The effect of essentiality on risk assessment.

Metals are ubiquitous in the human environment, making exposure inevitable and requiring scientifically sound risk assessment methodology to ensure adequate health protection. Within this area, as part of its ongoing efforts to improve and harmonize internationally approaches to risk assessments, the International Program on Chemical Safety (IPCS) has initiated work to improve the risk assessment procedures for essential trace elements (ETEs). Zn, Cu, Se, Cr, and MO are ETEs for humans, with increasing evidence of an essential role for boron (B). For ETEs, there is a range of daily intake within which the organism maintains homeostasis. At intakes below this range, there is an increased risk from deficiency, and at intakes above the range toxicity may develop. Obviously, for ETEs one cannot assume zero exposure is without risk. Adequate health protection will require the cooperative effort of scientists in nutrition and toxicology to develop the limits of the accepted range for ETEs considering such unique properties of metals as bioavailability, speciation, interactions, and biokinetics. Based on previous work by other groups and the recommendations of an IPCS consultation, a scientific monograph will be completed by IPCS. It will examine present risk assessment methodology for ETEs, and develop scientific principles supporting use of a homeostatic model for the development of dietary reference values and tolerable daily intakes. The objective is to develop an internationally accepted methodology for assessing ETEs as part of the IPCS effort to harmonize approaches to risk assessment worldwide. A recent IPCS Task Group on Zn highlighted some of the scientific issues that require resolution to avoid an overlap of the recommended daily intake based on nutritional needs with that based on toxicity and will serve as a case study.

International Programme on Chemical Safety (IPCS) environmental health criteria on boron human health risk assessment.

The International Program on Chemical Safety (IPCS), a collaborative program of the United Nations Environment Program, the International Labour Organization (ILO), and the World Health Organization (WHO), includes the Environmental Health Criteria (EHC) Program inaugurated in 1973 by WHO. These EHC are integrated evaluations of the human health and environmental risks from exposure to specific chemicals carried out by a group of international scientists. Boron (B) was evaluated at an IPCS Task Group (TG) convened in November 1996. All TGs are convened under WHO rules and procedures. These procedures relate the overall process used to prepare an EHC including transparency of the process, conflict of interest, the roles of Members and Observers, and the conduct of the TG. The scope and purpose of an EHC, for an element such as B, and its possible role in national and international chemical safety programs will be discussed. In the early 1990s, countries asked that IPCS request TGs to prepare, where data permit, health-based guidance values (GVs) (both total daily intake and to recommend health-based guidelines for various environmental media). This final evaluation in an EHC reflects the collective consensus view of the TG Members. To foster the use of consistent methodology by TGs, IPCS prepared in 1994 an EHC on the methodology for the preparation of GVs for human exposure limits (EHC 170). In developing their final evaluation, TGs have been asked to consider using this methodology. This was done by the TG on B, and a total daily intake for humans of 0.4 mg/kg body wt was derived from animal studies of reproductive and developmental effects in rodents and pharmacokinetic data from both animals and humans. The application of the methodology described in EHC 170 regarding choice of critical effect and uncertainty factors will be discussed.

The IPCS collaborative study on neurobehavioral screening methods.

The International Programme on Chemical Safety sponsored a collaborative study to evaluate the utility of neurobehavioral test methods for identifying neurotoxic chemicals. The protocol consisted of a functional observational battery and automated assessment of motor activity. The study involved four laboratories in the United States and four in Europe, each of which evaluated the dose- and time-related effects of seven prototypic chemicals following both single and 4-week repeated exposures. The protocol was designed to assess the general utility and reliability of neurobehavioral screening procedures in a diversity of testing situations. The results of chemical testing indicated that all participating laboratories generally could detect and characterize the effects of known neurotoxicants, despite some differences on specific endpoints. These data provide important information regarding the reliability and sensitivity of neurobehavioral screening methods over a range of laboratory conditions. The purpose of this workshop was to describe the background and study design of the collaborative effort, present the data (including comparison of results across laboratories), and discuss issues regarding the conduct and interpretation of these behavioral tests, as well as future directions for neurotoxicity screening.

The IPCS Collaborative Study on Neurobehavioral Screening. I. Background and genesis.

Numerous events over several years culminated in recognition of the need to explicitly evaluate the nervous system as a potential target for environmental chemicals. Based on recommendations from several international expert panels, the International Programme on Chemical Safety (IPCS) sponsored the Collaborative Study on Neurobehavioral Screening Methods. A Steering Committee was created to oversee the project, develop the testing protocol, recruit participating laboratories and review and analyze the data. The protocol specified the tests, the chemicals (supplied from a common source) and the exposure conditions (acute and repeated dosing). Test methods were based upon existing practices in toxicological screening as well as recent advances in neurotoxicity screening. Chemicals were selected to produce different profiles of neurobehavioral effects. Considerable latitude was afforded the participating laboratories in the choice of several key variables (e.g., strain of rat, testing device for motor activity assessment) that could potentially affect the results of the experiments. The approach therefore provided a standardized yet flexible protocol for evaluating the reproducibility of neurobehavioral screening data in diverse laboratory settings.

The IPCS Collaborative Study on Neurobehavioral Screening Methods: II. Protocol design and testing procedures.

This paper describes the development of the protocol for the International Programme on Chemical Safety (IPCS)-sponsored Collaborative Study on Neurobehavioral Screening Methods, including background on the methods and chemicals selected, as well as details concerning the conduct of the collaborative study, including proficiency testing, range-finding and main study. Participating laboratories in the collaborative study received training in the conduct and scoring of the behavioral tests and each laboratory received a video training film to train additional personnel as needed. Each of the eight laboratories that chose to participate in the study completed proficiency testing and assessed seven representative chemicals using a functional observational battery and automated motor activity assessment. The seven chemicals studied were acrylamide, bis-acrylamide, p,p'-DDT, lead acetate, parathion, toluene, and triethyl tin. Participants received coded samples of the chemicals from a common source. Each laboratory derived doses for single and repeated administration based on the determination of a within-laboratory acute "top dose." Animal strains were not standardized and laboratory conditions were standardized to a limited degree in order to judge the general utility and robustness of these procedures in a diversity of testing situations.

The IPCS Collaborative Study on Neurobehavioral Screening Methods: III. Results of proficiency studies. Steering Group.

The goal of the IPCS Collaborative Study on Neurobehavioral Screening Methods was to determine the intra- and inter-laboratory reliability of a functional observational battery (FOB) and an automated assessment of motor activity in eight laboratories world-wide. The first phase of the Collaborative Study involved training the participants: evidence of training was then evaluated using positive-control compounds. The positive-control studies required the laboratories to identify, using the FOB, specific neurotoxic syndromes produced by acute exposure to p,p'-DDT, parathion, and by short-term repeated dosing with acrylamide. For the sake of expediency, only one dose of each chemical was used instead of collecting dose-response data. Motor activity test chambers were not of uniform design. The laboratories were therefore required to demonstrate adequate sensitivity by the ability to detect statistically-significant activity increases and decreases produced by triadimefon and chlorpromazine, respectively, following acute administration of a range of doses. The resulting FOB and motor activity data showed variability in the magnitude of effects obtained: some of these differences were attributed to miscommunications, difficulties with the techniques or protocol, or the limitations of having only one dose. All laboratories, however, successfully met the criteria set forth by the Study Steering Committee.

The IPCS Collaborative Study on Neurobehavioral Screening Methods: IV. Control data. Steering Group.

The goal of the International Programme on Chemical Safety (IPCS) Collaborative Study on Neurobehavioral Screening Methods was to determine the intra- and inter-laboratory reliability of a functional observational battery (FOB) and an automated assessment of motor activity in eight laboratories worldwide. The control data were crucial to the outcome of the studies in terms of sensitivity and reliability of the test measures, which in turn impact on the between-laboratory comparisons of chemical effects. In addition, analyses of control data can aid in determining endpoints that may require modification to improve their sensitivity and reliability. The control data from the eight laboratories were examined in terms of the following parameters: 1) control variability within studies for each laboratory; 2) within-laboratory replicability of control values across studies; 3) within-laboratory stability of control values over the course of testing for a given study; and 4) between-laboratory comparisons of parameters (1), (2), and (3). The analyses indicated considerable differences across endpoints, wherein some measures showed high variability and little replicability, while others were extremely reproducible. Generally, there were similar ranges of variability and replicability of control data across laboratories, although in some cases one or two laboratories were markedly different from the others. The physiological (weight, body temperature) and neuromuscular (grip strength, landing foot splay) endpoints exhibited the least variability, whereas the subjective assessments of reactivity varied the most. These data indicate a reasonable degree of comparability in the data generated in the participating laboratories.

The IPCS Collaborative Study on Neurobehavioral Screening Methods: V. Results of chemical testing. Steering Group.

The IPCS Collaborative Study on Neurobehavioral Screening Methods was undertaken to determine the intra- and inter-laboratory reliability of a functional observational battery (FOB) and an automated assessment of motor activity in eight laboratories world-wide. Following the training phase and the conduct of proficiency studies in all laboratories, participants proceeded to test the effects of seven chemicals in both single dose and four-week repeated dosing scenarios. The chemicals studied were acrylamide, bisacrylamide, p,p'-DDT, lead acetate, parathion, toluene, and triethyl tin. Participants received coded samples from a common source. In order to judge the general utility of these procedures in a diversity of testing situations, laboratories conducted the studies under their standard conditions, using their choice of rat strain and test equipment. Chemical does and time of peak effect for acute testing were determined by each laboratory: these parameters were quite similar for some chemicals, but varied greatly for others. The results of the chemical tests indicated that while there was some variability in the data on specific endpoints, all laboratories detected and characterized the effects of all but one of the known neurotoxicants. The one exception (toluene) was probably due to other factors (e.g., dose level, route of administration) rather than lack of sensitivity of the test methods. This study provides extensive data regarding the use of neurobehavioral screening methods over a range of laboratory conditions as well as the reliability, sensitivity, and robustness of the tests to detect neurotoxic potential of chemicals.

The IPCS Collaborative Study on Neurobehavioral Screening Methods: VII. Summary and conclusions.

In the International Programme on Chemical Safety (IPCS) Collaborative Study on Neurobehavioral Screening Methods, eight participating laboratories used a standard battery of behavioral tests to determine, in rats, the effects of seven representative chemicals following acute and repeated dosing. The results of the collaborative study indicate good agreement across laboratories with regard to the data collected in vehicle controls. It was clear, however, that some behavioral measures had significantly more variability than other tests. The laboratories also demonstrated the ability to detect known neurotoxic chemicals and identify profiles of effects that differed from non-neurotoxic agents. The results of the study suggest that appropriate training of personnel is crucial to ensure the reliability of the test battery. The results also underscore the importance of dose selection in behavioral screening studies, since it is sometimes difficult to determine the specificity of behavioral changes in animals receiving high doses of some chemicals. The collaborative study also emphasizes the need to utilize a battery of tests in screening a wide range of potential neurotoxic agents. Analysis of data from such studies poses unique challenges due to the large number of tests and test times, and the consequent possibility of false positives. Some statistical concerns may be alleviated by grouping the results from tests that measure similar functions into neurobiological domains. Although this approach improves confidence in the biological relevance of chemical-induced changes in behavior, it may also lead to false negatives. The exploration of other statistical approaches to analyze data from experiments using a test battery is encouraged. Nevertheless, results of the collaborative study strongly support the use of behavioral tests in hazard identification.

Use of mechanistic information in risk assessment for toxic chemicals.

Risk assessment (RA) for toxic chemicals is assumed to be a scientific activity providing a framework of principles for the complication and evaluation of all available scientific information and the rational extrapolation to human health effects in as quantitative terms as possible and with a high degree of certainty. Sensible public health decisions are made more certain through the use of mechanistic information throughout the 4 steps in RA: hazard identification, dose-response assessment, exposure (dose) assessment and risk characterisation. Examples of the use of mechanistic information to assess risks of systemic, developmental/reproductive and neurotoxic effects show how to move away from the presently used threshold/no observable adverse effect/uncertainty factor default methodology towards an evaluation based on all available scientific data. The experience gained in cancer RA in the use of metabolic and tissue binding (receptor) models as well as physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PBPD) models can be transferred to non-cancer RA. A good example is the use of a PBPK model for the hepatoxicity of chloroform. As in cancer RA, as default positions are replaced by biological data the risk assessments become less uncertain when extrapolating between species. Combining information on tissue dosimetry and response data can also provide an estimate of variability within populations, which is impossible with present default type methodology but essential for adequate risk characterisation. Unlike the cancer field there is no single hypothesis for the mechanism of action for the multitude of non-cancer end-points studied.

Environmental monitoring for genotoxicity with plant systems. An introduction and study design.

Under the sponsorship of the International Programme on Chemical Safety (IPCS), 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic hazards of environmental chemicals. The bioassays included in this collaborative study were: Arabidopsis thaliana embryo and chlorophyll assay and Tradescantia stamen hair assay, Tradescantia paludosa micronucleus assay and Vicia faba root tip assay. Four to six laboratories participated in the performance of each of the bioassays. All laboratories participating in a particular bioassay were supplied with uniform plant material as well as standardized protocol. Five direct acting water soluble test chemicals, i.e. maleic hydrazide, methyl nitrosourea, ethyl methanesulfonate, sodium azide and azidoglycerol, were selected for this study. The study was designed to be completed in three phases. Ethyl methanesulfonate was used as a positive control and has already been reported earlier (Sandhu et al., 1991). The data from the remaining four chemicals used for the evaluation of four plant test systems in the first phase of the collaborative study are reported in this issue.

Environmental monitoring for genotoxicity with plant systems. Results and recommendations.

In the first phase of a collaborative study by the International Programme on Chemical Safety (IPCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trad-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN3 giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN3. For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN3, which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN3 to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.

Assessing the neurotoxic potential of chemicals--a multidisciplinary approach.

Since 1981, the development of methodology to assess the neurotoxic potential of chemicals has been a high priority within the International Programme on Chemical Safety (IPCS). Following the completion of an in-depth review of the scientific principles and methods for the assessment of neurotoxicity associated with chemical exposures, IPCS started to develop a multidisciplinary and comprehensive approach for neurotoxicity testing of chemicals. In view of the complexity of the nervous system and the variety of effects caused by chemicals, no single test or approach will be appropriate. Initially, IPCS evaluated neurobehavioral tests as well as in vitro procedures as screening tests, and an international collaborative study of neurobehavioral tests appropriate for screening chemicals for neurotoxicity is now in progress. Possible integration of higher level neurobehavioral tests with neurophysiological, biochemical, and pathological procedures in future testing strategies are discussed.

Methodology in neurotoxicology--activities within the World Health Organization and International Programme on Chemical Safety.

For many years, the potential deleterious effects of environmental factors on the human nervous system has been examined by several programmes within the World Health Organization (WHO). This presentation will concentrate on the development of methodology to determine the health risks from chemical exposures both in the work place, as well as, the general environment. IPCS as a scientifically based cooperative programme of the United Nations Environment Programme, International Labour Organisation, and WHO, has as one of its goals the development and use of methods to assess human health and environmental risks from chemicals. In this presentation, emphasis will be given to the development by IPCS of an integrated multidisciplinary approach for assessing the neurotoxic potential of chemicals and the risks to human health. The complexity of the nervous system and the variety of effects caused by chemicals will demand several tests in a multidisciplinary approach if the neurotoxic risk of any chemical is to be adequately characterized. Efforts by IPCS to evaluate the use of in vitro and neurobehavioural tests for screening purposes will be discussed as will the roles played by pathology, biochemistry, and neurophysiology in the characterization of neurotoxic risk. Finally, the WHO neurobehavioural core battery developed within the Occupational Health Programme will be described briefly.

Toxicological response and its reversibility in rats fed Lake Ontario or Pacific coho salmon for 13 weeks.

Lake Ontario coho salmon were known to contain a mixture of chemical contaminants. A previous study demonstrated that rats fed the Lake Ontario fish-supplemented diet for 28 days exhibited mild biochemical and histological changes. The purpose of the present study was to determine the effects due to a longer term of exposure and the reversibility of these effects. Growth rate and food consumption were not affected by feeding the animals with Lake Ontario or Pacific fish-supplemented diets for 13 weeks. No deaths were observed. Decreased spleen weights were observed in groups of males fed 1.45%, 5.8% Lake Ontario and 2.9% Pacific diet. After a 13 week recovery the spleen weights returned to normal. Decreased serum potassium was observed in male rats fed 2.9% Lake Ontario diet, and all levels of Pacific diet for 13 weeks, and was not evident following maintenance on normal diet. Serum glucose was not affected by the 13-week period of treatment, however; a reduction in this parameter occurred in male rats fed the two highest doses of Lake Ontario diet and all doses of Pacific diet following the 13-week recovery period. Minor hematological changes occurred only in the male rats fed either Lake Ontario or Pacific diet following a 13 week recovery period and included reduced marrow myeloid cells and myeloid/erythroid ratio. Hepatic microsomal ethoxyresorufin deethylase activity was significantly increased in rats ingesting Lake Ontario diet. Mild histological changes occurred in the liver and thyroid of the treated males, and in the liver and kidney of the treated females. These changes were attributed to the chemical residues and/or the fish diet. Data presented here indicated that the Lake Ontario fish-supplemented diet can cause mild biochemical, hematological and histological changes but most of these were reversible when exposure was terminated.

Trihalomethanes: II. Reversibility of toxicological changes produced by chloroform, bromodichloromethane, chlorodibromomethane and bromoform in rats.

Groups of 20 male and 20 female rats were fed 0, 5, 50, 500 or 2500 ppm trihalomethanes (chloroform, bromodichloromethane, chlorodibromomethane and bromoform) in their drinking water for 90 days. Ten rats from each group were killed at this time and the remaining animals were fed tap water for a further 90 days before they were sacrificed. Suppression of growth rate was observed in male and female rats fed 2500 ppm chloroform and bromodichloromethane (BDCM) for 90 days. After the subsequent 90-day recovery period, no effects on body weight gain in either sex were apparent. Food consumption was depressed in all groups of males and females receiving 2500 ppm chloroform, chlorodibromomethane (CDBM) and BDCM for 90 days and in these same groups during the recovery period. Bromoform at 2500 ppm suppressed food consumption of male rats but this effect was not evident 90 days after cessation of exposure. Decreased lymphocyte counts were only observed in the recovery groups fed chloroform (500 ppm, male), CDBM (2500 ppm, female) and bromoform (2500 ppm, females). Histological changes in the livers and thyroids of male and female rats were qualitatively and quantitatively similar for the four trihalomethanes. These changes were mild in nature, and were not evident 90 days after cessation of exposure. These data indicate that trihalomethanes can produce biochemical hematological and histological changes but most of these were reversible when exposure had terminated.

Toxicity of trihalomethanes: I. The acute and subacute toxicity of chloroform, bromodichloromethane, chlorodibromomethane and bromoform in rats.

In an acute study, groups of 10 male and 10 female rats were given single oral doses of chloroform, bromodichloromethane (BDCM), chlorodibromomethane (CDBM), or bromoform and were observed for clinical symptoms for the following 14 days. Median lethal doses (LD 50) of the four trihalomethanes were found to be between 848 and 1388 mg/kg. Some groups which survived the treatment for 14 days showed reduced food intake, growth retardation and increased liver and kidney weight. Elevated serum cholesterol levels were observed in the surviving male rats treated with chloroform and CDBM, and in the females treated with chloroform. Decreased liver protein content occurred in male but not female rats fed chloroform and bromoform. In contrast, increase aniline hydroxylase activity was observed in female rats fed chloroform but not bromoform. Hematological values which were altered by the four trihalomethanes were hemoglobin, hematocrit, RBC, WBC, neutrophil and lymphocyte counts. Treatment-related histologic changes were observed in the liver and kidney of rats. These changes were qualitatively and quantitatively similar for the four trihalomethanes. These data indicate that trihalomethanes at large single oral doses can produce a wide range of toxic changes in the rat. In a subacute study, groups of 10 male rats were fed four trihalomethanes at 0, 5, 50 or 500 ppm in their drinking water for 28 days. The growth rate and food intake were not affected by treatment. A slight increase in relative kidney weight was observed in the groups fed 5 ppm chloroform; 500 ppm bromoform, 5 and 500 ppm BDCM. The animals fed the highest dose of chloroform showed decreased neutrophils. Serum biochemical parameters and hepatic microsomal enzyme activities were not altered by any of the four trihalomethanes. No histopathological changes were seen in the tissues examined.