ILAR Journal Online, Volume 34(3) 1992: ILAR's Fortieth Anniversary

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ILAR Journal V34(3) 1992 [FORMERLY ILAR NEWS]
ILAR's Fortieth Anniversary

Special Insert

Alternatives to the Use of Live Vertebrates in Biomedical Research and Testing: An Annotated Bibliography

Compiled by:

George J. Cosmides

Robert S. Stafford

Po-Yung Lu

NATIONAL ACADEMY PRESS
Washington, D.C. 1992

This bibliography is an edited, condensed version of quarterly bibliographies prepared by the National Library of Medicine in cooperation with the Oak Ridge National Laboratory. Quarterly bibliographies can be obtained by contacting:

Dr. Po-Yung Lu
Toxicology Information Response Center
Oak Ridge National Laboratory
P.O. Box 2008, MS6050
Oak Ridge, TN 37831-6050
615/574-7587

Reprints of this annual, condensed bibliography are available from:

Institute of Laboratory Animal Resources
National Research Council
2101 Constitution Avenue, NW
Washington, DC 20418

Introduction

Because of considerable interest from Congress, the National Institutes of Health (NIH), and the public about animal weftare and alternatives to animal testing, the National Library of Medicine (NLM) searches its online data bases and prepares quarterly annotated bibliographies on alternative or in vitro methods for toxicity testing and biomedical research. The objective is to present current literature, organized as citations with brief annotations, for easy scanning.

The Institute of Laboratory Animal Resources (ILAR) has invited NLM to publish in ILAR News an annual supplement, like this one, which is an edited, concatenated version of the quarterly bibliographies. The ILAR News Editorial Panel and outside reviewers will edit and condense the quarterly bibliographies so the entries are appropriate for the ILAR News audience.

The scientific community is concerned with humane animal care and is sensitive to public concerns about how and why animals are used in biomedical research and toxicity testing. The following events reflect the involvement of the public and the U.S. government in this issue: an array of federal legislation related to animal welfare and the use of laboratory animals, including the U.S. Public Health Seaice Policy on the Humane Care and Use of Laboratory Animals, and efforts at NIH to promote and support a search for alternative methods to the use of animals in biomedical research and testing.

Scientists generally view the use of laboratory animals in biomedical research and toxicity testing as necessary except where valid, scientific alternatives are available. It is unlikely that in vitro methods will satisfy all testing requirements. However, when animals must bc used, the scientific community supports careful consideration of the number of animals used and encourages reductions when they are scientifically feasible. Indeed, three Rs have emerged in the area of animal testing alternatives: replacement, refinement, and reduction.

Therefore, by providing the scientific community with access to its bibliographic databases and by producing bibliographies on animal alternatives, NLM is supporting efforts by the NIH to increase the knowledge needed to develop methods of biomedical research and experimentation that

do not require the use of vertebrate animals,
reduce the number of vertebrate animals used in research,
produce less pain and distress in vertebrate animals than do current methods,
validate or demonstrate the reliability of nonanimal methods, and
increase the use of nonvertebrate animal research methods that have been found to be valid and reliable.

The NLM anticipates an acceleration in the development of alternative or in vitro methods used in toxicity testing and biomedical experimentation, which will result in more articles about these methods in the literature.

The NLM hopes the bibliographies, compiled from its databases, will help ILAR News readers keep abreast of the new literature. Information on ordering the unedited, quarterly bibliographies or reprints of this edited annual supplement can be found on the inside cover of this insert. Please direct any comments or suggestions to Dr. George J. Cosmides, National Library of Medicine, Bethesda, MD 20894.

Carcinogenicity

Parke, D. V., C. loamfides, and D. E V. Lewis. 1990. Computer modeling and in vitro tests in the safety evaluation of chemicals--strategic applications. Toxicol. in Vitro 4(4-5):680-685.

A strategy based on cytochrome P 450s resulted in the development of a computer program (COMPACT) and of a program of enzyme induction studies as short-term tests to predict the potential toxicity/carcinogenicity of chemicals. COMPACT was validated against the rodent tumorigenicity assay and the Ames test in a study of 100 miscellaneous chemicals and showed excellent correlations. A QSAR for a series of 14-methylbenzanthracenes shows good correlation of mutagenicity with the energy of the lowest empty MO [E(LEMO)] values of the chemicals. A strategy for COMPACT and P 450 induction studies in chemical safety evaluation is presented.
Rosenkranz, H. S., and G. Klopman. 1990. New structural concepts for predicting carcinogenicity in rodents: An artificial intelligence approach. Teratog. Carcinog. Mutagen. 10(2):73-88.

The Computer Automated Structure Evaluation (CASE) method for studying structure-activity relationships has been applied to a data base of rodent carcinogens. It has been demonstrated that CASE is able to identify determinants embedded in the molecular structure which, with a high probability, predict rodent carcinogenicity. CASE has also identified determinants associated with the activity of non-genotoxic carcinogens, thereby suggesting that there is a structural commonality in the activity of these molecules. The present study reveals that there are "universal" as well as species-specific structural determinants of carcinogenicity. CASE was able to predict the carcinogenicity in rodents of certain endogenous pesticides in edible plants.

Cardiotoxicity

Loew-Friedrich, I., E Von Bredow, and W. Schoeppe. 1991. A cell culture assay for the detection of cardiotoxicity. J. Pharmacol. Methods 25(2):144-145.

The use of shock protein formation, a cellular response to cell-damaging stress, is proposed as an assay to monitor cardiotoxicity. Isolated, cultured cardiac myocytes were prepared by trypsin digestion method from 18-day fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Drugs relevant in transplant medicine were tested for possible cardiotoxic effects. The ability to induce shock protein synthesis seems to be restricted to toxic drugs. The proposed in vitro model system for cardiotoxicity is animal-saving and sensitive.

Cytotoxicity

Babich H., and E. Borenfreund. 1991. Cytotoxicity and genotoxicity assays with cultured fish cells: A review. Toxicol. in Vitro 5(1):91-100.

This is a review of the use of fish cell lines for in vitro cytotoxicity and genotoxicity assays of chemical test agents.
Brun, H. P., J. F. Leonard, V. Moronvalle, J. M. Calllaud, C. Melcion, and A. Cordier. Pig Leydig cell culture: A useful in vitro test for evaluating the testicular toxicity of compounds. Toxicol. Appl. Pharmacol. 108(2):307-320.

In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, the authors developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with different mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt [3-(4.5-dimethylthiazol)-2.5-diphenyltetrazolium bromide] which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. This in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.
Loew-Friedrich, I., and W. Schoeppe. 1991. Effects of calcium channel blockers on stress protein synthesis in cardiac myocytes. J. Cardiovasc. Pharmacol. 17(5):800-806.

The detection of stress proteins, certain protein groups of molecular weight 70 or 30 kilodaltons synthesized de novo under stress conditions, served as an assay for monitoring cellular toxicity. Typical toxins inducing stress protein formation in fetal mouse cardiac myocytes are cadmium chloride and hydrogen peroxide.
Rahn, C. A., D. W. Bombick, and D. J. Doolittle. 1991. Assessment of mitochondrial membrane potential as an indicator of cytotoxicity. Fundam. Appl. Toxicol. 16(3):435-448.

The objective of this study was to characterize a procedure for evaluating mitochondrial membrane potential, an integral component of cellular energy homeostasis and normal cellular function, as an in vitro indicator of chemically induced cytotoxicity. Rhodamine 123 was used to evaluate disturbances in mitochondrial membrane potential. Cultured rat liver epithelial cells or human skin fibroblasts treated with the oxidative phosphorylation uncoupler 2,4-dinitrophenol or the cytochrome oxidase addition, acetaldehyde was used to demonstrate the specificity of this assay system. This assay provides a tool for evaluating the effect of chemical treatments on mitochondrial membrane potential, and is also an indicator of cytotoxicity that does not require the use of animals.
Zhang, S.-Z, M. M. Lipsky, B. E Trump, and I.-C. 1990. Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes. Cell Biol. Toxicol. 6(2):219-234.

The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin-B1 (AFB1) were examined in hepatocytes using uptake of neutral-red dye (NR) as the endpoint. The effects on cell viability were determined by measuring uptake of NR spectrophotometrically. The concentrations of DMN or AFB1 required to reduce NR absorbency to half of the control value were determined. Leakage of lactate-dehydrogenase from the cultures into the medium was examined for comparison purposes. Experiments leading to optimization of the NR uptake test are described. The authors conclude that the NR uptake test is a sensitive, simple, and reproducible technique for rapidly determining the cytotoxicity of xenobiotics under controlled in vitro conditions.

Dental

Mcryon, S. D., and A. M. Brooks. 1990. In vitro comparison of the cytotoxicity of t~elve endodontic materials using a new technique. Int. Endod. J. 23(4):203-210.

aims

Developmental Toxicity

Bantie, J. A., D. J. Fort, J. R. Rayburn, D. J. DeYoung, and S. J. Bush. 1990. Further validation of FETAX: Evaluation of the developmental toxicity of five known mammalian teratogens and non-teratogens. Drug Chem. Toxicol. 13(4):267-282.

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Peters, P. W. J., and A. H. Piersma. 1990. In vitro embryotoxicity and teratogenicity studies. Toxicol. in Vitro. 4(4-5):570-576.

This publication is a review and discussion with. 74 references on the design and choice of suitable in vitro and nonmammalian tests for evaluating embryotoxicity and teratogenicity, particularly with regards to potential teratogenic hazard to man.

Eye

Reader, S. J., V. Blackwell, R. O'Hara, R. H. Clothier, G. Grifl'm, and M. Balls. 1990. Neutral red release from pre-loaded cells as an in vitro approach to testing for eye irritancy potential. Toxicol. in Vitro 4(4-5):264-266.

Preliminary studies were made on the exposure of fibroblast-like cells, pre-loaded with a vital dye (neutral red), to high concentrations of test materials for brief periods. Measurement of loss of neutral red from damaged cells provides a readily quantifiable endpoint. When the results obtained with eight detergent formulations were compared with maximum Draize eye scores, a better correlation was obtained with conjunctival and iris effects than with corneal scores or average Draize scores.
Roberts, C. D. 1991. Statistical model in tests for eye irritants. Food Chem. Toxicol. 29(7):463-468.

A test used to classify substances for eye irritancy, as required by the Consumer Product Safety Commission, is performed on one to three groups of six albino rabbits in a sequential manner. When the statistical implications of the test are realized, it is possible for a substance to be classified as an irritant with fewer reactions than the number required for it to be classifted as not an irritant. Probability models and expected sample size calculations have been derived. A procedure is given for correcting the inconsistency in the current test, and an alternative test, which considerably reduces the number of animals required, is proposed.

Hepatotoxicity

Azri, S., A. J. Gandolfi, and K. Brendel. 1990. Carbon tetrachloride toxicity in precision-cut rat liver slices. In Vitro Toxicol. J. Mol. Cell. Toxicol. 3(2):127-138.

The hepatotoxicity of carbon-tetrachloride (CCLA) was investigated in rat liver slices. Liver slices were prepared from adult male Sprague-Dawley rats, some of which had been pretreated for enzyme induction. Hepatotoxicity was assessed by determining the loss of intracellular potassium-ion (K+). The liver slices were assayed for isocitrate-dehydrogenase and alanine-aminotransferase activity. The extent of covalent binding of CCIA to protein and lipid macromolecules was determined. The authors conclude that the liver slice system is a useful technique for investigating cellular mechanisms involved in CCL4 hepatotoxicity.
Bridges, J.W. 1990. Specific organ/system toxicity: The liver. Scope 41(Short-Term Toxic. Tests Non-Genotoxic Eff.):lll-133.

No single test is now or is ever likely to be able to provide all the information that is required to assess the hepatotoxic properties of a chemical. However, the rapid developments in methodology for handling isolated cells indicate that, in the near future, such in vitro preparations may be suitable for the initial screening of chemicals for acute hepatotoxic properties and for obtaining information on the relative response to a chemical of human liver compared with other species.

Irritancy

Segal, L., D. Riedel, and L. Ritter. Evaluation of normal human epidermal keratinocyte cultures as a test system for the assessment of the dermal irritancy of pesticides. 1990. Todcol. in Vitro 4(4-5):277-279.

Cultured normal human epidermal keratinocytes (NHEK) have been proposed as a good model system for the assessment of dermal irritancy and toxicity because they participate in dermal inflammation, and therefore constitute valid components of the reactions of human skin to toxins. The usefulness of this approach for dermal irritancy/toxicity evaluation of pesticide ingredients and formulations will be assessed by measuring the release of [3H]arachidonic acid from prelabeled NHEK cultures in response to pesticides added to the tissue culture medium.
Sterzal, W., F. G. Bartnik, W. Matthies, W. Kaestner, and K. Kuenstler. 1990. Comparison of two in vitro and two in vivo methods for the measurement of irritancy. Todcol. in Vitro 4(4-5):698-701.

In order to validate in vitro assays for local tolerance testing, results from the following experiment were compared: (1) cytotoxicity testing in cell culture (neutral red assay); (2) tests with the chorioallantoic membrane of fertilized chicken eggs; (3) rabbit eye mucous membrane tests; and (4) occluded epicutaneous testing in human volunteers. The data indicate that in vitro testing with the first two methods yields reliable results with respect to the eye and human skin irritation data within homologous substance classes. A test procedure is proposed that involves comparative testing of the chemicals with unknown irritant properties together with known weak and strong irritants from the same class of chemicals as standards in the test series. This procedure seems to be suitable as a preliminary screen for identifying severe irritants prior to the performance of any in vivo studies. It would reduce the number of animals to be used in vivo and lead to the avoidance of exposure of animals to harmful substances.

Lung

Mossman, B.T. 1990. In vitro studies on the biologic effects of fibers: Correlation with in vivo bioassays. Environ. Health Perspec. 88:319-322.

Data were summarized from work with asbestos in vitro that have elucidated physicochemical features of asbestos which are important in the induction of toxicity, proliferation, and the morphologic transformation of cells in vitro. The reported studies have been useful in defining mechanisms contributing to the causation of fiber associated lung diseases. The author concludes that in order to evaluate whether in vitro bioassays are amenable to predicting the pathogenic potential of synthetic and naturally occurring fibers comparatively, it will be necessary to develop in vitro models employing target cells of the lung including mesothelial cells, tracheobronchial epithelial cells, and lung fibroblasts.

Metabolism

Barr, J., A. J. Weir, K. Brendel, and I. G. Sipes. 1991. Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue. Xenobiotica 21(3): 341-350.

Precision cut human liver slices in dynamic organ culture were used to study the integrated metabolism of 7-ethoxycoumarin and the conjugate of 7-hydroxycoumarin. A cold-storage transit buffer has been described and used to maintain levels of drug metabolism in both rat and human tissue for periods of up to 6 hours. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.

Mutagenesis

Nestmann, E. R., R. L. Brillinger, J. P. Gilman, C. J. Rudd, and S. H. Swierenga. 1991. Recommended protocols based on a survey of current practice in genotoxicity testing laboratories. II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung, and L5178Y mouse lymphoma cells. Mutat. Res. 246(2):255-284.

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.

Pyrogens

Hansen, E. W., and J. D. Christensen. 1990. Comparison of cultured human mononuclear cells, Limulus Amebocyte Lysate, and rabbits in the detection of pyrogens. J. Clin. Pharm. 15(6):425-433.

Isolated human mononuclear cells were exposed to either lipopolysaccharide or Staphylococcus aureus secreted interleukin-1 like material. The secretion was concentration dependent. The sensitivity and specificity of the Limulus Amebocyte Lysate test, the rabbit pyrogen test, and the monocyte test are compared. The monocyte test is proposed as an alternative in vitro test to the rabbit pyrogen test.

Skin

Dykes, P. J., M. J. Edwards, M. R. O'Donovan, V. Merritt, H. E. Morgan, and R. Marks. 1991. In vitro reconstruction of human skin: The use of skin equivalents as potential indicators of cutaneous toxicity. Toxicol. in Vitro 5(1):1-8.

GalIi, C. L., and M. Marinovich. 1990. In vitro biochemical markers of skin toxicity. NATO ASI Ser., Ser. A; 181 (Skin Pharmocol. Toxicol.:Recent Adv. 181:165-179.)

For the skin, there are many in vitro systems proposed for the evaluation of toxicity with different results. Each in vitro model has its specific limitations, and therefore it is important to select the optimum model for each hypothesis under investigation. In skin organ culture, tissue organi?ation and morphology, cellular dynamics, and cell interactions are preserved in a setting comparable to the in vivo situation. The absence of immunologic and inflammatory reactions, tissue innervation, and sufficient oxygen and nutrient supply are limitations of this model. This is a review with 57 references.
Li, L., L. B. Margolis, and R. M. Hoffman. 1991. Skin toxicity determined in vitro by three-dimensional native-state histoculture. Proc. Natl. Acad. Sci. 88(5):1908-1912.

Described is a gel-supported in vitro system for culturing skin samples in a three-dimensional native state. The culture system is used for toxicity measurements of ascertaining cell viability using two fluorescent dyes: (1) 2',7;-bis-(2-carboxyethyl) -5(and-6) carboxyfluorescein acetoxymethyl ester, specific for living cells, and (2) propidium iodide, specific for dead cells. Cell staining with the dyes is measured throughout the tissue block by confocal scanning fluorescence microscopy. It is also demonstrated that the end point of (3H)thymidine incorporation measured by histological autoradiography can be used to measure toxicity. The native-state model for skin may be an effective replacement for animal systems and superior to the dispersed skin cell systems used previously. It can allow rapid, inexpensive measurements of the effect of manufactured products, drugs, and pollutants on the skin.
Nakamura, M., T. Rikimaru, T. Yano, K. G. Moore, P. J. Pula, B. H. Schofield, and A. M. Dannenberg, Jr. 1990. Full-thickness human skin explants for testing the toxicity of topically applied chemicals. J. Invest. Dermatol. 95(3):325-332.

Surber, C., K. -P. Wilhelm, H. I. Maibach, L. I. Hall, and R. H. Guy. 1990. Partitioning of chemicals into human stratum corneum: Implications for risk assessment following dermal exposure. Fundam. Appl. Toxicol. 15(1):99-107.

A particularly important question is to what extent can the skin permeability of a given compound be predicted from simple experiments. The literature on percutaneous absorption identifies two key observations: (1) the stratum corneum, the skin's outermost layer, is the major barrier to chemical transport, and (2) there are qualitative correlations between penetrant permeability and various oil/water partition coefficients. After obtaining relevant data it is suggested that measured partition coefficient values may be useful predictors of in vitro and in vivo skin transport and valuable assets, therefore, in the evaluation of risk following dermal exposure.

Teratogenicity

Hulme, L. M., K. A. Atkinson, R. H. Clothier, and M. Balls. 1990. The potential usefulness of a differentiating teratocarcinoma cell line in in vitro toxicity testing. Toxicol. in Vitro 4(4-5):598-592.

Described is an investigation into the possible use of a permanent cell line that possessed many of the properties of embryonic cells, i.e., a differentiating cell line, F9 (derived from a mouse teratocarcinoma), in the development of an in vitro teratogenicity test. In a preliminary study, six chemicals were tested for their modulating effects on differentiation in undifferentiated, differentiating, and differentiated F9 cells. These effects were assessed morphologically and by measuring the production of laminin (a biochemical marker of F9 differentiation). The potential of the F9 cell line in in vitro teratogenicity testing is discussed.
Lachinski, G., R. Vogel, and H. Spielmann. 1991. Cytotoxicity test using blastocyst-derived euploid embryonai stem cells: A new approach to in vitro teratogenesis screening. Reprod. Toxicol. 5(1):57-64.

To develop a mammalian in vitro system for teratogenicity testing, cytotoxicity of ~enobiotics was evaluated in pluripotent euploid embryonal stem cells (ESC) derived from mouse blastocysts. The dimethyl-thiazol-diphenyl tetrazolium bromide (MTn) assay was the most appropriate test system for cytotoxicity determinations with ESC. Although some ~enobiotics had to be classified as false negatives in this system, the ESC cytotoxicity assay holds promise as a new in vitro screening assay in teratology.

Toxicity

van den Heuvel, M. J., D. G. Clark, R. J. Fielder, P. P. Koundakjian, G. J. Oliver, D. Pelling, N. J. Tomlinson, and A. P. Walker. 1990. The international validation of a fixed-dose procedure as an alternative to the classical LD₅₀ test. Food Chem. Toxicol. 28(7):469-82.

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Zbinden, G. 1986. Invited contribution: Acute toxicity testing, public responsibility, and scientific challenges. Cell Biol. Toxicol. 2(3):325-335.

This paper reviews the possibilities of using fewer animals to obtain relevant information on the acute hazards of chemical substances, but it also identifies the reasons why the traditional testing approaches cannot be changed immediately. Proposals are presented on how pain and suffering of the animals included in acute toxicity tests can be reduced. The use of in vitro systems for the evaluation of the hazardous properties of chemicals is discussed.

Toxicology

Warren M., K. Atkinson, and S. Steer. 1990. The Ergatt/Frame data bank of in vitro techniques in toxicology. Toxicol. in Vitro 4(4-5):707-710.

The computerized INVITTOX data bank of in vitro toxicological techniques comprising four interrelated libraries are described. Aims, contacts, services, funding, and expansion are discussed.