Animals and the 3Rs in toxicology research and testing: The way forward.

Despite efforts to eliminate the use of animals in testing and the availability of many accepted alternative methods, animals are still widely used for toxicological research and testing. While research using in vitro and computational models has dramatically increased in recent years, such efforts have not yet measurably impacted animal use for regulatory testing and are not likely to do so for many years or even decades. Until regulatory authorities have accepted test methods that can totally replace animals and these are fully implemented, large numbers of animals will continue to be used and many will continue to experience significant pain and distress. In order to positively impact the welfare of these animals, accepted alternatives must be implemented, and efforts must be directed at eliminating pain and distress and reducing animal numbers. Animal pain and distress can be reduced by earlier predictive humane endpoints, pain-relieving medications, and supportive clinical care, while sequential testing and routine use of integrated testing and decision strategies can reduce animal numbers. Applying advances in science and technology to the development of scientifically sound alternative testing models and strategies can improve animal welfare and further reduce and replace animal use.

Recent progress and future directions for reduction, refinement, and replacement of animal use in veterinary vaccine potency and safety testing: a report from the 2010 NICEATM-ICCVAM International Vaccine Workshop.

Veterinary vaccines contribute to improved animal and human health and welfare by preventing infectious diseases. However, testing necessary to ensure vaccine effectiveness and safety can involve large numbers of animals and significant pain and distress. NICEATM and ICCVAM recently convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing, and to identify priority activities to advance new and improved methods that can further reduce, refine and replace animal use. Rabies, Clostridium sp., and Leptospira sp. vaccines were identified as the highest priorities, while tests requiring live viruses and bacteria hazardous to laboratory workers, livestock, pets, and wildlife were also considered high priorities. Priority research, development and validation activities to address critical knowledge and data gaps were identified, including opportunities to apply new science and technology. Enhanced international harmonization and cooperation and closer collaborations between human and veterinary researchers were recommended to expedite progress. Implementation of the workshop recommendations is expected to advance new methods for vaccine testing that will benefit animal welfare and ensure continued and improved protection of human and animal health.

ICCVAM evaluation of the murine local lymph node assay. The ICCVAM review process.

New test methods are being developed to improve the prediction of human and environmental risks and to benefit animal welfare by reducing, refining, and replacing animal use. Regulatory adoption of new test methods is often a complex and protracted process, requiring test method validation, regulatory acceptance, and implementation. Assessments of new test methods have not always been uniform within or among regulatory agencies. Thus, there have been increased pressures for a harmonized approach to test method evaluation and acceptance. In 1997, in response to these pressures and to U.S. Public Law 103-43, the National Institute of Environmental Health Sciences (NIEHS) established the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to coordinate interagency consideration of new and revised test methods. This article describes the validation and acceptance criteria and process used for the first test method evaluated by ICCVAM, the murine local lymph node assay (LLNA). Based on ICCVAM's conclusions and recommendations, the LLNA has been accepted by U.S. regulatory agencies as a stand-alone assay for allergic contact dermatitis. Two related articles in this series of three present the results of the independent peer review evaluation of the LLNA and summarize the performance characteristics of the database substantiating the validity of the LLNA.

ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel.

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose-response information and statistical analyses. A standardized LLNA protocol is provided.

ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods.

To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.

Animals as sentinels of human health hazards of environmental chemicals.

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environment," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and nonmammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Workshop overview: scientific and regulatory challenges for the reduction, refinement, and replacement of animals in toxicity testing.

Public concern for animal welfare has been expressed through legislative control of animal use for experimental purposes since the first legislation was introduced in 1876 in the United Kingdom. Legislative control of animal use has been introduced in virtually every developed country, with major initiatives in Europe (1986) and the United States (1966 and 1985). Advances in scientific thinking resulted in the development of the concept of the three Rs--refinement, reduction, and replacement--by Russell and Burch in 1959. The field has expanded substantially since, with specialist scientific journals dedicated to alternatives, World Congresses organized to discuss the scientific and philosophical issues, and European and U.S. validation organizations being launched. Current scientific attention is focused on validation of alternative methods. The underlying scientific principles of chemical toxicity are complicated and insufficiently understood for alternative methods for all toxicity endpoints of importance in protecting human health to be available. Important lessons have been learned about how to validate methods, including the need to have prediction models available before the validation is undertaken, the need to understand the variability of the animal-based data which is to be used as the validation standard, and the need to have well-managed validation programs. Future progress will depend on the development of novel methods, which can now be validated through international collaborative efforts.

Validating new toxicology tests for regulatory acceptance.

Before a new or revised toxicology test is considered acceptable for safety evaluation of new substances, the test users and the industrial and regulatory decision makers must feel comfortable with it, and the decisions it supports. Comfort with, and the acceptance of, a new test comes after knowing that it has been validated for its proposed use. The validation process is designed to determine the operational characteristics of a test, that is, its reliability and relevance, in addition to its strengths and limitations. The reliability of a test is measured by its reproducibility. Its relevance is judged by its mechanistic relationship to the health effects of concern, and its ability to predict or identify those effects. The U.S. government has recently formed the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to work with federal agencies and test developers to coordinate the evaluation and adoption of new test methods. The ICCVAM will provide guidance to agencies and other stakeholders on criteria and processes for development, validation, and acceptance of tests; coordinate technical reviews of proposed new tests of interagency interest; facilitate information sharing among agencies; and serve as an interagency resource and communications link with parties outside of the federal government on matters of test method validation.

Introduction and summary of the 13th meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies.

A workshop on alternative toxicological testing methodologies was convened by the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC) 26-31 January 1997 in Ispra, Italy, at the European Centre for the Validation of Alternative Methods. The purpose of the workshop was to assess the current status of alternative testing methodologies available to evaluate adverse human health and environmental effects of chemicals. Another objective of the workshop was to identify and recommend research needed to fill knowledge gaps that would lead to new test methodologies. Four work groups were established to address conceptual issues, acute toxicity, organ toxicity, and ecotoxicology. A joint workshop report was prepared for each topic and included recommendations for the development and use of alternative methods. Participants concluded that alternative methods and approaches are available that can be incorporated into tiered strategies for toxicological assessments. Use of these methods will reduce the numbers of animals required, and in some instances reduce animal pain and distress. It was recommended that future efforts to develop test methods should emphasize mechanism-based methods that can provide improved predictions of toxicity. Continued international cooperation was encouraged to facilitate future progress in the development of alternative toxicological testing methods. These methods will provide for improvements in human health protection, environmental protection, and animal welfare.

13th meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies and conceptual issues.

Substantial world-wide resources are being committed to develop improved toxicological testing methods that will contribute to better protection of human health and the environment. The development of new methods is intrinsically driven by new knowledge emanating from fundamental research in toxicology, carcinogenesis, molecular biology, biochemistry, computer sciences, and a host of other disciplines. Critical evaluations and strong scientific consensus are essential to facilitate adoption of alternative methods for use in the safety assessment of drugs, chemicals, and other environmental factors. Recommendations to hasten the development of new alternative methods included increasing emphasis on the development of mechanism-based methods, increasing fundamental toxicological research, increasing training on the use of alternative methods, integrating accepted alternative methods into toxicity assessment, internationally harmonizating chemical toxicity classification schemes, and increasing international cooperation to develop, validate, and gain acceptance of alternative methods.

A standard procedure for measuring pellet hardness of rodent diets.

A Chatillon Model TCM-200 test stand with exchangeable flat horizontal or concave receptacle bases and a DFI-200 gauge load cell with multiple types of upper exchangeable test jaws (large round-flat, medium round-flat, chisel, bullet, and cone-shaped) were compared by using preautoclaved and autoclaved NIH-31 rodent diet pellets to determine which type of hardness testing system would give the most accurate and reproducible results for measuring pellet hardness. The type and size of the contact area of the upper jaws significantly affected the force required to break the pellets. Significant differences were observed between the flat-horizontal and concave receptacle bases in the force required to break the pellets when using the two round-flat upper jaws. In contrast, similar results were obtained with both bases when the bullet, chisel, or cone-shaped upper jaws were used. Autoclaved pellets were 69.4% (range, 49 to 94%) harder than preautoclaved pellets. These results suggest that different testing systems can be used for measuring pellet hardness and that a standard procedure must be used in order to compare pellet hardness results between different testing laboratories. It was concluded that the flat-horizontal base and the larger round-flat end upper jaw gave the most reproducible results for measuring pellet hardness.

Daily administration of the progesterone antagonist RU 486 prevents implantation in the cycling guinea pig.

Since progesterone is required to prepare the endometrium for implantation of an embryo, a progesterone antagonist may inhibit nidation and thus prevent pregnancy. We addressed this possibility in the guinea pig, the small laboratory animal whose reproductive physiology most resembles that of women. Daily administration of the antiprogestin RU 486 (0, 1, 2, or 3 mg/kg, subcutaneously) for 9 days after mating inhibited implantation in a dose-dependent fashion. When this compound was given daily throughout the estrous cycle, cyclic vaginal changes, ovulation, and mating were suppressed in up to 17%, 28%, and 55% of animals, respectively. Two of seven mated female animals receiving RU 486, 1 mg/kg/day, had implantation sites. Nidation was completely blocked at higher doses. Thus daily antiprogestin administration prevented pregnancy in sexually active, normally cycling guinea pigs. A similar strategy using a daily antinidatory dose of an antiprogestin may offer a novel approach to human fertility control.

Acute clinical malaria (Plasmodium inui) in a cynomolgus monkey (Macaca fascicularis).

Acute clinical malaria caused by Plasmodium inui was diagnosed in an adult female cynomolgus monkey (Macaca fascicularis) 4 years after importation into the United States. Stress and immunosuppression associated with experimentation completed 2 weeks earlier may have contributed to the development of severe clinical disease. Clinical findings included severe regenerative anemia, hepatosplenomegaly, weakness, lethargy, weight loss, and anorexia. The infection was treated and successfully eliminated with chloroquine hydrochloride administered intramuscularly at a dose of 5 mg/kg base given at 0, 6, 24, 48, and 72 hours. Treatment also included a blood transfusion and intensive supportive care.

Hematologic characterization of naturally occurring malaria (Plasmodium inui) in cynomolgus monkeys (Macaca fascicularis).

Twenty of 47 recently imported cynomolgus monkeys (Macaca fascicularis) were found to have malarial infections. The agent identified was Plasmodium inui. All infections were subclinical in nature. Parasitemias ranged from 10 to 900 parasites/mm3 of whole blood. Pre- and post-treatment hematologic values were evaluated following treatment with chloroquine. Treatment was effective in clearing parasitemias from 13 of 14 infected monkeys. Pretreatment values of hematocrit, hemoglobin, and mean corpuscular volume were significantly different in infected animals compared to noninfected animals. While post-treatment hemoglobin and hematocrit values returned to noninfected control levels, mean corpuscular volume values of infected animals remained significantly lower in the post-treatment period.