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Butadiene diepoxide (BDO2), a metabolite of 1,3-butadiene (BD) and potent mutagen, is suspected to be a proximate carcinogen in the multisite tumorigenesis in B6C3F1 mice exposed to BD. Rats, in contrast to mice, do not form much BDO2 when exposed to BD, and they do not form cancers after exposure to the low levels of BD at which mice develop lung and heart tumors. Tests were planned to determine the direct carcinogenic potential of BDO2 in similarly exposed rats and mice, to see if they would develop tumors of the lung (the most sensitive target organ in BD-exposed mice) or other target tissues. The objective of the current series of studies was to assess the acute toxicity and dosimetry to blood and lung of BDO2 administered by various routes to B6C3F1 mice and Sprague-Dawley rats. The studies were needed to aid in the design of the carcinogenesis study. Initial studies using intraperitoneal injection of BDO2 were designed to determine the rate at which each of the species cleared the compound from the body; the clearance was equally fast in both species. A second study was designed to determine if the highly reactive BDO2, when deposited in the lung, would enter the bloodstream from the lung; intratracheally instilled BDO2 did enter the bloodstream, indicating that exposure via the lungs would result in BDO2 reaching other organs of the body. In a third study, rats and mice were exposed by inhalation for 6 h to 12 ppm BDO2 to determine blood and lung levels of the compound. Concentrations of BDO2 in the lung immediately after the exposure were 2 to 3 times higher than in the blood in both species (approximately 500 and 1000 pmol/g blood in the rat and mouse, respectively). As expected, mice received a higher dose/g tissue than did rats, consistent with the higher minute volume/kg body weight of the mice. The inhalation dosimetry study was followed by a histopathology study to determine the acute toxicity to rodents following a single, 6-h exposure to 18 ppm BDO2. No clinical signs of toxicity were observed; lesions were confined to the olfactory epithelium where areas of necrosis were observed. Analysis of bronchoalveolar lavage fluid did not indicate pulmonary inflammation. Based on these findings, an attempt was made to expose rats and mice repeatedly (for 7 days) to 10 and 20 ppm BDO2, but these exposure concentrations proved too toxic, due to inflammation of the nasal mucosa and occlusion of the nasal airway, a lesion that cannot be tolerated by obligate nose breathers. Finally, the toxicity of rats and mice exposed 6 h/day for 5 days to 0. 2.5, or 5.0 ppm BDO2 was determined. The repeated exposures caused no clinical signs of toxicity, nor were any lesions observed in the respiratory tract or other major organs. Therefore, the final design selected for the carcinogenesis study comprised exposing the rats and mice for 6 h/day, 5 days/week for 6 weeks to 0, 2.5, or 5.0 ppm BDO2. [ABSTRACT FROM PUBLISHER]

Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile compound used as a chemical intermediate, a catalyst, and an antiknock additive in gasoline. This organometallic chemical is of particular interest because of its structural similarities to other metallocenes, some of which are carcinogenic. F344/N rats and B6C3F mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m, 6 hr/day, 5 days/week, for 13 weeks. During these exposures, no rats or mice died, nor were any clinical signs of ferrocene-related toxicity observed. At the end of the exposures, male rats exposed to the lowest and highest level of ferrocene had decreased body weight gains compared to filtered-air-exposed control male rats, while body weight gains for all groups of both ferrocene- and filtered-air-exposed female rats were similar. Male mice exposed to ferrocene had no differences in body weight gains, compared to controls, but female mice had decreases in body weight gains at the 10 and 30 mg/m exposure levels. There were exposure concentration- and exposure-time-related increases in lung burdens of iron. The mean iron lung burden in rats exposed to 30 mg ferrocene vapor/m for 90 days was four times greater than the burden in control rats. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen, clinical chemistry, and hematology parameters were observed. This suggests that the accumulations of iron in lung did not cause an inflammatory response nor any functional impairment of the lung. There were no indications of developing pulmonary fibrosis nor of any hematologic toxicity. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Exposure-related histopathologic alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium where pigment accumulation, necrotizing inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. Histochernical stains of these target tissues showed the presence of iron ions. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalyzed lipid peroxidation of cellular membranes or the iron-catalyzed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA. [ABSTRACT FROM PUBLISHER]

Two-Week, Repeated Inhalation Exposure of F344/N Rats and B6C3F Mice to Ferrocene. SUN, J. D., DAHL, A. R., GILLETT, N. A., BARR, E. B., CREWS, M. L., EIDSON, A. F., BECHTOLD, W. E., BURT, D. G., DIETER, M. P., AND HOBBS, C. H. (1991). . . 17, 150-158. Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile, organometallic compound used as a chemical intermediate, a catalyst, and as an antiknock additive in gasoline. It is of particular interest because of its structural similarities to other metallocenes that have been shown to be carcinogenic. F344/N rats and B6C3F, mice were exposed to 0, 2.5, 5.0, 10, 20, and 40 mg ferrocene vapor/m3, 6 hr/day for 2 weeks. During these exposures, there were no mortality and no observable clinical signs of ferrocene-related toxicity in any of the animals. At the end of the exposures, male rats exposed to the highest level of ferrocene had decreased body-weight gains relative to the weight gained by filtered air-exposed control rats, while body-weight gains for all groups of both ferrocene- and filtered air-exposed female rats were similar. Male mice exposed to the highest level of ferrocene also had decreased body-weight gains, relative to controls, while female mice had relative decreases in body-weight gains at the three highest exposure levels. Male rats had a slight decrease in relative liver weight at the highest level of exposure, whereas no relative differences in organ weights were seen in female rats. Male mice had exposure-relative decreases in liver and spleen weights, and an increase in thymus weights, relative to controls. For female mice, relative decreases in organ weights were seen for brain, liver, and spleen. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Histopathological examination was done only on the nasal turbinates, lungs, liver, and spleen. The only exposure-related finding was histopathologic lesions in the nasal turbinates of both species. These lesions were primarily centered in the olfactory epithelium and were morphologically diagnosed as subacute, necrotizing inflammation. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. In vitro metabolism studies of ferrocene showed that nasal tissue, particularly the olfactory epithelium, had 10 times higher “ferrocene hydroxylating” activity than did liver tissue from the same animals. These results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by iron-catalyzed lipid peroxidalion of cellular membranes. [ABSTRACT FROM PUBLISHER]

Effect of Inhaled Azodicarbonamide on F344/N Rats and B6C3F, Mice with 2-Week and 13-Week Inhalation Exposures. MEDINSKY, M. A., BECHTOLD, W. E., BIRNBAUM, L. S., BOND, J. A., BURT, D. G., CHENG, Y. S., GILLFTT, N. A., GULATI, D. K., HOBBS, C. H., AND PICK-RELL, J. A. (1990). Fundam. Appl. Toxicol 15, 308/319. Azodicarbonamide (ADA), a compound used in the baking and plastics industries, has been reported to cause pulmonary sensiti-zation and dermatitis in people. Two-week repeated and 13-week subchronic inhalation exposures of F344/N rats and B6C3F, mice to ADA were conducted to determine the toxicity of inhaled ADA. The mean air concentrations of ADA in the 2-week studies were 207, 102, 52, 9.4, or 2.0 mg/m3. No exposure-related mortality nor abnormal clinical signs were observed in rats or mice during or after exposure. The terminal body weights were slightly depressed in the highest exposure group. Liver weights were lower in male rats exposed to 200 mg ADA/m3. No significant lesions were noted on either gross or histologic evaluation of rats or mice. In the 13-week subchronic study, the mean air concentrations of ADA were 204, 100, or 50 mg/m3. No mortality or clinical signs related to exposure were observed. The terminal body weights of exposed rats were not significantly different from those of control rats but were significantly depressed in mice exposed to 100 or 200 mg ADA/m3. No histopathological lesions were noted in mice. Lung weights were increased and enlarged mediastinal and/or tracheobronchial lymph nodes were noted in rats exposed to 50 mg ADA/m3. No exposure-related lesions were observed microscopically in rats exposed to 100 or 200 mg ADA/m3. All rats in the 50 mg ADA/m3 exposure group only had lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia, suggesting a possible immune reaction to an antigen in the lung. Viral titers for rats exposed to 50 mg ADA/m3 were negative for Sendai virus and pneumonia virus of mice, which produce similar lesions. The possibility of an unknown viral antigen causing this lesion cannot be eliminated. Lung tissue from male rats was analyzed for ADA and biurea, the major metabolite of ADA. No ADA was detected. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration, suggesting that clearance was somewhat impaired with repeated exposures. However, even at the highest exposure concentration, this amount of biurea was less than 1 % of the estimated total ADA deposited over the exposure period. In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m3. Exposure to ADA for up to 13 weeks did not appear to be toxic to rodents [ABSTRACT FROM PUBLISHER]

The Fate of Inhaled Azodicarbonamide in Rats. Mewhinney, J. A., AYRES, P. H., BECHTOLD, W. E., DUTCHER, J. S., CHENG, Y. S., BOND, J. A., MEDINSKY, M. A., HENDERSON, R. F., AND BIRNBAUM, L. S. (1987). . , 372–381. Azodicarbonamide (ADA) is widely used as a blowing agent in the manufacture of expanded foam plastics, as an aging and bleaching agent in flour, and as a bread dough conditioner. Human exposures have been reported during manufacture as well as during use. Groups of male F344/N rats were administered ADA by gavage, by intratracheal instillation, and by inhalation exposure to determine the disposition and modes of excretion of ADA and its metabolites. At 72 hr following gavage, 30% of the administered ADA was absorbed whereas following intratracheal instillation, absorption was 90%. Comparison between groups of rats exposed by inhalation to ADA to achieve body burdens of 24 or 1230 μg showed no significant differences in modes or rates of excretion of [C]ADA equivalents. ADA was readily converted to biurea under physiological conditions and biurea was the only C-labeled compound present in excreta. [C]ADA equivalents were present in all examined tissues immediately after inhalation exposure, and clearance half-times on the order of 1 day were evident for all tissues investigated. Storage depots for [C]ADA equivalents were not observed. The rate of buildup of [C]ADA equivalents in blood was linearly related to the lung content as measured from rats withdrawn at selected times during a 6-hr inhalation exposure at an aerosol concentration of 25 μg, ADA/liter. In a study extending 102 days after exposure, retention of [C]ADA equivalents in tissues was described by a two-component negative exponential function. The results from this study indicate that upon inhalation, ADA is rapidly converted to biurea and that biurea is then eliminated rapidly from all tissues with the majority of the elimination via the urine. [ABSTRACT FROM PUBLISHER]

Benzene is a ubiquitous environmental pollutant that is known to cause hematotoxicity and leukemia in humans. The initial oxidative metabolite of benzene has long been suspected to be benzene oxide (3,5-cyclohexadiene-1,2-oxide). During in vitro experiments designed to characterize the oxidative metabolism of [14C]benzene, a metabolite was detected by HPLC-radioactivity analysis that did not elute with other known oxidative metabolites. The purpose of our investigation was to prove the hypothesis that this metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM), and NADPH (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) and then extracted with methylene chloride. Gas chromatography-mass spectrometry analysis of incubation extracts for mice, rats, and humans detected a metabolite whose elution time and mass spectrum matched that of synthetic benzene oxide. The elution time of the benzene oxide peak was approximately 4.1 min, while phenol eluted at approximately 8 min. Benzene oxide also coeluted with the HPLC peak of the previously unidentified metabolite. Based on the 14C activity of this peak, the concentration of benzene oxide was determined to be approximately 18 microM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes with 1 mM benzene. The metabolite was not observed in incubations using heat-inactivated microsomes. This is the first demonstration that benzene oxide is a product of hepatic benzene metabolism in vitro. The level of benzene oxide detected suggests that benzene oxide is sufficiently stable to reach significant levels in the blood of mice, rats, and humans and may be translocated to the bone marrow. Therefore benzene oxide should not be excluded as a possible metabolite involved in benzene-induced leukemogenesis. [ABSTRACT FROM PUBLISHER]

Hemoglobin (Hb) and albumin (Alb) adducts of the benzene metabolites benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) were analyzed by gas chromatography-mass spectrometry in 43 exposed workers and 44 unexposed controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. When subjects were divided into controls (n = 44) and workers exposed to 31 ppm (n = 22) of benzene, median 1,4-BQ-Alb adducts were 2110, 5850, and 13,800 pmol/g Alb, respectively (correlation with exposure: Spearman r = 0.762; P < 0.0001); median BO-Alb adducts were 106, 417, and 2400 pmol/g Alb, respectively (Spearman r = 0.877; P < 0.0001); and median BO-Hb adducts were 37.1, 50.5, and 136 pmol/g Hb, respectively (Spearman r = 0.757; P < 0.0001). To our knowledge, this is the first observation that adducts of 1,4-BQ are significantly correlated with benzene exposure. When compared on an individual basis, Alb adducts of 1,4-BQ and BO and Hb adducts of BO were highly correlated with each other and with urinary phenol and hydroquinone (P < 0.0001 for all of the comparisons). Although detectable in the assays, Hb adducts of 1,4-BQ and both Hb and Alb adducts of 1,2-BQ produced erratic results and are not reported. Interestingly, cigarette smoking increased Alb adducts of 1,4-BQ but not of BO, suggesting that benzene from cigarette smoke was not the primary contributor to the 1,4-BQ adducts.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is weakly carcinogenic in Sprague-Dawley rats after chronic exposures to concentrations of 1000 and 8000 ppm. Conversely, in B6C3F1 mice, tumors occur after chronic exposures to concentrations as low as 6.25 ppm. Previously, we have shown that tissue concentrations of the mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), are present in greater concentrations in mice than in rats following acute exposures to low levels (100 ppm or less). This disparity was particularly significant for the diepoxide. We hypothesized that if these epoxides are involved in the carcinogenic response of BD, then they will also be present in rat tissues at relatively high concentrations following exposures to 8000 ppm BD. In the present study, concentrations of the BD epoxides, BDO and BDO2, were determined in blood of female Sprague-Dawley rats following a single 6-h exposure and 10 repeated exposures to a target concentration of 8000 ppm BD. Concentrations of these epoxides were also determined in a number of other tissues, including the primary rat target organ-mammary gland-following 10 repeated exposures. Blood concentrations of BDO were 4030 pmol/g +/- 191 following a 6-h exposure and were 18% lower following the 10-day exposure. Blood concentrations of BDO2, following the 8000 ppm exposures, were very similar to those previously observed after exposures to 62.5 ppm BD (11 +/- 1 and 17 +/- 1 pmol/g following exposures of 6h and 6h/day for 10 days, respectively.) Concentrations of BDO ranged from 740 +/- 110 (femur) to 8990 +/- 1150 (fat) pmol/g tissue. Concentrations of BDO2 were similar among eight tissues analyzed, ranging from 5 +/- 1 (femur) to 17 +/- 3 (heart) pmol/g tissue. Tissue concentrations of butadiene monoepoxide were increased by 17- to 50-fold in tissues from rats exposed by inhalation to 8000 ppm BD as compared to tissues from rats exposed to 62.5 ppm BD. Based on earlier studies at our institute the internal dose of BD increases approximately 14-fold in the 8000 ppm-exposed rats compared to rats exposed to 62.5 ppm BD. Concentrations of butadiene diepoxide in rat tissues following an exposure to 8000 ppm BD were similar to those observed in rat tissues following exposures to 62.5 ppm BD. This study shows that pathways responsible for the accumulation of BDO2 in rats are saturated following low-level BD exposures. This suggests that the primary determinant of BD tumorigenicity in rats is not butadiene diepoxide. The high levels of BDO observed in rat mammary tissue suggest that this metabolite may be a more important determinant of BD carcinogenesis in the rat., (Copyright 1998 Academic Press.)

Exposure to high concentrations of butadiene has been shown to cause cancer among exposed workers. We have conducted a biomarker study to elucidate whether current butadiene exposure conditions are hazardous to workers. Twenty-four workers exposed consistently to butadiene were matched with 19 co-workers who had much less contact with butadiene and who served as our controls. In the standard cytogenetic assay, there was no difference in chromosome aberration frequencies between the exposed and control groups. In the challenge assay, the exposed group shows a consistent, but non-significant, increase in chromosome aberrations indicating some abnormality in DNA repair response. The observed dicentric frequency in the challenge assay (indicative of abnormal repair of damaged chromosomes) is significantly correlated with a butadiene metabolite, 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, in urine (r = 0.52; p = 0.04). Furthermore, cigarette smokers had consistently abnormal repair response compared with non-smokers for both the control and exposed groups. A small subset of the studied workers were evaluated for toxicant-induced DNA repair deficiency using an independent cat-host cell reactivation (CAT-HCR) assay. When cigarette smokers and non-smokers were combined in our analysis, we observed that the exposed group (n = 9) had a significant reduction of DNA repair activities (p = 0.009) compared with the control group (n = 6). Cigarette smoking contributed significantly to the effect as exposed smokers (n = 4) had a significant reduction in DNA repair activities (p = 0.04) compared with exposed non-smokers. The results from the two independently conducted assays support each other and confirm the previously reported abnormal DNA repair response in another group of butadiene workers. In conclusion, our data indicates that exposure to environmental toxicants, such as butadiene, can cause DNA repair defects. Therefore, the current butadiene exposure conditions are still hazardous to workers. However, our data indicates that butadiene is not a potent genotoxic agent. Furthermore, the butadiene-induced effect is significantly enhanced by the cigarette smoking habit.

The use of biological markers in the evaluation of human exposure to hazardous agents has increased rapidly in recent years. Because 1,3-butadiene is a mutagenic carcinogen, existing occupational levels of exposure may be appropriately evaluated using somatic cell mutation as a biomarker. Previously, we have described a biomarker study of workers in a butadiene monomer plant (Ward et al., 1994). We now report results from a second study of the same group of workers, conducted after plant modernization, and present preliminary results from a study of exposures in a styrene butadiene rubber (SBR) plant. Air levels of butadiene were determined using either charcoal tubes with air pumps or passive badge dosimeters. The quantity of a butadiene metabolite in the urine was used as a biomarker of exposure and the mutagenic effects of exposure were measured using the autoradiographic hprt mutant lymphocyte assay. In all three studies, the frequencies of hprt mutants were significantly elevated in workers from the areas of highest exposure when compared to workers from lower exposure areas or non-exposed subjects. The concentration of the urinary metabolite was significantly increased in high-exposed workers in the first study of monomer plant workers but not in the second. In the first monomer plant study, historical air concentrations of butadiene were higher in the production units than in the central control unit. While concurrent determined air concentrations were not elevated in the second monomer plant study, they were elevated in high exposure areas in the SBR plant study. Mutant frequencies in the lower-exposure and the non-exposed groups were consistent with historical values for non-smoking individuals who were not exposed to known mutagens. The use of biomarkers, including the hprt mutant lymphocyte assay, may be of great value in determining an appropriate occupational exposure limit for butadiene.

Hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation frequency (M(f)) was studied in workers at a polybutadiene rubber production facility in Yanshan, China. Exposed workers included for study were active either as process analysts, who sampled butadiene production process lines and analyzed product by gas chromatography, or as process operators, who did routine process control, minor maintenance and, as needed, major repair operations. For process analysts at the polymer and dimethyl formamide (DMF) facilities, the median air levels of BD were 1.0 and 3.5 ppm, respectively. Among process operators, air levels of 1.1 ppm were found during routine activities, while the median air level during pump repair and related operations was 45 ppm (6-h time-weighted average). Overall, M(f) was similar in unexposed (mean M(f) = 20.2 x 10(-6)) and butadiene-exposed (mean M(f) = 21.6 x 10(-6)) workers (P = 0.68). M(f) decreased with cloning efficiency, increased with age, and was moderately greater in women than in men. After adjustment by multiple regression analysis for mean age, sex, and cloning efficiency, the adjusted mean M(f)(Xadj) was 13.6 x 10(-6) in unexposed and 18.0 x 10(-6) in butadiene-exposed. This 32% difference was, however, not statistically significant (P = 0.13). Butadiene exposure was associated with a modest, if any, increase in hprt M(f) in this population of Chinese workers.

Levels of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2) were compared in tissues of male Sprague-Dawley rats and male B6C3F1 mice and in tissues of male and female Sprague-Dawley rats following inhalation exposures to 62.5 ppm 1,3-butadiene (BD). In male rats, BDO2 levels were highest in blood and were present at a concentration of only 5 +/- 1 pmol/g. Following a 6-h exposure, the concentration of BDO2 in the blood, femurs, lung and fat of female rats was 3 to 7-fold that of male rats. Levels of BDO were similar in tissues of female and male rats. Generally, levels of BDO were approximately 3 to 8-fold greater in mouse tissues as compared with rat tissues following 4-h exposures to BD. In blood, 204 +/- 15 pmol/g BDO2 was detected in male mice, while in rats, blood BDO2 levels were 5 +/- 1 pmol/g. This study shows marked species differences in tissue levels of BD epoxides, particularly BDO2, in rats and mice, and is the first to show gender differences in BD metabolism.

Species differences in the metabolism of 1,3-butadiene (BD) have been studied in an effort to explain the major differences observed in the responses of mice, the sensitive species, and rats, the resistant species, to the toxicity of inhaled BD. BD is metabolized by the same metabolic pathways in all species studied, but there are major species differences in the quantitative aspects of those pathways. Of the species studied, mice are the most efficient at metabolizing BD to the initial metabolite, the monoepoxide (BDO). Mice either convert most of the BDO to the diepoxide (BDO2), the most mutagenic of the BD metabolites, or form conjugates of the BDO with glutathione (GSH). Rats, on the other hand, are less active at forming BDO, oxidize very little of the BDO to BDO2, and form GSH conjugates with either the BDO or its hydrolysis product, butenediol. Primates convert even less of inhaled BD to BDO and hydrolyze most of the BDO to the butenediol. The extent to which primates form BDO2 is unknown. Because of the association of high levels of the highly mutagenic BDO2 with the sensitive rodent strain, it is important to determine the production of this metabolite in primates, particularly humans.