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ILAR Journal V33(3) 1991

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

NATIONAL ACADEMY PRESS Washington, D.C. 1991

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 alternatives 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 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.

Behavior

Perigo, G., W. C. Bryant, L. Belvin, and F.S. vom Saal. 1989. The use of live pups in a humane, injury-free test for infanticidal behavior in male mice. Anim. Behav. 38:897-8.

The objective was to determine whether one could accurately predict the behavior of a male mouse toward an unprotected pup based on his behavior toward a protected pup within a tube made of wire-mesh window/ fly screen. Ninety percent of the predictions based on a male's behavior toward the protected pup matched with his behavior toward the unprotected pup. Results demonstrate that the behavioral measurements necessary for infanticidal experiments can be accomplished humanely and accurately without risk or injury to live pups.

Carcinogenesis

Atchison, M., M. L. Atchison, and B. L. Van Duuren. 1985. Cocarcinogenesis in vitro using Balb/3t3 cells and aromatic hydrocarbon cocarcinogens. Cell Biol. Toxicol. 1(4):323-31.

The mouse skin cocarcinogens fluoranthene, pyrene, and undecane were used with the indirect-acting carcinogen, benzo(a)pyrene, and the direct-acting alkylating carcinogen, beta-propiolactone, in an in vitro transformation assay. Dose response, cytotoxicity, and transformation studies with these compounds were performed with a subclone (A31-1-1 ) of the Balb/3T3 cell line. All cells isolated from transformed foci showed characteristics of transformed cells including anchorage-independent growth.

Brock, W. A., F. L. Baker, and L. J. Peters. 1989. Radiosensitivity of human head and neck squamous cell carcinomas in primary culture and its potential as a predictive assay of tumor radiocurability. Int. J. Radiat. Biol. 56(5):751-60.

In this study, the tumors from 72 patients with head and neck squamous carcinoma have been grown in an adhesive tumor-cell assay system and radiosensitivity (S2: survival at 2.0 Gy) values have been measured. The characteristics of these cultures, such as growth rate, clonogenicity and growth enhancement by epidermal growth factor, do not correlate with S2. The average S2 value of the 72 cultures is 0.33, which is lower than for cultures derived from melanomas and lung adenocarcinomas. Twenty-six patients followed up for at least 15 months have been evaluated for local tumor control. The average S2 value of the seven patients with recurrences in this group is slightly higher (0.43) than that from the other patients (0.30). There is considerable overlap of S2 values in the two groups, and more patients must be evaluated before the groups can be compared statistically.

Fitzgerald, D. J., C. Piccoli, and H. Yamasaki. 1989. Detection of non-genotoxic carcinogen in the Balb/ C 3t3 cell transformation/mutation assay system. Mutagenesis 4(4):286-91.

Balb/C 3T3 cells were used in a simultaneous cell transformation and mutation assay protocol. Three known mutagenic animal carcinogens tested positive for transforming activity. Three nonmutagenic compounds, two human carcinogens and a possible human carcinogen, were tested. All three displayed transforming activity. These results support the validity of this system as a reliable short-term in vitro test for carcinogens.

Hanauske, U., A. R. Hanauske, G. M. Clark, D. Tsen, J. Buchok and D. D. Von Hoff. 1989. A new in vitro screening system for anticancer drugs for the treatment of non-small-cell lung cancer. Sel. Cancer Ther. 5(3):97-111.

A semiautomated radiometric assay (BACTEC 460 system) was examined for screening of activity of anticancer drugs against human non-small-cell lung cancer cell lines. Two cell lines (CALU-3, SK-MES-1) exhibited in vitro drug sensitivity patterns closest to those observed in clinical studies. These two cell lines might be most useful for screening in this system which has advantages over other, more time-consuming screening systems.

Ito, N., K. lmaida, R. Hasegawa, and H. Tsuda. 1989. Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis. CRC Crit. Rev. Toxicol. 19(4):385-415.

Medium term bioassay methods for hepatocarcinogens and nonhepatic organ systems have been developed as a bridge to overcome the disadvantages of in vitro short term tests and the longer 2 year assays. Male F344 rats were initiated with 0 or 200 mg/kg diethylnitrosamine. The test compound was given 2 weeks later, the rats killed at week 8 and the liver assayed for placental glutathione-S-transferase. Using 136 chemicals, 41 tested positive. Twenty-seven of 30 hepatocarcinogens gave positive results. None of the noncarcinogens gave positive results. This method uses fewer animals and is shorter than the conventional 2 year in vivo assay.

Kurian, P., S. Nesnow, and G. E. Milo. 1990. Quantitative evaluation of the effects of human carcinogens and related chemicals on human foreskin fibroblasts. Cell Biol. Toxicol. 6(2):171-84.

Ten compounds representative of diverse classes of chemicals were evaluated for their cytotoxicity and transforming ability to human skin fibroblasts in vitro. Anchorage-independent growth of exposed cells in soft agar was used as a biological endpoint for the expression of chemical transformation. The data suggest that anchorage-independent colony forming ability of exposed cells is a reliable marker to measure the carcinogenic potential of various hazardous chemicals.

Mason, J. M., R. Langenbach, M. D. Shelby, E. Zeiger, and R. W. Tennant. 1990. Ability of short-term tests to predict carcinogenesis in rodents. Annu. Rev. Pharmacol. Toxicol. 30:149-68.

This is a review in which in vitro genotoxicity assays, in vivo genotoxicity assays, and assays for nongenotoxic carcinogens are discussed.

Mothersill, C., A. Cusack, and C. B. Seymour. 1989. Enhanced proliferation of cells from human tissue explants following irradiation in the presence of environmental carcinogens. Radiat. Environ. Biophys. 28(3):203-12.

A method which allows growth of normal human tissue to be studied in vitro is used to investigate possible interactive effect of radiation and environmentally important in vivo carcinogens on esophageal and urothelial cell growth. Greatly enhanced cell proliferation could be detected at precise combinations of radiation and carcinogen, suggesting a synergistic interaction between the two agents. The results may have implications for the design and interpretation of experiments aimed at elucidating early or premalignant changes in epithelial tissues.

Rao, G. N., and J. Huff. 1990. Refinement of long-term toxicity and carcinogenesis studies. Fundam. Appl. Toxicol. 15(1):33-43.

Continual refinement of animal toxicity and carcinogenesis studies can be an effective means of reducing the numbers of animals used and conserving time and resources without compromising scientific quality. Adequate care of animals with control of variables such as light, temperature, diet, bedding, diseases, and genetic characters of laboratory animals will decrease the variability. Other considerations for refinement of carcinogenesis studies and the alleviation of pain and distress are given. One being the selection of genetically engineered animal models with known oncogenes to reduce the duration and increase the sensitivity of carcinogenesis studies with a reduction in the use of animals.

Sakai, A., and M. Sato. 1989. Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method. Mutat. Res. 214(2):285-296 (52 refs.).

Cultures of BALB/3T3-mouse clone A31-1-1 cells were treated with a test chemical dissolved in dimethyl sulfoxide for 72 hours, transferred to fresh normal medium and incubated for 3 days, treated with 12-O-tetradecanoylphorbol- 13-acetate for 2 weeks, grown in fresh normal medium for another 3 weeks, and finally fixed with methanol, stained with Giemsa, and examined for transformed foci. Results of experiments described in this publication lead the authors to conclude that the two stage BALB/3T3 transformation system appears to be sensitive to a wider range of potential carcinogens than the standard assay.

Strobl, J. S., K. L. Kirkwood, T. K. Lantz, M. A. Lewine, V. A. Peterson, and J. F. Worley III. 1990. Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine. Cancer Res. 50(17):5399-405.

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, measurement of [3H]thymidine incorporation and cell numbers were used to characterize effects of pimozide, thioridazine, W- 13, and W- 12 on MCF-7 human breast cancer cell growth. The authors conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of estrogen receptor positive and estrogen receptor negative human breast tumors.

Commentary

Balls, M., and R. Colthier. 1987. Validation of alternative toxicity test systems: lessons learned and to be learned. Mol. Toxicol. 1(4):547-59.

Validation in the context of in vitro toxicity tests is defined, and various aspects of the validation process are discussed. It is concluded that a thorough reevaluation of current practice is essential if the promise and potential of nonanimal toxicity tests are to be fully realized and if valid alternative tests acceptable to regulatory agencies are to be developed.

Comments on the LDs0 and acute eye and skin irritation tests. The animals in research committee of the society of toxicology and approved by the SOT council. 1989. Fundam. Appl. Toxicol. 13(4):621-3.

Conduct of any form of testing of potentially hazardous materials in animals, including lethality or eye and skin irritation testing, should be undertaken only after careful consideration of the necessity for, the objectives behind, and the possible alternatives to, such testing. Acute toxicity testing to determine an approximate lethal dose provides a basis for a comparison of the relative toxicities of different materials. These data are used to classify materials for transportation and labeling, to provide information for treatment of acute intoxications, to aid in dose selection for subsequent toxicity studies, and to provide comparison data for evaluation and validation of alternative methods in toxicology. Although the classical LDs0 test provides a general estimate of the quantity of chemical likely to cause death, much of the same information can be provided by other forms of testing in which significantly fewer numbers of animals are employed. Acute eye and skin irritation tests on chemical substances are conducted in order to characterize the hazards associated with ocular or dermal exposure. At present, tests in intact animals are the only means of assessing the potential hazard from such exposure other than direct testing in man. Although validated in vitro alternatives to eye and skin irritation tests in animals are not available currently, many tests under development show promise and may be useful as initial screening techniques. Complete validation of these alternate forms of testing for irritation may reduce the need to use whole animals. Until these procedures have been thoroughly tested and validated, the investigator will have to rely on conventional methods.

DePass, L. R. 1989. Alternative approaches in median lethality (LDs0) and acute toxicity testing. Toxicol. Lett. 49(2-3):159-70 (29 refs.).

The LDs0 test was introduced by Trevan in 1927 for biological standardization of dangerous drugs. Since then, the LDs0 has gained wide acceptance as a measure of acute toxicity of all types of substances. Recently, however, the LDs0 test has been criticized as an unnecessary waste of resources. Therefore, efforts have been made to reduce the number of animals used in such tests and to avoid using this test unless required by regulations. Future approaches include in vitro cytotoxicity methods and computer-based structure-activity models. It is felt that, when LDs0 tests are required, the most economical approach should be used, without undue concern for statistical precision.

Dresser, R. 1988. Standards for animal research: looking at the middle. J. Med. Philos. 13(2):123-43.

Much of the public debate over laboratory animal use has focused on either the scientist's demand for absolute freedom of inquiry, or the abolitionist's demand for an end to animal use in science. Yet many recent proposals for reform seek instead to balance the interests of laboratory animals in avoiding harm against the interests of research beneficiaries in continued animal use. This essay is an analysis of the intermediate reform positions and their underlying ethical principles.

Hampson, J. E., and S. R. Silcock. 1985. Animal protection and toxicity testing regulations. Food Chem. Toxicol. 23(2): 183-6.

Safety testing illustrates a very real ethical dilemma between moral obligations to protect humans and to protect animals from harm. The pragmatic approach to resolving this conflict is to pursue the 3 Rs of Replacement, Reduction and Refinement. Safety tests set out in regulation protocols often conflict with this ethic, being wasteful in numbers of animals used, having unnecessarily severe endpoints, and resulting in duplication. Suggestions for refinement of these tests are discussed. It is concluded that such refinement could lead to the generation of more meaningful data, thus improving both animal protection and human safety.

Lansdell, H. 1988. Laboratory animals need only humane treatment: animal "rights" may debase human rights. Int. J. Neurosci. 42(3-4):169-78.

Arguments for animal "rights" confuse the issue of what rights are about and, in the context of the care of laboratory animals, are misleading. Only human beings have rights and they should be cherished and extended. Consideration of the welfare of animals is important, but the context is that it is for the benefit of human beings and the animals serving humanity. Scientists need to explain the worth of animal research, particularly in regard to psychological studies. They also need to expose the fallacies in the animal rightists' arguments as one of the means to help diminish the threat to science.

Loeb, J. M., W. R. Hendee, S. J. Smith, and M. R. Schwartz. 1989. Human vs. animal rights: in defense of animal research. J. Am. Med. Assoc. 262(19):2716-20.

Recently, the American Medical Association prepared a detailed analysis of the controversy over the use of animals in research, and the consequences for research and clinical medicine if the philosophy of animal rights activists were to prevail in society. This article is a condensation of the Association's analysis.

Marcus, E. 1990. New research methods seen unlikely to eliminate animal testing. The Washington Post Aug. 28:A3.

Methods to replace animals in drug and cosmetic testing are discussed. Scientists who work with these methods say such tests can replace some animal testing now and that in coming years new tests will replace more animal testing. But they say it may never be possible to replace animals completely. Toxicologists give different reasons for why animal tests cannot be eliminated. Animal rights advocates contend that animal tests have major limitations and that the public does not need many of the products being tested.

Mehlman, M. A., E. A. Pfitzer, and R. A. Scala. 1989. A report on methods to reduce, refine and replace animal testing in industrial toxicology laboratories. Cell Biol. Toxicol. 5(3):349-58.

The Committee to Promote Principles of Reduction, Refinement and Replacement of Animal Testing in Industrial Toxicology Laboratories was established in 1987 to work toward industry-wide improvements in laboratory animal testing methods. This is the first in a continuing series of reports the committee plans to produce as part of an ongoing program to promote communication among industrial toxicologists about successful methods of reducing, refining and replacing animal testing.

Meier, J., and K. Stocker. 1989. On the significance of animal experiments in toxicology. Toxicon 27(1):91-104.

In increasing areas of the world, there is considerable controversy over the use of animals in scientific research. Ethical, scientific and economic reasons require a reduction in animal experiments as well as animal numbers, a refinement of existing methods and, wherever possible, a replacement of in vivo methods by in vitro test systems. By scoping on papers published in Toxicon between 1980 and 1986, the current situation of animal experimentation is evaluated. Methods are suggested to bring a reduction in the number of animal experiments.

Pakes, S. P. Contributions of the laboratory animal veterinarian to refining animal experiments in toxicology. Fundam. Appl. Toxicol. 15(1):17-24.

The role of the veterinarian in working with toxicologists to refine animal studies includes such activities as assisting in the planning stages of studies and in presubmission review of protocols, providing training in anticipation and recognition of pain and distress, providing information concerning pharmacologic and nonpharmacologic methods for alleviating or minimizing pain and distress, providing a high quality animal care and health program to support studies, and assisting the toxicology staff in monitoring all phases of the study and in addressing problems in a timely manner. Additional research is required to develop more objective methods for recognizing pain and for providing better methods for prevention and alleviation.

Poulsen, E. 1989. Influence of legislation in toxicology. Biomed. Environ. Sci. 2(1):30-5.

It is suggested that legislation for the protection of experimental animals has a profound influence in toxicology and accelerates the use of in vitro and other alternative methods.

Rabouhans, M. L. 1986. Reduction of animal usage: British Pharmacopoeia Commission policy. Dev. Biol. Stand. 64:11-6.

The British Pharmacopoeia Commission is committed to working towards the replacement or modification, wherever possible, of those assays and tests of the Pharmacopoeia that are performed in vivo and which may involve pain. An alternative or modified method is adopted by the Commission once it has been clearly demonstrated that it offers satisfactory control for pharmacopoeial purposes of the potency or other specific property of the preparation concerned. While in vitro alternatives remain the long term objective, the modification of existing in vivo methods to permit non-lethal endpoints and the possibility of using analgesics are also considered.

Rhodes, C. 1988. Current status of in vitro toxicity tests in the industrial setting: a European viewpoint. In Vitro Toxicol. 2(3):151-60.

The use of in vitro studies was reviewed and discussed in relation to approaches to reducing the cost of regulatory toxicology in Europe. Refinement of current methodologies was one obvious approach to reducing use of animals in toxicity testing. At present, there are few in vitro alternatives for in vivo studies in regulatory guidelines.

Steiger, A. 1989. [Animal welfare legislation and animal experiments in Switzerland--effects and challenges] Schweiz. Arch. Tierheilkd. 131(7):435-56.

This paper (written in German) describes Swiss animal welfare legislation, which restricts experiments on animals. It became effective in 1981. The discussions, the legal prescriptions, the official guidelines and the ethical principles for animal experimentation issued by scientists are treated. The legislation has slowly brought about positive effects. Animal experimentation has for several reasons shown a remarkable decline in the last few years. Research on alternative methods to animal experiments was substantially promoted. Further improvements in the field of animal experimentation are requested.

Zbinden, G. 1988. Reduction and replacement of laboratory animals in toxicological testing and research. Interim report 1984-1987. Biomed. Environ. Sci. 1(1):90-100.

The reasons that reduction and replacement of laboratory animals are advancing rapidly in basic biomedical research and why progress is much slower in industrial toxicology are analyzed. Encouraging developments concerning acceptance of new concepts in acute toxicity testing by various regulatory agencies are reviewed.

Cytogenetics

Eastmond, D. A., and J. D. Tucker. 1989. Identification of aneupioidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ. Mol. Mutagen. 13:34-43 (38 refs.).

A method was developed for the identification of possible aneuploidy inducing agents and rapid assay using cytokinesis blocked human lymphocytes and an antikinetochore antibody. The assay involved the chemical or radiation induced formation of micronuclei in the lymphocytes and the use of the antibody to determine whether the micronuclei contained centromeres, an indicator of high potential for aneuploidy. The authors conclude that the assay described in this publication can discriminate between aneuploidogens and clastogens and may allow a more rapid identification of chemicals with aneuploidy inducing potential.

Migliore, L., M. Nieri, S. Amodio, and N. Loprieno. 1989. The human lymphocyte micronucleus assay: a comparison between whole-blood and separated-lymphocyte cultures. Mutat. Res. 227(3):167-172 (6 Refs.).

The chemical induction of micronuclei was examined in whole blood cultures and separated lymphocyte cultures. With both assays the cytokinesis block method was used to discriminate binucleate cells after the first cell cycle from undivided cells. Blood samples from the same healthy female donor were used for each set of experiments. The results of these assays suggested that whole blood cultures may be considered a better experimental condition for the detection of micronuclei induced by chemicals in vitro.

Nusse, M., M. Kramer, S. Viaggi, A. Bartsch, and S. Bonatti. 1987. Antikinetochore antibodies and flow karyotyping: new techniques to detect aneuploidy in mammalian cells induced by ionizing radiation and chemicals. Mol. Toxicol. 1(4): 393-405 (24 Refs.).

Aneuploidy induction by chemicals and ionizing radiation was studied in vitro in Chinese-hamster-V79 cells and Chinese-hamster-CCHE/27 embryo cells. After treatment the cells were stained with antibodies to kinetchores using the serum of a scleroderma patient. The DNA in the nuclei and micronuclei were counter-stained with propidium-iodide. The frequency of micronuclei, the fraction of kinetochore positive and negative micronuclei per cell, and the fraction of kinetochore positive micronuclei were determined using an immunofluorescence technique. Cultures of CHE-O cells, a clone isolated from CCHE/27 cells, were irradiated with 0 or 15Gy gamma rays. Surviving cells were stained with propidium-iodide and the fluorescence distribution of the chromosomes was determined by a flow cytometric technique. The authors conclude that the in vitro micronuclei kinetochore assay appears to be a useful technique for distinguishing between micronuclei containing acentric fragments and those containing whole chromosomes or centtic fragments.

Park, E. H., J. S. Lee, A. K. Yi, and H. Etoh. 1989. Fish cell line (ULE-23HU) derived from the fin of the central mudminnow (umbra linti): suitable characteristics for clastogenicity assay. In Vitro Cell. Dev. Biol. 25(11):987-994.

A cell line (ULF-23HU) from the fin of the central mudminnow (Umbra limi) was characterized and tested for its suitability to assess cytogenetic damages induced by chemicals in fish. The results on the growth kinetics and cytogenetic characteristics offered the high possibility of the use of this cell line as a suitable in vitro model for clastogenicity studies in fish.

Petcharuttana, Y., G. R. Cutter, R. G. Meeks, and A. E. Lorincz. 1989. Fluorescence microscopy of DES-induced morphologic transformation in unfixed cultured cells. J. Oral Pathol. Med. 18(8):451-456.

Cultured baby Syrian hamster kidney cells (BHK 21), synchronized with hydroxyurea, were treated with varying amounts of the carcinogen diethylstilbestrol during the first mitosis. DES-induced morphologic transformation was assessed by fluorescence microscopy of acridine orange, supravitally stained cells. Traditional karyotyping techniques were used to monitor DES-induced aneuploidy in parallel cell cultures. A definite dose-response relationship for both morphologic transformation, as well as for aneuploidy was observed. These findings portend a significance for use of fluorescence microscopy of morphologic transformation of unfixed supravitally stained mammalian cells, for the rapid assessment of compounds that promote aneuploidization and carcinogenesis.

Roy, A. K., G. Talukder, and A. Sharma~ 1990. Effects of aluminum sulfate on human leukocyte chromosomes in vitro. Mutat. Res. 244(2):179-184.

Information on the effects of aluminum on chromosomes in vitro in mammalian systems is relatively meager. The present work was undertaken to study the effect of aluminum sulphate on human lymphocytes stimulated to proliferate in culture, as associated with factors such as the age and sex of the donor.

Cytology

Lirsac, P. N., D. Nolibe, and H. Metivier. 1989. Immobilization of alveolar macrophages for measurement of in vitro dissolution of aerosol particles. Int. J. Radiat. Biol. 56(6):1011-21.

The dissolution of aerosol 57Co304 particles was investigated by exposing baboon alveolar macrophages (AM) to the particles and immobilizing the AM in alginate macrobeads crosslinked with Ca2+ ions. The rate obtained with immobilized AM is very close to the rates previously found for adherent AM in culture. Moreover, AM that had phagocytosed PuO₂ particles before immobilization by the same method gave a mean daily dissolution rate close to that obtained in vivo with baboons. It is also demonstrated that immobilization did not affect AM viability. The present method of immobilization seems very promising compared to conventional methods because it facilitates manipulation, allows the use of cells at a high density in a small volume, and gives results for particles with a very low dissolution rate.

Osteen, K. G., G. A. Hill, J. T. Hargrove, and F. Gorstein. 1989. Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil. Steril. 52(6):965-72.

This report describes an improved method for isolation of endometrial epithelial and stromal cells, using biopsy specimens as a tissue source. Separated cells were obtained using selective enzymatic digestion in conjunction with physical separation procedures. Using this system, isolated cells from normal endometrium can readily be obtained for in vitro studies. Within the defined conditions of a culture system, important areas of current concern in the endometrium such as ectopic endometrial growth and implantation can be addressed.

Shaw, K. Y., J. S. Tang, and C. F. Chao. 1989. Functional evaluation of cultured rabbit osteoblast-like cells. Proc. Natl. Sci. Counc. Repub. China lB] 13(3): 192-200.

Using methods described in this publication it was indicated that the cells isolated from young rabbit long bone endosteal were osteoblast-like and still maintained their biological function. The system for culturing osteoblast-like cells is described as a successful attempt at growing bone tissue in vitro starting from isolated bone cells. Therefore, this modified method for bone cell culture on collagen precoated culture flasks could be used as the experimental model in studies concerning the osteoblasts in vitro.

Cytotoxicity

Arenholt-Bindslev, D., P. Horsted-Bindslev, and H. P. Philipsen. 1987. Toxic effects of two dental materials on human buccal epithelium in vitro and monkey buccai mucosa in vivo. Scand. J. Dent. Res. 95(6):467-74.

Confluent cultures of human buccal epithelial cells were exposed to graded dilutions of Gluma Bond or 3M Etching Liquid for 5 minutes. In vivo, monkey buccal mucosa was exposed to the same agents for 5 minutes. Data obtained suggest that the in vitro model may be useful in assessing mucosal toxicity and in studying mechanisms of toxic action.

Acosta, D., G. C. Hsieh, and P. J. Davis. 1990. Cultured eukaryotic fungal cells as an alternative test system for accessing the cytotoxicity of drugs and chemicals. The Toxicologist 10(1):327(Abstr. 1308).

In this study, the cytotoxicity of bromobenzene and amitriptyline, well-known hepatotoxicants, was compared in primary cultures of postnatal rat (8-10 days old) hepatocytes and cultured fungal cells of Cunninghamella echinulata (ATCC 9244). Reduction of tetrazolium MTT, leakage of cytosolic lactate dehydrogenase (LDH), of cellular alkaline phosphatase (ALPase) activity, and changes in lactate (L)/pyruvate (P) ratios were used for toxicity evaluation. Growth inhibition was determined in fungal cell cultures. The data suggest that eukaryotic fungal cells may be utilized as an experimental model to screen for compounds potentially toxic to humans and/or animals.

Babich, H., S. H. Goldstein, and E. Borenfreund. 1990. In vitro cyto- and genotoxicity of organomercurials to cells in culture. Toxicol. Lett. 50(2-3): 143-9.

BG/F epithelioid cells derived from fin tissue of bluegill sunfish were used to study cytotoxic effects for a series of mercury compounds. The in vitro cytotoxicity was similar to that observed in a 48-h LCs0 in vivo acute toxicity assay with rainbow trout. Using induction of micronuclei as an indicator of genetic damage, the organomercurials, but not mercuric chloride, were noted to be clastogenic to the BG/F cells.

Bombick, D. W., and D. J. Doolittle. 1990. Plasma membrane characteristics as indices of in vitro toxicity. The Toxicologist 10(1):328(Abstr. 1309).

The cellular plasma membrane is one potential site of action for chemically induced cytotoxicity. The objective of this study was to evaluate the relative sensitivity of several indicators of plasma membrane function as indicators of in vitro cytotoxicity. Plasma membrane parameters examined include assessments of cell shape, membrane fluidity, LDH release and ability to exclude a fluorescent dye. The development of mechanistically oriented cytotoxicity indices should be valuable for establishing strategies for toxicological evaluation.

Borenfreund, E., and O. Borrero. 1984. In vitro cytotoxicity assays. Potential alternatives to the draize ocular allergy test. Cell. Biol. Toxicol. 1(1):55-65.

A short-term cytotoxicity assay carried out in multiwell test plates and a supplementary colony forming assay are both useful for screening and range finding of toxic concentrations of test agents. The highest tolerated dose (HTD), a concentration at which only minimal morphological changes were observed, was designated as endpoint in the assay. Epithelial rabbit cornea cells, murine fibroblasts, Chinese hamster lung cells, human heparoma cells and mouse macrophage cultures were used as targets. An ID50 of colony formation was used as a quantitative corroborating test. This easily reproducible, rapid in vitro test is cost-effective and can be used for preliminary large scale screening of potential toxicants.

Chiba, K., and K. Tohyama. 1989. Safety evaluation of oil ingredients for cosmetics using cell culture system. Nippon Koshohin Kagakkaishi 13(4):194-202.

This article (in Japanese) describes cytotoxicity tests for the safety evaluation of 42 oil ingredients for cosmetics carried out in vitro by using cultured epithelial cells derived from human skin (JTC-17). The cells were exposed to the test substances which were dispersed in the cultured medium using an ultrasonic emulsion method, and cell viability was estimated by Neutral Red dye uptake. Comparison was made between the results of in vitro tests and primary irritancy tests on rabbit skin. Though not all of the primary skin irritants show toxicity to the cultured cells, the in vitro cytotoxicity test described here seems to be simple and useful as a first screening test for primary skin irritants of oil ingredients for cosmetics.

Clothier, R. H., L. M. Hulme, M. Smith, and M. Balls. 1988. Comparison of the in vitro cytotoxicities and acute in vivo toxicities of 59 chemicals. Mol. Toxicol. 1(4):571-7.

The in vitro cytotoxicities of 59 chemicals, expressed as ID50 (i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control cultures) and obtained using murine 3T3-L1 cells and the FRAME kenacid blue method, were compared with rat oral and mouse ip LDs0. A better in vivo/in vitro correlation was obtained for the chemicals with mouse ip LDs0 (r = 0.80) than with rat oral LD50 (r = 0.76). The best correlation was found when the most toxic values were used in the comparison (r = 0.81).

Dal Pozzo, O., P. A. Bernabei, V. Santini, R. Saccardi, S. Santini, and P. Rossi Ferrini. 1989. A comparison of two assays for the in vitro chemosensitivity testing of human acute non-lymphoblastic leukemia cells. J. Chemother. 1(5):318-23.

In vitro chemosensitivity of blast cells from 19 patients, affected by acute non-lymphocytic leukemia, to cytosine arabinoside and daunorubicin was investigated. A semiautomated P-iodonitrotetrazolium violet (INT) colorimetric assay, based on the ability of viable cells to cleave piodonitrotetrazolium violet into a red formazan derivative and a short-term antimetabolic assay based on the uptake inhibition of [3H]-thymidine were compared in terms of their ability to predict clinical outcome. Both methods were able to discriminate between sensitive and resistant patients by in vitro cytosine arabinoside testing. A good correlation was demonstrated between the results of the two different methodological approaches, validating the recently described INT sensitivity test.

Ford, C. H. J., V. J. Richardson, and G. Tsaltas. 1989. Comparison of tetrazolium colorimetric and [3H]uridine assays for in vitro chemosensitivity testing. Cancer Chemother. Pharmacol. 24(5):295-301.

A colorimetric assay for assessing chemosensitivity with the advantages of safety, cost, and simplicity was previously described and relies on the ability of living cells to reduce a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MMT), into an insoluble formazan precipitate. A modified MTT assay for chemosensitivity screening is described but further modifications seem necessary to improve the sensitivity and decrease the problem of cell loss after washing.

Hanson, J. A., E. A. Bean, S. R. Nute, and J. L. Moore. 1989. A novel dye exclusion assay for measurement of cell response following in vitro exposure to radiation or drugs. Leuk. Res. 13(10):943-7.

A novel dye exclusion assay based on differential staining of fixed cells has been evaluated using the MOLT4 (T-lymphoblastic leukaemia) and DAUDI (Burkitt lymphoma) cell lines. Reproducible differential staining was found with fixed samples kept for up to 2 months. Reproducible dose-response data were obtained with cells treated with radiation or drugs and assayed after 4 days in culture. This ability to fix cells prior to measurement of viability is of general use particularly where time constraints prevent haemocytometer counting. More specifically, it may have potential use in the field of chemosensitivity testing particularly where large sample numbers require rapid processing.

Hanson, J., E. A. Bean, and J. L. Moore. 1989. The MTT assay used to assess in vitro xenograft cell response to radiation. Int. J. Radiat. Biol. 56(5):846.

The basic principle of the MTT assay is the reduction by viable cells of a tetrazolium salt producing formazan crystals. These are solubilized and the resultant absorbance measured. The MTT assay was evaluated with the aim of measuring the radiosensitivity of new xenograft cell lines. The assay has advantages in terms of speed and simplicity over other assays measuring cell response and providing the cell conditions and time of assay are optimized for each cell line. It is a useful additional test system.

Hoh, A., K. Maier, and R. M. Dreher. 1987. Multilayered keratinocyte culture used for in vitro toxicology. Mol. Toxicol. 1(4):537-46.

Human keratinocytes are the most appropriate target cells for evaluating mechanisms of skin cytotoxicity and pharmacology of chemical agents. The advantage of the test described here is that it uses adequate target cells and that it evaluates both the ability of the test chemical to penetrate several cellular layers as well as the ability to interfere with cellular function. The endpoints are cell viability and cell metabolism, which are determined by neutral red uptake and MTT reduction, respectively. This test system might be in its present stage a supplement to current tests which may replace in vivo irritation tests like the Draize test.

Hulme, L. M., H. L. Reeves, R. H. Clothier, M. Smith, and M. Balls. 1987. Assessment of two alternative methods for predicting the in vivo toxicities of metallic compounds. Mol. Toxicol. 1(4):589-596 (17 refs.).

The ability of two in vitro techniques, consisting of the FRAME cytotoxicity test and calculating the softness of the metal, to predict in vivo toxicity of metallic compounds was evaluated. The FRAME test consists of incubating the test compound with mouse-3T3-L1 fibroblasts and determining the concentration required to reduce the cellular protein concentration by 50 percent. Metal softness of the metal ion in the test compound is calculated from the values of the coordinate bond energies for forming the iodide and fluoride. The authors conclude that of the two methods, the FRAME cytotoxicity test is the better predictor of in vivo toxicity.

Hulme, L. M., H. L. Reeves, R. H. Clothier, M. Smith, and M. Balls. 1988. Assessment of two alternative methods for predicting the in vivo toxicities of metallic compounds. Mol Toxicol. 1(4):589-96.

The FRAME in vitro cytotoxicity assay and a physicochemical parameter for metal ions were assessed for their ability to predict the in vivo acute toxicities of 52 metallic compounds. The in vitro toxicity values (expressed as IDs0 i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control culture values) correlated better with mouse ip LDs0 values than with rat oral LDs0 values.

Iselt, M., W. Holtei, and P. Hilgard. 1889. Tetrazolium dye assay for rapid in vitro assessment of cytotoxicity. Arzneim. Forsch. 39(7):747-749 (9 refs.).

A new method for the solubilization of formazan crystals, which makes the tetrazolium dye assay more rapid, simpler and more reproducible, is described. The value of the improved assay for the in vitro determination of cytotoxicity is discussed.

Jaurand, M. C., I. Bastie-Sigeac, M. J. Paterour, A. Renier, and J. Bignon. 1983. Possibility of using rat mesothelial cells in culture to test cytotoxicity, clastogenicity, and carcinogenicity of asbestos fibers. Annals of the New York Academy of Sciences 407:409-411 (9 refs.).

The cytotoxicity, clastogenicity, and carcinogenicity of asbestos fibers were examined in rat pleural mesothelial cell cultures. Observed effects were cytoplasmic vacuolation, binucleated cell formation, growth inhibition, and sister chromatid exchange. From results of experiments reported in this publication the authors conclude that rat pleural mesothelial cells in culture provide a useful model for the study of the interactions between asbestos and mesothelial cells.

Larsson, R., and P. Nygren. 1990. Pharmacological modification of multi-drug resistance (MDR) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin A and verapamil on MDR leukemia T-cells. Int. J. Cancer 46(1):67-72.

A novel fluorometric microculture cytotoxicity assay, based on measurements of fluorescein diacetate hydrolysis and DNA staining by Hoechst 33342, was used for drug sensitivity testing and detection of resistance reversal in acute lymphoblastic leukemia cell lines. The 72-hour assay was found to be sensitive, reproducible and linearly related to the number of viable cells within a broad range of cell concentrations.

Lass, J. H., R. J. Mack, P. S. Imperia, K. Mallick and H. M. Lazarus. 1989. An in vitro analysis of aminoglycoside corneal epithelial toxicity. Curr. Eye Res. 8(3):299-304.

The cytotoxicity of four aminoglycoside agents was evaluated using an in vitro confluent rabbit corneal epithelial cell culture model. Cultures were established and cells replated. After 48 hours either vehicle or an antibiotic was added, each for 5, 30, or 60 minutes. 3H-thymidine was added and incorporation was measured. The in vitro data derived from these tests corroborates the animal and limited clinical data available for the aminoglycosides.

Maile, W., T. Lindl, and D. G. Weiss. 1987. New methods for cytotoxicity testing: quantitative video microscopy of intracellular motion and mitochondria-specific fluorescence. Mol. Toxicol. 1(4):427-437 (16 refs.).

The effects of 2-hydroxy-ethylmethacrylate on human-IMR-90 fibroblasts were examined. Cytotoxicity was assessed by staining the cells with a fluorescent dye and determining the velocity of lysosomal movement and observing changes in the mitochondria and fine structure by Allen video enhanced contrast differential interference contrast (AVEC/DIC) microscopy. Cytotoxicity was also evaluated by measuring lactate-dehydrogenase (LDH) release and trypan-blue exclusion and by morphometric analysis for comparison purposes. The authors conclude that the AVEC/DIC technique permits cell organelle dynamics to be quantitatively assessed. The technique is a more sensitive test system for assessing cytotoxicity than conventional techniques.

Marinovich, M., E. Tragni, A. Corsini, and C. L. Galli. 1990. Quantification of in vitro cytotoxicity of surfactants: correlation with their eye irritation potential. J. Toxicol. Cutaneous Ocul. Toxicol. 9(3): 169-78.

A number of surfactants were studied for their effects on lactate dehydrogenase (LDH), protein content, and de novo synthesis in a murine epidermal cell line (HEL/30). Protein synthesis, evaluated as [3H]leucine incorporation into cell proteins, was a more sensitive endpoint of toxicity than LDH leakage or protein content. Comparative surfactant toxicity followed the general order of cationic > anionic > nonionic > amphoteric. A good rank correlation was observed between their relative toxicity produced in vitro and the eye irritation produced in vivo in the Draize test. This sensitive and reproducible in vitro method may offer a means for screening potentially irritating water-soluble chemicals such as surfactants to quantify their toxicity.

Mossman, B. T., and A. M. Sesko. 1990. In vitro assays to predict the pathogenicity of mineral fibers. Toxicology 60(1-2):53-62.

In an effort to find alternative approaches to animal testing, cells (rat alveolar macrophages, hamster tracheal epithelial cells, rat lung fibroblasts) of the respiratory tract have been evaluated for cytotoxic and metabolic changes after exposure to fibers and particles. Release of superoxide from alveolar macrophages, release of 51 Chromium in hamster tracheal epithelial (HTE) cells, and an increase in the ratio of collagen to non-collagen protein in the cell line (RL-82) of rat lung fibroblasts are taken as indicators of the fibrogenic potential of minerals. Results suggest that a battery of in vitro assays can be used to screen the capability of minerals to elicit cell damage and inflammatory changes in the respiratory tract.

Naughton, B. A., R. A. Preti, and G. K. Naughton. 1989. Suspended nylon screen long-term bone marrow cultures as substrates for cytotoxicity determinations. Attem. Methods Toxicol. 7(In Vitro Toxicol:New Dir.) 245-53.

A long-term bone marrow culture system developed in the laboratory should serve as an excellent substrate for cytotoxicity determinations. Nylon filtration screen is inoculated with bone marrow stromal cells and suspended in flasks containing liquid medium. A second inoculation of hematopoietic cells is applied after approximately 70% of the mesh spaces are spanned by developing stroma. A three-dimensional growth pattern is observed and extensive hematopoietic colonization occurs in the stromal interstices. These cultures are easily manipulable and support multilineage hematopoietic expression. Assessment of cytotoxic effects can be performed by phenotypic and/or hematopoietic progenitor analysis of the adherent zone. It may also be possible to test the effects of potential therapeutic regimens on a patient's bone marrow cells in vitro before they are used in vivo.

Ponec, M., M. Haverkort, Y. L. Soei, J. Kempenaar, and H. Bodde. 1990. Use of human keratinocyte and fibroblast cultures for toxicity studies of topically applied compounds. J. Pharm. Sci. 79(4):312-6.

In order to obtain more insight into the potential skin toxicity of penetration enhancers, they were administered to cultured human keratinocytes and fibroblasts and the following cytotoxicity assays were performed: inhibition of the proliferation of fibroblasts and keratinocytes; inhibition of collagen contraction by fibroblasts; and cell morphology changes in confluent cultures of fibroblasts and keratinocytes. An obvious advantage of the in vitro model presented here is its immediate availability and reproducibility, which allows for the comparison of a large series of topical agents with respect to their cell toxicity under standardized conditions. However, this single-cell model lacks some of the properties found in intact skin, such as the stratum corneum barrier, and interactions between keratinocytes and other cells, such as Langerhans cells. Hence, extrapolation of these data to in vivo should be done with caution.

Rahn, C. A., D. W. Bombick, and D. J. Doolittle. 1990. Mitochondrial membrane potential as an indicator of in vitro cytotoxicity. The Toxicologist 10(1):328(Abstr. 1310).

In this study, mitochondrial membrane potential, an integral component of cellular energy, homeostasis, and normal cellular function, was examined for potential use in an in vitro assay to evaluate chemically induced cytotoxicity. Cultured rat liver epithelial cells or human skin fibroblasts were used to characterize the system. This assay system should provide a valuable tool for the evaluation of chemical treatments on mitochondrial membrane potential, as well as a mechanistically based indicator of cytotoxicity which does not require the use of animals.

Reinhardt, C. A. 1987. Do we find relevant parameters for in vitro cytotoxicity testing? Mol. Toxicol. 1 (4):383-91.

The current strategy for the design of cytotoxicity tests was reviewed in light of goals, and in the capacity of in vitro tests to fulfill expectations for alternative methods to animal testing. Past experience with in vitro and short-term tests for mutagenicity indicate that it is not wise to look for one single supertest. The relevance of an integrated approach must be aimed directly at the organisms that may be exposed.

Reubel, G. H., M. Gareis, and W. M. Amselgruber. 1989. Effects of the fusarium mycotoxins zearalenone and deoxynivalenol on the mitochondrial methylthiazoltetrazolium-cleavage activity of mono-layer cells. Toxicol. In Vitro 3(4):311-316.

The application of a modified colorimetric assay for the evaluation of mycotoxin-derived cytotoxicity is reported. Using four mammalian monolayer cell types (swine kidney, African green monkey kidney, Madin Darbin canine kidney and bovine embryonic lung cells), the influence of the mycotoxins zearalenone and deoxynivalenol on the cellular methylthi-azoltetrazolium (MTT)-cleavage activity was evaluated after different toxin exposure times. The yellow tetrazolium salt is converted by mitochondrial enzymes of metabolically active cells into purple formazan products. These crystals were dissolved in dimethyl sulphoxide to obtain a homogeneous solution, the optical density of which was suitable for precise spectrophotometric measurement by a plate reader. The results indicate that monolayers are useful targets in the MTT assay for investigating the action of mycotoxins and for performing comparative studies in mammalian cells.

Safavi, K. E., L. S. Spangberg, N. S. Costa, Jr., and G. Sapounas. 1989. An in vitro method for longitudinal evaluation of toxicity of endodontic sealers. J. Endod. 15(10):484-6.

An in vitro method for longitudinal evaluation of root canal sealers was developed and applied. A newly introduced cell culture chamber was used to evaluate the cytotoxic effects of test samples immediately after mixing and for an extended period of time thereafter. A ranking of the test materials, based on their cytotoxicity, was allowed by the method.

Seibert, H., M. Kolossa, and O. Wassermann. 1989. Bovine spermatozoa as an in vitro model for studies on the cytotoxicity of chemicals: effects of chlorophenols. Cell Biol. Toxicol. 5(3):315-30.

The suitability of ejaculated bovine spermatozoa as an in vitro model for the assessment of the cytotoxic potential of chemicals was evaluated using several endpoints: swimming activity, adenine nucleotide content, membrane integrity and oxygen consumption. The results indicate that bovine spermatozoa may become a useful in vitro model for the toxicological evaluation of chemicals providing quantitative as well as qualitative data.

Shopsis, C., E. Borenfreund, J. Walberg, and D. M. Stark. 1985. A battery of potential alternatives to the Draize test: uridine uptake inhibition, morphological cytotoxicity, macrophage cherootaxis and exfoliative cytology. Food Chem. Toxicol. 23(2):259-66.

Four assays that may serve as components of a battery of alternatives to the conventional Draize test are described. The exfoliative cytology assay is a refinement of the Draize test that may provide a more sensitive and more objective endpoint. The macrophage migration assay addresses the inflammatory aspects of the physiological response to irritation. The uridine uptake inhibition assay uses a quantitative, reversible endpoint to detect the short-term action of agents on cell membranes and cell phosphorylative potential. Finally, the cytological assay serves as a rapid, easily performed general indicator of cytotoxic action. The two latter assays have been demonstrated to correlate very well with Draize test results and with each other for a wide range of test agents.

Silber, P., T. J. Stephens, B. Reece, D. Long, and B. Bryan. 1990. The use ofBalb/C fibroblasts and the protozoan tetrahymena thermophila as in vitro alternatives to in vivo safety testing. The Toxicologist 10(1):327 (Abstr. 1306).

In this study, a battery of four in vitro cytotoxicity tests was examined as an alternative to oral, ocular and dermal toxicity testing in animals. The in vitro battery included the neutral red uptake, total protein and enzyme (LDH) release endpoints in Balb/C 3T3 fibroblasts, and a Tetrahymena thermophila motility assay. Data from these in vitro assays were compared to historical in vivo studies. Although there was significant correlation of in vitro and in vivo test data, the relative sensitivity of the battery was dependent on the class of test materials. Results showed that this battery of in vitro tests serves as a valuable toxicity screen.

Skehan, P., R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney, and M. R. Boyd. 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82(13):1107-12.

A rapid, sensitive, and inexpensive method was developed for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method relies on protein binding of the dye sulforhodamine B with determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The sulforhodamine B assay provides a colorimetric endpoint that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantRating clonogenicity, and is well suited to high-volume, automated drug screening. Sulforhodamine B fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

Srivastava, S., S. D. Gorham, and J. M. Courtney. 1990. Screening of in vitro cytotoxicity by the adhesive film test. Biomaterials l l (2): 133-7.

The adhesive film test is proposed as a screening method to predict potential cytotoxicity of biomaterials. This in vitro test utilizes a sterile strip of acrylate-based medical adhesive as an anchorage substrate in cytotoxicity studies. The adhesive film allows direct fixation of test samples to the base of the petri dish, ensuring close contact between sample and cells. The test is based on the principle that toxic components present in the test material will readily leach out into the culture medium and adversely affect the local cell population. The main advantage of the adhesive film test is that a viable cell population can be added directly to the test plate and after an incubation period of 24 hours, the cellular response can be recorded as either cytotoxic or cytocompatible. Microscopic examination can be followed by quantifying the results using a micrometer to measure cellular attachment areas, migration distances and zones of inhibition. In addition, the adhesive film used to attach the test samples is shown to support extensive fibroblast growth and attachment to its surface and hence can also function as a negative nontoxic control in cytotoxicity studies.

Tyson, C. A., S. J. Gee, K. Hawk-Prather, D. L. Story, and H. A. Milman. 1989. Correlation between in vivo and in vitro toxicity of some chlorinated aliphatics. Toxicol. In Vitro 3(2): 145-50.

The release of lactate dehydrogenase and aspartate aminotransferase from rat and mouse hepatocytes was used to rank the in vitro cytotoxic potential of 8 chlorinated aliphatic compounds. The rankings were compared with the maximum tolerated doses for each species. Significant correlations between the rankings were obtained. These data support the use of isolated hepatocytes for screening of chemicals for relative toxicity.

Yoshimoto, Y., Y. Hara, T. Abe, A. Akamine, K. Maeda, and M. Aono. 1989. Basic studies on calcium oxide-phosphorus pentoxide-magnesia-silica-calcium fluoride system glass ceramics. 1. Morphology under the phase-contrast microscope and growth of cultured cells. Nippon Shishubyo Gakkai Kaishi 31 (2):640-50.

This paper, written in Japanese, describes experiments to determine the biocompatibility of glass ceramics. In vitro studies were carried out by a cell culture method using four established cell lines. Phase-contrast microscopy was used to determine the condition of cells after periods of contact with glass ceramic disks. From the results of these in vitro experiments the authors conclude that glass ceramics possess the characteristics needed for bone grafts and implant materials.

Zhang, S. Z., M. M. Lipsky, B. F. Trump, and I. C. Hsu. 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):219-34.

Neutral red in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. Uptake was proportional to the concentration of the neutral red solution and the numbers of viable liver cells. Neutral red can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. The cytotoxic effects of dimethylnitrosamine and aflatoxin BI were examined by the neutral red assay on rat and human hepatocyte cultures and found to be dependent on dose and time of exposures. Compared with the lactate dehydrogenase leakage test, the neutral red assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Dental

Al-Nazhan, S., and L. Spangberg. 1990. Morphological cell changes due to chemical toxicity of a dental material: an electron microscopic study on human periodontal ligament fibroblasts and L929 cells. J. Endod. 16(3):129-34.

In order to study the morphological changes taking place in cells exposed to new endodontic materials with polymer bases, L929 cells and human periodontal fibroblasts were observed using scanning electron microscopic and transmission electron microscopic techniques. The morphological changes of the cells were correlated to the quantitative results observed simultaneously in cytotoxicity studies using the radiochromium release method. Results indicate that it may be proper to use periodontally derived cells for the study of cytotoxic mechanisms of polymer endodontic filling materials.

Arenholt-Bindslev, D., and P. Horsted-Bindslev. 1989. A simple model for evaluating relative toxicity of root filling materials in cultures of human oral fibroblasts. Endod. Dent. Traumatol. 5(5):219-26.

Test tubes filled with freshly mixed root filling materials were transferred into tissue culture flasks. Normal human oral fibroblasts were seeded in the flasks. Morphological cell changes were studied up to 15 days. It was concluded that the present model can be used as a simple and relatively cheap screening test for initial toxicity testing of dental materials.

Kasugai, S., N. Hasegawa, and H. Ogura. 1990. A simple in vitro cytotoxicity test using the MTT (3-(4,5)-dimethylthiazol-2-YL)-2,5-diphenyl tetrazolium bromide) colorimetric assay: analysis of eugenol toxicity on dental pulp cells (RPC-C2A). Jpn. J. Pharmacol. 52( 1 ):95-100.

A clonal fibroblastic cell line, (RPC-C2A), from rat incisal dental pulp was used to examine the effectiveness of the MTT colorimetric assay to test for the toxicity of eugenol. Dimethyl sulfoxide was used to attack the problem of insolubility of MTT formazan produced by the activity of mitochondria dehydrogenases. Although the correlation between spectrophotometric absorbance and cell numbers was not completely linear, this method could be used effectively as a simple preliminary assay to test for the toxicity of dental drugs and materials.

Developmental toxicity

Angle, C. R., D. J. Thomas, and S. A. Swanson. 1990. Lead inhibits the basal and stimulated responses of a rat osteoblast-like cell line ROS 17/2.8 To 1 alpha, 25-dihydroxyvitamin D3 and IGF-I. Toxicol. Appl. Pharmacol. 103(2):281-7.

Low-level exposure to lead impairs longitudinal growth in children and in experimental animals. The proposed mechanisms include decreased osteocalcin secretion in response to 1 alpha,25-(OH)2 vitamin D3 and decreased response to insulin-like growth factor-I. The interaction of lead, 1 alpha,25-(OH)2 vitamin D3, and insulin-like growth factor-I was investigated in an osteoblast-like cell line from rat osteosarcoma, ROS 17/ 2.8. The data are consistent with a toxic effect of lead on osteoblastic function and the cellular responses to 1 alpha,25-(OH)2 vitamin D3 and insulin-like growth factor-I. This interaction may be relevant to impaired childhood growth at low levels of lead exposure.

Bantie, J. A. 1990. Further development and validation of the frog embryo teratogenesis assay-xenopus (FETAX). Govt. Reports Announcements & Index (GRA&I) Issue 04:NTIS/AD-A213 868/3.

The goal of this project was to develop and validate the Frog Embryo Teratogenesis Assay Xenopus (FETAX). FETAX is a 96 hour whole embryo bioassay designed to rapidly assess the developmental toxicity of pure compounds or complex mixtures. The four specific objectives of this study were: 1) validate FETAX using direct-acting compounds of known mammalian developmental toxicity, 2) develop and validate an in vitro metabolic activation system using rat liver microsomes, 3) explore the use of three carrier solvents which will help solubilize non-polar compounds and, 4) develop an atlas of malformations which will assist users in performing FETAX. Endpoints of FETAX are mortality, malformation and growth inhibition.

Regan, C. M., A. M. Gorman, O. M. Larsson, C. Maguire, M. L. Martin, A. Schousboe, and D. C. Williams. 1990. In vitro screening for anticonvulsant-induced teratogenesis in neural primary cultures and cell lines. Int. J. Dev. Neurosci. 8(2):143-50.

To establish inherent potential for the induction of neural tube defects, the ability of selected anticonvulsant agents to interfere with cell division has been established in vitro using an antiproliferative assay in clonal cell lines and a cytotoxicity assay using primary cultures of cerebral cortex neurons at different stages of development. It is concluded that assessment of antiproliferative activity of anticonvulsant drugs may be one criterion for identification of teratogenic potential during neurulation.

Shi, M. and E. M. Faustman. 1989. Development and characterization of a morphological scoring system for reedaka (Oryzias latipes) embryo culture. Aquat. Toxicol. (Amst.) 15(2):127-40.

Interest in non-mammalian alternatives to in vivo animal teratogenicity testing has dramatically increased. However, few of these alternative screening systems have been critically evaluated or validated. In order to characterize such tests, there is a need for reproducible and standardized means of monitoring and scoring development. To address this need, a morphological scoring system was developed for medaka embryos. This paper presents this scoring system and demonstrates its application in developmental toxicity evaluations.

Staples, R. E., P. H. Lieder, C. A. Mauro, B. E. G. Gabel, and E. M Johnson. 1989. Hydra test modification to identify developmental hazards among volatile liquids and gases. Teratology 39(5):483.

As opposed to the mammalian test, the hydra test is much less costly, is more rapid to conduct, and requires only a very small amount of test chemical. The test can now be conducted in vials outfitted with teflon-lined screw-caps to provide a closed system from which samples can be taken for analytical purposes. The conclusion that FC-132B, a highly volatile liquid, was not uniquely sensitive to the mammalian conceptus was supported by the results of a preliminary test in the rat.

Endocrinology

Bestetti, G. E., C. E. Boujon, D. K. Tontis, U. Forster, S. Grimm, and G. L. Rossi. 1989. [Alternative methods: development and use of two in vitro models for endocrine studies]. Schweiz. Arch. Tierheilkd. 131(9):537-45.

Most endocrinological studies are normally performed on several animal groups. In order to overcome some of the drawbacks associated with in vivo experimentation, a method was developed using two in vitro models by means of which could be studied on the tissues from the same animal, (1) function and structure of the hypothalamus, and (2) function and structure of isolated pituitary cells. Using these models can considerably reduce the number of animals needed for the studies, replace the in vivo by the in vitro experiments and refine the methods avoiding, among others, the extrapolation of results. This publication is written in French.

Eye

Bracher, M., C. Faller, J. Spengler, and C. A. Reinhardt. 1987. Comparison of in vitro cell toxicity with in vivo eye irritation. Mol. Toxicol. 1(4):561-70.

The cytotoxicity potentials of 26 different cosmetic ingredients were ranked for each of four in vitro test and compared with the in vivo eye irritation in guinea pigs (Draize test) after application of 5% or 2.5% solutions of the same test batches. Strong irritants could be easily detected by most of the in vitro tests, but the neutral red uptake assay was the only one that was able to distinguish the minimally irritating test agents from strong irritants as well as from nonirritants. Other in vitro tests considered were cell detachment from culture dish, growth inhibition, and membrane permeability.

Bracher, M., C. Failer, J. Spengler, and C. A. Reinhardt. 1988. Comparison of in vitro cell toxicity with in vivo eye irritation. Mol. Toxicol. 1(4):561-70.

The effects of 26 different cosmetic ingredients were assessed by four endpoints indicative of qualitatively and quantitatively different cytotoxicities: (1) neutral red uptake reduction after 24 hours of treatment (NR-90 and NR-50); (2) cell detachment from culture dish after 4 hours of treatment (CD-25); (3) growth inhibition after 48 hours of treatment (GI-50); and (4) membrane permeability measured by fluorescent dye retention (fluorescence shift FS-25) and dye exclusion (viability ratio VR-25). The cytotoxicity potentials of the test agents were ranked for each in vitro test and compared with the in vivo eye irritation in guinea pigs (Draize test) after application of 5 or 2.5% (wt./vol.) solutions of the same test batches. The neutral red uptake assay was the only one that was able to distinguish the minimally irritating test agents from strong irritants as well as from nonirritants. At least two of the other cell tests (CD-25, GI-50, FS-25, and VR-25) had to be considered to allow an adequate interpretation of in vitro cytotoxicity.

Bruner, L. H. and R. D. Parker. 1990. Evaluation of six in vitro alternatives for ocular irritancy testing. The Toxicologist 10(1):258(Abstr. 1029).

Six in vitro assays were evaluated to determine if any are useful as screening procedures in ocular safety assessments. The six assays evaluated included the Microtox Assay, Light Addressable Potentiometric Sensor, Neutral Red Assay, Total Protein Assay, Tetrahymena thermophila motility assay, and the EYTEX System. There was a significant correlation between the in vivo irritancy and the in vitro data for all the tests except the EYTEX System. These results suggest it may be possible to classify materials into broad irritancy categories and thereby use some of these assays as prescreens prior to in vivo confirmation in the ocular safety assessment process.

Conduzorgues, J. P., M. Hagege, M. Dumas, and A. Meybeck. 1989. Participation of the membrane effect in the cytotoxicity of surfactants comparison with eye irritation in vivo. Lens Eye Toxic. Res. 6(1-2):375-8.

Several surfactants were tested in 3 systems, i.e. rabbit erythrocytes for membrane effects, rabbit cornea fibroblasts to determine toxicity after 3 days, and the rabbit eye (Draize test) and the results compared. Good correlations were found for most of the chemicals among the tests.

Damji, K. F., J. Rootman, B. Palcic, and G. Thurston. 1990. Pharmacological modulation of human subconjunctival fibroblast behavior in vitro. Ophthalmic Surg. 21(1):31-43.

The response of human subconjunctival fibroblasts to a variety of pharmacological agents was evaluated utilizing a novel in vitro wound assay and a separate proliferation assay. The authors conclude that the wound assay is well suited for rapid screening of drugs for their effect on fibroblast morphology, motility, and proliferation, and that colchicine and cytochalasin B, in doses well below those documented to produce ocular toxicity, are effective in inhibiting directed migration and proliferation of subconjunctival fibroblasts in vitro. Differences in mechanism, onset of action, therapeutic range, and cytotoxicity of drugs could be exploited in controlling ocular fibroblast behavior in vivo.

Dierickx, P.J. 1989. Glutamic acid uptake inhibition assay in cultured Hep G2 cells as an alternative method for evaluating potential in vivo eye irritation. Alternatives to Laboratory Animals 16:344-52.

Glutamic acid (GA) content was measured in cultured Hep G2 cells, after treatment of the cells with test compounds. Results with 37 chemicals were compared with their respective rabbit eye irritation data. The xenobiotics were applied to the cells for 4 hours at 5 different concentrations. The cells were then incubated for 15 minutes with tritiated GA and GA uptake inhibition was measured. A linear correlation coefficient of r = 0.66 was found between the in vitro and the mean corneal opacity scores. However, r = 0.81 was found when the in vitro results were compared with range-finding scores, indicating that a closer relationship exists between cytotoxicity and the latter.

Gordon, V. C., and C. P. Kelley. 1989. An in vitro method for determining ocular irritation. Cosmet. Toiletries 104(10):69-74.

The EYTEX method, a corneal opacification assay for prediction of ocular irritation, based on alterations in a protein matrix reagent, is discussed as an alternative to the Draize test. Jack bean powder was extracted to obtain a liquid protein matrix for experiments. Ocular irritants were tested for their ability to produce opacity in vitro.

Gordon, V. C., and C. P. Kelly. 1989. Validation of an in vitro method for determining ocular irritation of cosmetic ingredients and products. Cosmet. Toilet. 104:67,69-70,72 (6 refs.).

Evaluation of the Eyetex method, an in vitro method used for determining ocular irritation of cosmetic products and raw materials, is discussed. Complete results for a validation study of 921 compounds using this method are summarized.

Kennah, H. E., S. Hignet, P. E. Laux, and C. S. Barrow. 1990. Reducing the number of rabbits required for eye irritation testing utilizing corneal swelling. The Toxicologist 10(1): 155(Abstr. 618).

The objective of this study was to determine whether the number of rabbits employed in Draize eye irritation studies could be reduced and still maintain correct conclusions regarding the eye irritation of various substances. An alternative to the Draize scoring system was used which involves measurement of increases in corneal thickness accompanying eye irritation. The corneal swelling technique can provide statistically-analyzable eye irritancy data that will quantitate irritancy potential from three rabbit subsets rather than the traditional six-animal data sets.

Lawrence-Beckett, E. M., J. T. James, and C. C. Lee. 1990. Initial in vitro eye irritation testing using the EYTEX (trademark) system. Govt. Reports Announcements & Index (GRA&I), Issue 10. NTIS/AD-A217 208/8, 23p: Proj. 1C162622A554.

The goal of this project was to determine how accurate the EYTEX data were when compared to the standard in vivo eye irritation data (Draize test) and also for inter- and intralaboratory variability. Two assays employed for this purpose were the Standard Assay and the Membrane Partition Assay. The approach was to evaluate the chemicals at concentrations comparable to potential human exposures. In Phase I of the project, six toxicology laboratories were contracted to participate in an external evaluation of the Standard EYTEX System. In Phase II, the EYTEX System was used to evaluate a select group of military compounds and compare the results with Draize scores. The data and equivalence values indicate that the EYTEX System is a suitable prescreening tool, and a need exists for the development of a broader data base.

Lazarus, H. M., P. S. Imperia, R. E. Botti, R. J. Mack, and J. H. Lass. 1989. An in vitro method which assesses corneal epithelial toxicity due to antineoplastic, preservative and antimicrobial agents. Lens Eye Toxic. Res. 6(1-2):59-85.

An in vitro model for studying the cytotoxicity of pharmacological agents on primary rabbit corneal epithelium employing (3H)thymidine incorporation was developed. The in vitro cytotoxicity data derived from these tests correlate well with previous in vivo and pre-clinical corneal epithelial toxicity studies. This model may be useful in the toxicological study of future topical ophthalmic agents.

Pape, W. J., U. Pfannenbecker, and U. Hoppe. 1987. Validation of the red blood cell test system as in vitro assay for the rapid screening of irritation potential of surfactants. Mol. Toxicol. 1(4):525-36.

The in vitro red blood cell assay (RBC assay) allows the estimation of irritation potentials of tensides and detergents. The estimation is based on the differentiation between cell membrane lysis and cell protein denaturation. All data were compared with in vivo data on eye irritancies. The good correlation of in vivo and in vitro data (r = .806, p < .0001) allows one to predict eye irritation. The RBC assay is an inexpensive, rapid, irritancy screening test that provides reliable results with good reproducibility. The test helps to reduce or even avoid animal testing in this application.

Reader, S. J., V. Blackwell, R. O'Hara, R. H. Colthier, G. Griffin, and M. Balls. 1989. A vital dye release method for assessing the short-term eytotoxic effects of chemicals and formulations. Alternatives to Laboratory Animals 17:28-37.

In the field of in vitro toxicology, attention is being focused on the development and validation of potential alternatives to the Draize eye irritancy test. As one alternative mouse embryonic fibroblast (3T3-L1) cells were cultured. The cells were incubated at 37°C in 95% air/5% CO₂ for 4 days until confluent, the medium replaced with medium containing neutral red, and after 3 hours the neutral red medium was removed. The test was carried out within 1 hour. At the end of the treatment period (usually 1 minute), the test sample was removed and neutral red destain solution was added. The neutral red released by the treated cells was measured and correlated with the known in vivo irritancy to the eye.

Reinhardt, C. A., M. Aeschbacher, M. Bracher, and J. Spengler. 1987. Validation of five alternative methods (4 cell tests plus hen's egg test) for predictive eye irritation testing (Draize test). Experientia 43(6):708.

Fifty-one different cosmetic ingredients were tested in five alternative methods to animal experimentation and compared with in vivo guinea pig eye irritation data. The cell tests included cell detachment, growth inhibition, and membrane permeability by dye retention and by dye exclusion. The hen's egg test was performed on the chorioallantoic membrane of 10-day-old chicks. A good correlation between the four alternative in vitro tests and the in vivo irritation data was obtained which allowed a prediction of irritation potential of unknown test chemical based on cell toxicity. The hen's egg test was shown to be of limited value.

Shopsis, C. 1989. Validation study: ocular irritancy prediction with the total cell protein, uridine uptake, and neutral red assays applied to human epidermal keratinocytes and mouse 3T3 cells. Altem. Methods Toxicol. 7(In Vitro Toxicol:New Dir.):273-87.

Ocular irritancy may be predicted in vitro using normal human epidermal keratinocytes (NHEK) cells and the cell protein or neutral red assays. Alkalinity or acidity of test agents cannot be predicted with cell cultures, and these properties of test agents should, therefore, be measured by titration. For the nonalkaline and nonacidic agents, these assay systems correlated well with Draize scores and irritancy evaluations.

Tachon, P., J. Cotovio, K. G. Dossou, and M. Prunieras. 1989. Assessment of surfactant cytotoxicity: comparison with the Draize eye test. Int. J. Cosmet. Sci. 11 (5):233-244.

We have compared the in vitro cellular toxicity and the in vivo ocular irritation potency of 16 surfactants. In vitro, the cellular toxicity was estimated on Chinese hamster lung fibroblasts (V79) using a cell mortality test and a cell growth inhibition test. In vivo, each surfactant was applied directly to the cornea of six albino rabbits. The results suggest that the use of cell culture tests as prescreening systems to measure eye irritation potency of surfactants could be a reliable alternative method in order to reduce the use of the Draize rabbit eye test and can provide a better knowledge of the irritative process.

Triglia, D., P. T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989. Interlaboratory validation study of the keratinocyte neutral red bioassay from clonetics corporation. Altern. Methods Toxicol. 7(In Vitro Toxicol:New Dir.):357-65.

The performance characteristics of the keratinocyte neutral red bioassay, an in vitro alternative to the Draize test for cytotoxicity evaluation, were similar from laboratory to laboratory and correlated well with the in vivo ocular irritancy data for 12 surfactant-based test agents. The bioassay is based on standardized reagents and culture conditions in order to minimize interassay variation. It is sensitive and simple to perform, uses no radioactive materials, obviates the need for cell counting, eliminates interfering substances which are present in serum, and allows the user to obtain a quantitative endpoint using normal human epidermal keratinocytes.

Vercoutre, M.P. 1990. The importance of being mildest. Comun. J. Com. Esp. Dererg. 21:159-66.

To show that cosmetics are safe, some governments, such as France require demonstration of the mildness of a product by in vivo rabbit-eye and rabbit-skin tests. The in vitro Red Blood Cells assay is not only a good tool to predict the eye-irritation potential of a surface-active product, such as a shampoo, but also is a fast and low-cost analytical method to screen prototype formulations during product development.

Watanabe, M., K. Watanabe, K. Suzuki, O. Nikaido, I. Ishii, H. Konishi, N. Tanaka, and T. Sugahara. 1989. Use of primary rabbit cornea cells to replace the Draize rabbit eye irritancy test. Toxicol. in Vitro 3(4):329-34.

The Draize ocular irritancy test was compared with cytotoxicity determined from colony forming ability in cells freshly isolated from rabbit cornea (RC). Chemicals used in consumer products were used as test agents. There was a close correlation between the cytotoxicity in RC cells in vitro and the Draize score in vivo in response to the chemicals. The data suggest that the cytotoxicity test in vitro using primary RC cells may be useful as a substitute for the Draize eye irritancy test.

Genotoxicity

Quillardet, P., G. Frelat, V. D. Nguyen, and M. Hofnung. 1989. Detection of ionizing radiations with the SOS chromotest, a bacterial short-term test for genotoxic agents. Murat. Res. 216(5):251-257 (43 refs.).

The ability of the SOS chromotest, developed for use with chemical agents, to detect exposure to ionizing radiation was evaluated. Its basis relied on test agents activating the sfiA gene in bacteria. This evaluation was conducted in Escherichia-coli K12 strain PQ37 bacteria. The test appears to be a sensitive and simple assay for quantitating the genotoxic effects of ionizing radiations and chemicals.

Hepatotoxicity

Kohira, T., and T. Nakamura. 1989. Use of primary cultured hepatocytes for toxicology. J. Toxicol. Sci. 14 (Suppl. 3):108-14.

Toxicological studies using primary cultured hepatocytes were reviewed in Japanese. Primary cultures of mature hepatocytes retain many liver functions and various hormonal responses for long periods. Toxicological studies using primary cultured hepatocytes show a high correlation with some in vivo studies. Some of the shortcomings such as lack of enzyme production stability are being worked out. It is suggested that in the future it may be possible to culture hepatocytes for long periods or subculture them and still have them maintain liver function. Not only rodent hepatocytes but also human hepatocytes may become more useful for toxicological assessments and screening of drugs used for treating liver diseases.

Liu, J., W. C. Kershaw, and C. D. Klaassen. 1990. Rat primary hepatocyte cultures are a good model for examining metallothionein-induced tolerance to cadmium toxicity. In Vitro Cell. Dev. Biol. 26(1): 75-79.

The effect of Zn-induced metallothionein on the toxicity, uptake, and subcellular distribution of cadmium was examined in rat primary hepatocyte cultures and compared to results obtained earlier in this laboratory from intact animals. Cytotoxicity was assessed by enzyme leakage, intracellular potassium loss, and cellular glutathione content. These results indicate that cultured hepatocytes are a model for examining metallothionein-induced tolerance to cadmium hepatotoxicity.

Lamb, R. G., J. F. Borzelleca, L. W. Condie, and C. Gennings. 1989. Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes. Toxicol. Appl. Pharmacol. 101(1):106-13.

Primary cultures of adult rat hepatocytes were incubated with carbon tetrachloride and/or chloroform. Agent-dependent alterations in hepatocyte functions were assessed by measuring (1) [3H]choline incorporation into phosphatidylcholine (endoplasmic reticulum), (2) MTT (tetrazolium salt) reduction (mitochondria), and (3) AST release into medium (plasma membrane). These effects of carbon tetrachloride and/or chloroform on liver cell functions in vitro are consistent with liver alterations observed in vivo. Therefore, primary cultures of adult rat hepatocytes may be an appropriate model in vitro to assess the hepatotoxic potential of agents alone or in combination.

Immunology

Coghlan, L.G., and M. Hanausek. 1990. Subcutaneous immunization of rabbits with nitrocellulose paper strips impregnated with microgram quantities of protein. J. Immunol. Methods 129(1): 135-8.

This report describes the production of polyclonal antibodies against microgram quantities of a weakly immunogenic tumor-associated protein of canine origin. The technique employs the subcutaneous implantation of nitrocellulose electroblot strips without the use of adjuvant. The method is simple, appears reliable, can be used to improve antigen purity, and is applicable for either polyclonal or monoclonal antibody production in different host species. In addition, because traditional adjuvants are not required with this system, severe inflammatory responses and associated animal discomfort are reduced.

Immunotoxicity

Murray, M. J., and J. H. Dean. 1989. Characterization of in vivo and in vitro chemical exposure and immunization approaches for assessing effects on acquired immunity. Altern. Methods Toxicol. 7(In Vitro Toxicol:New Dir.): 141-9.

Many approaches for primary immunotoxicity assessment include a limited evaluation of in vitro immune function. For screening purposes, and where little data exist concerning the toxicity of the chemical or compound of interest, in vivo exposure in conjunction with the in vitro immune function assays is preferable to in vitro exposure. Immune function assays with appropriate sensitivity and comprehension in terms of the cell types, interactions and functions they assess, are best suited for screening purposes. Two such assays are the antibody plaque assay and the cytotoxic T lymphocyte assay. These assays provide important measures of functional competence in the two major branches (cellular and humoral) of the immune response. They are antigen-driven responses and immunization with the appropriate antigen can be either in vivo or in vitro. For a more detailed understanding and dissection of mechanisms associated with chemically induced immunotoxicity, in vitro systems may provide advantages over in vivo models.

Tam, P. E., and R. D. Hinsdill. 1990. Screening for immunomodulators: effects of xenobiotics on macrophage chemiluminescence in vitro. Fundam. Appl. Toxicol. 14(3):542-53.

Macrophage chemiluminescence was evaluated as a primary screening assay by assessing the modulatory activity of 17 different chemicals. Elicited mouse peritoneal macrophages were exposed to the chemicals in vitro, and chemiluminescence was measured in response to an opsonized yeast stimulus. Macrophage chemiluminescence is suggested as a simple, quantitative, yet sensitive immunotoxicologic screening assay capable of identifying many known immunomodulatory drugs.

Irritancy

Deleo, V. A., D. Hanson, S. Scheide, B. J. Kong, and S. Desalva. 1989. The effect of surfactants on metabolism of choline phospholipids in human epidermal keratinocytes in culture. J. Toxicol. Cutaneous Ocul. Toxicol. 8(2):227-40.

Human epidermal keratinocytes were prelabeled with (3H)choline and then treated with four surfactants. Media were assayed for release of labeled choline metabolites. Irritancy of these surfactants was determined in two animal test systems: the guinea pig immersion assay and the guinea pig dermal irritancy assay. The rank order response for the choline release assay was analogous to the rank order irritancy in animal skin. This positive correlation suggests that this system merits further investigation as an alternative in vitro assay for human skin irritancy.

Gajjar, L. and D. J. Benford. 1987. Irritancy testing in cultured keratinocytes. Mol. Toxicol. 1 (4):513-523 (9 refs.).

An in vitro system for assessing skin and eye irritancy was evaluated. The system used a keratinocyte line derived from explant cultures of rat sublingual epithelium. Cytotoxicity was indicated by the cells releasing alkaline-phosphatase and by determining the extent of uptake of neutral-red and kena-blue dyes. Although there are insufficient in vivo skin irritancy data to obtain a meaningful comparison, the test data correlate well with data obtained in other in vitro and in vivo tests used to assess eye irritancy.

Garcia Ramon, M. T., I. Ribosa, J. Sanchez Leal, and J. L. Parra. 1989. Comparison of diffusion by anionic surfactants through cellulose acetate and collagen membranes. Int. J. Cosmet. Sci. 11(3):121-8.

Recent research trends have been oriented towards the establishment of new 'in vitro' techniques that will avoid animal experimentation. Some results on the rate of diffusion of different anionic surfactants through both cellulose acetate and collagen membranes are described. A correlation between results of diffusion through the protein membrane and results published on the same surfactants and their irritation potential during 'in vivo' testing appears possible.

Wilson, S. A., M. L. Jasiewicz, F. W. Bonnet, S. Brown, and C. J. Potter. 1989. An in vitro screening test for the assessment of the irritant potential of parenteral formulations. Toxicol. in Vitro 3(4):335-9.

A cytotoxicity assay based on the uptake of Neutral Red by HeLa cells was investigated as an in vitro screen for assessing the irritancy potential of parenteral formulations. Three irritants and two nonirritants were examined for cytotoxic effects in this system. The ranking of the injectables tested, in order of decreasing cytotoxic effects, correlated with both previously reported qualitative in vitro data and their reported irritancy when administered to man. The HeLa cell/Neutral Red cytotoxicity assay may provide an alternative to animal studies for the assessment of the irritancy potential of parenteral formulations during the preliminary stages of their development.

Lung

Fisher, G. L. 1989. Use of pulmonary macrophages in metal toxicity: a brief overview. Journal of the American College of Toxicology 8(7): 1247-1250 (4 refs.).

Pulmonary macrophages were used to assess metal and complex mixture toxicity by measuring the extent of cellular adherence to a foreign substrate, phagocytosis of inert particles, release of lysosomal or cytoplasmic enzymes, metabolic alterations such as changes in oxygen consumption or free radical production, or changes in cytokine production. The author concludes that when they are performed carefully, alveolar macrophage assays can be used to predict pulmonary toxicity.

Fisher, G. L., K. L. McNeill, and J. T. Smith. 1989. In vitro effects of fibrous and nonfibrous silicon nitride on bovine pulmonary macrophages. Environ. Res. 50(2):279-288.

Bovine pulmonary macrophages were exposed in vitro to a fibrous silicon nitride, nonfibrous (milled) silicon nitride cornminuted from the fibrous powder, alpha-quartz (an active control), or glass beads (an inert control). Functional evaluation, biochemical studies (increased release of lactate dehydrogenase and acid phosphatase, consistent with both cell membrane and lysosome lysis, and depression of total protein levels, suggesting significant impairment of cellular synthetic processes), and scanning electron microscopy were used to assess the effects.

Metabolism

Bisgaard, H. C., and H. R. Lam. 1989. In vitro and in vivo studies on the metabolism of 1,3-diaminobenzene: comparison of metabolites formed by the perfused rat liver, primary rat hepatocyte cultures, hepatic rat microsomes and the whole rat. Toxicol. In Vitro 3(3): 167-74.

The metabolism of the aromatic amine, 1,3-diaminobenzene was studied in vitro by use of the perfused rat liver, primary rat hepatocyte cultures and hepatic rat microsomes. The metabolites formed were compared with urinary metabolites excreted by the rat in vivo. The results show that in addition to the metabolites that can be detected in the urine in vivo, several other derivatives, including glucuronic acid conjugates are formed by the rat liver. The results suggest that primary rat hepatocyte cultures are the preferred model system in metabolic studies in vitro.

Delraso, N. J., D. R. Mattie, and Godin. 1989. In vitro toxicity of solubilized 2,3,4-trimethylpentane. I. Cytotoxicity and metabolism of TMP using primary hepatocytes. In Vitro Cell. Dev. Biol. 25(11):1031-8.

Metabolite analysis by gas chromatography-mass spectrometry of supernatant and cell extracts from hepatocyte cultures exposed to 2,3,4-trimethylpentane for four hours indicated the presence of three metabolites: 2,3,4-trimethyl- 1 -pentanol, 2,3,4-trimethyl-2-pentanol, and 2,3,4-trimethyl-1-pentanoic acid. This study indicates that primary hepatocyte suspension cultures provide a useful system for rapidly identifying liver metabolites of selected test compounds.

Henze, E., K. Schoser, R. Lietzenmayer, G. Schnur, M. Sauer, R. Kunz, M. Clauser and W. E. Adam. 1989. [Studies of myocardial metabolism using P-31-NMR spectroscopy and a simple heart perfusion model of slaughtered animals--an approach to avoiding in vivo animal experiments] Z. Kardiol. 78(9):553-60.

In research cardiology large numbers of in vivo animal experiments, mostly with dogs or pigs, are necessary. It was the aim of this study (described in German) to test a simple in vitro perfusion heart model from swine harvested in the slaughterhouse as a potential substitute for in vivo animal studies.

Kasai, K., M. T. Hori, and W. G. Goodman. 1990. Characterization of the transferrin receptor in UMR-106-01 osteoblast-like cells. Endocrinology 126(3):1742-1749.

UMR-106-01, an osteoblast-like cell line, was used in an effort to determine the mechanism by which the accumulation of iron or aluminum might cause metabolic bone disease. The results indicate that osteoblast-like cells have a single class of membrane receptors for transferrin and that the regulation of transferrin receptor expression in UMR-106-01 cells is similar to that in other cell types. The uptake of iron and gallium via the Tf-receptor complex can affect osteoblast proliferation, and such a mechanism may contribute to the bone cell toxicity of various metals.

Kelner, M. J., and R. Bagnell. 1989. Paraquat resistance associated with reduced NADPH reductase in an energy-dependent paraquat-accumulating cell line. Arch. Biochem. Biophys. 274(2):366-74.

In vitro screening of potential antidotes that act by blocking paraquat uptake requires a cell line that accumulates paraquat by an energy-dependent mechanism. Various lymphoblastoid cell lines were screened until one was found that accumulated paraquat by an energy-dependent mechanism. During study of this cell line, a marked resistance to paraquat developed in a clone. The resistance was associated with a reduction in NADPH reductase activity, confirming the original report (using microsomal preparations) that intracellular reduction of paraquat occurs primarily by this enzyme. Thus, this cell line will be useful for studying other toxins where the involvement of NADPH reductase is suspected, but not proven.

Knasmueller, S., A. Szakmary, and A. Wottawa. 1988. Microbial host-mediated assays, a convenient method to investigate the biotransformation of xenobiotic compounds to genotoxins in vivo. Bull. Poi. Acad. Sci.: Biol. Sci. 36(10-12):225-34 (43 refs.).

This publication is a review on the compounds tested so far in different host-mediated assay procedures. The advantages of this approach were discussed in comparison to conventional in vitro testing. Some recent developments are communicated such as the development of repair tests in which fruit flies can be used as host animals.

Paolini, M., E. Sapigni, P. Hrelia, S. Grilli, G. Lattanzi, M. Scotti, and G. Cantelli Forti. 1990. Strategies for optimization of short-term genotoxicity tests: the synergistic effect of NADPh and NADH on P450 function in processing pre-mutagens. Mutagenesis 5(1):51-4.

The synergistic effect of NADPH and NADH on P450 functions upon pre-mutagens requiring metabolism during the incubation conditions used in the liver microsomal assay was studied. The findings presented in this publication lead the authors to suggest the routine use of both NADPH and NADH in order to increase the 'sensitivity' of in vitro mutagenicity screens.

Safe, S., T. Zacharewski, L. Safe, M. Harris, C. Yao, and M. Holcomb. 1989. Validation of the AHH induction bioassay for the determination of 2,3,7,8-TCDD toxic equivalents. Chemosphere 18(1-6):941-6.

The response of the aryl hydrocarbon-hydroxylase (AHH) induction in rat hepatoma cells to polyhalogenated aromatic hydrocarbons was examined. For comparison, test chemicals were injected into male rats and the rats were sacrificed 2 weeks later. Good correlation was found between the median effective concentration (EC50) values for hepatic microsomal AHH and ethoxyresorufin o-deethylase (EROD) induction, body weight loss and thymic atrophy determined from the rat model and EC50 values for AHH and EROD induction in rat hepatoma cell cultures.

Shirley, M. A., and R. C. Murphy. 1990. Metabolism of leukotriene B4 in isolated rat hepatocytes. Involvement of 2,4-dienoyl-coenzyme a reductase in leukotriene B4 metabolism. J. Biol. Chem. 265(27): 16288-95.

Radiolabelled leukotriene (LT) B4 was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed.

Methodology

Eckert, J. 1988. Cryopreservation of parasites. Experientia 44(10):873-7.

It is concluded that cryopreservation of certain stages of helminth and protozoan parasites is a useful technique for long-term storage of defined isolates, which can contribute considerably to reducing the number of experimental animals usually required for serial passages.

Ennever, F. K., and H. S. Rosenkranz. 1989. Application of the Carcinogenicity Prediction and Battery Selection method to recent National Toxicology Program short-term test data. Environ. Mol. Mutagen. 13(4):332-8.

The Carcinogenicity Prediction and Battery Selection (CPBS) methodology was applied to data on the genotoxicity and carcinogenicity of 73 chemicals published by the National Toxicology Program. Initial analysis was limited to testing in rodent bioassays and the four short term tests: Salmonella microsome mutagenicity, cytogenetic aberrations in vitro, sister chromatid exchanges in vitro, and mutagenicity in the mouse lymphoma cell line L5178Y. The results indicate that the CPBS methodology could be applied to a single self-containing set of data. This method provides a marked improvement in predictions.

Gad, S. C. 1990. Recent developments in replacing, reducing, and refining animal use in toxicologic research and testing. Fundam. Appl. Toxicol. 15(1):8-16.

Review of annual reports of the numbers of animals used in testing in the United States, the United Kingdom, and Japan shows a continuing reduction in the numbers for all species. Multiple in vitro systems have been developed for screening/testing for eye and skin irritation, skin sensitization, teratology, and other endpoints and a scientific consensus has been formed on requirements and process for validation. However, the use of these test systems in place of existing in vivo tests is minimal. At the same time, innovative designs have been developed (and are in wide use) for in vivo tests which reduce both the numbers and the pain and distress of animals used in testing. Progress and dialogue continue on modification of both U.S. and international requirements and guidelines for testing, and for defining an "approval" process for alternatives and innovations.

Rubinstein, L. V., R. H. Shoemaker, K. D. Paull, R. M. Simon, S. Tosini, P. Skehan, D. A. Scudiero, A. Monks, and M. R. Boyd. 1990. Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J. Natl. Cancer Inst. 82(13):1113-18.

A detailed comparison of data generated by the assay based on the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and the protein-binding dye sulforhodamine B assay was undertaken by the National Cancer Institute (NCI). The results indicate that under the experimental conditions used and within the limits of the data analyses, the assays perform similarly. Because the sulforhodamine B assay has practical advantages for large-scale screening, however, it has been adopted for routine use in the NCI in vitro antitumor screen.

Umminger, B. L. 1990. Unconventional organisms as models in biological research. J. Exp. Zool. Suppl. 4:2-5.

Many recent reports have recommended a reduction in the use of mammals in biomedical research to promote animal welfare by utilizing alternative methods and unconventional model organisms. Currently we lack adequate models for all the biological phenomena worthy of study. Biologists must continue to take advantage of the functional biodiversity of organisms not only to identify new model organisms for studying more optimally those life processes we know something about, but also to discover entirely new biological principles, processes, and products. Increased emphasis on the need to preserve the diversity of life also requires more knowledge of the comparative biology of processes such as reproduction, growth, defense mechanisms, and nutrition. There is a critical need of commercial biotechnology for basic functional information on more species, especially microorganisms and plants.

Zbinden, G. 1990. Alternative methods, the present and future. Therapie 45(4):347-50.

This paper, written in French, states that alternative methods are widely used in fundamental biological research. Among 6,649 papers given at the Federation of American Societies for Experimental Biology 1989 meeting, nearly 60% deal with experimental data obtained without the use of live animals. Alternative methods are less frequently utilized in industrial toxicology. The reason for this discrepancy is discussed as well as the efforts made to reduce the numbers of animals for acute toxicity studies (LDs0) and cutaneous and eye tolerance. Present scientific developments as well as the possible agreement by regulatory agencies of the newly developed tests are presented. In spite of the increasing acceptance of alternative methods, large numbers of animals are still used. The reasons for this situation are analyzed, as are the efforts made to encourage research for the replacement of animals in programs traditionally based on animal experimentation.

Mutagenesis

Clay, P., and M. F. Cross. 1990. Microwell mutation assays: evaluation of ethylmethanesulphonate, benzo[a]pyrene and benzidine using the tk locus in L5178Y mouse lymphoma cells. Mutagenesis 5(Suppl.):45-54.

As part of the third UKEMS collaborative trial, ICI Central Toxicology Laboratory (CTL) tested three chemicals in a microwell adaptation of the L5178Y gene mutation assay using the thymidine kinase (tk) locus. Cofactors for metabolic activation were optimized prior to testing the chemicals under differing expression times and S9 levels. The results for zero hour survival, relative total growth and mutant frequency were subjected to extensive statistical analyses. It was concluded that the methods described produce a robust and reliable assay and that for routine use a single expression time and S9 level are sufficient, provided true independent replicates are used.

McPherson, M. F., and E. R. Nestmann. 1990. The SIMULTEST approach for testing mutagens in the Salmonella microtitre fluctuation assay. Environ. Mol. Mutagen. 16(1):21-5.

The concept of combining several histidine-dependent Salmonella strains in a single test, the SIMULTEST, has been applied to the microtitre fluctuation test. The activity of five mutagens was determined in strains TA97, TA98, TA100, and TA102 individually as well as in a SIMULTEST mixture. All five compounds were mutagenic in the SIMULTEST, demonstrating the utility of this time and labor-saving approach of combining strains for testing with this method. The microtitre fluctuation SIMULTEST results were quantitatively comparable to those of the SIMULTEST salmonella/microsome plate test.

Norimura, T., V. M. Maher, and J. J. McCormick. 1990. A quantitative assay for measuring the induction of mutations in human peripheral blood T-lymphocytes. Mutat. Res. 230(1):101-9.

T-lymphocytes stored in liquid nitrogen and returned to culture shortly before mutagen exposure exhibited the same sensitivity as freshly-isolated T-cells to killing by the agents tested. It was shown that if mutagenized populations frozen during the expression period were thawed and assayed, they exhibited the same cloning efficiencies and frequencies of 6-thioguanine-resistant cells as did the corresponding populations that had been assayed directly without freezing. Use of these procedures should facilitate investigation of the frequency and kinds of mutations induced in the HPRT gene of peripheral blood T-lymphocytes in vivo and in vitro.

Schreiner, C. A. 1983. Application of short-term tests to safety testing of industrial chemicals. Annals of the New York Academy of Sciences 407:367-373 (23 refs.).

The use of short term testing batteries to predict the mutagenicity and carcinogenicity of industrial chemicals and the use of mutagenic screens and teratogenesis were reviewed. The author concludes that short term mutagenicity tests are of value in predicting risk to humans, but recommends caution in interpreting in vitro mutagenicity test results applied to industrial compounds, and that in vitro activity be correlated to in vivo response by comparing results at similar endpoints, as well as the nature of specific compounds types.

Nephrotoxicity

Boogaard, P.J., G.J. Mulder, and J.F. Nagelkerke. 1989. Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. II. Alpha-methylglucose uptake as a sensitive parameter for mechanistic studies of acute toxicity by xenobiotics. Toxicol. Appl. Pharmacol. 101 (1): 144-157 (38 refs.).

In order to investigate whether alpha-methylglucose uptake by isolated cells was suitable as an early index for cytotoxicity, a comparison was made of the effects on alpha-methylglucose uptake with those on intracellular ATP concentrations following treatment with a number of compounds chosen on the basis of their different mechanisms of nephrotoxic effect. The authors conclude that alpha-methylglucose is a sensitive parameter to assess cytotoxicity in vitro.

Bruggeman, I. M., J. J. W. M. Mertens, J. H. M. Temmink, M. C. Lans, R. M. E. Vos, and P. J. van Bladeren. 1989. Use of monolayers of primary rat kidney cortex cells for nephrotoxicity studies. Toxicol. In Vitro 3(4):261-270.

Kidney cells were isolated from rat kidney cortex and maintained in short-term monolayer cultures. A number of important parameters were studied in order to establish the usefulness of these cells for toxicity studies. Many relevant enzyme systems remained present and functional. This monolayer culture system, which can be used in combination with filters, seems to be suitable for studying various mechanisms of nephrotoxicity.

Hazen-Martin, D. J., D. A. Sens, J. G. Blackburn, M. C. Flath, and M. A. Sens. 1989. An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro. In Vitro Cellular and Developmental Biology 25(9):791-799 (32 refs.).

Evidence was offered of ultrastructural changes in response to very low dosages of ionic cadmium in human proximal tubule cells in vitro. Transport function and subtle ultrastructural modifications in membrane structure can be observed at these concentrations and may more realistically portray the cadmium induced events resulting in human proximal tubule dysfunction. According to the authors, these studies clearly indicate that very low cadmium concentrations, below those eliciting proximal tubule cell death or destruction of tight junctions and the epithelial sheet, elicit subtle fine structural changes coupled with alterations in transport function. The in vitro system used mimics many of the well defined cadmium elicited responses reported in vivo systems.

Hazen-Martin, D. J., D. A. Sens, J. G. Blackburn, and M. A. Sens. 1989. Cadmium nephrotoxicity in human proximal tubule cell cultures. In Vitro Cellular and Developmental Biology 25(9):784-790 (23 refs.).

A study was conducted to observe alterations in cellular viability and fine structure in response to exposure to cadmium as cadmium-chloride in vitro. The human proximal tubule cell culture model, as used in this study, offered a method of identifying the early or acute effects of cadmium in the human kidney. Cells treated with each of the cadmium doses exhibited some evidence of cell death. Cadmium treated tubule cells demonstrated fine structural alterations including condensation of nuclear chromatin, loss of microvilli structure, disorganization of lateral membrane interdigitation, and decreased uptake of aminoglycoside antibiotics as evidenced by decreased numbers of myeloid bodies in these cells. The authors conclude that the use of the human proximal tubule culture system may be helpful in discerning structural and functional effects of exposure to cadmium and other nephrotoxicants on the kidney.

Neurology

Biondi, C., E. Fabbri, M. E. Ferretti, D. Sonetti, and A. M. B. Fantin. 1989. Effects of lead exposure on cyclic AMP and correlated enzymes in viviparus ater (MoUusca gastropoda) nervous system. Comp. Biochem. Physiol. C. Comp. Pharmacol. Toxicol. 94(1):327-334.

This publication describes the use of histochemical and biochemical assays to study in vivo and in vitro interactions of lead with cyclic AMP in the ganglionic complex of Viviparus ater (Mollusca Gastropoda).

Chiba, A., D. Shepherd, and R. K. Murphey. 1988. Synaptic rearrangement during postembryonic development in the cricket. Science 240(4854):901-5.

Synaptic rearrangement during development is a characteristic of the vertebrate nervous system and was thought to distinguish vertebrates from the invertebrates. However, examination of the wind-sensitive cercal sensory system of the cricket demonstrates that some identified synaptic connections systematically decrease in strength as an animal matures, while others increase in strength over the same period. Moreover, a single sensory neuron could increase the strength of its synaptic connection with one interneuron while decreasing the strength of its connection with another interneuron. Thus, rather than being a hallmark of the vertebrate nervous system, synaptic rearrangement is probably characteristic of the development of many if not all nervous systems.

Neurotoxicity

Atterwill, C. K. 1987. Brain reaggregate cultures in neurotoxicological investigations: adaptational and neuroregenerative processes following lesions. Mol. Toxicol. 1(4):489-502.

In vitro neural systems can be predictive for CNS neurotoxicity. Using rat whole-brain reaggregate cultures, good in vitro/in vivo correlations for the cholinergic neurotoxicant ethylcholine mustard aziridinium have been demonstrated. Treatment of reaggregates lesioned with the cholinotoxin with a neurotropic factor, nerve growth factor (NGF), did not reverse the lesion but treatment of control cells with NGF elevated both choline acetyltransferase activity and muscarinic receptor binding. The "lesioned" reaggregate culture system may thus be of future value in evaluating potential therapeutic agents that could reverse such lesions in the CNS.

Audesirk, G., and T. Audesirk. 1990. Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis. Neurotoxicology 10(4):659-69.

The effects of acute in vitro lead exposure on slowly inactivating voltage-sensitive calcium channels in central neurons of the freshwater pond snail Lymnaea stagnalis were studied under voltage clamp. Three physiologically distinct cell types were used: two subsets of the B cell cluster (Bpos and Bneg) and the pedal giant neuron (RPeD 1). These data indicate that (1) voltage-sensitive calcium channels in Lymnaea neurons are inhibited by nanomolar concentrations of free Pb2+; (2) there are multiple types of calcium channels in Lymnaea neurons; and (3) the effects of in vitro lead exposure differ qualitatively among channel types.

Davenport, C. and K. T. Morgan. 1988-89. In vitro neurotoxicology: industrial applications. In Vitro Toxicol. 2(3):207-218

Evaluation of neurotoxicity is heavily dependent upon the use of live animals. One alternative procedure is a cytotoxicity assay using neuronal cell culture. Appli-cations discussed for this in vitro technique include: concentration-response relationships, mechanisms of action, toxicity of parent compound or metabolites, structure-activity relationships, and pharmacologic intervention. This work demonstrates some of the advantages and disadvantages of in vitro alternatives to whole animal testing.

Macklis, J. D. and R. D. Madison. 1990. Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. J. Neurosci. Methods 31(1):43-6.

Described is a visual assay of neuronal electrophysiologic status for use with cultured neurons, based on the exclusion of propidium iodide by intact cellular membranes. This fluorescent dye binds to nucleic acids at concentrations suitable for long-term exposure to neurons without toxicity. The progressive loss of resting membrane potential and the progressive inability to generate stimulated action potentials by cultured mouse dorsal root ganglion neurons is correlated with increasing incorporation of propidium iodide. The scoring system used to gauge incorporation of propidium iodide is rapid and highly reproducible using a standard fluorescence microscope. Applications exist for studies of neuronal toxicity, survival, and electrophysiology in vitro.

Mueller, L. J., C. M. Moorer-Van Delft, R. Zijl, and E. W. Roubos. 1990. Use of snail neurons in developing quantitative ultrastructural parameters for neurotoxic side effects of Vinca antitumor agents. Cancer Res. 50(6): 1924-8.

The central nervous system of the snail Lymnaea stagnalis was studied in order to develop a test system to predict the neurotoxic side effects of the three cytostatic Vinca alkaloids, vincristine, vindesine, and vinblastine. On the basis of a comparison of the data presented in this publication with clinical data on Vinca-induced neurotoxicities, it is proposed that neurons of the snail L. stagnalis may be suitable for the development of a test system to predict the degree of clinical neurotoxicity induced by Vinca antitumor drugs.

Schaad, U. B., K. Guenin, C. Steffen, and N. Herschkowitz. 1988. Effects of antimicrobial agents used for therapy of CNS infections on dissociated brain cell cultures. Pediatr. Res. 24(3):367-72.

Experiments were undertaken to determine if in vitro exposure of cultured brain cells to antibacterial drugs could predict neurotoxicity in man. Effects of antibiotics used for therapy of bacterial CNS infections on growth and differentiation in dissociated rat brain cell cultures were studied. Results demonstrated a reversible inhibition of cerebral sulfate transferase activity and to a lesser extent of DNA synthesis in brain cell cultures by the highest concentrations studied of amikacin, cefuroxime, and ceftazidime which correspond to peak cerebrospinal fluid values attained by intraventricular therapy in patients. Accumulation of DNA reflects brain cel. l growth whereas cerebral sulfate transferase activity parallels brain cell differentiation. Thus, this brain cell culture model might become a supplement, complement, or even alternative technique for neurotoxicity assessment of antibiotics with proven or potentiai value for therapy of CNS infections.

Seegal, R. F., K. Brosch, B. Bush, M. Ritz, and W. Shain. 1990. Effects of Aroclor 1254 on clopamine and norepinephrine concentrations in pheochromocytoma (PC-12) cells. Neurotoxicology 10(4):757-64.

Pheochromocytoma (PC-12) cells synthesize, store, release, and metabolize dopamine and norepinephrine in a manner analogous to that observed in the mammalian central nervous system. These cells were used to develop and validate an alternate method to animal testing to assess the effects of a complex environmental mixture of polychlorinated biphenyls (Aroclor 1254) on cellular catecholamine function. The results indicate that PC-12 cells may be useful for neurochemical evaluation of neurotoxicants with particular reference to effects on catecholaminergic systems.

St. Clair, M. B., D. C. Anthony, C. J. Wikstrand, and D. G. Graham. 1990. Neurofilament protein cross-linking in gamma-diketone neuropathy: in vitro and in vivo studies using the seaworm Myxicola infundibulum. Neurotoxicology 1990(Winter); 10(4): 743-56.

Myxicola infundibulum is a marine worm which is gaining popularity in neuroscience research because of its large syncytial axon. A model system using Myxicola has been developed to investigate neurofilament crosslinking in worms exposed to neurotoxic agents whose putative mechanisms involve covalent crosslinking of neurofilament proteins. These studies support previous observations which suggest that neurofilament crosslinking is the molecular event which initiates neurofilament aggregation in gamma-diketone neurotoxicity, and establish Myxicola infundibulum as a useful species in which to study certain neurotoxic compounds.

Nucleic acids

Singhal, R. N., H. B. Sarnat, and R.W. Davies. 1989. Unimpaired RNA synthesis in neurons and epithelial cells in a freshwater leech exposed to the organophosphate insecticide chlorpyrifos. Sci. Total Environ. 83(1-2): 195-202.

This study used the dominant freshwater leech Nephelopsis obscura as a representative aquatic invertebrate to test toxicity of chlorpyrifos on a nontarget species. RNA synthesis in neurons of the cerebral ganglion, epithelial cells of the intestine, and the tegument of small immature and large mature N. obscura was examined histochemically with acridine orange fluorochrome after exposure to chlorpyrifos.

Pathology

Thornton, D. H., R. A. Nicholas, and G. W. Wood. 1986. Development of in vitro tests for detection of extraneous agents. Dev. Biol. Stand. 64:195-8.

Tests have been carried out on a variety of potential contaminating avian pathogens to determine whether tests in chicks offer any advantage over tests in embryos or cell cultures. In many cases, but not all, in vitro tests were shown to be more sensitive. The use of fluorescent antibody or enzyme-linked assays serves to enhance the sensitivity of the tests. In the future it may be possible to adapt techniques such as nucleic acid hybridization to the detection of extraneous agents.

Tzianabos, A. O., and F. G. Rodgers. 1989. Pathogenesis and chemotherapy of experimental Legionella pneumophila infection in the chick embryo. Int. J. Med. Microbiol. 271(3):293-303.

The pathogenicity of Legionella pneumophila, serogroup 1, strain Nottingham N7, was assessed in terms of LDs0 data and the ability of the organism to induce pathological lesions in the fertile hen's egg. Results reported here indicate that the fertile hen's egg may be a useful alternative to other animal systems for the in vivo testing of clinically putative antimicrobial agents in the treatment of Legionnaires' disease.

Phototoxicity

Duffy, P. A., A. Bennett, M. Roberts, and O. P. Flint. 1987. Prediction of phototoxic potential using human A431 cells and mouse 3T3 cells. Mol. Toxicol. 1(4):579-87.

An assay using established cell lines, human A431 epidermal cells and mouse 3T3 fibroblasts, has been developed to predict the phototoxic potential of compounds. The test determines the viability in response to UV light in both the presence and absence of the test compound. The system has been validated with 30 compounds classified according to reactions in humans.

Long, D., T. Stephens, B. Reece, B. Bryan, and P. Silber. 1990. Evaluation of the yeast phototoxicity assay as an in vitro alternative to in vivo phototoxicity testing. The Toxicologist 10(l):326(Abstr. 1304).

In this study the responsiveness of the in vitro yeast phototoxicity test to a variety of fragranced and unfragranced cosmetic formulations was compared with the in vivo guinea pig phototoxicity models. The results of these experiments show that the in vitro yeast assay is more sensitive to phototoxicants than either the guinea pig or human phototoxicity models. Therefore, the yeast assay can serve as a rapid and inexpensive screening in vitro assay for phototoxicity evaluation, so long as the sensitivity of this model is considered.

Tenenbaum, S., J. DiNardo, W. E. Morris, B. A. Wolf, and R. W. Schnetzinger. 1984. A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials. Cell Biol. Toxicol. 1(1): 1-9.

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. Activity is quantified by cytotoxicity measured as zones of inhibition. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

Physiology

Erickson-Lamy, K. A. The perfused trabecular mesh-work as a model for outflow (human). Crisp Data Base, National Institutes Of Health.

The experimental system to be used in these studies is the perfused human outflow tissue model. This newly developed model involves placement of the eviscerated (e.g. lens and uveal tissue are removed) anterior corneoscleral shell with attached trabecular meshwork (TM) onto a specialized perfusion apparatus. The TM and associated outflow tissues are perfused with culture medium at a physiologically-relevant perfusion pressure in a 5% CO₂ environment at 37°C. Under these conditions, the perfused TM is similar to the human outflow system in vivo for several days with regard to morphology as well as functional parameters. Of considerable interest is the finding that epinephrine increases outflow facility in the perfused TM model. The ability to study the epinephrine responsive human o