Incorporating potency into EU classification for carcinogenicity and reproductive toxicity.

Although risk assessment, assessing the potential harm of each particular exposure of a substance, is desirable, it is not feasible in many situations. Risk assessment uses a process of hazard identification, hazard characterisation, and exposure assessment as its components. In the absence of risk assessment, the purpose of classification is to give broad guidance (through the label) on the suitability of a chemical in a range of use situations. Hazard classification in the EU is a process involving identification of the hazards of a substance, followed by comparison of those hazards (including degree of hazard) with defined criteria. Classification should therefore give guidance on degree of hazard as well as hazard identification. Potency is the most important indicator of degree of hazard and should therefore be included in classification. This is done for acute lethality and general toxicity by classifying on dose required to cause the effect. The classification in the EU for carcinogenicity and reproductive toxicity does not discriminate across the wide range of potencies seen (6 orders of magnitude) for carcinogenicity and for developmental toxicity and fertility. Therefore potency should be included in the classification process. The methodology in the EU guidelines for classification for deriving specific concentration limits is a rigorous process for assigning substances which cause tumours or developmental toxicity and infertility in experimental animals to high, medium or low degree of hazard categories by incorporating potency. Methods are suggested on how the degree of hazard so derived could be used in the EU classification process to improve hazard communication and in downstream risk management.

A proposal to improve clarity and communication in the EU Classification process for chemicals for carcinogenicity and reproductive and developmental toxicity.

There is an issue in the EU classification of substances for carcinogenicity and for reproductive or developmental toxicity which has brought difficulties to those involved in the process. The issue lies in the inability of the classification system to distinguish between carcinogens and reproductive toxicants with different levels of concern. This has its origins in the early years of toxicology when it was thought that a relatively small number of chemicals would be either carcinogens or reproductive toxicants, but this has turned out not to be the case. This can cause problems in communicating to the users of chemicals, including the public, the nature of the hazard presented by chemicals. Processes have been developed within the classification system for setting specific concentration limits which assess the degree of hazard for carcinogens and reproductive toxicants as high, medium or low. However these categories are not otherwise used in classification. It is proposed that their wider use would bring the advantages of transparency, clarity of communication, certainty of the process and would allow chemicals with a high degree of hazard to be identified and managed in an appropriate way.

Hazard characterisation of chemicals in food and diet. dose response, mechanisms and extrapolation issues.

Hazard characterisation of low molecular weight chemicals in food and diet generally use a no-observed-adverse-effect level (NOAEL) or a benchmark dose as the starting point. For hazards that are considered not to have thresholds for their mode of action, low-dose extrapolation and other modelling approaches may be applied. The default position is that rodents are good models for humans. However, some chemicals cause species-specific toxicity syndromes. Information on quantitative species differences is used to modify the default uncertainty factors applied to extrapolate from experimental animals to humans. A central theme for extrapolation is unravelling the mode of action for the critical effects observed. Food can be considered as an extremely complex and variable chemical mixture. Interactions among low molecular weight chemicals are expected to be rare given that the exposure levels generally are far below their NOAELs. Hazard characterisation of micronutrients must consider that adverse effects may arise from intakes that are too low (deficiency) as well as too high (toxicity). Interactions between different nutrients may complicate such hazard characterisations. The principle of substantial equivalence can be applied to guide the hazard identification and hazard characterisation of macronutrients and whole foods. Macronutrients and whole foods must be evaluated on a case-by-case basis and cannot follow a routine assessment protocol.

Prenatal toxicity of inhaled polymeric methylenediphenyl diisocyanate (MDI) aerosols in pregnant wistar rats.

Mated Wistar rats, 25/group, were exposed to polymeric methylenediphenyl diisocyanate (MDI) aerosol of respirable size for 6 h/day, on gestational days (gd) 6 through 15, at 0, 1, 4, and 12 mg/m3. Maternal clinical signs, body weights, and feed and water consumption were measured throughout gestation. At scheduled sacrifice on gd 20, maternal body, gravid uterine, liver, and paired lung weights were documented. Corpora lutea were counted, implantation sites were identified: resorptions, dead and live fetuses, and placentas were weighed. All live fetuses were counted, sexed, weighed, and examined for external alterations; approximately 50% of the live fetuses/litter were preserved in Bouin's fixative and examined for visceral alterations, and the remaining live fetuses/ litter were cleared and stained with alizarin red S and examined for ossified skeletal alterations. Maternal toxicity was observed at 12 mg/m3, including mortality (2 of 24 pregnant), damage to the respiratory tract, reduced body weights and weight gain, reduced liver and increased lung weights, and reduced gravid uterine weight (the last not statistically significantly different from the control value). Developmental toxicity was also observed at 12 mg/m3, including reduced placental and fetal body weights and an increased incidence of fetal skeletal variations and skeletal retardations. There was no evidence of maternal or developmental toxicity at 1 or 4 mg/m3. The no observed adverse effect concentration for maternal and developmental toxicity was therefore 4 mg/m3. There were no treatment-related teratogenic effects at any concentrations evaluated.

Health risk of low-dose pesticides mixtures: a review of the 1985-1998 literature on combination toxicology and health risk assessment.

A literature review covering the last 14 yr has been performed in the field of combination toxicology and human risk assessment from exposure to chemical mixtures, with special emphasis on mixtures of pesticides at low doses, that is, at levels likely to occur in human diet and environment. Despite a large body of knowledge in the field of risk assessment methodologies for exposure to chemical and pesticide mixtures, there is no single methodological approach in "combination toxicology" and health risk assessment of chemical mixtures, and therefore professional judgment is still required. Generally, the dose or response additivity approach that may be applied to evaluate potential risk for chemical mixtures in human toxicology overestimates the risk of a combination of chemicals. The recent endocrine disrupter issue demonstrated the difficulty of reproducibility of data when testing environmental toxicants at very low levels, and the need for more basic work in this field. The use of integrated methodological approaches may provide more reliable predictive data in the risk assessment of chemical mixtures in future. Yet data have demonstrated that exposure to a combination of compounds does not cause effects stronger than the ones of their most active component, provided components are present at low concentration levels, like acceptable daily intake (ADI) or reference dose (RfD) levels, well below their respective no-observed-adverse-effect levels (NOAELs). Although it has been demonstrated that a combination of compounds with the same target organ and the same or very similar mechanisms of action may cause additive or synergistic effects, the chance of such effects will most likely diminish with decreasing exposure levels to such combinations. Synergism and antagonism may both occur at the same time at different organs or targets in the same organism. However, and despite some exceptions, it has been demonstrated that interaction between components is not a common event at low levels of human exposure such as those that may occur through pesticides residues in food or drinking water. The introduction of a special safety factor as a standard for mixtures in addition to those normally used for deriving ADIs, RfDs, or minimal risk levels is not supported by data. It can be concluded from our review that, as a general rule, exposure to mixtures of pesticides at low doses of the individual constituents does not represent a potential source of concern to human health.

Respiratory hypersensitivity to diphenylmethane-4,4'-diisocyanate in guinea pigs: comparison with trimellitic anhydride.

Published evidence demonstrates successful induction and elicitation of respiratory hypersensitivity in guinea pigs by the known human respiratory allergens trimellitic anhydride (TMA) and diphenylmethane-4,4'-diisocyanate (MDI). From these data it is apparent that TMA-related respiratory hyperresponsiveness can be elicited readily in guinea pigs upon inhalation challenge with the free chemical. Despite the interlaboratory variability in methodological procedures used for the sensitization as well as elicitation of response and the wide range of concentrations of TMA employed for challenge exposures (6-57 mg/m(3) air), TMA had been unequivocally identified as a benchmark respiratory sensitizer by measurements of the respiratory rate during challenge. The protocols were duplicated to examine the respiratory sensitizer MDI. In intradermally sensitized guinea pigs, changes in immediate-onset-like respiratory response were observed when MDI challenge concentrations exceeded approximately 30 mg MDI/m(3) air. Collective experimental evidence suggests that the respiratory responses observed upon challenge with TMA were markedly more pronounced and easier to identify than those recorded following challenge with MDI or MDI conjugate. In contrast to TMA, irritant concentrations of MDI had to be used to elicit any respiratory response and the differentiation of irritant and allergic responsiveness became increasingly difficult. Despite the absence of unequivocal changes in breathing patterns upon MDI challenge, MDI-sensitized animals displayed elevated anti-MDI immunoglobulin G1 (IgG1) antibodies, and a significant influx of eosinophilic granulocytes in the bronchial wall and lung-associated lymph nodes. Therefore, it is believed that the robustness of this animal model to identify low-molecular-weight agents as respiratory sensitizer is increased when several endpoints are considered. These are (1) positive respiratory response upon challenge with the hapten, and if negative, also challenge with the conjugate of the hapten; (2) an influx of eosinophilic granulocytes; and (3) increased specific IgG1 response. Furthermore, it appears that particles in the range of approximately 2-6 microm evoke more consistent respiratory response upon challenge exposure than particles in the 1-2 microm range.

Development of FlexSure HP--an immunochromatographic method to detect antibodies against Helicobacter pylori.

We describe a solid-phase immunochromatographic serologic test, FlexSure HP, to detect IgG antibodies against Helicobacter pylori. H. pylori colonize the stomach and proximal duodenum, cause ulcer disease and mucosa-associated lymphoid tissue lymphoma, and have a role in the development of other disorders, including gastric adenocarcinoma. FlexSure HP consists of a test strip, conjugate pad, and absorbent pad, in a novel reverse-flow chromatography format. In these studies, FlexSure HP was demonstrated to be specific for IgG antibodies against H. pylori. The reactive cutoff of the test was consistent with [13C]urea breath test and commercially available ELISAs. FlexSure HP had 94% sensitivity, 88% specificity, and 91% accuracy relative to [13C]urea breath test; and 95% sensitivity, 94% specificity, and 95% overall agreement relative to high-molecular-mass cell-associated protein enzyme immunoassay (HM-CAP EIA). FlexSure HP is a simple-to-perform, visually read test requiring no specialized training, equipment, or instrumentation, and yields rapid, accurate, qualitative results.

Toluene diisocyanate: an assessment of carcinogenic risk following oral and inhalation exposure.

Although respiratory sensitization and pulmonary irritation have been the subject of particular studies with toluene diisocyanate (TDI), in recent years the potential carcinogenicity of TDI has been a reason for concern and speculation. This has arisen from the expectation that following exposure to TDI the chemical would hydrolyze at aqueous tissue surfaces to give rise to toluene diamine (TDA), a mutagen and rodent carcinogen. The chemistry of TDI suggests that the reaction with biological NH2 groups such as those on proteins, and polymerization to oligoureas, will compete with the hydrolysis reaction. This has been shown with results of in vitro studies where conjugation to protein occurs without detectable formation of TDA when protein solutions in saline are exposed to TDI vapor. Lower pH levels leading to high protonation of biological NH2 groups facilitate hydrolysis of TDI to TDA and subsequent formation of polyureas. These observations are consistent with comparative toxicokinetic studies in rats, which demonstrate significant levels of TDA following oral dosing with TDI--due to the acidic environment in the stomach--but not after inhalation. These results provide an explanation for the tumors observed in rodents after oral dosing of TDI in corn oil, but not after inhalation. Inhalation is the relevant route of human exposure for TDI and the toxicokinetics of TDI exposure at occupational exposure limits have been studied. These data provide a means by which quantitative estimates of the risk of carcinogenicity possibly resulting from the intermediate formation of TDA during TDI exposure can be obtained. Several calculations have been made, all of which lead to the conclusion that TDI exposure by inhalation at the recommended occupational limits will not give rise to significant carcinogenic risk.

The evaluation of carcinogenic risk to humans: occupational exposures in the spraying and application of insecticides.

The International Agency for Research on Cancer (IARC) has reviewed the carcinogenic risk to man of the occupation of spraying or applying insecticides. IARC was unable to conclude that a causal relationship had been established (category 1), but there was some evidence which led them to consider the occupation to be probably carcinogenic (in category 2A). These conclusions have been reviewed by a working group of the International Group of National Associations of Manufacturers of Agrochemical Products (GIFAP) to determine what steps needed to be taken which might improve safety for those working with insecticides. The working group reinforced the view that a causal relationship could not be established. In addition, the usage rate of insecticides has decreased since the time of the studies that IARC considered, their general toxicity has decreased, and the products themselves have been the subject of close regulatory scrutiny. It is considered that these changes are addressing any concerns that may be raised by the IARC report.

Affiliations among steroid receptors as revealed by multivariate analysis of steroid binding data.

To illustrate the informative value of descriptive multivariate analysis in biochemical screening, we have analyzed several data matrices relating to the binding of steroids to the estrogen, progestin, androgen, glucocorticoid and mineralocorticoid receptors in different organs and species. We first compared dendrograms of steroid hormone receptors, that were obtained by an automatic hierarchical classification analysis of the binding data, to published phylogenetic trees of nuclear receptors based on amino-acid sequence analysis. The former classification describes the affiliations among the receptors as given by the binding specificity of a population of 187 steroids in a traditional cytosol binding assay (an indirect comparison of ligand binding sites); the latter describes the affiliations among the receptors as given by a comparison of selected primary sequences involved in ligand-dependent regulation of transactivation and dimerization. A similar hierarchical classification was also performed on the binding data of 62 steroids to myometrium cytosol from different species in order to show to what extent the progesterone-binding proteins in these species are affiliated. Hierarchical clustering methods classify each type of variable (receptor or steroid) independently. In order to be able to correlate both types of variable (receptors and steroids) on single-display graphs, it is necessary to resort to correspondence factorial analysis (CFA). CFA ranks the information content within the experimental system, highlighting major correlations and disclosing secondary correlations by eliminating redundant information and background noise. This multivariate method, applied to the analysis of published data, illustrated the particular specificity of estrogen binding in human vagina and raised the question of the nature of the binding protein in this tissue. Our examples are based on small data tables that can and have been analyzed de visu. However, it is certain that such descriptive multivariate techniques are indispensable for the analysis of large data banks not only to define structure-activity relationships but to estimate the degrees of affiliation among the biological variables being measured. Knowledge of such affiliations will help to organize available information in a context where the complexity of the biological systems under study is becoming increasingly apparent.

Olfactory and hepatic changes following a single inhalation exposure of 3-trifluoromethyl pyridine in rats: concentration and temporal aspects.

The effects of a single exposure to 3-trifluoromethyl pyridine (3FMP), were investigated in two studies. In the first study, rats were exposed nose only to 0, 50 or 800 ppm 3FMP for periods of 15 min to 4 h. Half were sacrificed on day 3 and the remainder on day 10. In the second study, rats were exposed whole body to 0, 0.1, 1.0, 10 or 50 ppm 3FMP for 6 h, with sacrifices immediately after exposure (6 h), 24 h and on days 3 (48 h after exposure started) 5, 8, 11, 35, 70 and 157. Effects were seen in the olfactory epithelium at concentrations of 1 ppm and above and in the liver at concentrations of 50 ppm and above. In the olfactory epithelium the earliest changes were seen immediately after exposure and by 24 h this progressed to extensive necrosis with sloughing of the epithelium. By day 3, the epithelium was replaced by undifferentiated basophilic cells, considered to reflect early regeneration. Regeneration progressed to complete recovery between days 70 and 157, no changes were seen in the nasal respiratory epithelium, an olfactory function test on rats exposed for 6 h to 50 or 10 ppm 3FMP showed a reduced sense of olfaction at days 3 and 5 with complete recovery on subsequent days, indicating functional recovery in advance of histological normality. Single cell necrosis was seen in the liver at day 3 after 30 min exposure and immediately after 6 h exposure to 50 ppm 3FMP. At 24 h after a 6 h exposure to 50 ppm this had progressed to necrosis, haemorrhage and moderate cytoplasmic hepatocyte vacuolation in centrilobular areas. The lesion had completely recovered by day 5.

Assessment of the reproductive toxicology of bromochlorodifluoromethane (BCF, halon 1211) in the rat.

The effect of bromochlorodifluoromethane (BCF) on reproduction in the rat has been investigated in two studies. Pregnant female rats were exposed by inhalation to 1000, 10,000, or 50,000 ppm BCF for six hours a day on days six to 15 of gestation (day of mating = day 0). Exposure to 50,000 ppm BCF caused a reduction in maternal weight gain over the exposure period but there was no evidence of either teratogenicity or embryo/fetotoxicity at any concentration. In a study designed to assess the potential effect of BCF during a complete reproductive cycle male and female rats were exposed to 5000 ppm or 25,000 ppm BCF for six hours a day for five days a week for 10 weeks (males) or three weeks (females) before mating. Exposure to BCF continued during mating and up to day 20 of gestation for half the females which were subsequently allowed to litter and the development of their offspring monitored. The remaining females were removed from exposure to BCF after mating and killed on day 20 of gestation for examination of their uterine contents. There were no effects on adult fertility, pup numbers, survival, or pup development. It was concluded that BCF had no reproductive toxicity potential in the rat.

Inhalation hazard test. Interlaboratory trial with OECD method 403.

Six industrial laboratories validated the Inhalation Hazard Test (OECD Method 403) using eight volatile chemicals. The test gave similar results in all laboratories, despite variation in inhalation exposure systems and strain of rat used. Detailed atmosphere analyses are not necessary, since nominal atmosphere concentrations were close to the analysed values. The method gives reproducible results directly applicable to hazard evaluation, and is quicker and cheaper and uses fewer animals than the conventional LC50 test.