Tier Testing Research Articles

Factors considered in using birds for evaluating endocrine-disrupting chemicals.

Documented effects on fish and wildlife populations, coupled with evidence from human poisonings, epidemiology, and experimental toxicology, led to the formation of the Endocrine Disruptor Screening Program within the US Environmental Protection Agency. The main objectives of the program are to validate and implement the screens and tests that have been proposed for evaluating possible endocrine-disrupting activity of chemicals. An avian two-generation test is one of the recommended higher tier tests currently undergoing prevalidation. The advantages and disadvantages of the two species of quail considered as candidates, the northern bobwhite (Colinus virginianus) and the Japanese quail (Coturnix japonica), are described as well as the basis for final selection of the Japanese quail. Among the numerous considerations necessary for ultimately optimizing a two-generation test method using birds, the following key factors are discussed: the number of birds used in the test, when to begin exposure of the P generation, selection and exposure of the F1 generation, and endpoints.

The benefits of the 3T3 NRU test in the safety assessment of cosmetics: long-term experience from pre-marketing testing in the Czech Republic

We have introduced the 3T3 NRU cytotoxicity test for methodological, economical and ethical reasons as a regular part of tier pre-marketing testing to assess local tolerance of raw materials for cosmetics, household chemicals and final cosmetic products. Using the 3T3 cell line according to the standard INVITTOX protocol No.64 (NRU Assay) the borderline concentration, relevant to the highest tolerated dose, is determined for each material. The toxic effect is reached at different concentration levels specific for individual cosmetics categories, depending on their chemical characteristics. Typical ranges of cytotoxicity for specific categories of cosmetics were established after testing of hundreds of materials. The range lies between 1 μg/ml (anti-dandruff shampoos), up to 2000 μg/ml (toothpastes and mouthwashes). The 3T3 NRU cytotoxicity test is a sensitive tool able to identify more agressive products, that are also more likely to evoke irritation in human skin. It was even possible to detect protective effects of one natural herbal ingredient. The comparative study of cytotoxicity test results and human patch test results from a group of essential oils is presented. Cytotoxicity tests represent a highly ethical approach for estimation of irritancy. On the basis of in vitro test results suggesting low risk we can proceed to confirmatory tests in human volunteers.

Environmental risk assessment scheme for plant protection products

Environmental risk assessment scheme for plant protection products

Ecological risk assessment and testing for endocrine disruption in the aquatic environment

Field observations of endocrine disruption (ED) in fish and other aquatic species have contributed to a global effort to establish test methods for detecting ED effects in wildlife species. For ecological risk assessment, validated tests are needed for Amphibia, fish and aquatic invertebrates, supported by a tiered approach incorporating mechanistic data and exposure characterisation. The potential for extrapolation of ED data from mammalian to aquatic species may be limited, however, due to significant physiological differences in function and regulation of hormone systems in (aquatic) lower vertebrates and invertebrates. Presently, the OECD is considering a tiered approach for ED risk assessment, incorporating a fish 14-day screening assay (Tier 1); fish development and reproduction tests (both Tier 2); and a fish full life-cycle test (Tier 3). For detection of (anti-)oestrogens, the yolk-precursor protein vitellogenin is an ideal biomarker of exposure and functionally equivalent biomarkers are being sought for (anti-)androgens in fish. At the two higher tiers, impacts are assessed in terms of apical endpoints (e.g. development, breeding behaviour and fecundity) and also gonadal histopathology. Validation of these higher tier tests should include comparison of sensitivity of biochemical and apical endpoints to optimise the value of biomarkers for predicting adverse health effects (e.g. impaired reproduction). The specificity of future OECD fish and amphibian test guidelines for endocrine disrupters needs further consideration through inclusion of mechanistic endpoints based on state-of-the-art molecular endocrinology.

Possibilities for Refinement and Reduction: Future Improvements Within Regulatory Testing

Approaches and challenges to refining and reducing animal use in regulatory testing are reviewed. Regulatory testing accounts for the majority of animals reported in the most painful and/or distressful categories in the United States and Canada. Refinements in testing, including the use of humane endpoints, are of increasing concern. Traditional approaches to reduction (e.g., improving experimental design) are being supplemented with complementary approaches, such as the use of tier testing to eliminate some chemicals prior to in vivo testing. Technological advances in telemetry and noninvasive techniques will help decrease either the demand for animals in testing or animal suffering. Further decreases in animal use will stem from international harmonization and coordination of testing programs. Progress in refinement and reduction faces a variety of broad challenges, including limited funding for research. In the specific area of refinement, a key challenge is the issue of distress (as distinct from pain). In the area of reduction, the practice of using unjustifiably high numbers of animals from small species (e.g., rodents) should be challenged. One case study of the use of carbon dioxide as a euthanasia agent illustrates the need for further analysis and research. Notwithstanding the complexities and challenges, the potential for refinement and reduction in regulatory testing is encouraging.

Ecological Risk Assessment of Endocrine Disruptors

The European Centre for Ecotoxicology and Toxicology of Chemicals proposes a tiered approach for the ecological risk assessment of endocrine disruptors, integrating exposure and hazard (effects) characterization. Exposure assessment for endocrine disruptors should direct specific tests for wildlife species, placing hazard data into a risk assessment context. Supplementing the suite of mammalian screens now under Organization for Economic Cooperation and Development (OECD) validation, high priority should be given to developing a fish screening assay for detecting endocrine activity in oviparous species. Taking into account both exposure characterization and alerts from endocrine screening, higher tier tests are also a priority for defining adverse effects. We propose that in vivo mammalian and fish assays provide a comprehensive screening battery for diverse hormonal functions (including androgen, estrogen, and thyroid hormone), whereas Amphibia should be considered at higher tiers if there are exposure concerns. Higher tier endocrine-disruptor testing should include fish development and fish reproduction tests, whereas a full life-cycle test could be subsequently used to refine aquatic risk assessments when necessary. For avian risk assessment, the new OECD Japanese quail reproduction test guideline provides a valuable basis for developing a test to detecting endocrine-mediated reproductive effects; this species could be used, where necessary, for an avian life-cycle test. For aquatic and terrestrial invertebrates, data from existing developmental and reproductive tests remain of high value for ecological risk assessment. High priority should be given to research into comparative endocrine physiology of invertebrates to support data extrapolation to this diverse fauna.

Ecological risk assessment of endocrine disruptors.

The European Centre for Ecotoxicology and Toxicology of Chemicals proposes a tiered approach for the ecological risk assessment of endocrine disruptors, integrating exposure and hazard (effects) characterization. Exposure assessment for endocrine disruptors should direct specific tests for wildlife species, placing hazard data into a risk assessment context. Supplementing the suite of mammalian screens now under Organization for Economic Cooperation and Development (OECD) validation, high priority should be given to developing a fish screening assay for detecting endocrine activity in oviparous species. Taking into account both exposure characterization and alerts from endocrine screening, higher tier tests are also a priority for defining adverse effects. We propose that in vivo mammalian and fish assays provide a comprehensive screening battery for diverse hormonal functions (including androgen, estrogen, and thyroid hormone), whereas Amphibia should be considered at higher tiers if there are exposure concerns. Higher tier endocrine-disruptor testing should include fish development and fish reproduction tests, whereas a full life-cycle test could be subsequently used to refine aquatic risk assessments when necessary. For avian risk assessment, the new OECD Japanese quail reproduction test guideline provides a valuable basis for developing a test to detecting endocrine-mediated reproductive effects; this species could be used, where necessary, for an avian life-cycle test. For aquatic and terrestrial invertebrates, data from existing developmental and reproductive tests remain of high value for ecological risk assessment. High priority should be given to research into comparative endocrine physiology of invertebrates to support data extrapolation to this diverse fauna.

Yeasts as a model for assessing the toxicity of the fungicides Penconazol, Cymoxanil and Dichlofluanid

In the present work the sensitivity of yeast strains of Kluyveromyces marxianus, Pichia anomala, Candida utilis, Schizosaccharomyces pombe and Saccharomyces cerevisiae, to the fungicides cymoxanil, penconazol, and dichlofluanid, was evaluated. Dichlofluanid induced the most negative effects, whereas penconazol in general was not very toxic. Overall, our results show that the parameters IC 50 for specific respiration rates of C. utilis and S. cerevisiae and C D for cell viability of S. cerevisiae can be applied to quantify the toxicity level of the above compounds in yeast. Hence, could be explored as an alternative or at least as a complementary test in toxicity studies and, therefore, its potential for inclusion in a tier testing toxicity test battery merits further research.

Experimental studies on immunosuppression: how do they predict for man?

The ultimate goal of any animal model in immunotoxicity testing is that it be a sensitive predictor of xenobiotic-induced immune dysfunction in humans. Such models should be capable of identifying the target(s) within the immune system affected by the xenobiotic. In particular the tier testing models have been successfully used to identify and characterize a variety of different immunotoxicants in animals as it pertains to immunosuppression and reduced resistance to infectious diseases. These tier models in mice and rats have been validated in interlaboratory studies. Although these protocols were designed for studies of rats and mice, some have been applied succesfully for studying immunotoxicity in other animal species, including non-human primates. A great amount of data has been generated by the application of these models, which demonstrate that xenobiotics alter the immune system of animals. In man, the database on chemical-induced immunosuppression is limited, as the use of markers of immunotoxicity has received little attention in clinical and epidemiological studies. Such studies have not been performed frequently, and their interpretation often does not permit unequivocal conclusions to be drawn, due for instance to the presence of confounding factors and the uncontrolled nature of exposure. Also, testing possibilities in humans are limited and immune function changes by chemical exposure are often subtle. In humans, a number of agents have been shown to have immunosuppressive properties (including PCBs, PCDDs, PCDFs, oxidant gases, and ultraviolet radiation), but the strongest evidence stems from the clinical use of immunosuppressant drugs in transplant patients. These human data do in general terms confirm the data gained with experimental animals. Immunotoxicity assessment in rodents therefore adequately forms the basis for human risk assessment. Knowledge on the predictability of these animal models and immune assays can be further improved by comparison of the human and animal data obtained in the development of drugs.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

A Tier-Testing Strategy for Managing Programs of Environmental Effects Monitoring

Abstract A good environmental effects monitoring program (EEM) provides answers to clear, well-defined questions. The answers should be quantitative and lead directly to decisions about the effluent being studied and about further EEM studies. Preliminary studies should trigger more in-depth monitoring only when predefined thresholds are exceeded. When in-depth work suggests that further studies are unnecessary, there should be a return to a lower level of effort. These criteria lead directly to a tiered strategy for managing EEM that defines the sequence and choice of studies through successive cycles. The benefits of a tiered approach are (1) rules that all stakeholders can understand and comply with; (2) monitoring limited to that which is essential; and (3) assurance that all appropriate elements are included, but only when necessary. Within the context of the new pulp and paper EEM program, tier testing is an efficient, logical and systematic strategy for managing monitoring.

Use and misuse of biomarker tests in “environmental conditions”

Use and misuse of biomarker tests in “environmental conditions”

An immunotoxicity assessment of food flavouring ingredients

A rapid screening protocol incorporating key elements of the US National Toxicology Program's immunotoxicity tier testing strategy was used to evaluate the effects of 35 commonly used food flavouring ingredients on humoral and cell-mediated immune responses. The test compounds were administered intragastrically on a daily basis for 5 days at three dose levels to female CD-1 or B6C3F 1 mice, 6–8 wk old. A host resistance assay ( Listeria monocytogenes bacterial challenge) was conducted to assess cell-mediated immunity. Humoral immunity was measured by the antibody plaque-forming cell (PFC) response to sheep erythrocytes. Body weights, lymphoid organ weights and spleen cellularity were also measured. Cyclophosphamide (80 mg/kg) served as an immunosuppressive positive control agent. The results indicated that the majority of the flavouring ingredients tested did not modulate the cell-mediated or humoral immune response. However, at very high dose levels, two of the materials tested, peppermint oil and citral dimethyl acetal, did increase mortality rate and reduce survival time in the host resistance assay. Neither of these materials significantly altered the PFC response. This rapid, economical screening battery for potential immunotoxicants proved to be a useful means of evaluating a large number of structurally diverse compounds and mixtures to prioritize them for more definitive testing.

New approaches to the assessment of eye and skin irritation

Assessment of eye and skin irritation potential is an important part of any comprehensive toxicology programme for new chemicals and consumer products. The original skin and eye irritation assessment methods described by Draize et al. are still widely used for regulatory purposes with only slight modification. These methods have been re-evaluated in the light of the modern approach to toxicological testing requiring refinement of protocols, reduction in animal numbers, and replacement of in vivo tests by in vitro assays. A refinement in the original Draize skin irritation test accepted by regulatory authorities is reduction of test substance exposure time from 24 to 4 h. This makes the test less stressful. The Low Volume Eye Irritation Test is also a less stressful, more predictive refinement of the Draize eye irritation procedure. Many in vitro models are being developed for use in the assessment of eye and skin irritation potential in the hope that the animal tests may some day be replaced. In order for alternative tests to be incorporated into safety assessment, they must undergo a defined evaluation that allows toxicologists to determine whether the new procedures provide relevant information. As the validity of the methods is determined, the new procedures can be incorporated into safety assessment using a tier assessment process. The first step in tier testing is a thorough analysis of existing in-house and published data on the test substance or similar material. A review of physical/chemical characteristics can also provide valuable information. If more data are needed in order to complete the safety assessment, the next step is to evaluate the test substance in in vitro assays. The in vitro data may provide sufficient information to make a safety assessment. If additional information is required, then in vivo testing may be conducted using a limited number of animals or human clinical studies. If alternative procedures are to be ultimately accepted for use in safety assessment, regulatory authorities must become active participants in programmes designed to assess and validate the new tests.

Strategy for and Experience with Neurotoxicity Testing of New Pharmaceuticals

In response to present and anticipated regulatory needs for routine screening of pharmaceuticals for assessment of their neurotoxic potential, a primary tier screen for rodents, consisting of a functional observational battery (FOB) and an automated test of motor activity, has been developed at Searle. Additionally, an FOB for dogs currently is being developed. The rodent FOB assess such functions as home cage and open field activity, stimulus reactivity, and neuromuscular function. The dog FOB emphasizes evaluation of gait, postural reactions, and reflex function. The strategy taken has been to incorporate the primary rodent tier screen into repeated dose preclinical studies. Positive findings would trigger a secondary tier of testing, which would involve the use of more complex and integrated tests of neurobehavioral function. The methodologies for both rodent and nonrodent primary tier screens, validation study results, and the scientific criteria that would trigger secondary tier testing are discussed.

A toxicological screen to determine in vitro survival and competition of environmentally applied pseudomonads with mouse fecal microbiota

Engineered bacterial strains have been developed for several environmental uses, including bioremediation and application as biopesticides. Previous in vivo work in this laboratory examined the colonization and competition potential of Pseudomonas spp., isolated from a commercial product for PCB degradation with the intestinal flora of CD-1 mice. In the present study, the same pseudomonads and two additional Escherichia coli strains were evaluated by an in vitro tier testing approach developed to screen environmentally relevant microorganisms for their ability to colonize the mammalian intestinal tract and compete with the normal intestinal tract flora. In the initial tier, the competitor strains were tested for their ability to survive in anaerobic pure culture. Next, they were cultured anaerobically in the presence of mouse fecal microbiota. Finally, serial transfers were performed to monitor the survival and competition potential of each strain with the fecal microbiota. All of the tested strains survived for 48 h in pure culture, and all survived for 24 h in culture with the mouse fecal microbiota. Escherichia coli strains survived serial transfer through five cultures, although in declining numbers. Environmental Pseudomonas spp. survived transfer through three cultures but were not detected after four transfers. No effects of the competitor strains on the fecal microbiota were found. This in vitro system eventually will be used with human fecal microbiota. The potential consequences of human exposure to environmentally released microorganisms can then be extrapolated by comparison of human in vitro results with rodent in vitro and in vivo results in a parallelogram approach.

Two tier sight testing

Two tier sight testing

Evolution of testing strategies for genetic toxicity

Shortly following the inception of genetic toxicology as a distinct discipline within toxicology, questions arose regarding the type and number of tests needed to classify a chemical as a mutagenic hazard or as a potential carcinogen. To some degree the discipline separated into two sub-specialties, (1) genetic risk assessment and (2) cancer prediction since data from experimental oncology also supports the existence of a genotoxic step in tumor initiation. The issue of which and how many tests continued to be debated, but is now focused more tightly around two independent phenomena. Tier or sequential testing was initially proposed as a logical and cost-effective method, but was discarded on the basis that the lower tier tests appeared to have too many false responses to force or exclude further testing of the test agent. Matrix (battery) testing was proposed for screening on the hypothesis that combinations of endpoints and multiple phylogenetic target organisms were needed to achieve satisfactory predictability. As the results from short-term test ‘validation’ studies for carcinogen prediction and evaluations of EPA's Gene-Tox data accumulated, it became obvious that qualitative differences remained between predictive and definitive tests and by assembling different combinations of short-term assays investigators did not appear to resolve the lack of concordance. Recent trends in genetic toxicology testing have focused on mathematical models for test selection, and standardized systems for multi-test data assessment.

A new approach for using short-term tests to screen complex mixtures

This paper suggests a framework for developing parsimonious batteries of rapid, inexpensive biological assays which provide quantifiable estimates of human risk. The framework for the statistical design is tier testing. The framework develops an unusual perspective for using chemical spiking to evaluate the toxicity of a complex mixture to a battery of bioassays. This framework allows immediate evaluation of hazardous wastes and identifies important problems to provide focus for basic research.

Growth and photosynthetic response of a freshwater alga,Selenastrum capricornutum, to an oil shale by-product water

Several recent studies have focused on toxic effects of various oil shale waters to freshwater algae (e.g., Cleave et al. 1980; McKnight et al. 1983). Algal bioassays are ecologically significant, since algae are the dominant primary producers in most freshwater environments. Furthermore, algae have been shown to be more sensitive to complex wastes than fish or invertebrates. Using a standard algal species provides a systematic approach to toxicant evaluation. One such species is Selenastrum capricornutum Printz, a freshwater unicellular green alga. A strategy to assess the hazards of complex effluents should include a battery of rapid, easily conducted, predictive tests within the larger framework of tier testing. This approach is used in this study to compare the growth and photosynthetic response of S. capricornutum to an oil shale by-product water and its organophilic and hydrophilic subfractions.