Craig S. Barrow

Benzene disposition in the rat after exposure by inhalation

Abstract Little information is available on benzene disposition after exposure by inhalation despite the importance of this route in man. Benzene metabolites as a group have been measured in bone marrow, but quantitation of individual metabolites in this target tissue has not been reported. Male Fischer-344 rats were exposed to 500 ppm benzene in air and the uptake and elimination was followed in several tissues. Concentrations of free phenol, catechol, and hydroquinone in blood and bone marrow were also measured. Steady-state concentrations of benzene (11.5, 37.0, and 164.0 μg/g in blood, bone marrow, and fat, respectively) were achieved within 6 hr in all tissues studied. Benzene half-lives during the first 9 hr were similar in all tissues (0.8 hr). A plot of amount of benzene remaining to be excreted in the expired air was biphasic with t 1 2 values for the α and β phases of 0.7 and 13.1 hr, respectively. Phenol was the main metabolite in bone marrow at early times (peak concentration, 19.4 μg/g). Catechol and hydroquinone predominated later (peak concentrations, 13.0 and 70.4 μg/g, respectively). Concentrations of these two metabolites declined very slowly during the first 9 hr. These data indicate that free catechol and hydroquinone persist in bone marrow longer than benzene or free phenol.

Respiratory tract lesions induced by sensory irritants at the RD50 concentration

Exposure of mice to airborne sensory irritants causes a concentration-dependent depression of respiratory rate. The RD50 concentration (that concentration which elicits a respiratory rate decrease of 50%) has been predicted to be an unacceptable occupational exposure concentration due to intolerable sensory irritation and possible respiratory tract injury in humans. The purpose of this study was (1) to determine whether lesions occur in the respiratory tract of Swiss-Webster mice after exposure to the RD50 concentrations of ten sensory irritants and (2) to compare these changes with respect to type and severity. The RD50 values (ppm) of the chemicals studied are as follows: 2,4-toluene diisocyanate (0.4), acrolein (1.7), formaldehyde (3.1), chloropicrin (8.0), chlorine (9.3), sulfur dioxide (117), ammonia (303), hydrogen chloride (309), dimethylamine (511), and epichlorohydrin (687). After exposure of mice for 6 hr/day for 5 days, the respiratory tract was examined for histopathologic changes. All irritants produced lesions in the nasal cavity with a distinct anterior-posterior severity gradient. There was considerable variation in the extent, and nature of the lesions. The lesions ranged from slight epithelial hypertrophy or hyperplasia to epithelial erosion, ulceration, and necrosis with variable inflammation of the subepithelial tissues. Only chlorine, chloropicrin, and epichlorohydrin induced lesions in the lower respiratory tract. These findings give additional support to the potential value of the RD50 model for setting occupational exposure guidelines and predicting the risk of injury to the respiratory tract from exposure to airborne sensory irritants.

Effect of single or repeated formaldehyde exposure on minute volume of B6C3F1 mice and F-344 rats☆

Abstract To characterize species differences in sensory irritation to formaldehyde (HCHO) and to determine if tidal volume ( V T ) compensation parallels the respiratory rate (ƒ) depression, ƒ, V T , and minute (V E ) of B6C3F1 mice and F-344 rats were measured during 10-min, head-only HCHO exposures. Comparisons also were made between naive (no previous HCHO exposure) and pretreated animals (HCHO, 2, 6, or 15 ppm, 6 hr/day for 4 days). HCHO exposure elicited immediate and simultaneous decreases in ƒ and V E in a concentration-dependent manner. No significant compensation in V T was observed in either species except the 15-ppm-pretreated rats. While ƒ and V E remained maximally depressed in mice for the remainder of the exposure period, recovery occurred in rats. In addition to tolerance development, rats were also less sensitive to HCHO. The RD50 (concentration at which ƒ decreased by 50%) of naive rats was 31.7 ppm vs 4.9 ppm for mice. Similar RD50 values and time-response curves for V E and ƒ were observed among naive and pretreated groups, although the baseline ƒ of pretreated animals was significantly depressed as compared to naive animals. In summary, HCHO exposure depressed V E in both mice and rats. But, because of greater sensitivity and lack of tolerance, mice were able to minimize inhalation of HCHO more than rats. This species difference may contribute to differences in respiratory tract toxicity from inhaled HCHO.

Sensory irritation structure-activity study of inhaled aldehydes in b6c3f1 and swiss-webster mice.

The sensory irritation potential of a series of saturated and unsaturated aliphatic and cyclic aldehydes was investigated in B6C3F1 and Swiss-Webster mice. With the reflex decrease in respiratory rate as the endpoint response, alpha, beta-unsaturated aliphatic aldehydes yielded RD50 values (concentration which elicits a 50% decrease in respiratory rate) ranging from 1 to 5 ppm while saturated aliphatic aldehydes with two or more carbons produced RD50 values from 750 to 4200 ppm. Cyclic aldehydes produced intermediate RD50 values which ranged from 60 to 400 ppm. No statistically significant differences were found between concentration-response curves of B6C3F1 and Swiss-Webster mice. Saturated aliphatic aldehydes with two or more carbons were nearly 1000 times less potent than formaldehyde. Although the mechanisms responsible for stimulation of trigeminal nerve endings by airborne chemicals are poorly understood, several hypotheses may help to explain the differences seen in this study. For example, the sensory irritation potency of the saturated aliphatic aldehydes diminished with their reported dehydration constants which may determine the degree to which these aldehydes crosslink with receptor proteins. The sensory irritation potency of acrolein and crotonaldehyde was probably due to 1,2 or 1,4 addition reactions. Additionally, molecular conformation and a recently published physical mechanism may contribute to sensory irritation responses, particularly for the less reactive aldehydes. Tentative threshold limit values (TLVs), based upon prevention of sensory irritation, were extrapolated from the RD50 values of Swiss-Webster mice. With the exception of crotonaldehyde, good agreement was found with currently published TLVs.

Sensory irritation response to inhaled aldehydes after formaldehyde pretreatment

Pretreatment of Fischer-344 (F-344) rats with formaldehyde (HCHO) induces significant cross tolerance to the sensory irritation properties of Cl2. The purpose of this study was to determine if HCHO pretreatment would cause sensory irritation cross tolerance to other inhaled aldehydes. Male F-344 rats, weighing 190 to 210 g, were pretreated with 15 ppm HCHO, 6 hr/day for 9 days, and challenged on the 10th day with a saturated (acetaldehyde, propionaldehyde, and butyraldehyde), unsaturated (acrolein and crotonaldehyde), or cyclic (cyclohexanecarboxaldehyde, 3-cyclohexene-1-carboxaldehyde, and benzaldehyde) aldehyde. The sensory irritation response in these animals was quantified by measuring respiratory rate depression in a head-only inhalation chamber using plethysmographic techniques. Control animals were challenged identically without prior pretreatment. In naive (nonpretreated) animals, the concentration eliciting a 50% decrease in respiratory rate (RD50) was 23 ppm or less for unsaturated aliphatic aldehydes. For cyclic and saturated aliphatic aldehydes, the RD50 ranged from 600 to 1000 ppm and 3000 to 6800 ppm, respectively. Formaldehyde pretreatment resulted in cross tolerance only with acetaldehyde (RD50 increased 3.5-fold) and acrolein (RD50 increased 5-fold). These results indicate that the development of cross tolerance following HCHO pretreatment is not a general phenomenon. Prediction of acceptable concentrations of occupational exposure for the prevention of sensory irritation in humans has been based primarily on RD50 data for mice. Comparison of the RD50 values obtained for rats in this investigation with previously published results for mice varied by over one-half an order of magnitude, thereby disputing the usefulness of data from F-344 rats in setting threshold limit values for the prevention of sensory irritation.

Two-Year Inhalation Exposure of Female and Male B6C3F1 Mice and F344 Rats to Chlorine Gas Induces Lesions Confined to the Nose

Chlorine gas is a respiratory irritant in both animals and humans that produces concentration-dependent responses ranging from minor irritation to death. Female and male B6C3F1 mice and F344 rats were exposed to chlorine gas for up to 2 years to determine chronic toxicity and carcinogenicity. Groups of approximately 70 each of female and male mice and rats were exposed to 0, 0.4, 1.0, or 2.5 ppm chlorine gas for 6 hr/day, 5 days/week (mice and male rats), or 3 alternate days/week (female rats) for 2 years, with an interim necropsy of rats at 12 months (10 rats/sex/concentration group). A complete necropsy was performed on all animals. Histological examination was performed on all organs from high-concentration and control animals and selected target organs from mid- and low-concentration groups. Exposure-dependent lesions were confined to the nasal passages in all sex and species groups. Chlorine-induced lesions, which were most severe in the anterior nasal cavity, included respiratory and olfactory epithelial degeneration, septal fenestration, mucosal inflammation, respiratory epithelial hyperplasia, squamous metaplasia and goblet cell hypertrophy and hyperplasia, and secretory metaplasia of the transitional epithelium of the lateral meatus. Intracellular accumulation of eosinophilic proteinaceous material was also a prominent response involving the respiratory, transitional, and olfactory epithelia, and in some cases the squamous epithelium of the nasal vestibule. Many of these nasal lesions exhibited an increase in incidence and/or severity that was related to chlorine exposure concentration and were statistically significantly increased at all chlorine concentrations studied. Male mice and female rats appeared more sensitive to chlorine than female mice and male rats, respectively. The reasons for the sex differences within a species were not determined. Interspecies differences in regional dosimetry and site-specific tissue susceptibility to chlorine exposure should be taken into account when using these data for accurate assessment of potential human health risks. The incidence of neoplasia was not increased by exposure, indicating that inhaled chlorine in rats and mice is an upper respiratory tract toxicant but not a carcinogen.

Ammonia production in inhalation chambers and its relevance to chlorine inhalation studies

Abstract Inhalation exposure of animals to low concentrations of chlorine (ca. 1 ppm) may be complicated by the formation of chloramines from the reaction of ammonia, evolved from animal urine and feces, with chlorine. To address this problem, ammonia levels were determined by an indophenol method during a 6-hr period, in inhalation chambers using male, Fischer 344 rats under varying conditions of air flow and animal loading. At chamber air flows of 13, 26, or 40 liters/min, ammonia concentrations were obtained at 2, 4, and 6 hr for 1, 3, or 5% animal loading. Ammonia concentrations increased with time, peaking at 6 hr; they increased with increased percentage animal loading and decreased with increased chamber air flow. At 6 hr, for 13 liters/min, and 1, 3, or 5% animal loading, ammonia concentrations were 0.46, 1.91, and 2.42 ppm, respectively. At 40 liters/min, the ammonia levels at 6 hr were 0.22, 0.39, and 1.30 ppm for 1, 3, or 5% animal loading. Chloramines were detected in a chamber containing low concentrations of chlorine and ammonia (ca. 2 ppm each) by using a modified methyl orange method. These results suggest that, depending upon the variables indicated, sufficient ammonia may be evolved from animals in an inhalation chamber to reduce the amount of free chlorine present. Taken in conjunction with the toxic action of chloramines, such reduction may affect the outcome of chronic, low-level exposures to chlorine.

One-year inhalation toxicity study of chlorine in rhesus monkeys (Macaca mulatta).

Chlorine (Cl2) gas is a potentially lung-damaging irritant which is used in the chemical, plastics, and paper industries. There are no data published using experimental animals on the chronic inhalation toxicity of chlorine. The purpose of this study was to investigate the chronic effects of Cl2 inhalation in Rhesus monkeys (Macaca mulatta). Rhesus monkeys were exposed to concentrations of 0, 0.1, 0.5, or 2.3 ppm Cl2 for 6 hr per day. 5 days per week for 1 year. Pulmonary physiology (pulmonary diffusing capacity and distribution of ventilation), body weights, urinalysis, electrocardiographs, hematology, and clinical chemistry were evaluated monthly during the study. Blood gas evaluations were performed at 3-month intervals during the study. Histopathologic, ophthalmologic, and neurologic parameters were evaluated after the 1-year exposure period. Monkeys exposed to 2.3 ppm Cl2 exhibited signs of ocular irritation during the daily exposures and a superficial conjunctival irritation was present in the 2.3 ppm group after the 1-year exposure regimen. Treatment-induced lesions revealed by histopathology were confined to the respiratory tract. Lesions associated with the nasal parasite Anatrichosoma spp. were present in the region of squamous epithelium of the nasal vestibule and did not interfere with interpretation of Cl2-induced effects. Treatment-induced histopathologic changes were found in the respiratory epithelium of the nasal passages and trachea and were limited to focal, concentration-related epithelial hyperplasia with loss of cilia and decreased numbers of goblet cells in affected areas. These changes in the nose and trachea were focal and mild in monkeys exposed to 2.3 ppm and were not found in all animals in these exposure groups. Tracheal lesions were confined to the 2.3 ppm group. The lesions observed at 2.3 ppm were not present in all animals. At the lower Cl2 concentrations, similar though less prominent respiratory epithelial lesions were observed. The latter changes were very minimal and were confined to the nasal passages of some treated monkeys and one male control animal. The results of this study indicate that 2.3 ppm chlorine acts as an upper respiratory irritant in monkeys, while 0.5 and 0.1 ppm induce changes of questionable clinical significance. Furthermore, the monkey appears to be less sensitive than the rat to chlorine toxicity.

Accelerating the Development of 21st-Century Toxicology: Outcome of a Human Toxicology Project Consortium Workshop

The U.S. National Research Council (NRC) report on “Toxicity Testing in the 21st century” calls for a fundamental shift in the way that chemicals are tested for human health effects and evaluated in risk assessments. The new approach would move toward in vitro methods, typically using human cells in a high-throughput context. The in vitro methods would be designed to detect significant perturbations to “toxicity pathways,” i.e., key biological pathways that, when sufficiently perturbed, lead to adverse health outcomes. To explore progress on the report’s implementation, the Human Toxicology Project Consortium hosted a workshop on 9–10 November 2010 in Washington, DC. The Consortium is a coalition of several corporations, a research institute, and a non-governmental organization dedicated to accelerating the implementation of 21st-century Toxicology as aligned with the NRC vision. The goal of the workshop was to identify practical and scientific ways to accelerate implementation of the NRC vision. The workshop format consisted of plenary presentations, breakout group discussions, and concluding commentaries. The program faculty was drawn from industry, academia, government, and public interest organizations. Most presentations summarized ongoing efforts to modernize toxicology testing and approaches, each with some overlap with the NRC vision. In light of these efforts, the workshop identified recommendations for accelerating implementation of the NRC vision, including greater strategic coordination and planning across projects (facilitated by a steering group), the development of projects that test the proof of concept for implementation of the NRC vision, and greater outreach and communication across stakeholder communities.

Perinatal toxicity and metabolism of n-hexane in Fischer-344 rats after inhalation exposure during gestation

Abstract Repeated exposure of adult rats to n -hexane produces a central and peripheral neuropathy which may be mediated through metabolic activation to methyl n -butyl ketone (MBK) and 2,5-hexanedione (2,5-HD). The perinatal toxicity of n -hexane and its metabolism in the pregnant rat have not been investigated. Pregnant rats were exposed for 6 hr per day to 1000 ppm n -hexane on Days 8–12, 12–16, or 8–16 of gestation. No significant alterations in fetal resorptions, body weights, visible anomalies, and the incidence of soft tissue and skeletal anomalies were noted in any of the treatment groups. The postnatal growth of pups born from dams exposed to 1000 ppm n -hexane 6 hr/day on Days 8–16 of gestation was significantly depressed compared to controls up to 3 weeks after birth (mean treated litter weight 13.9% less than control at 3 weeks). Litter weights of treated pups had returned to control values by 7 weeks after birth. n -Hexane was metabolized to MBK and 2,5-HD in pregnant rats exposed to 1000 ppm n -hexane on Day 20 of gestation. Concentrations of the three compounds in the fetus were approximately equal to those in maternal blood at all times after exposure. The half-life of 2,5-HD in maternal blood was significantly greater than n -hexane and MBK (3.90 hr vs 1.24 and 0.99 hr, respectively). Thus, n -hexane and its metabolites MBK and 2,5-HD may have only a minimal potential to alter perinatal development of rats.