Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance.

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.

Inhalation toxicity of 1,6-hexamethylene diisocyanate homopolymer (HDI-IC) aerosol: results of single inhalation exposure studies.

The early acute pulmonary response of female Wistar rats exposed nose-only to a mixture of 1,6-hexamethylene diisocyanate homopolymer (HDI-IC) aerosol was examined. This study was designed to investigate the time course of the relationship between acute pulmonary irritation and ensuing disturbances of the air/blood barrier in rats exposed to concentrations of 3.9, 15.9, 54.3, or 118. 1 mg HDI-IC/m(3). The duration of exposure was 6 h, followed by serial sacrifices 0 h, 3 h, 1 day, 3 days, and 7 days postexposure. Concentrations were selected based on the results of a 4-h acute inhalation study in rats (LC(50) = 462 mg/m(3)). Bronchoalveolar lavage (BAL) fluid was analyzed for markers indicative of injury of the bronchoalveolar region, including phospholipids as proxy of altered surfactant homeostasis. Glutathione (GSH) was determined in BAL fluid and lung tissue. BAL cells with increased intracellular phospholipids were observed on day 1 and especially day 3, with some residual increase on day 7. Increased intracellular phospholipids and activity of acid phosphatases appear to suggest that phagocytized phospholipids may transiently affect lysosomal function. Following exposure to 15.9 mg/m(3), changes returned almost entirely to the level of the air-exposed control on day 7. Especially at higher exposure concentrations, lung weights and total number of cells in BAL were still statistically significantly elevated at this time point. Experimental evidence suggests that markers indicative of a dysfunction of the air/blood barrier, such as angiotensin-converting enzyme, total protein, and phospholipids engulfed by alveolar macrophages, were most sensitive to probe this type of changes. Although GSH in BALF was increased following exposure, there was an apparent depletion of tissue GSH immediately after cessation of exposure. In summary, this study suggests that respirable HDI-IC aerosol appears to cause a transient dysfunction of the air/blood barrier indicated by an increased extravasation of plasma constituents. Despite the remarkable extent of effects observed, most changes were reversible within a postexposure period as short as 7 days. First evidence of increased leakage of pulmonary epithelial barrier was observed at 3.9 mg/m(3). With respect to changes of early markers of pulmonary epithelial barrier dysfunction, approximately 3 mg HDI-IC/m(3) was considered to be the threshold concentration for acute pulmonary irritation.

Respiratory sensitization to diphenyl-methane-4,4'-diisocyanate (MDI) in guinea pigs: impact of particle size on induction and elicitation of response.

The impact of particle size of aerosolized polymeric diphenylmethane-4,4'-diisocyanate (MDI) for the induction and elicitation of respiratory sensitization was evaluated. Four groups of 16 female guinea pigs each received either the vehicle, repeated intradermal (id) injections (3 x 0.3% MDI), one high-level inhalation exposure of 15 min to 135 mg MDI/m(3) air using a small aerosol (MMAD approximately 1.7 microm) or large aerosol (MMAD approximately 3.8 microm). Three weeks later, animals were challenged subsequently with two ramped concentrations of MDI aerosol (average concentrations 16 and 49 mg/m(3) air, each for 15 min) and two different particle sizes, i.e., the MMAD was either approximately 1.6 microm or approximately 5.1 microm for the small- and large-size aerosol, respectively. Respiratory sensitization was assessed by two endpoints: the measurement of respiratory rate, and examination of influx of eosinophilic granulocytes into the mucosa and submucosa of the trachea, bronchi, and lung-associated lymph nodes (LALN). The recruitment of eosinophilic granulocytes into bronchial tissues was subdivided as follows: muscularis mucosae, submucosa, and perivascular. From measurements of respiratory rate, it would appear that guinea pigs sensitized by id injections or by inhalation exposure with the large aerosol tended to display a higher responsiveness than naive controls when challenged with the small aerosol. The recruitment of eosinophilic granulocytes in the bronchial tissue was greater in both inhalation induction groups as compared to the vehicle control. It appears that there was a somewhat greater response in animals sensitized by id injections or by inhalation exposure with the large aerosol and challenged with the small aerosol. Topographically, this difference was apparent only at the bronchial perivascular level and lung-associated lymph nodes (LALN), whereas at the submucosal and muscularis mucosae level the impact on particle size tended to be less pronounced. In summary, this study suggests that a brief, high-level inhalation exposure of MDI aerosol caused a sensitization of bronchial tissues in guinea pigs. The higher sensitization potency of the large aerosol may possibly be related to a dosimetric phenomenon because of the greater fraction of deposition of large particles within the upper respiratory tract. Overall, challenge exposures with this type of irritant aerosol appear to evoke more consistent effects when the MMAD is in the range of approximately 2 rather than approximately 5 microm.

Assessment of respiratory hypersensitivity in guinea-pigs sensitized to diphenylmethane-4,4'-diisocyanate (MDI) and challenged with MDI, acetylcholine or MDI-albumin conjugate.

Guinea-pigs were sensitized to monomeric diphenylmethane-4,4'-diisocyanate (MDI) by two intradermal injections (1-10% MDI, injection volumes of 50-100 microliters/day, on days 0, 2 and 4) or by a single brief high-concentration inhalation exposure (135 or 360 mg/m3, 15 min). Starting with day 21 following sensitization the animals were subjected to inhalation-challenge exposures (30 min) with non-irritating and irritating concentrations of the hapten (MDI). MDI-challenge concentrations ranged from 3.4 +/- 0.9 to 60 +/- 14.3 mg/m3 air. In some groups guinea-pigs were also challenged with acetylcholine (ACh) aerosol or the MDI-guinea pig serum albumin (GPSA) conjugate. Experimental findings indicated that from intradermally sensitized animals an immediate onset respiratory hypersensitivity response could only be elicited with concentrations exceeding the irritant threshold concentration for MDI, i.e. with concentrations greater than approximately 20 mg/m3 air. Guinea-pigs challenged with the MDI-GPSA conjugate (35.3 +/- 2.8 mg/m3 air) also experienced a weak immediate-type respiratory hypersensitivity response. An increased non-specific airway hyper-responsiveness following ACh-challenge was only observed from animals challenged with approximately 60 mg MDI/m3 air. The histopathological evaluation of lungs and lung-associated lymph nodes revealed an association of the increase in eosinophilic granulocytes and concentration of MDI used for challenge exposures. It appeared, in most instances, that this influx was more pronounced in animals sensitized with MDI as compared with concurrent controls challenged with the same MDI concentration. Guinea-pigs sensitized by a single 15-min inhalation exposure to either 135 or 360 mg MDI/m3 air were challenged sequentially with 12 +/- 2.1 mg MDI/m3 air, ACh and MDI-GPSA conjugate. Following the inhalation-induction, an airway hyper-responsiveness was elicited both after challenge with MDI and with the MDI-GPSA conjugate. The influx of eosinophilic granulocytes was more pronounced from animals sensitized by inhalation when compared with guinea-pigs sensitized intradermally and challenged with the same concentration of MDI. Thus, experimental findings suggest that elicitation of respiratory hypersensitivity is concentration-dependent and that challenge concentrations should slightly exceed the threshold concentration for irritation (approximately 20 mg/m3). Sensitization by inhalation increased the susceptibility to irritant stimuli and thus confounds the selection of the most appropriate concentration for challenge. However, the combined assessment of specific pathologic features such as airway eosinophilia and the evaluation of several breathing parameters during hapten- and ACh-challenge make it easier to distinguish effects caused by irritation and respiratory hypersensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of inhaled cholinesterase inhibitors on bronchial tonus and on plasma and erythrocyte acetylcholine esterase activity in rats.

Young adult male and female Wistar rats were inhalationally exposed head-only for 1 or 4 h to different anticholinesterase aerosols. The compounds tested were dichlorvos, fenamiphos, methamidophos, parathion, a pyrimidine thiophosphate and the carbamate propoxur. These compounds are direct or indirect inhibitors of cholinesterase activity. Immediately after termination of exposure to the compounds, the rats were anesthetized with barbiturate and subjected to pulmonary function tests. An acetylcholine provocation test was performed to correlate the effect of the cholinesterase inhibition and lung resistance. The results basically revealed that by inhalation exposure bronchoconstriction in the absence of acetylcholine provocation did not occur at toxicologically significant doses of the pesticides. An increase in lung resistance was observed only after provocation. However, measurements of plasma cholinesterase activity proved to be more sensitive than the provocation test. With regard to their diagnostic value, the results of the reported study may be summarized as follows (beginning with the most sensitive parameter): plasma cholinesterase activity depression greater than or equal to acetylcholine-induced bronchoconstriction greater than or equal to cholinergic symptoms greater than erythrocyte cholinesterase activity depression greater than pulmonary resistance without acetylcholine provocation.

Rat model of lung fibrosis: comparison of functional, biochemical, and histopathological changes 4 months after single irradiation of the right hemithorax.

This study investigated changes in lung function, hydroxyproline (OH-pro) content of lung tissue and histopathology in anesthetized, spontaneously breathing rats after a single, selective irradiation of the right hemithorax with a single dose of 20 Gy. The objective of this animal model was to examine as to whether non-invasive lung function measurements (LFM) could be used to analyze the magnitude of the irradiation-related pneumonitis and its long-term sequel occurring in the right lung in the presence of a normal left lung. Four months after irradiation, the OH-pro content in the irradiated right lung was determined and compared with the non-irradiated contralateral left lung, as well as lungs from non-irradiated sham controls. LFM revealed significantly depressed flow-volume curves and reduced quasistatic compliance, suggesting a marked diminution of elastic recoil of the lung. Total lung capacity (TLC) was significantly decreased, while the residual volume (RV) and functional residual capacity (FRC) remained almost unchanged. One of the most predominant dysfunction of the lung was a severe maldistribution of ventilation shown by the single-breath N(2)-wash-out test. Single-breath carbon monoxide diffusing capacity (Dlco) was significantly decreased. The content of OH-pro, a marker of increased collagen, was significantly increased in the irradiated right lung but was indistinguishable from sham controls in the non-irradiated left lung. Histopathological examinations provided evidence of both inflammatory and fibrotic lesions in the irradiated lobes, including bronchiolo-alveolar hyperplasia. No changes were observed in the non-irradiated left lung. In summary, effects observed in the irradiated right lung were largely consistent with effects described in other animal models of human interstitial pulmonary fibrosis. Non-invasive LFM were considered to be particularly sensitive to study the overall extent of changes, however, the interpretation of findings appears to be complicated by the lobar heterogeneity of tissue- and flow-related functional end points.

Hazard identification and risk assessment of pyrethroids in the indoor environment.

Household insecticide products raise several important considerations concerning safety. These are related to the use of insecticides by untrained individuals, the difficulty of controlling the use of these products once purchased by the consumer and the potential exposure of the very young and very old, possibly with or without pre-existing pulmonary disease. Exposure to pyrethroids contained in mats or vaporizers, being slow release systems, have particular potential for long-term low-level exposure whilst for foggers, spray-cans or sprayed formulations the short-term high-level exposures may be of more concern. According to the volatility of the active ingredient contained in the household insecticide, its persistence in a non-inhalable matrix, i.e. sedimented house dust, may be short or long for highly volatile or low volatile active ingredients, respectively. On the other hand, the potential of exposure is apparently just reciprocal. This demonstrates that the extent and duration of exposure may be highly product-specific. Accordingly, the extent of exposure has to be accounted for and for risk assessment both concentration-dependent (e.g. sensory irritation) as well as concentration x time (= dose) related effects have to be considered and addressed in adequate bioassays. The issue as to whether pyrethroids adhering to house dust is of concern has been addressed in a model study using carpets treated with pyrethroids. This study has demonstrated that the total mass of pyrethroid applied to the carpet and that brushed off within an 18-h period is too small to be of any relevance for risk assessment. Therefore, assessment of health hazards in the indoor environment based simply on methodologies of emptying the household vacuum cleaner and analysing its content, which addresses contamination only, rather than examination of the actual airborne concentration, including other relevant airborne materials, is prone to tremendous errors and misjudgments. Due to the many substances potentially present in house dust and indoor air, e.g. bioaerosols originating from animals, pests and microorganisms, volatile organic substances (VOCs) or metals, prudent expert judgment is needed to assess the relevance of analytical findings. The complex indoor exposure scenario makes it especially difficult to causally relate clinical and epidemiological findings to arbitrarily selected indicator substances contained in a matrix not readily available to inhalation exposure.

Modeling of toxicological effects of fire effluents: prediction of toxicity and evaluation of animal model.

Methodology for the prediction of the toxic effects of fire effluents has made considerable progress [1,2]. As emphasized by Hartzell [2] a limiting factor has often been the availability and quality of analytical input data. The Finney model [3] was used to predict the lethal potency and would appear to have utility as a tool in reducing the number of experimental animals used in material testing. However, pilot bioassay data are indispensable to validate the prediction and to categorize fire effluents into narcosis inducing, irritant or of causing unusual or unexpected toxicity. The comparison of predicted and actually observed carboxy-hemoglobin levels is considered to be a sensitive but indirect tool to assess whether major effects on respiration occurred. All laboratory combustion toxicity methods suffer from several types of limitations. However, they might be expected to be relatable to at least some stages of actual fires. Due to a lack of a clearly defined 'generation process'--if compared with conventional inhalation toxicity studies--the classification into broad categories of relative toxic potency seemed to be more appropriate than an absolute classification scheme.

Risk assessment of pyrethroids following indoor use.

One notable form of toxicity associated with exposure to high concentrations of synthetic pyrethroids has been a cutaneous paresthesia. This strong excitatory action on the sense organs in the vertebrate skin and upper respiratory tract is characteristic of synthetic pyrethroids, whereas the cyano pyrethroids evoke more intense neuroexcitatory activities than the noncyano pyrethroids. Such facial sensations and irritative symptoms appear to be produced by direct stimulation of peripheral sensory nerve endings rather than by inflammatory mechanisms. Effects related to sensory irritation can be evoked by a wide variety of substances occurring in the indoor environment, and analysis of the etiopathological relationships presents difficult and complex medical and scientific issues. For the appropriate assessment of pyrethroids in the indoor environment, it would be helpful to have an objective laboratory assay to confirm and quantitate the degree of sensory irritation evoked by airborne pyrethroids. A bioassay was established using the nociceptive system of mice and rats to assess the extent of pyrethroid-related sensory irritation to the respiratory tract. For analysis, aerosolized Cyfluthrin was selected due to the greater potency of the alpha-cyano pyrethroids to evoke sensory irritation. Additionally, this pyrethroid was tested in a carpet-model to assess the extent to which pyrethroid-laden dust from carpets is likely to become airborne following continuous brushing. Comparative evaluations of the sensory irritation potential of aerosolized Cyfluthrin in mice and rats revealed that for assessment of the sensory irritant threshold concentration, rats appeared to be more susceptible than mice. Measurements performed repeatedly during subacute exposure to the pyrethroid (6 h/day, 5 days/week for 4 consecutive weeks) did not indicate any alteration in responsiveness, and the magnitude of changes in breathing patterns was similar to those observed following acute 1-h exposure. These findings confirm the conclusion that alpha-cyano-pyrethroids appear to act as "pure" sensory irritants and that the effects observed are non-cumulative and transient in nature. Concomitant respiratory tract inflammation and ensuing changes in susceptibility-common findings in chemical sensory irritants-did not occur. From the studies addressing the dislodgeability of pyrethroid containing dust from carpets, it is apparent that measurement of deposited dust is a poor substitute for airborne dust. Even under worst-case testing conditions (continuous brushing of the carpet for approximately 19 h in a bias-flow compartment), only a very small fraction of the pyrethroid laden dust particles charged to the carpet could be recovered airborne (0.04%/m2 per h). Thus, experimental findings support the conclusion that such agents cannot be dislodged from carpets to an extent that toxicologically significant airborne concentrations are attained. Therefore, assessment of health hazards in the indoor environment based solely on "vacuum cleaner" sampling is prone to a high level of errors and misjudgment.

Predictive testing for respiratory sensitisation.

A rat bioassay has been developed to provide an objective approach for the identification and classification of upper and lower respiratory tract irritants, with particular emphasis on the concentration-dependent induction and regression of lesions characteristic of asthma, such as persisting non-specific airway hyperreactivity, inflammation and ensuing mismatch of the ventilation-perfusion relationship. For the identification of respiratory allergy, the established guinea-pig bioassay has been further refined. Refinement focused on procedures making this animal model more robust to changes in study design. Attempts were made to allow differentiation of non-specific and specific bronchial hyperresponsiveness and to minimise the use of hapten-protein conjugates for elicitation of respiratory allergy. It appears that the combined assessment of specific pathologic features such as airway eosinophilia and the evaluation of several breathing parameters during hapten and acetylcholine bronchoprovocation challenge make it easier to distinguish effects caused by irritation and respiratory hypersensitivity. Findings support the conclusion that current guinea-pig models require specific optimisation of sensitisation and challenge procedures for each chemical class tested.

Assessment of pyrethroid-induced paraesthesias: comparison of animal model and human data.

The quantification of upper respiratory tract (URT) sensory irritation is considered to be important in rodent inhalation studies, since it may be also used as an endpoint mimicking trigeminal paraesthesias observed in humans. URT sensory irritation is known to be associated with rodent-specific secondary physiological effects such as depression of body temperature and changes in heart rate. In acutely exposed rats, these endpoints have been addressed by telemetrical measurements. The analysis of the ventilation pattern during acute inhalation studies of rats exposed to the alpha-cyano-pyrethroid cyfluthrin demonstrates that concentration-dependent URT sensory irritation was associated with a hypothermic response. The no-effect levels (NO(A)EL) based on the URT sensory irritation endpoint following acute inhalation exposure for 1 h and following a repeated 4-week or 13-week inhalation exposure for 6 h/day on 5 days week were virtually identical (approximately 0.1 mg/m3 air). An additional objective was to examine whether human volunteers experience comparable signs when acutely exposed for 1 h to airborne concentrations slightly above or in the range of the NO(A)EL. In human volunteers there were no clinically significant or pyrethroid related abnormalities in vital signs, ECG's or in any clinical laboratory tests after either exposure, although transient effects related to URT (sensory) irritation were reported. In conclusion, an initial actual exposure concentration of approximately 0.1 mg cyfluthrin/m3 air appears to be in the range of the sensory irritant threshold concentration for both rats and humans. Thus, with regard to physiological afferent portal-of-entry effects, the interspecies response was consistent.

Assessment of chemicals for their potential to induce respiratory allergy in guinea pigs: A comparison of different routes of induction and confounding effects due to pulmonary hyperreactivity.

Guinea pigs were sensitized either to selected low molecular weight chemicals known to induce respiratory allergy in humans, trimellitic anhydride (TMA), toluene diisocyanate (TDI), or diphenylmethane-4,4'-diisocyanate (MDI), or to ovalbumin (OA) as a positive control. In most instances, sensitization was induced either by repeated intradermal injections or by a single brief (15-min) high-concentration inhalation exposure. For TMA the repeated high dose intradermal injection regimen was compared with a single low dose intradermal injection regimen. Additionally, the effectiveness of the single 15-min induction protocol was compared with that of five consecutive inhalation exposures each of 3 hr/day. Animals were challenged 2-3 wk later by exposure to the substance used for induction, either as the free chemical or as a hapten-protein conjugate, and with increasing concentrations of acetylcholine (ACh). Challenge with the parental or conjugated hapten was used to assess compound-specific immediate-onset respiratory hyperreactivity, while ACh challenges were used to identify non-specific airway hyperreactivity. After intradermal sensitization with either MDI or TMA guinea pigs challenged with the corresponding hapten-protein conjugate showed a moderate incidence of immediate-type respiratory responses. However, the highest incidence of unequivocal allergic responses was evident from challenge with the hapten rather than with the protein conjugate, although these responses were only elicited with slightly irritant concentrations. After challenge with irritant concentrations of TDI, animals sensitized intradermally did not experience characteristic changes in respiratory patterns. On challenge with Ach and the TDI-protein conjugate these same animals showed an increased airway hyperresponsiveness although characteristic stereotypic breathing patterns, as observed in sensitized animals challenged with TMA, TMA-protein conjugate, or OA, were not detected. Comparison of the intradermal and inhalation induction regimens indicated that prior encounters with irritant haptens by inhalation reduces the concentration required to elicit airway hyperresponsiveness. This finding supports the conclusion that in animals sensitized and challenged by inhalation, irritant respiratory responses may be misconstrued as immediate-onset allergic responses. It appeared that the low dose single intradermal injection protocol is more effective in sensitizing guinea pigs than the high dose repeated injection protocol.

Analysis of alternative methods for determining ocular irritation.

According to classification and labelling requirements, chemicals, dyes, agrochemicals and pharmaceutical formulations have to be evaluated for their potential to induce eye irritancy or corrosion. An attempt was made to analyse the predictive power of the bovine eye-chicken egg chorioallantoic membrane (BE-CAM) assay in comparison with results obtained using the conventional Draize method. In summary, results showed limited correlation between reactions in vitro and responses of eyes in vivo. In a pilot study, ultrasonic pachymetry showed high sensitivity and fairly good correlation between corneal thickness and clinical observations in eyes.

Repeated 4-week inhalation exposure of rats: effect of low-, intermediate, and high-humidity chamber atmospheres.

A subacute nose-only inhalation study with low (approximately 3%), medium (approximately 40%), and high humidity (approximately 80%) has been performed on young adult Wistar rats. Exposure was 6-hr/day on 5 days/week for 4 consecutive weeks. Rats housed individually in the animal holding room, deprived of feed and water during exposure of the remaining groups, served as concurrent controls (sham controls). This study served the purpose to assess whether toxicologically significant effects occur when rats are repeatedly exposed to lower or higher humidity chamber atmospheres than proposed by current testing guidelines. For analysis, conventional end-points as required by common testing guidelines were considered, i.e., clinical observations before and after exposure, rectal temperatures, body weights, feed and water consumption. At the end of the 4-week exposure period, ophthalmological and gross pathological examinations were made and major organ weights determined. The histopathological examinations comprised the nasal cavities, larynx, trachea, and lungs. There was no apparent evidence of humidity-related effects on nose-only exposed rats. When compared with non-exposed sham controls, however, body weights, water and feed consumption were markedly reduced in all nose-only exposure groups. In summary, it can be concluded that rats tolerated either humidity atmosphere without any specific effects. As far as there were differences to sham controls they appear to be more controlled by the differences in the exposure patterns (nose-only versus normal housing) than differences in the humidity of chamber atmospheres. Thus, deviations of current testing guidelines for repeated exposure inhalation studies with regard to humidity, do not appear to have any appreciable impact on the study outcome.

Household insecticides: evaluation and assessment of inhalation toxicity: a workshop summary.

Particularly in tropical countries household insecticides are used on a day-by-day basis to control mosquitoes, other crawling and flying insects to prevent the spread of vector-borne diseases. The products used most often are spray-cans, oil-sprays, mosquito coils as well as slow-release vaporising systems such as mats and liquid vaporiser. The extent and duration of exposure of humans is highly dependent on the type of product used. The objective of this workshop was to analyse the necessity and feasibility of inhalation studies with household insecticides taking into account the specific constrains associated with each type of end-use product. The standardisation of inhalation studies with regard to the generation of test atmospheres, mode and duration of exposure, and selection of adequate toxicological endpoints were addressed. Due to the complex nature of exposure atmospheres generated by some household insecticides, viz. mosquito coils, it is scientifically challenging to characterise the pathomechanism of most concern, since irritant combustion gases, volatile and semi-volatile organic substances, particulates (soot), condensation aerosols and re-condensed substances onto particulates may act independently, synergistically or mixture specific. It has been concluded that for the comparative safety evaluation and risk assessment of indoor insecticide end-use products generally recognised guidance for harmonised inhalation testing is required: 1) For high-dose release products, such as spray-cans, acute inhalation testing appears to be most relevant. 2) For low-dose, slow-release devices, subchronic inhalation studies of 13-weeks, duration of exposure 6 hours/day for 5 consecutive days per week, should be performed on rats preferably with the end-use product. A dose-range finding study of 2-weeks duration, daily exposure, should be available for the justification of dose selection and to demonstrate that the findings of 5 days/week exposure is not different from that utilising a daily exposure. 3) In certain instances, inhalation testing of shorter duration, for example 2 or 4 weeks, may be appropriate if the inhalation toxicity of the basic formula has already been characterised or the major constituents contained in the formulation have been examined in extant studies. The duration of study should also depend on actual use and exposure pattern, the accumulation potential and the mode of action of the ingredients of concern. 4) Particularly for mosquito coil smokes, the possible physico-chemical interactions of particulates, potentially irritant water soluble or nonsoluble vapours and aerosol or vapours of the active ingredient(s) are virtually impossible to predict without appropriate inhalation testing. 5) Physiological measurements may be useful for the detection and quantification of the respiratory tract sensory irritation potency. Biochemical endpoints in lung lavage and conventional histopathology of the lungs and extrathoracic airways are considered to be important. 6) It has been agreed that the nose-only exposure technique provides the most cost-effective and controlled means to expose small laboratory animals to well defined atmospheres as long as the limitations of this technique are taken into account.

Validation of a solvent-free sampler for the determination of low molecular weight aliphatic isocyanates under thermal degradation conditions.

During the thermal degradation of 1,6-hexamethylenediiso- cyanate-based (HDI) car paint, the eight most abundant isocyanates generated are isocyanic acid, methyl isocyanate, ethyl isocyanate, propyl isocyanate, butyl isocyanate, pentyl isocyanate, hexyl isocyanate, and 1,6-hexamethylenediisocyanate. For the first time, a method using solvent-free samplers is proposed and validated for the simultaneous sampling of all these isocyanates. The sampling efficiency during thermal degradation of car paint can be affected by the formation of dust and aerosols and by the emission of many chemicals, such as isocyanic acid, anhydrides, amines, and alcohols that consume the reagent or interfere in the derivatization procedure. Sampling was performed using cassettes containing two 1-(2-methoxyphenyl)piperazine (MOPIP)-coated glass fiber filters (MFs) (approximately 4.9 mg per filter) and compared with bubblers containing 15 mL of MOPIP solution in toluene (1.0 mg/mL(-1)) and with bubblers backed with MFs. A DIN 53436 laboratory scale furnace was used to generate the isocyanates under thermal degradation conditions. For an aliphatic isocyanate concentration of approximately 42 microg(NCO) m(-3), no significant difference in sampling efficiency was observed between the three techniques studied, thus confirming the sampling efficiency of the MFs. The samples were analyzed using high-performance liquid chromatography coupled with electrospray/tandem mass spectrometry. Quantification was performed in daughter mode monitoring (MOPIP+H)(+) fragments. For concentrations between 0.013 microg(NCO) mL(-1) and 0.52 microg(NCO) mL(-1) for the monoisocyanates, and between 0.026 microg(NCO) mL(-1) and 1.04 microg(NCO) mL(-1) for the HDI, the correlation coefficients were in the 0.9974-0.9996 range (n = 18). Analytical reproducibility and precision were better than 95.4% and 94.9%, respectively, for all the isocyanates. The instrumental detection limits, defined as three times the standard deviation measured at the lowest point on the calibration curve were in the 1.8-3.0 ng(NCO) mL(-1) range (n = 8), which corresponds to about 0.37-0.60 microg(NCO) m(-3) for a 15-L air sample when the filters are desorbed in 3 mL.

Repeated inhalation challenge with diphenylmethane-4,4'-diisocyanate in brown Norway rats leads to a time-related increase of neutrophils in bronchoalveolar lavage after topical induction.

Diphenylmethane-4,4'-diisocyanate (MDI) is a low-molecular-weight chemical known to cause occupational asthma. The objective of this study was to evaluate the topical and inhalation routes of sensitization on the elicitation response of MDI in the Brown Norway (BN) rat model following repeated challenge exposures. BN rats were either induced topically (150 microl MDI on the flanks, booster administration to the skin of the dorsum of both ears using 75 microl/dorsum of each ear) or by inhalation (5x3 h/d, 28.3+/-3.0 mg MDI/m3 [+/-SD]). Inhalation challenge exposures with MDI (15.7+/-1.4 mg/m3, duration 30 min) were made on d 21, 35, 50, and 64. One day after each challenge, rats were rechallenged with methacholine (MCh) aerosol. Respiratory changes were monitored during challenges. One day after the MCh challenge, selected endpoints in bronchoalveolar lavage (BAL), the weights of lungs, and auricular and lung-associated lymph nodes were determined. After the first and last challenge, lymph nodes and lungs were examined by histopathology. Repeated challenge with MDI or MCh did not elicit marked changes in respiratory patterns at any time point. Mild but consistent time-related increased BAL neutrophils and slightly increased lung and lymph-node weights occurred in topically sensitized rats as compared to the remaining groups. In topically sensitized rats, in the lung histopathology revealed activated lymphatic tissue and an increased recruitment of airway eosinophils. Immunoglobulin (Ig) E determinations (serum and BAL) did not show any differences amongst the groups. Thus, high-dose topical induction with MDI was associated with a neutrophilic and eosinophilic inflammatory response in the lung after repeated inhalation challenge with MDI, with magnitude of effect dependent on the specific methodology used.

Validation of an improved nose-only exposure system for rodents.

Several types and modifications of nose-only inhalation chambers for exposing rodents are described. The improvement of this 'flow-past' -like nose-only exposure system is that it is modular, i.e. it can be used for acute studies with a maximum of 20 rodents (one segment) or for chronic inhalation studies with 100 (or more) rodents per chamber with five (or more) segments. Another goal was to design a nose-only exposure system that provides maximal computer support and automatization, as well as robust aerosol collection conditions. The evaluation of the five-segment chamber, charged with 98 rats, revealed that a flow rate of 0.75 l air min-1 or approximately 2.5 times the rat minute ventilation volume per exposure port is sufficient to provide homogeneous temporal and spatial exposure conditions. Also, the aerosol size distribution was constant throughout the chamber. Experimental data suggest that computer-controlled sampling of the test atmosphere up to ca. 6 l air min-1 did not alter the flow dynamics of the exposure system. The nose-only inhalation chamber developed is suitable for short-term and long-term inhalation toxicity studies using small laboratory rodents with minimal consumption of test compound.

Assessment of respiratory hypersensitivity in guinea pigs sensitized to toluene diisocyanate: improvements on analysis of respiratory response.

Groups of guinea pigs of the Hartley strain were sensitized to toluene diisocyanate (TDI) by combined single intradermal injection and repeated inhalation exposure (3 h/day for 5 consecutive days) to 0, 3.8, 11, 26, 46, and 51 mg TDI/m3 air. One group of animals was sensitized by intradermal injection only. Sham-exposed and TDI-polyisocyanate resin-sensitized guinea pigs served as controls. Three weeks after the first encounter with the inducing agent, animals were challenged with the free TDI (approximately 0.5 mg/m3) and 1 week later with TDI-guinea pig serum albumin conjugate. Breathing patterns were analyzed by objective mathematical procedures taking into account the intensity and duration of the respiratory rate exceeding +/- 3 standard deviations of the individual prechallenge exposure period. In none of the animals challenged with TDI were conclusive immediate-onset respiratory responses identified. During the TDI conjugate challenge a characteristic increase in respiratory rate was observed in all groups sensitized with TDI. In each of the sham and TDI-resin control groups, 1 of 16 animals responded mildly to the conjugate challenge. With regard to analysis of the development of asthma-like dyspnea, the results obtained suggest that respiratory response can suitably be defined by objective mathematical analysis of breathing patterns. Moreover, the "duration" of response exceeding +3 x standard deviation of prechallenge baseline data appears to show less variability when compared to the "intensity" of response (area). It can be concluded that this method of evaluation of respiratory response may be useful to compare more quantitatively this type of data and serves the objective of decreasing potential interlaboratory variability.

Acute inhalation toxicity of polymeric diphenyl-methane 4,4'-diisocyanate in rats: time course of changes in bronchoalveolar lavage.

The early acute pulmonary response of Wistar rats exposed nose-only to respirable polymeric diphenylmethane 4,4'-diisocyanate (MDI) aerosol was examined. This study investigated the time course of the relationship between acute pulmonary irritation and ensuing disturbances of the air/blood barrier in rats exposed to concentrations of 0.7, 2.4, 8, or 20 mg MDI/m3. The duration of exposure was 6 h. The time-response relationship of MDI-induced acute lung injury was examined 0 h (directly after cessation of exposure), 3 h, 1 day, 3 days, and 7 days after exposure. Bronchoalveolar lavage (BAL) fluid was analyzed for markers indicative of injury of the bronchoalveolar region, i.e., angiotensin-converting enzyme, protein, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, and sialic acid. Phosphatidylcholine and acid phosphatase were determined in BAL fluid and cells. Glutathione was determined in BAL fluid and lung tissue. This analysis revealed no latent period of effects except a transiently delayed influx of cells and increased lung weights on postexposure days 1 and 3. Markedly loaded BAL cells with phosphatidylcholine were observed on day 1 only. In most instances, changes returned to the level of the air exposed control on day 7, except increased glutathione in lung tissue. The findings suggest that the most sensitive markers of dysfunction of the air/blood barrier are angiotensin-converting enzyme and protein, including alkaline phosphatase. The statistically significant increase in intracellular phosphatidylcholine and decreased intracellular acid phosphatase on the exposure day suggest that increased amounts of phospholipids are phagocytized by alveolar macrophages, associated with protracted lysosomal catabolism. Partially glutathione-depleted rats exposed to 20 mg/m3 experienced a more pronounced increase in BAL protein than normal rats. In summary, this study suggests that respirable polymeric MDI aerosol interacts directly with the air/blood barrier causing increased extravasation of plasma constituents as a result of increased permeability of capillary endothelial cells. Overall, a transient dysfunction of the pulmonary epithelial barrier occurred at level as low as 0.7 mg/m3 and appears to be related a dysfunction of pulmonary surfactant. Nonprotein sulfhydryl constituents appear to play a role as portal-of-entry specific modifying factors.