Developmental toxicity evaluation of inhaled citral in Sprague-Dawley rats.

Citral is a commonly used fragrance and flavour ingredient that has demonstrated a potential for teratogenicity in chick embryo screening studies. To investigate potential mammalian developmental toxicity, pregnant Sprague-Dawley rats were exposed to citral by inhalation for 6 hr/day on gestation days 6-15 at mean concentrations of 0, 10 or 34 ppm as vapour, or 68 ppm as an aerosol/vapour mixture. Dams were killed on gestation day 20 and the foetuses were removed and evaluated for gross, visceral and skeletal malformations. Exposure to 68 ppm was maternally toxic, with reduced body-weight gains, ocular opacity, breathing difficulty, nasal discharge and salivation noted in the dams. No maternal toxicity was seen at the lower vapour exposure levels. The number of corpora lutea, implantations, resorptions, foetal viability, litter size, and sex ratio were not adversely affected by citral at any exposure level tested, and no exposure-related malformations were observed. At a maternally toxic exposure level, a slight reduction in mean foetal body weight and a slight increase in the incidence of hypoplastic bones were noted. Results of this study indicate that citral does not produce developmental toxicity in the rat when administered by inhalation at concentrations up to a maternally toxic exposure level.

Evaluation of the developmental toxicity of caffeine and caffeine metabolites using the frog embryo teratogenesis assay--Xenopus (FETAX).

The developmental toxicities of caffeine and 13 metabolites, including theophylline, and paraxanthine and a synthetic methylxanthine analogue 3-isobutyl-methylxanthine (IBMX) were evaluated using the Frog Embryo Teratogenesis Assay Xenopus (FETAX). Young X. laevis embryos were exposed to these compounds in each of two separate concentration-response experiments with and without an exogenous metabolic activation system (MAS). Results obtained from these studies indicated that relative teratogenic potencies of caffeine and each of its di- and monomethylxanthine metabolites were similar. Representatives of both the substituted uric and uracil metabolites were less developmentally toxic on an equimolar basis than the methylxanthines, suggesting that they may have represented detoxification metabolites. IBMX, a phosphodiesterase inhibitor also known to be an adenosine receptor antagonist, was the most potent developmental toxicant of the materials evaluated. In conclusion, none of the caffeine metabolites tested was found to be significantly more potent than caffeine itself in the FETAX assay.

An immunotoxicity assessment of food flavouring ingredients.

A rapid screening protocol incorporating key elements of the US National Toxicology Program's immunotoxicity tier testing strategy was used to evaluate the effects of 35 commonly used food flavouring ingredients on humoral and cell-mediated immune responses. The test compounds were administered intragastrically on a daily basis for 5 days at three dose levels to female CD-1 or B6C3F1 mice, 6-8 wk old. A host resistance assay (Listeria monocytogenes bacterial challenge) was conducted to assess cell-mediated immunity. Humoral immunity was measured by the antibody plaque-forming cell (PFC) response to sheep erythrocytes. Body weights, lymphoid organ weights and spleen cellularity were also measured. Cyclophosphamide (80 mg/kg) served as an immunosuppressive positive control agent. The results indicated that the majority of the flavouring ingredients tested did not modulate the cell-mediated or humoral immune response. However, at very high dose levels, two of the materials tested, peppermint oil and citral dimethyl acetal, did increase mortality rate and reduce survival time in the host resistance assay. Neither of these materials significantly altered the PFC response. This rapid, economical screening battery for potential immunotoxicants proved to be a useful means of evaluating a large number of structurally diverse compounds and mixtures to prioritize them for more definitive testing.

Measurement of respiratory tract ammonia in the rabbit and implications to sulfuric acid inhalation studies.

Ammonia (NH3) in the respiratory tract has the potential to neutralize inhaled acid vapors and aerosols. Levels of exhaled (nasal) NH3 were measured in rabbits at different times on the same day, on different days, and in rabbits in a normal fed state, or in a fasted or fed state in which the teeth were brushed and the mouth cleansed. The variability of NH3 levels within any individual rabbit was found to be of the same order as the variability found between different animals. In addition, rabbits which were fasted and had their teeth brushed exhaled significantly less NH3 than did fed animals. Levels in the former group ranged from 4 to 236 micrograms/m3, while those in the latter group ranged from 10 to 758 micrograms/m3. Although brushing the teeth of fed animals compressed the observable range of NH3 levels (22-404 micrograms/m3), this was not a significant reduction compared to fed, unbrushed animals. Thus, fasting likely minimized foodstuff in the mouth; the latter may contribute to NH3 formation through bacterial degradation, which appears to be a significant source of NH3 exhaled through the nose. The NH3 concentrations observed may produce variable degrees of neutralization of inhaled H2SO4 droplets before they deposit in the lungs.

Effect of repeated exposures to nitrogen dioxide and sulfuric acid mist alone or in combination on mucociliary clearance from the lungs of rabbits.

The biological response to ambient air pollution may be a function of specific combinations of pollutants. Groups of rabbits were exposed to NO2 (0.3 ppm or 1 ppm) with and without H2SO4 (0.5 mg/m3) for 2 hr/day for up to 14 days for assessment of effects upon mucociliary clearance of tracer particles from the tracheobronchial tree. Exposure to NO2 did not alter clearance, while exposure to H2SO4 produced a retardation toward the middle of the exposure series. The combination of 0.3 ppm NO2 with H2SO4 resulted in a speeding of clearance, while no change from control was seen with the mixture employing 1 ppm NO2 with acid. These results emphasize the importance of performing studies with pollutant mixtures, since it is not always possible to extrapolate responses from studies examining effects of individual pollutants.

Acute and subchronic ozone inhalation in the rabbit: response of alveolar macrophages.

Ozone is a potent oxidant gas and a common constituent of photochemical smog. This investigation evaluated the numbers and functional capabilities of alveolar macrophages (AM) recovered from rabbits undergoing acute and subchronic ozone exposure. Bronchoalveolar lavage was performed immediately, 24 h, and 7 d after acute (2-h) exposure to 0.1 or 1.2 ppm ozone, and on d 3, 7, and 14 during subchronic (2 h/d X 13 d) exposure to 0.1 ppm ozone. After acute exposure to 1.2 ppm, a marked increase in lavaged neutrophils was observed at 24 h. A single exposure to 0.1 ppm resulted in increased AM at 7 d, while repeated exposures resulted in an increase in AM and neutrophils on d 7 and 14. AM phagocytosis was depressed immediately and 24 h after acute exposure to 0.1 ppm, and at all time points after exposure to 1.2 ppm. Repeated exposures to 0.1 ppm produced reductions in the numbers of phagocytically active AM on d 3 and 7, with a return to control levels by d 14. Substrate attachment by AM was impaired immediately after exposure to 1.2 ppm; AM mobility was not altered by any of the ozone exposures. The results of these studies demonstrated significant alterations in the numbers and functional properties of AM as a result of single or repeated exposure to 0.1 ppm ozone, a level below the current National Ambient Air Quality Standard. These findings indicate that levels of ozone frequently encountered in areas of high photochemical air pollution can elicit a pulmonary inflammatory response and can impair pulmonary defense capabilities.

Early alveolar clearance of particles in rabbits undergoing acute and subchronic exposure to ozone.

The concentration-response relationship for changes in early alveolar clearance resulting from O3 inhalation were investigated. Groups of five rabbits were exposed to 0.0, 0.1, 0.6, or 1.2 ppm O3, 2 hr/day X 1 day; or to 0.0, 0.1, or 0.6 ppm O3, 2 hr/day X 13 days. Following the initial O3 exposure all rabbits inhaled an aerosol of 85Sr-tagged, 3.0-micron, polystyrene latex particles and particle retention was determined daily for the following 14 days. Single exposures to O3 produced a concentration related trend from accelerated clearance at 0.1 ppm to a transient impairment of clearance at 1.2 ppm. Subchronic exposure resulted in a significant acceleration of particle clearance at both 0.1 and 0.6 ppm, with the effect more pronounced at the higher level. These results demonstrate that single or repeated exposure of rabbits to 0.1 ppm O3, a level below the current National Ambient Air Quality Standard, produces alterations in early alveolar clearance. The effects observed at higher levels of O3 appeared to depend on both the concentration and exposure regime; acute exposures produced a concentration related trend toward an initial retardation of clearance, which was followed by an acceleration in particle removal, while repeated exposures resulted in a persistent acceleration of clearance.

Changes in early alveolar particle clearance due to single and repeated nitrogen dioxide exposures in the rabbit.

To better understand the potential health risks associated with short-term NO2 exposures, a study was conducted to examine the effects of single and repeated NO2 exposures on the clearance of inert tracer particles from the alveolar region of rabbit lungs. Single 2-h exposures to 0.3, 1.0, 3.0, and 10.0 ppm produced a concentration-related acceleration in alveolar particle clearance, which resulted in greater particle removal when compared to control. The greatest response was produced at the lower NO2 levels, where as much as 40% more particles were cleared when compared to control. Fewer particles were cleared following a 10.0-ppm NO2 exposure when compared to the lower NO2 levels, and there were indications from the clearance pattern that the higher level was beginning to slow clearance, although an actual retardation was not found. Repeated 14-d exposures (2 h/d) to 1.0 or 10.0 ppm NO2 produced a response similar to a single exposure at the same concentration, suggesting a certain degree of adaptation was produced after the initial exposures. Possible mechanisms for these differences in clearance patterns are discussed. The results of this study demonstrated altered alveolar clearance following short-term NO2 exposure at environmentally relevant concentrations; changes in this important host defense mechanism may be indicative of some underlying pathologic condition.

Measurement of particle clearance from the alveolar region of the rabbit respiratory tract.

This paper describes a method for the measurement of the clearance of inert, insoluble radioactively tagged tracer particles from the alveolar region of the rabbit respiratory tract. The technique uses a fixed detector for noninvasive, external monitoring. Validation of the method is presented, as is the clearance pattern of 3.5-micron latex particles as measured for 56 days postexposure. The clearance of latex deposited within the alveolar region can be described by two phases within this time frame, having half-times of 4.9 and 30.1 days, respectively. Clearance of latex initially deposited on the tracheobronchial tree was essentially complete by 2 days after exposure.

A parallelogram approach for safety evaluation of ingested acetaldehyde.

Route-specific carcinogenicity data are often lacking for compounds of regulatory importance. Acetaldehyde (AA), for example, a natural constituent in foods, is a rodent carcinogen via the inhalation route, but oral carcinogenicity data are not available. In the absence of such data, a parallelogram approach can be used to estimate the oral carcinogenic potency of this chemical. The relative potency of AA to the structurally related compound formaldehyde (FA) in the nose and the relative potency of FA in the nose and stomach serve as the basis for estimating the potency of AA in the stomach. On a dosimetric basis, inhaled AA is 14- to 35-fold less potent than FA in producing nasal DNA-protein crosslinks (DPX), subchronic tissue injury, and tumors. Ingested AA is also considerably ( approximately 5-fold) less potent than FA in producing gastric injury and DPX. Compared to the nose, the stomach is 10- to 60-fold less sensitive to both AA- and FA-induced DPX and subchronic tissue injury. The parallelogram approach will not supplant long-term oral carcinogenicity studies with AA; however, the consistent pattern of decreased sensitivity of acetaldehyde compared to formaldehyde, lower sensitivity of the stomach to the nose, and the lack of gastric tumorigenicity of orally ingested formaldehyde strongly suggests that ingested acetaldehyde is not likely to be carcinogenic. Successful estimation of the carcinogenic potency of ingested glutaraldehyde, for which chronic oral and inhalation data are available, provides further support that the parallelogram approach can provide a reasonable estimate of the carcinogenic potency of closely related aldehydes, such as AA.