Dose response for formaldehyde-induced cytotoxicity in the human respiratory tract.

Human studies of the sensory irritant effects of formaldehyde are complicated by the subjective nature of some clinical endpoints. This limits the usefulness of such studies for quantitative noncancer risk assessment of airborne formaldehyde. Objective measures of the noncancer effects of formaldehyde, such as the rate of regenerative cellular proliferation (RCP) secondary to cytolethality, can be obtained from laboratory animals but present the challenge of interspecies extrapolation of the data. To the extent that uncertainties associated with this extrapolation can be reduced, however, dose-response data obtained in laboratory animals are a viable alternative to clinical studies. Here, we describe the extrapolation of dose-response data for RCP from F344 rats to humans. Rats inhaled formaldehyde (0, 0.7, 2.0, 6.0, 10, and 15 ppm) 6 h/day, 5 days/week for up to 2 years. The dose response for RCP was J-shaped, with the rates of RCP at 0.7 and 2.0 ppm below but not statistically different from control, while the rates at the higher concentrations were significantly greater than control. Both the raw J-shaped data and a hockey-stick-shaped curve fitted to the raw data were used for predicting the human dose response for RCP. Cells lining the nasal airways of F344 rats and rhesus monkeys are comparably sensitive to the cytolethal effects of inhaled formaldehyde, suggesting that the equivalent human cells are also likely to be comparably sensitive. Using this assumption, the challenge of rat-to-human extrapolation was reduced to accurate prediction of site-specific flux of formaldehyde from inhaled air into the tissue lining the human respiratory tract. A computational fluid dynamics model of air flow and gas transport in the human nasal airways was linked to a typical path model of the human lung to provide site-specific flux predictions throughout the respiratory tract. Since breathing rate affects formaldehyde dosimetry, cytotoxicity dose-response curves were predicted for three standard working levels. With the most vigorous working level, the lowest concentrations of formaldehyde predicted to exert any cytotoxic effects in humans were 1.0 and 0.6 ppm, for the J-shaped and hockey-stick-shaped RCP curves, respectively. The predicted levels of response at the lowest effect concentrations are smaller than can be measured clinically. Published literature showing that the cytotoxicity of inhaled formaldehyde is related to exposure level rather than to duration of exposure suggests that the present analysis is a reasonable basis for derivation of standards for continuous human exposure.

Respiratory tract toxicity in rats exposed to Mexico City air.

The rat has been used extensively as a health sentinel, indicator, or monitor of environmental health hazards, but this model has not been directly validated against human exposures. Humans in Mexico City show upper respiratory tract lesions and evidence of pulmonary damage related to their environmental inhalation exposure. In this study, male and female F344 rats were exposed (23 hr/day) in Mexico City to local Mexico City air (MCA)* for up to seven weeks. Controls were maintained at the same location under filtered air. Prior to these exposures, several steps were taken. First, the nasal passages of normal male rats shipped from the United States and housed in Mexico City were examined for mycoplasma infection; no evidence of infection was found. In addition, a mobile exposure and monitoring system was assembled and, with an ozone (O3) exposure atmosphere, was tested along with supporting histopathology techniques and analysis of rat nasal and lung tissues. Last, the entire exposure model (equipment and animals) was transported to Mexico City and validated for a three-week period. During the seven-week study there were 18 one-hour intervals during which the average O3 concentration of MCA in the exposure chamber exceeded the US National Ambient Air Quality Standard (NAAQS) of 0.120 ppm 03 (hourly average, not to be exceeded more than once per year). This prolonged exposure of healthy F344 rats to MCA containing episodically low to moderate concentrations of 03 (as well as other urban air pollutants) did not induce inflammatory or epithelial lesions in the nasal airways or lung as measured by qualitative histologic techniques or quantitative morphometric techniques. These findings agree with those of previous controlled O3 inhalation studies, but they are in contrast to reports indicating that O3-polluted MCA causes significant nasal mucosal injury in adults and children living in southwestern Mexico City. Taken together, these findings may suggest that human airways are markedly more susceptible to the toxic effects of MCA than are the airways of the F344 rat.

alpha 2u-Globulin nephropathy, renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-344 rats.

tert-Butyl alcohol (TBA) has been shown to cause kidney tumors in male rats following chronic administration in drinking water. The objective of the present study was to determine whether TBA induces alpha 2u-globulin (alpha 2u) nephropathy (alpha 2u-N) and enhanced renal cell proliferation in male, but not female, F-344 rats, and whether the dosimetry of TBA to the kidney is gender specific. Male and female F-344 rats were exposed to 0, 250, 450, or 1750 ppm TBA vapors 6 h/day for 10 consecutive days to assess alpha 2u-nephropathy and renal cell proliferation and for 1 and 8 days to evaluate the dosimetry of TBA following a single and repeated exposure scenario. Protein droplet accumulation was observed in kidneys of male rats exposed to 1750 ppm TBA, with alpha 2u-globulin immunoreactivity present in these protein droplets. A statistically significant increase in alpha 2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to 1750 ppm TBA with a exposure-related increase in renal cell proliferation. Renal alpha 2u concentration was positively correlated with cell proliferation in male rat kidney. No histological lesions or increased renal cell proliferation was observed in female rats exposed to TBA compared to controls. The TBA kidney:blood ratio was higher at all concentrations and time points in male rats compared with female rats, which suggests that TBA is retained longer in male rat kidney compared with female rat kidney. Together these data suggest that TBA causes alpha 2u-N in male rats, which is responsible for the male rat-specific increase in renal cell proliferation.

Induction and time-dependent accumulation of micronuclei in peripheral blood of transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation.

In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.

Haber's rule: a special case in a family of curves relating concentration and duration of exposure to a fixed level of response for a given endpoint.

The concept that the product of the concentration (C) of a substance and the length of time (t) it is administered produces a fixed level of effect for a given endpoint has been ascribed to Fritz Haber, who was a German scientist in the early 1900s. He contended that the acute lethality of war gases could be assessed by the amount of the gas in a cubic meter of air (i.e. the concentration) multiplied by the time in min that the animal had to breathe the air before death ensued (i.e. C x t=k). While Haber recognized that C x t=k was applicable only under certain conditions, many toxicologists have used his rule to analyze experimental data whether or not their chemicals, biological endpoints, and exposure scenarios were suitable candidates for the rule. The fact that the relationship between C and t is linear on a log-log scale and could easily be solved by hand, led to early acceptance among toxicologists, particularly in the field of entomology. In 1940, a statistician named Bliss provided an elegant treatment on the relationships among exposure time, concentration, and the toxicity of insecticides. He proposed solutions for when the log-log plot of C and t was composed of two or more rectilinear segments, for when the log-log plot was curvilinear, and for when the slope of the dosage-mortality curve was a function of C. Despite the fact that Haber's rule can underestimate or overestimate effects (and consequently risks), it has been used in various settings by regulatory bodies. Examples are presented from the literature of data sets that follow Haber's rule as well as those that do not. Haber's rule is put into perspective by showing that it is simply a special case in a family of power law curves relating concentration and duration of exposure to a fixed level of response for a given endpoint. Also shown is how this power law family can be used to examine the three-dimensional surface relating C, t, and varying levels of response. The time has come to move beyond the limited view of C and t relationships inferred by Haber's rule to the use of the broader family of curves of which this rule is a special case.

Effect of different sampling designs on outcome of endocrine disruptor studies.

In this article, we demonstrate how sampling strategy can influence the outcome of endocrine disruptor studies. In a study of the weak xenoestrogen bisphenol A (BPA), possible treatment-related effects on ventral prostate (VP) fresh weight were found in rat offspring at 6 months of age when only one or two male pups were randomly selected from each litter. In subsequent BPA and di-n-butyl phthalate studies, large intralitter variability of this specific end point was apparent when the VP weights from entire litter complements were examined. We modeled the effects of sampling 1, 2, or 3 pups from each litter using the full-litter complement data. When one pup was randomly selected, a substantial percentage of incorrect conclusions about the presence or absence of treatment effects occurred. These statistical modeling analyses raise significant concern about the selection of one pup per litter for highly variable end points.

Promotion by sodium barbital induces early development but does not increase the multiplicity of hereditary renal tumors in Eker rats.

Induced cell proliferation is important in the mode of action of many non-genotoxic renal carcinogens. Since Tsc2 mutant (Eker) rats are genetically predisposed to the development of renal cell tumors, they provide a useful animal model in which to study the action of renal carcinogens. Sodium barbital was used as a model non-genotoxic renal carcinogen to test whether a concentration that increased renal tubular proliferation without severe nephrotoxicity would enhance tumor induction in a hereditary tumor model. First, a subchronic concentration-response study was conducted in wild-type male Long-Evans rats to determine increased cell proliferation without severe nephrotoxicity. Rats were dosed with sodium barbital in the feed at 0, 50, 250, 500, 1000, 2000 or 4000 p.p.m. for 3 or 8 weeks. Cell proliferation within the cortex and nephrotoxicity were quantitated. Enhanced proliferation with minimal nephrotoxicity occurred at 500 p.p.m. A second study was conducted in male Tsc2 mutant rats given sodium barbital in the feed at 0, 100 or 500 p.p.m. from 9 weeks of age to either 6 or 12 months of age. An additional group of rats was treated with sodium barbital for 6 months and then provided control feed until 12 months of age. Rats necropsied at 6 months of age had a concentration-dependent increase in preneoplastic and total renal lesions. Sodium barbital-treated rats necropsied at 12 months of age had numbers of lesions that were not different from controls. Total combined preneoplastic and neoplastic lesions in the 6 month, high dose group was the same as the 12 month control group. These data show that sodium barbital caused progression to the stage of spontaneous renal lesions in Tsc2 mutant rats but did not increase their overall number. These data suggest that enhanced cell proliferation without significant cytotoxicity exerted a promotional influence in this hereditary model.

Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Comparison of pulmonary and pleural responses of rats and hamsters to inhaled refractory ceramic fibers.

The present study was designed to determine whether pleural fiber burdens or subchronic pleural fibroproliferative and inflammatory changes can help explain the marked interspecies differences in pleural fibrosis and mesothelioma that are observed following long-term inhalation of RCF-1 ceramic fibers by rats and hamsters. Fischer 344 rats and Syrian golden hamsters were exposed to RCF-1 for 4 h per day, 5 days per week, for 12 consecutive weeks. Lung and pleural fiber burdens were characterized during and after exposure. For all time points, approximately 67% of fibers associated with lung tissues from both rats and hamsters were longer than 5 microns in length. In comparison, fibers longer than 5 microns recovered from the pleural compartment, following a 12-week exposure and 12 weeks of recovery, accounted for 13% (hamsters) and 4% (rats) of the distribution. In the 12 weeks after the cessation of exposure, the number of fibers longer than 5 microns in length remained constant in the hamster at approximately 150 fibers per cm2 pleura. This was 2 to 3 times the corresponding fiber surface density in the rat. Significant pulmonary and pleural inflammation was detected at all time points and for both species. DNA synthesis by pleural mesothelial cells was quantified by bromodeoxyuridine uptake following 3 days of labeling. Labeling indices were higher in hamsters than in rats, both for RCF-1-exposed and filtered air-control animals and was highest for the parietal surface of the pleura. Significantly greater collagen deposition was measured in the visceral pleura of hamsters 12 weeks post-exposure but was not significantly elevated in rats. These findings demonstrate that subchronic inhalation exposure to RCF-1 induces pleural inflammation, mesothelial-cell turnover, pleural fibrosis, and an accumulation of fibers with a length greater than 5 microns in the hamster. The accumulation of long fibers in the pleural space may contribute to the pathology observed in the hamster following chronic inhalation of RCF-1, whereas the presence of short, thin fibers may play a role in the acute-phase biological response seen in both species.

Comparison of pleural responses of rats and hamsters to subchronic inhalation of refractory ceramic fibers.

In the present subchronic study, we compared pleural inflammation, visceral pleural collagen deposition, and visceral and parietal pleural mesothelial cell proliferation in rats and hamsters identically exposed to a kaolin-based refractory ceramic fiber, (RCF)-1 by nose-only inhalation exposure, and correlated the results to translocation of fibers to the pleural cavity. Fischer 344 rats and Syrian golden hamsters were exposed to 650 fibers/cc of RCF-1, for 4 hr/day, 5 days/week for 12 weeks. Following 4 and 12 weeks of exposure, and after a 12-week recovery period, pleural lavage fluid was analyzed for cytologic and biochemical evidence of inflammation. Visceral and parietal pleural mesothelial cell proliferation was assessed by immunocytochemical detection of bromodeoxyuridine incorporation. Pleural collagen was quantitated using morphometric analysis of lung sections stained with Sirius Red. Fiber-exposed rats and hamsters had qualitatively similar pleural inflammation at each time point. Mesothelial cell proliferation was more pronounced in hamsters than in rats at each time point and at each site. In both species, the mesothelial cell labeling index was highest in the parietal pleural mesothelial cells lining the surface of the diaphragm at each time point. Hamsters but not rats had significantly elevated collagen in the visceral pleura at the 12-week postexposure time point. Fibers were found in the pleural cavities of both species at each time point. These fibers were generally short and thin. These results suggest that mesothelial cell proliferation and fibroproliferative changes in the pleura of rodents following short-term inhalation exposure are associated with fiber translocation to the pleura and may be predictive of chronic pleural disease outcomes following long-term exposure.

Evaluation of the metabolism and hepatotoxicity of styrene in F344 rats, B6C3F1 mice, and CD-1 mice following single and repeated inhalation exposures.

Styrene is used for the manufacture of plastics and polymers. The metabolism and hepatotoxicity (mice only) of styrene was compared in male B6C3F1 mice, CD-1 mice, and F344 rats to evaluate biochemical mechanisms of toxicity. Rats and mice were exposed to 250 ppm styrene for 6 h/day for 1 to 5 days, and liver (mice only) and blood were collected following each day of exposure. Mortality and increased serum alanine aminotransferase (ALT) activity were observed in mice but not in rats. Hepatotoxicity in B6C3F1 mice was characterized by severe centrilobular congestion after one exposure followed by acute centrilobular necrosis. Hepatotoxicity was delayed by 1 day in CD-1 mice, and the increase in ALT and degree of necrosis was less than observed for B6C3F1 mice. Following exposure to unlabeled styrene for 0, 2, or 4 days, rats and mice were exposed to [7-14C]-styrene (60 microCi/mmol) for 6 h. Urine, feces, and expired air were collected for up to 48 h. Most styrene-derived radioactivity was excreted in urine. The time-course of urinary excretion indicates that rats and CD-1 mice eliminated radioactivity at a faster rate than B6C3F1 mice following a single 250 ppm exposure, consistent with a greater extent of liver injury for B6C3F1 mice. The elimination rate following 3 or 5 days of exposure was similar for rats and both mouse strains. Following three exposures, the total radioactivity eliminated in excreta was elevated over that measured for one exposure for both mouse strains. An increased excretion of metabolites on multiple exposure is consistent with the absence of ongoing acute necrosis following 4 to 5 daily exposures. These data indicate that an induction in styrene metabolism occurs after multiple exposures, resulting in an increased uptake and/or clearance for styrene.

Effects of immobilization restraint on Syrian golden hamsters.

Rodent nose-only inhalation toxicology systems comprise whole-body immobilization in plastic restraint tubes. This method of restraint is known to have a variety of effects on animals. In the studies reported here, two independent toxicology laboratories examined the effects of inhalation tube restraint in Syrian golden hamsters, a species that has recently gained importance in inhalation studies of fibrous particulates. Body weight, food and water consumption, core body temperature, and plasma cortisol and corticosterone concentrations were assessed in animals immobilized in nose-only inhalation tubes, and the results were compared with those from unrestrained cage-control animals. Animals were immobilized for either 6 h/ day, 5 days/week for 13 weeks (subchronic), or 4 h/day for 14 consecutive days (subacute), mimicking exposure conditions commonly used in nose-only inhalation studies. Tube restraint was found to induce a marked decrease in body weight, which increased in response to cessation of restraint. The body weight decrement was associated with significant differences in food and water consumption between the restrained and control groups in the subacute study and only food consumption in the subchronic study. During the restraint period, core body temperature in the immobilized animals increased slightly but not above the normal range for this species. Plasma cortisol and corticosterone concentrations were not significantly increased with use of restraint, compared with values in controls. Immobilization-associated body weight depression in Syrian golden hamsters is important for the evaluation of nose-only inhalation study results because many normal physiologic parameters, as well as toxicant-induced effects, are associated with body weight status.

Neurotoxicological evaluation of ethyl tertiary-butyl ether following subchronic (90-day) inhalation in the Fischer 344 rat.

The purpose of this study was to evaluate whether repeated 6-h exposure (65 exposures over a 14- week period) of male and female Fischer-344 rats (n = 12 rats/sex/concentration) to ethyl tertiary-butyl ether (ETBE) atmospheres at 500, 1750, or 5000 ppm would result in neurotoxicity. Neurotoxicity was assessed by a blinded functional observational battery (FOB), motor activity, and terminal neuropathology. Motor activity was assessed 4 days prior to ETBE exposure and following 20, 42, and 65 days of exposure. The FOB was assessed 4 days prior to ETBE exposure and following 1, 6, 10, 20, 42, and 65 days of exposure. Transient ataxia, a sign of narcosis, was noted in male rats immediately following the 6-h exposure to 5000 ppm ETBE. Statistically significant treatment effects on motor activity were not observed. Minor changes in grip strength and hindlimb splay were observed; however, none demonstrated a dose-response relationship or a consistent pattern of neurological dysfunction. No gross or microscopic abnormalities were observed in the central, peripheral, or autonomic nervous systems of rats exposed to 5000 ppm ETBE. No statistically significant differences in brain weight or size were observed in ETBE-exposed rats. A statistically significant increase in body weight was observed in female rats exposed to 5000 ppm following 42 and 65 exposure days. Although ataxia was a common feature of acute ETBE neurotoxicity in rats following high-level exposure, adverse neurological effects are not expected in the general public at the anticipated exposure levels associated with automotive refueling.

Pharmacokinetics of methanol and formate in female cynomolgus monkeys exposed to methanol vapors.

The 1990 Clean Air Act Amendments contain mandates for reduced automotive emissions and add new requirements for the use of alternative fuels such as methanol to reduce certain automotive pollutants. Methanol is acutely toxic in humans at relatively low doses, and the potential for exposure to methanol will be increased if it is used in automotive fuel. Formate is the metabolite responsible for neurotoxic effects of acute methanol exposure. Since formate metabolism is dependent on folate, potentially sensitive folate-deficient subpopulations, such as pregnant women, may accumulate formate and be at higher risk from low-level methanol exposure. Our objective was to determine the pharmacokinetics of 14C-methanol and 14C-formate in normal and folate-deficient monkeys after exposure to 14C-methanol vapors at environmentally relevant concentrations: below the threshold limit value (TLV), at the TLV of 200 parts per million (ppm), and above the TLV. Four normal adult female cynomolgus monkeys were individually anesthetized with isoflurane, and each was exposed by endotracheal intubation to 10, 45, 200, or 900 ppm 14C-methanol for 2 hours. Concentrations of the inhaled and exhaled 14C-methanol, blood concentrations of 14C-methanol and 14C-formate, exhaled 14C-carbon dioxide (14CO2), and respiratory parameters were measured during exposure. After exposure, 14C-methanol and 14CO2 exhaled, 14C-methanol and 14C-formate excreted in urine, and 14C-methanol and 14C-formate in blood were quantified. The amounts of exhaled 14C-methanol and 14CO2, blood concentrations of 14C-methanol and 14C-formate, and 14C-methanol and 14C-formate excreted in urine were linearly related to methanol exposure concentration. For all exposures, blood concentrations of 14C-methanol-derived formate were 10 to 1000 times lower than endogenous blood formate concentrations (100 to 200 mM) reported for monkeys and were several orders of magnitude lower than levels of formate known to be toxic. Since the metabolism of formate in primates depends on the availability of tetrahydrofolate, the same four monkeys were next placed on a folate-deficient diet until folate concentrations in red blood cells consistent with moderate folate deficiency (29 to 107 ng/mL) were achieved. Monkeys were then reexposed to the highest exposure concentration, 900 ppm 14C-methanol, for a similar 2-hour period, and again the pharmacokinetic data described above were obtained. Even with a reduced folate status, monkeys exposed to 900 ppm methanol for 2 hours had peak concentrations of methanol-derived formate that were well below the endogenous levels of formate. Although these results represent only a single exposure and therefore preclude broad generalizations, they do suggest the body contains sufficient folate stores to effectively detoxify small doses of methanol-derived formate from exogenous sources, such as those that might occur during normal use of automotive fuel.

Metabolic interactions of 1,3-butadiene and styrene in male B6C3F1 mice.

Butadiene and styrene are a mixture of hazardous air pollutants found in the workplace of industries producing polymers such as styrene-butadiene rubber. Both butadiene and styrene require metabolic activation to exert their genotoxic effect; therefore metabolic interactions may influence their genotoxicity. Our objective was to quantitate potential metabolic interactions in mice exposed to a mixture of butadiene and styrene. The rate of metabolism of butadiene and styrene was estimated from the steady-state rate of uptake of the chemicals by male B6C3F1 mice exposed for 8 hr in a dynamic, whole-body inhalation system to 100 or 1000 ppm butadiene in combination with 0, 50, 100, or 250 ppm styrene. Styrene, styrene oxide, 1,2-epoxy-3-butene, and 1,2:3,4-diepoxybutane concentrations in blood were measured by gas chromatography-mass spectrometry at 2, 4, 6, and 8 hr of exposure. As the styrene concentration in the mixture increased, the rate of butadiene metabolism was inhibited up to 48%. 1,2-Epoxy-3-butene blood concentrations were increased by approximately 1.5-fold; however, 1,2:3,4-diepoxybutane blood concentrations were unaffected. Styrene uptake in the inhalation system was inhibited slightly by exposure with butadiene, but styrene blood concentrations increased significantly as the butadiene concentration in the mixture increased to 1000 ppm. Blood concentrations of styrene oxide increased approximately 1.6-fold for the 250-ppm styrene exposures when the butadiene concentration was increased from 0 to 1000 ppm. The data suggest that metabolic interactions occurred among the reactive metabolites (e.g., competition for detoxication pathways) as well as between butadiene and styrene in mice exposed to mixtures of butadiene and styrene. However, metabolic interactions were significant only at concentrations of butadiene and styrene higher than those typically observed in the workplace of industries producing polymers of butadiene and styrene.

Benzene-induced micronuclei in erythrocytes: an inhalation concentration-response study in B6C3F1 mice.

High concentrations (300-1000 p.p.m.) of benzene have been shown to induce an increase in the frequency of micronucleated erythrocytes in mice. This study investigated the mutagenicity of benzene at lower concentrations, including the current limit for occupational exposure, 1 p.p.m. The frequencies of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood of male B6C3F1 mice were measured following inhalation of benzene at 0, 1, 10, 100 or 200 p.p.m. during an 8 week exposure period. Only 100 and 200 p.p.m. benzene induced a statistically significant increased frequency of micronucleated erythrocytes in the bone marrow and blood. The frequency of MPCE plateaued at week 2 with 43/1000 (100 p.p.m.) and 86/1000 (200 p.p.m.) in the bone marrow as compared with 10/1000 for controls. The frequency of MNCE in the blood progressively increased to 13.4/1000 (100 p.p.m.) and 32.5/1000 (200 p.p.m.) at week 8 as compared with 1.8/1000 for controls. Cytotoxicity of replicating and maturing erythrocytes by 100 and 200 p.p.m. benzene delayed the accumulation of MNCE in the blood. There was not a statistically significant increase in the frequency of micronucleated erythrocytes, as an indicator of mutagenicity, with inhalation of 1 or 10 p.p.m. benzene over an 8 week period. A quadratic curve fit the bone marrow MPCE data of mice exposed to up to 200 p.p.m. benzene with a high correlation (R2 = 0.94) and could not be rejected based on lack of fit.

Mortality of iron foundry workers: IV. Analysis of a subcohort exposed to formaldehyde.

In the final phase of the mortality study of workers at an automotive iron foundry, a subset (N = 3929) of the original cohort of 8147 men, consisting of those exposed to formaldehyde during the period from January 1960 through May 1987, was analyzed. In addition to the external US population, an internal population (N = 2032), consisting of men who had worked in the same foundry during the same time period but not in formaldehyde-exposed jobs, was also used as a referent. Follow-up continued through December 31, 1989. Smoking status was ascertained for 65.4% of the exposed and for 55.1% of the unexposed cohorts. Detailed work histories and evaluation of occupational exposures by an industrial hygienist enabled us to categorize cumulative formaldehyde and silica exposures. Standardized mortality ratios were used to compare the mortality experience of the exposed cohort with the US population and, because of concerns about the healthy worker effect, with an occupational referent population. Relative risks for race, formaldehyde exposure status, smoking status, and silica exposure level were estimated by fitting a Poisson regression model to four causes of death: cancers of the buccal cavity and pharynx, lung cancer, diseases of the respiratory system, and emphysema. No association between formaldehyde exposure and deaths from malignant or nonmalignant diseases of the respiratory system was found. Cigarette smoking and silica exposure were found to be significantly associated with deaths attributed to lung cancer and disease of the respiratory system.

An in vitro method for determination of tissue partition coefficients of non-volatile chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and estradiol.

The development of an in vitro vial equilibration technique for determining tissue and liquid partition coefficients for non-volatile chemicals is described. Radiolabeled chemical dissolved in propylene carbonate is equilibrated with tissues or liquid at 37 degrees C in a vial system. The solvent must be essentially immiscible with the test material. The amount of chemical movement to the tissue or liquid is compared to an appropriate reference vial, and tissue or liquid:solvent partition coefficients are calculated. Tissue:solvent values divided by blood:solvent values provide tissue:blood partition coefficients required for developing physiologically based pharmacokinetic models for chemicals. These models are useful for estimating internal tissue doses to assess human risk from exposure to chemicals. Partition coefficients for various rat tissues, 0.9% saline solution and olive oil were determined in this study for radiolabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and for the less fat-soluble compound, estradiol. The TCDD tissue:propylene carbonate partition coefficients were found to be: blood, 0.091; fat, 17.02; liver, 0.419; brain, 0.632; kidney, 0.305; muscle, 0.408. For estradiol, the tissue:propylene carbonate partition coefficients were: blood, 0.286; fat, 0.169; liver, 1.032; brain, 0.554. The TCDD results compared well with values reported and estimated from a more protracted in vivo approach. Thus, this current technique offers a simpler and time-saving alternative to in vivo approaches for determining the partition coefficients of non-volatile compounds.

Mortality of iron foundry workers. III. Lung cancer case-control study.

A nested case-control study was undertaken to identify the determinants of lung cancer mortality in a cohort of 8147 foundry men among whom an excess of lung cancer deaths was previously observed. The present study consisted of all lung cancer deaths (N = 220) that occurred within this cohort between 1950 and 1989. both living and dead controls, matched on race and attained age, were selected in the ratio of 10:1 (N = 2200) by means of the incidence density sampling procedure. All cases and two controls per case, randomly selected from each case's 10 controls, were included in a smoking history survey. Basic smoking history information was obtained for about 71% of these study subjects. For the purpose of this study, formaldehyde exposure levels were categorized as high, medium, low, and none. Airborne silica exposure was categorized only as high, medium, and low levels, because all foundry workers were known to be exposed to silica. Conditional logistic regression analyses indicated that cigarette smoking was a strong predictor of lung cancer mortality in this cohort. Neither exposure to formaldehyde nor silica exposure level, nor employment in any of the six major work areas within the foundry, showed an association with lung cancer.