analyzed in our laboratory and the efficacy of the system for hepatocarcinogens has thereby been well established. This bioassay is increasingly becoming regarded as an appropriate alternative test system for carcinogenicity risk assessment. It is particularly useful for studies which require many experimental groups and for evaluation of chemopreventive agents.

Alternative Test Systems RFA

Alternative Test Systems RFA

An alternative study of the skin irritant effect of an homologous series of surfactants

An attempt has been made to differentiate between in vivo and in vitro skin reactions to a homologous series of surfactants (sodium alkyl sulfate, R-OSO 3 Na) and to determine the usefulness of percutaneous absorption in vitro as an alternative test system. Sodium alkyl sulfate showed considerable biological activity by virtue of its polar head groups. The length of the lipophilic chain in the surfactants was an important factor in their overall activity. The following in vivo tests were performed: a primary skin irritation test in guinea pigs, a primary eye irritation test in rabbits and a closed patch test in humans. Peak skin irritation occurred with C 10-C 16 sodium alkyl sulfate, which had lipophilic groups of different alkyl chain lengths. Cell injury was also evaluated by the neutral red dye uptake assay in rabbit corneal (RC) cells. C 4 and C 6 compounds had no effect, while maximal effects occurred with C 18. Protein denaturation and haemolysis occurred with C 10-C 16 compounds. In the percutaneous absorption test in guinea pig skin, permeation was low for the C 18 compound and high for the C 4 compound. The results with the C 18 compound suggest that differences between cell injury and skin irritation result from skin permeation. Although the C 18 compound caused cell injury, membrane destruction and protein denaturation were more severely with the C 10-C 16 compounds, owing to their strong haemolytic and protein-denaturation action.

Human monocyte-derived macrophage cultures: An alternative test system for the detection of pulmonary toxicity induced by inhaled particulate pollutants

Alveolar macrophages are major target cells for toxicity from inhaled particulates. To investigate pulmonary toxicity induced by airborne particulates a phagocytosis assay using human monocyte-derived macrophage cultures was used. Monocytes were isolated from peripheral blood and cultured for 10–14 days. During this period the monocytes differentiated to macrophages. After treatment with various concentrations of different samples of airborne particulate matter, phagocytosis was induced by the addition of Polychromatic Fluoresbrite Microspheres at a cell/particle ratio of 1:10. Phagocytosis was assessed from the determination of phagocytic activity (% cells showing phagocytosis) and of phagocytic capacity (number of phagocytized particles/cell). A concentration-dependent reduction of phagocytic activity and capacity was observed, while cell viability was not greatly affected as compared with the control. These results were in good agreement with those obtained from in vivo inhalation experiments with rodents as well as with those using rat alveolar macrophages obtained after bronchoalveolar lavage and treated in vitro with extracts of particulate matter. Therefore, it is suggested that the use of human monocyte-derived macrophage cultures represents a promising in vitro test system for the evaluation of pulmonary toxicity induced by particulate pollutants.

Test guideline development and animal welfare: Regulatory acceptance of in vitro studies

Toxicity studies are necessary in order to be able to identify the potential hazards of chemical interference with human reproduction. Until today, most useful contributions to the assessment of possible human reproductive toxicity are considered to be made by animal studies. The OECD Test Guidelines provide the international standards for safety testing and, consequently, have traditionally focused on animal studies. However, the OECD Member countries consider the welfare of laboratory animals also of importance and are of the opinion that animal welfare considerations should significantly influence the work in the OECD Chemical Programme. A constant effort is being made to discover alternative testing systems and to achieve their regulatory acceptance. However, activities predominately focus on finding alternatives to existing animal studies, rather than developing nonanimal tests that could contribute significantly to the hazard identification process. An approach based on the selection of endpoints essential for hazard identification that would focus on demonstrating similarities with the “real life” target events is considered likely to achieve regulatory acceptance much earlier than an approach based on high correlations between the alternative method and the existing animal study it is supposed to replace, simply because it is never better than the existing method, but at most “almost as reliable.”

Nonhuman Primates and Teratological Research

Nonhuman primates were first recognized as models for the study of developmental toxicity (teratology) following the thalidomide tragedy. Since that time they have played important roles in both testing of drugs for human safety and as models for studying specific malformations commonly seen in children. Although in vitro and alternative test systems using lower animal forms or simplified test systems have been incorporated into developmental toxicity studies, whole animal testing will be required for the foreseeable future because of the complex relationship of the maternal/embryofetal/placental unit. The nonhuman primate will be particularly valuable where equivocal results are experienced in other commonly used laboratory species, when the drug/chemical is likely to be used during pregnancy, and for human-derived biotechnical products which often are not bioactive in nonprimate species.

Effects of family size in selection and testing in a closed dam line of pigs

The objective of this study was to evaluate alternative selection and testing systems in closed dam lines of pig breeding programmes. A stochastic simulation model was used to study the effects of alternative systems on variances in family size, rate of inbreeding and response to selection for production and reproduction traits. Traits were assumed to be affected by many unliked loci, each of small additive effect. Differences in the selection response between alternative selection systems were small. A restriction on the number of boars selected per litter (within-full-sib family selection) had little influence on the rate of inbreeding and selection response. A restriction on the number of boars per sire (within-paternal half-sib family selection) gave a small reduction in the rate of inbreeding and response to selection. Increasing the number of boars was a better option for limiting the rate of inbreeding than within-family selection. Two alternative testing systems were compared. A system of one boar tested per litter gave ∼ 10% lower response to selection than a system of two boars tested per litter.

Progress and problems in evaluating and validating alternative assays in toxicology

Various techniques have been proposed and subsequently developed as potential replacements for whole-animal topical toxicology assays. Comparisons of in vitro and in vivo endpoints, the solubility of test agents, solvent effects, interactions between components of complex test samples and the standardization of data presentation are factors that influence not only individual laboratory results but interlaboratory reproducibility and the acceptance of newly developed alternative methods. Representative alternative test systems for evaluating topical toxicity are discussed. Examples of problems, data and solutions based on the authors' experience and on experience in other laboratories are reviewed. Specific problems in choosing endpoints, comparing results, recognizing responses of different types of target cell, evaluating the influences of support media in culture and interpreting published animal-based data are included.

Evaluation of chicken embryo, brine shrimp, and bacterial bioassays for saxitoxin.

The chicken embryo, brine shrimp (Artemia salina) and selected bacteria (Staphylococcus epidermidis, Micrococcus flavus, and Bacillus subtilis) were evaluated as alternative test systems for the determination of paralytic shellfish poisoning toxicity (saxitoxin). Dose levels ranging from 0.045 to 0.3 microgram were administered to the developing embryo through the air cell at either 0 or 96 h following incubation. Embryos dosed 96 h after incubation were the most sensitive, with 100% mortality at the 0.11-microgram dose level 24 h following exposure. Embryo mortality was 42% at to 0.3-microgram dose level when the toxin was administered at 0 h. Some embryonic malformations were observed in the 0-h treatment group. Brine-shrimp larvae were sensitive to saxitoxin at a dose level of 5 micrograms. A dose response based on mortality was apparent only 48 h after dosing. Limited growth inhibition was observed with the bacteria tested at concentrations between 0.0035 and 0.35 microgram and 0.35 microgram/well.

Reproductive Toxicity Testing: Evaluating and Developing New Testing Systems

Reproductive toxicity testing systems are used by national and international regulatory agencies. Protocols have not been standardized between agencies or even within certain agencies. Although there have been efforts at standardization, a certain amount of the differences between testing protocols is a reflection of the needs of the particular agency. New developments in in vitro techniques might lead to new test systems, but reproductive function is dependent upon the interaction of various cells and organs that cannot presently be copied in the test tube; this makes whole-animal testing systems a necessity. The present whole-animal models used by the Food and Drug Administration include the 3 segment reproduction studies used for testing drug safety and the multigeneration studies used for food additives. The Environmental Protection Agency has adopted 2 similar versions of a 2-generation study for the Office of Pesticide Programs and the Office of Toxic Substances. The National Toxicology Program, although not a regulatory agency, has taken a prominent role in reproductive toxicity testing, test system development, and test system evaluation. A new testing system, Fertility Assessment by Continuous Breeding (FACB), is currently being studied as a cost-effective and reliable alternative test system. The FACB protocol houses male and female mice as breeding pairs and removes offspring as soon as they are born during the first 14 weeks to allow continuous mating. Each breeding pair normally has up to 5 litters, and the last litter is saved to evaluate the second generation. The efficiency, reliability, and expense of the protocol are being compared to the existing testing systems.

The indirect rat mast cell degranulation test reconsidered.

The indirect rat mast cell degranulation test for the detection of human reaginic antibodies was reinvestigated. When the mast cells were incubated with human reaginic serum at concentrations exceeding approximately 3%, a massive, non-specific histamine release occurred, irrespective of the presence or absence of allergen. At tolerated serum concentrations (below 3%), the allergen concentration did not influence the release process. We conclude that the indirect rat mast cell test is not applicable as an alternative test system for the study of immediate hypersensitivity in humans.