ILAR Journal Online, Volume 31(1) 1989: Perspectives on Animal Use

Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V31(1) 1989
Perspectives on Animal Use

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

Compiled by:
George J. Cosmides, Deputy Director, Toxicology Information Program, National Library of Medicine, National Institutes of Health, Bethesda, Maryland
Robert S. Stafford, Information Analyst, Oak Ridge National Laboratory, Oak Ridge, Tennessee
Po-Yung Lu, Director, Chemical Hazard Evaluation Program, Oak Ridge National Laboratory, Oak Ridge, Tennessee

Reprinted from ILAR News, Volume 31, Number 1, 1989

NATIONAL ACADEMY PRESS Washington, D.C. 1989

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 welfare and alternatives to animal testing, the National Library of Medicine (NLM) searches its online databases 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, U.S. Public Health Service 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 alternative techniques are available. It is unlikely that in vitro methods will satisfy all testing requirements. However, when animals must be 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 nonani-mal 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 about the bibliographies to Dr. George J. Cosmides, National Library of Medicine, Bethesda, MD 20894.

Carcinogenicity

Santella, R. M. 1987. In vitro testing for carcinogens and mutagens. Pp. 39-46 in Occupational Medicine: State of the Art Reviews. Occupational Cancer and Carcinogenesis, vol. 2, no. 1, P. W. Brandt-Rauf, ed. Philadelphia: Hanley and Belfus (27 refs.).

In vitro testing for carcinogens and mutagens is discussed and contrasted with animal tests. Requirements of an ideal in vitro assay are listed. No single in vitro bioassay system can meet all the requirements. In vitro assays, when applied to chemicals of unknown carcinogenicity, must be used in a battery or tiered testing protocol. While negative results in a battery of in vitro assays may indicate low risk, a chemical with widespread human exposure may still be worth evaluating in a long-term animal bioassay.

Cytotoxicity

Bertolero, F., et al.. 1987. Quantitative studies on cytotoxicity and neoplastic transformation of BALB/ 3T3 cells by trace metals. Pp. 478-483 in Occupational and Environmental Chemical Hazards. Cellular and Biochemical Indices for Monitoring Toxicity, V. Foa, E. A. Emmett, M. Maroni, and A. Colombi, eds. Chichester, England: Ellis Horwood (5 refs.).

The BALB/3T3-C1-A31-1-1 cell line was tested to determine its potential use for the study of the effects of various metal salts in vitro. The authors conclude that the BALB/3T3 cell system is a valuable model for the study of correlations between cellular uptake, metabolism, cytotoxicity, and neoplastic transformation induced by metals in vitro.

Borenfreund, E., and C. Shopsis. 1985. Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15(8-9):705-711.

In vitro tests were developed as potential alternatives to in vivo eye irritancy testing. The emphasis was on finding a replacement for the Draize eye irritancy test. BALB/c mouse, hamster V79, rabbit epithelial cornea, human hepatoma, or mouse RAW 264.7 cells were incubated with 22 chemical agents to determine the highest tolerated doses. The authors note that results of attempts to design in vitro assays are encouraging. Large-scale validation tests are in progress.

Christensen, C., et al. 1987. The use of a human endometrial carcinoma cell line (RL-95) for in vitro testing of chemotherapeutic agents. Gynecol. Oncol. 28(1, Sept.):25-33.

RL-95 can be used as a model for testing chemotherapeutic agents in vitro. Effects of Adriamycin and cis-platinum were measured by cell counts and DNA synthesis. Cell kill was obtained with levels of drugs that are clinically achievable. Evidence is presented for increased cytotoxicity with concomitant, rather than sequential, chemotherapy. Results are also confirmed by testing with MCF-7, a well-known breast-cancer cell line. The results indicate that (1) endometrial carcinoma responds to Adriamycin and cis-platinum chemotherapeutic agents in vitro, and (2) RL-95 can be used as a model for testing varying concentrations, time of exposure, and combinations of chemotherapeutic agents.

van Lambalgen, R., and P. Lelieveld. 1987. The pit method: An automated in vitro technique for drug toxicity testing. Invest. New Drugs 5(2): 161-165.

An automated in vitro technique for drug toxicity testing using a human tumor cell culture and a DNA-specific fluorescing stain (propidium iodide) is described. Microtiter plates were read on a computer-controlled automated microscope with a photomultiplier. The results showed a linear relationship between cell number and fluorescence intensity. Reproducible dose-response curves were obtained for six drugs tested. A computer program calculated LD50 values, making use of adequate-growth and no-growth controls.

Developmental Toxicity

Johnson, E. M. 1987. A tier system for developmental toxicity evaluations based on considerations of exposure and effect relationships. Teratology 35(3):405-427 (54 refs.).

In vitro assays have been devised to quantitatively rank chemicals according to their developmental hazard index. Using these assays along with conventional in vivo methods and appropriate considerations of exposure, a tier approach to establish testing priorities that markedly reduce the time, cost, and number of laboratory animals needed for evaluation of developmental toxicity is described.

Johnson, E. M., et al. 1987. Use of the adult developmental relationship in prescreening for developmental hazards. Teratogenesis Carcinog. Mutagen. 7(3):273-285 (30 refs.).

A proposed tier system establishes priorities of testing chemicals already in commerce or brought into use each year for potential to adversely affect in utero development. Testing would be based on exposure and the concept of target organ toxicity applied to the embryo. It provides intensive in vivo evaluations of those chemicals for which developmental effects testing is most needed and avoids use of resources and animals for unnecessary testing of agents that do not pose threats to the conceptus.

DNA Effects

Hildebrand-Zanki, S. U., and D. H. Kern. 1987. In vitro assays for new drug screening: Comparison of a thymidine incorporation assay with the human tumor colony-forming assay. Int. J. Cell Cloning 5(5):421-43 i.

Efforts were made to determine the feasibility of using a thymidine incorporation assay for new drug screening. Concordance was obtained between the thymidine incorporation assay and the human tumor colony-forming assay with 20 coded compounds. Growth rates were higher and assays could be completed more quickly using the thymidine incorporation assay than with the human tumor colony-forming assay. It is concluded that the thymidine incorporation assay is a useful adjunct to in vivo tumor models for screening new anticancer agents.

Rosanda, C., et al. 1987. A short-term in vitro drug sensitivity assay in pediatric malignancies. Anticancer Res. 7(3) pt. B:365-367.

A short-term in vitro chemosensitivity test was applied to 131 specimens from 109 children with various malignancies. The clinical outcome was correlated with in vitro percentage inhibition of DNA synthesis by the drug tested. This test can be useful particularly in predicting chemoresistance in relapsing patients, to avoid unnecessary toxicity.

Eye

Morgan, R. L., et al. 1987. Prediction of ocular irritation by corneal pachymetry. Food Chem. Toxicol. 25(8):609-613.

Corneal pachymetry performed 3 days after application of a variety of test materials to the rabbit eye was found to be predictive of the eye irritation classification determined by observing the ocular response for 21 days. Both the left and right eyes of each rabbit were evaluated for irritation and corneal thickness for up to 21 days using a slit-lamp biomicroscope with a pachymeter attachment. Corneal pachymetry for determining eye irritation classification is presented as an alternative to the current 21-day test. It is more objective and requires a shorter observation period. Therefore, this method should lessen the cost of eye irritation testing and decrease the duration of discomfort that may occur among the test animals. The greater objectivity may also reduce the intra- and interlaboratory variation and the number of rabbits required for each testing.

Simmons, S. J., et al. 1987. Corneal epithelial wound closure in tissue culture: An in vitro model of ocular irritancy. Toxicol. Appl. Pharmacol. 88(I):13-23.

The influence of 13 test agents on the ability of cultured rabbit corneal epithelial cells to migrate and recover a wound was used to evaluate an in vitro model of ocular irritancy. The test ranks irritants in an order similar to that described in the literature for both in vivo and in vitro tests. This tissue culture model is conceptually simple, quantitative, and an alternative to the corneal component of whole animal testing for ocular irritancy.

Wallin, R. F., et al. 1987. The agarose diffusion method for ocular irritancy screening: Cosmetic products: Part I. J. Toxicol. Cutaneous Ocul. Toxicol. 6(4):239-250.

The agarose diffusion test has been scientifically validated and is well established as an in vitro alternative test to screen the toxicity of plastics in medical devices. Results reported in this publication indicate a high degree of correlation between the agarose diffusion test and the Draize eye irritancy test and permit testing of oil-in-water emulsions (both pigmented and nonpigmented), water-based suspensions, petroleum distillate-based suspensions, solutions, physical mixtures of waxes, and physical mixtures of dry powders in this alternative test system.

Genotoxicity

Butterworth, B. E., et al. 1987. A protocol and guide for the in vitro rat hepatocyte DNA-repair assay. Mutat. Res. 189(2): 113-121.

The in vitro rat hepatocyte DNA-repair assay is a valuable tool in assessing the genotoxic activity of chemical agents. An advantage of the assay is that the target cells themselves are metabolically competent, so that the patterns of metabolic activation and detoxification closely reflect those in the whole animal. This article provides a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA-repair assay.

Rossberger, S., et al. 1987. Comparison of the continuous rat hepatoma cell line 2SFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo(A)pyrene and hydroxyurea. Mutat. Res. 182(1):41-51 (51 refs.).

The suitability of the continuous rat hepatoma cell line 2sFou was compared with that of primary rat hepatocyte cultures (HPC) for determination of the genotoxicity of chemicals. The induction of DNA repair synthesis and inhibition of replicative DNA synthesis were assessed in the two cell systems. The authors conclude that 2sFou cells are sensitive indicators of DNA damage and are capable of activating hepatotropic promutagens, such as the nitrosamines, and therefore offer a potential alternative to HPC for testing genotoxicity, particularly in terms of long-range consequences.

Hepatotoxicity

Stacey, N. H. Assessment of the toxicity of chemical mixtures with isolated rat hepatocytes: Cadmium and chloroform. Fundam. Appl. Toxicol. 9(4):616-622.

The suitability of isolated rat hepatocytes for the investigation of the toxicity of chemical mixtures was studied. Cadmium chloride and chloroform were used because both had been previously investigated in hepatocytes and both produce hepatotoxicity after in vivo administration. The data support the proposed role of isolated hepatocytes in screening chemical mixtures for toxic effects.

Immunology

Tjernlund, U., et al. 1987. In vitro testing of contact sensitivity. Acta Derm. Venereol. (Stockh.)67(5):417-421.

A new in vitro technique that tests for contact sensitivity is described. The method is based on the fact that in response to immunologic stimuli, keratinocytes can start to synthesize and express class-II histocompatability antigens. Nickel sulphate stimulated lymphocytes from nickel-sensitive persons, and the supernatant of these cells thus induced expression of HLA-DR on keratinocytes when co-cultured with autologous normal skin biopsies. This result was in contrast to that in healthy controls.

Lung

Shami, S. G., et al. 1984. Organotypic culture of fetal lung type II alveolar epithelial cells: Applications to pulmonary toxicology. Environ. Health Perspect. 56:87-94.

A description is offered of techniques used for the isolation and culture of fetal type-II alveolar epithelial cells along with the morphologic and biochemical characteristics of these histotypic cultures. Not only has the type-II cell become important in the response of the lung to many toxic and carcinogenic agents, it has also been found to be involved in various respiratory diseases, including respiratory distress syndrome of newborns. Use of the model in studies of pulmonary toxicant and carcinogen effects is discussed.

Neurotoxicity

Costa, L. G. 1987. Peripheral models for the study of neurotransmitter receptors: Their potential applications to occupational health and occupational and environmental chemical hazards. Pp. 524-528 in Cellular and Biochemical Indices for Monitoring Toxicity, V. Foe, E. A. Emmett, M. Maroni, and A. Colombi, eds. Chichester, England: Ellis Horwood (17 refs.).

The study of enzymes and receptors in tissues to demonstrate that neurochemical parameters measured in peripheral tissue are valid markers of the same parameters in the brain or other organs is reviewed. The author concludes that using biochemical markers to detect toxic effects of chemicals on the nervous system has good potential for many applications in occupational health.

Model Systems in Neurotoxicology. Alternative Approaches to Animal Testing. 1987. Proceedings of the 31st OHOLO Conference, Tiberias, Israel, November 3-7, 1986. Dedicated to Professor H. Edery. Prog. Clin. Biol. Res. 253: 1-432.

Phototoxicity

Przybilla, B., et al. 1987. Phototoxicity of non-steroidal anti-inflammatory drugs demonstrated in vitro by a photo-basophil-histamine-release test. Photodermatology 4(2, Apr.):73-78.

The phototoxic activity of several nonsteroidal antiinflammatory drugs was evaluated in vitro by a newly developed photo-basophil-histamine-release test performed with human leukocytes. The results indicate that many nonsteroidal antiinflammatory drugs can exert phototoxic effects, which confirms clinical observations as well as other in vitro experiments. The photo-basophil-histamine-release test seems to be a promising new method for the identification of phototoxic compounds.

Statistics

Talsma, D. M., et al. 1988. Reducing the number of rabbits in the Draize eye irritancy test: A statistical analysis of 155 studies conducted over 6 years. Fundam. Appl. Toxicol. 10(1):146-153.

It is suggested that a suitable alternative is not yet available to the Draize eye irritancy test in rabbits. Therefore, a study was made of the adequacy of reducing the number of rabbits used per test. Data generated from six-rabbit eye irritation tests of 155 various materials were used to determine the ability of irritation scores from all possible combinations of five-, four-, three-, or two-rabbit subsets to predict the Draize score derived from six rabbits. This study indicates that a high level of accuracy can be obtained with reduced numbers of rabbits per test.

Teratology

Lin, G. H. Y. 1987. Prediction of teratogenic potential and a proposed scheme for teratogenicity screening of industrial research and development materials. In Vitro Toxicol. 1(3):203-217.

The possibility of using a battery of short-term in vitro assays as a method of screening industrial materials for teratogenicity is examined. Seven assays for which correlation studies have been reported are reviewed. It is suggested that a battery of three to four tests appear to be sufficient to predict teratogenic potential. The author concludes that the use of a battery of selected in vitro teratogenicity tests can both achieve cost effectiveness and reduce the number of animal tests required in safety screening of research and development materials.

Welsh, J. J. 1987. Teratological research using in vitro systems. IV. Cells in culture. Environ. Health Perspect. 72:225-235 (40 refs.).

Several in vitro cellular systems designed to screen agents for teratogenic potential are described in this report. Included are cell-based systems that use analysis of tumor cell attachment; intercellular communication; growth of human embryonic palatal mesenchyme cells; progesterone production in porcine granulosa cells; differentiation of embryonic neural crest, limb bud, midbrain, and Drosophila cells; and differentiation of tumor cells. This list represents several of the more prominent systems now being evaluated by the scientific community.

Whitby, K. E. 1987. Teratological research using in vitro systems. III. Embryonic organs in culture. Environ. Health Perspect. 72:221-223.

Published literature identifies use of the following embryonic organs in culture: kidney, pancreas, skin, palate, craniofacial tissue, tooth, lens, bones, digits, and liver. However, only the in vitro organ culture of the palate and tooth are reviewed as potential teratogenic screens in this paper.

Toxicity

Oliver, G. J., et al. 1986. An in vitro skin corrosivity test--modifications and validation. Food Chem. Toxicol. 24(6-7):507-512.

An in vitro rat epidermal slice technique was developed for identifying chemicals with potential to cause a corrosive lesion in animal skin in vivo. This potential was correlated with the ability to lyse stratum corneum and was measured as a lowering of the electrical resistance of the skin slice. The results illustrate the usefulness of this in vitro technique for identifying skin-corrosive chemicals. Overall, the model has considerable potential as a pre-screen for conventional animal tests with the additional advantage of providing objective and quantifiable information.

Rhodes, C., et al. 1987. A balanced approach to the detection, characterisation and mechanism of the toxicity of industrial chemicals. J. Toxicol. Sci. 12(2):243-251.

Although animal toxicity studies reasonably predict the probable human response following exposure, they are a focus of a strong public lobby supported by many scientists to curtail studies using experimental animals. Much effort is devoted toward the development of "alternative" in vitro and ex vivo procedures. More attention needs to be directed toward the design requirements of the validation studies needed to test performance and reproducibility and the evaluation of the parameters of sensitivity and specificity of these in vitro and ex vivo procedures.