Spacecraft Maximum Allowable Concentrations for Selected Airborne Contaminants: Volume 3 (1996)

John T. James, Ph.D.

Johnson Space Center Toxicology Group

Biomedical Operations and Research Branch

National Aeronautics and Space Administration

Houston, Texas

Ethanol is a clear, colorless, flammable liquid with an odor threshold of approximately 80 ppm (0.15 mg/L) (Amoore and Hautala, 1983) and a ''minimum identifiable odor level'' of approximately 350 ppm (0.66 mg/L) (Scherberger et al., 1958).

Ethanol is used as a fuel additive and in the manufacture of chemicals and medicines. It is used as a solvent in the manufacture of explo-

sives, plastics, resins, cosmetics, adhesives and preservatives. Ethanol is present in alcoholic beverages, which are widely consumed, and most toxicological concerns stem from the consequences of that consumption.

Ethanol has been found in almost every sample of shuttle air, but at concentrations that seldom exceed 0.01 mg/L (James et al., 1994). Concentrations in the Mir space station are often above 0.01 mg/L; however, concentrations as high as 0.107 mg/L have been reported (James and Coleman, 1994). Ethanol enters the spacecraft atmosphere by off-gassing from hardware and from its use as a cleaner and disinfectant in operational procedures. Dilutions of liquid ethanol are frequently used in payload experiments; occasionally, it is used in undiluted form, and as such, it could pose a significant eye hazard upon escape.

The absorption of ethanol has been most thoroughly studied by the oral ingestion route; however, absorption in the respiratory tract has been investigated in a few studies. It is important to compare the relative ability of the two routes of absorption to deliver ethanol to the bloodstream because that is where most of the distribution, metabolism, and excretion of ethanol occurs. Furthermore, specific toxic effects are often reported in terms of their probability of occurrence at known ethanol blood concentrations. Hence, it is important to know the efficiency of the inhalation route in delivering ethanol to the blood.

Orally ingested ethanol is absorbed rapidly from the gastrointestinal tract by simple diffusion; peak blood concentrations are reached in 0.5 to 1.5 h after ingestion has ended. Food in the stomach delays gastric emptying and consequently delays the delivery of ethanol to the small intestine, where absorption is more rapid than it is in the stomach (Rall, 1990). In experimental animals, absorption of an oral dose of 6.4 g/kg has been shown to be much slower in rats than in guinea pigs (Strubelt et al., 1974). That absorption rate results in the maximum blood concentrations in rats being about one third the maximum in guinea pigs; this species difference is less pronounced at lower doses.

In a human inhalation study, Lester and Greenberg (1951) showed

that the respiratory system absorbed about 62% of the ethanol from inspired air at concentrations ranging from 11 to 19 mg/L. This percentage appeared to be independent of ventilation rates, which ranged from 7 to 25 L/min. In the three test subjects, the blood alcohol concentrations averaged 4 mg/dL (0.004%) when inhaling ethanol at 15 to 16 mg/L for 3 h at a ventilation rate of 7 to 8 L/min. In two of the subjects tested at a ventilation rate of 15 L/min, the blood concentrations of ethanol after 3 h were approximately 9 mg/dL (0.009%). The concentration profiles suggested that the blood concentrations had reached equilibrium values during the 3 h of exposure. At a ventilation rate of 22 L/min, the blood concentrations, which averaged about 35 mg/dL (0.035%), did not appear to be at equilibrium after 3 h. The authors concluded that a ventilation rate above 14 L/min would be necessary to achieve a continuously increasing blood concentration of ethanol during an exposure at 15 mg/L for several hours.

In an experiment designed to test the hypothesis that an individual inhaling ethanol vapors from an open liquid source could have significant blood concentrations of alcohol, Mason and Blackmore (1972) showed that, in a warm room with a maximum concentration of 17 mg/L (estimated from the amount of ethanol evaporated from the source), the ethanol concentration in the blood of the four subjects was below 5 mg/dL (0.005%) when tested at various intervals during the exposure. Although the subjects experienced no subjective symptoms of intoxication, those entering the room for the first time found the atmosphere intolerable at the end of the exposure. That information is consistent with the findings of Lester and Greenberg (1951) described above and underscores the difficulty in reaching significant blood concentrations in practical situations where inhalation is the only route of entry. It has been shown in vitro that rat lung tissue can metabolize ethanol to carbon dioxide at a capacity roughly one fifth of that of liver slices (Masoro et al., 1953).

Some inhalation uptake data are available in rodents from experiments that were designed to develop models for evaluation of alcohol dependence and withdrawal. These inhalation models were developed to solve problems associated with variable blood alcohol concentrations after repeated oral or intravenous administrations. After the initial rise in blood ethanol concentration, continued inhalation of ethanol at a fixed concentration will result in gradually decreasing blood concentrations unless alcohol dehydrogenase (ADH) activity is inhibited (e.g., by

pyrazole) or the vapor concentration is increased during the exposure. Mice exposed to ethanol at 12 mg/L for 96 h, with daily injections of pyrazole, showed stable blood concentrations after about 20 h of exposure. The equilibrium blood concentration was approximately 170 mg/dL (0.17%) (Goldstein and Pal, 1971). A survey of rodent exposures to ethanol for up to 21 d suggested that the blood alcohol concentrations varied considerably with species, strain, and sex of the rodent (Goldstein, 1980). When exposures were conducted without an ADH inhibitor, the blood alcohol could be kept reasonably constant by doubling the exposure concentration progressively during the first 10 d of exposure. In rats exposed for 24 h at 28 mg/L, the blood concentration reached 340 mg/dL (0.34%). This exposure conferred tolerance to a hypothermia-inducing injection of ethanol administered 48 h after the exposure (Mullin and Ferko, 1981). Blood alcohol concentrations were stabilized at approximately 150 mg/dL (0.15%) by continuous exposures of rats that were individually exposed to air concentrations of 22 to 28 mg/L on the basis of their blood alcohol concentrations (Rogers et al., 1979).

The arterial concentration of ethanol is significantly higher than the venous concentration during the absorption phase (Harger and Forney, 1967; Keiding, 1979). Ethanol diffuses slowly across cellular membranes but passes rapidly into the brain (Crone, 1965). The tissue-to-blood-concentration ratios in cat tissue were found to be about 0.85 in all tissues studied except in fat, where the ratio was only 0.2 (Eggleton, 1940, as cited in Rowe and McCollister, 1982). The placental transfer of ethanol is rapid in the many species studied, and the concentrations follow those in maternal blood (IARC, 1988).

Once it reaches the blood, ethanol is removed at a constant rate in a given individual over a wide concentration range (zero-order kinetics); however, there is considerable evidence that the removal rate is lower at very low concentrations of ethanol. Above a peripheral blood con-

centration of about 2 mm (0.009%), the human liver eliminates ethanol at a constant rate; however, below that concentration, the rate of elimination decreases with concentration following Michaelis-Menton kinetics (Keiding, 1979; Bosron et al., 1988). Chronic alcoholics are able to eliminate ethanol from their blood almost twice as fast as the average person (Harger and Forney, 1967).

Ethanol is mainly eliminated from the body by oxidation in the liver to acetate, which is distributed to other tissues and metabolized to carbon dioxide for excretion by the lungs (see Metabolism below). A small fraction of ethanol is excreted without change by the kidneys (0.5 to 2%), lungs (5%), and sweat (0.5%); elimination rates range from 70 to 180 mg/kg·h or 170 to 410 mg/kg·h, depending on the method of calculation (Haggard and Greenberg, 1934; von Wartburg, 1989). A small amount has been reported to be conjugated and excreted in the kidney as ethyl glucuronide (Kamil et al., 1953).

Ethanol is certainly one of the most often studied toxicants and probably one of the earliest to receive serious toxicological evaluation and metabolic study. By 1900, it was known that nearly all of an ingested dose was metabolized by the test animal; this knowledge enabled use of ethanol as a source of energy (Jacobsen, 1952). The accumulation of fat in tissues of animals fed alcohol over long periods of time had also been demonstrated, even when the intake of food in the ethanol-fed animals and control animals was the same. Several investigators showed that the overall metabolic rate of an organism was not altered by ingestion of ethanol (Jacobsen, 1952).

Investigations during the next half century resulted in much more detailed understanding of ethanol metabolism in experimental animals and humans. The state of knowledge at that time was reviewed by Jacobsen (1952), and most of the information below is from his review.

Although some controversy existed about the shape of the blood-concentration curve after ethanol administration, the majority of available data indicated a linear decrease in concentration with time. A relatively high preconditioning dose of alcohol causes a second smaller dose to be removed faster than a dose given without preconditioning. With blood alcohol levels in the range of 15 to 94 mg/dL (0.015-0.094%), constant infusion of ethanol was shown to maintain constant blood levels in humans. The capacity to metabolize alcohol appeared to vary less than 25% among individuals. The prevailing opinion at that time was that long-time use of alcohol does not increase the rate of metabolism and that exercise, except for increased evaporation, does not increase the removal rate of alcohol. Starved animals have a lower rate of alcohol metabolism than well-fed animals.

Long before 1950, investigators conducted studies using hepatectomized dogs, liver slices, and perfused livers to show that the liver was the predominant organ to metabolize ethanol. Some metabolism of alcohol appeared to occur in muscles also (Bartlett and Barnet, 1949). Data were not consistent on whether the kidney could metabolize ethanol; however, it was apparent that little, if any, metabolism occurred in brain tissue.

The biochemical pathway for ethanol metabolism was reasonably well demonstrated to be as follows:

Acetaldehyde was found in the blood of animals and humans given sufficient doses of ethanol. Typically, the acetaldehyde concentration was less than 1% of the concentration of ethanol. Premedication with Anabuse was found to increase the blood concentrations of acetaldehyde up to 10 times over those in unmedicated animals after ethanol ingestion. Following up on evidence of the formation of acetate from ethanol, Bartlett and Barnet (1949) used ¹⁴C-labeled ethanol (1 g/kg) administered to rats to show that the acetate formed is rapidly oxidized to carbon dioxide, because 75% of the label was recovered in 5 h and 90% was recovered in 10 h.

The enzymes involved in the first step of the pathway were known to be ADH and, to a much reduced extent, catalase. ADH catalyzes the transfer of hydrogen to nicotinamide adenine dinucleotide (NAD+).

Catalase facilitates the transfer of hydrogen from ethanol by reduction of peroxide to water. Jacobsen (1952) speculated that the second step in the pathway was catalyzed by either a flavoprotein or another dehydrogenase, but no evidence indicated which type of aldehyde-oxidizing enzyme might actually be involved.

In general, the view of ethanol metabolism has not fundamentally changed from the pathway illustrated above. There have been significant new insights into ethanol's metabolism that have facilitated an understanding of the pathogenesis of ethanol-induced liver injury and carcinogenesis. Data now available on the location and enzymatic function of subcellular organelles in hepatocytes and how they respond to repeated insults from ethanol present a complex picture; the major features of that picture are described below. In addition, some of the early concepts about ethanol metabolism, especially the degree of variability among individuals, have been corrected by new data. Genetic variations in the enzymes involved in ethanol metabolism control the immediate response to ingested ethanol; however, no studies could be found in which differences in susceptibility to inhaled ethanol were evaluated.

The cascade of effects induced by ethanol can be partially understood by refinements in the pathway shown above. Many of the effects occur as a result of the changed redox state (NADH/NAD ratio). The excess of hydrogen equivalents from ethanol metabolism are used by mitochondria instead of those equivalents originating from the citric-acid cycle by metabolism of 2-carbon fragments from fatty acids. This process facilitates decreased fatty-acid metabolism and causes deposition of dietary fat in the liver. Chronic administration of ethanol to rats or baboons results in attenuation of this change in redox state, so continued accumulation of fat must depend on another mechanism, possibly one involving changes in the mitochondria (Lieber, 1979).

The first reaction in the metabolic pathway is primarily catalyzed by ADH in the cytosol. This step is affected by the presence of acetaldehyde, the transport of reducing equivalents, and the ability of the mitochondria to oxidize those reducing equivalents. ADH activity is affected by hormonal status, dietary status, and age of the subject (IARC, 1988). ADH is a dimer with multiple molecular forms determined by

at least five gene loci (ADH1 to ADH5). An atypical form (ADH2*2) has been identified at a high frequency (33 % to 81%) in Asian populations compared with a much lower frequency (1% to 12%) in Caucasian populations (Agarwal and Goedde, 1992). The atypical subunit of ADH2 has a 20-fold higher capacity than the ADH2*1 subunit to oxidize ethanol. That difference might explain the two-to threefold variability in alcohol elimination observed in humans given identical ethanol doses (Bosron et al., 1988).

Two other enzymes have been shown to be capable of catalyzing the first step in the pathway. Cytochrome P-450 (the microsomal ethanol-oxidizing system) depends on nicotinamide adenine dinucleotide phosphate (NADPH), oxygen, and hydrogen to oxidize ethanol to acetaldehyde. One isoenzyme of cytochrome P-450, IIE1, is induced by ethanol, and that induction might explain ethanol's ability to act as a cocarcinogen (see below). In addition, it has been argued that this enzyme is important in ethanol oxidation at high concentrations and may become more important with chronic ingestion (Lieber, 1984). Catalase, a hemoprotein located in the peroxisomes of many tissues, can also catalyze the first reaction, but it depends on a hydrogen peroxide generating system to control the rate (IARC, 1988). When ethanol is present at low concentrations, neither of these enzymes appears to be nearly as important as ADH in ethanol oxidation. In vitro studies of ethanol metabolism in rat and monkey liver slices have shown that more than 89% of the metabolism is due to ADH alone (Havre et al., 1977).

The second step in the pathway is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme present primarily in mitochondria with broad specificity for aldehydes. The mitochondrial form (ALDH2) is thought to be most important in oxidizing acetaldehyde, and because approximately 50% of the Japanese and Chinese lack an active form of this enzyme, they experience much higher blood acetaldehyde concentrations after ethanol ingestion (Bosron and Li, 1986). It was suggested that Asian individuals who exhibit the "flush" reaction to ethanol have the highly active ADH2 and the deficient ALDH2 enzymes (Yoshida et al., 1983). In fact, Shibuya et al. (1989) showed that of nine Japanese who flushed, all were at least heterozygous for the atypical allele (ALDH1*2 or ALDH2*2), and of six who did not flush, five were homozygous for the usual allele (ALDH1*1). Thomasson et al. (1993) showed that the atypical ALDH2*2 in a large population of Chinese men was associated with the most intense flushing (as measured by in-

creased facial blood flow). In individuals with the usual allele, the metabolic rate is very fast, so little acetaldehyde accumulates to reach the blood; however, in those who flush, the concentration of acetaldehyde may reach 100 µm (Bosron et al., 1988). Several isoenzymes of ALDH have been identified, and ALDH is present in tissues other than the liver (IARC, 1988). Acetaldehyde produced in the mitochondria during chronic ethanol intake appears to cause the biochemical changes and ultrastructural damage apparent in test animals and alcoholics (Lieber, 1979).

According to several reviews, the third step occurs mostly outside the liver when acetate is released into the circulation and delivered to peripheral tissues (Lieber, 1979; Weiner et al., 1988; Bosron et al., 1988). No specific data are cited by any of the reviewers to support this conclusion. Early studies using liver slices from rats and ¹⁴C-labeled ethanol suggested that the liver alone converted a sizable portion of ethanol to carbon dioxide (Bartlett and Barnet, 1949). A later study in 10 human subjects infused to an average plasma concentration of ethanol at 3.6 mm (0.017%) showed that the output of acetate from the splanchnic area was 50% to 100% of the ethanol disappearance rate (Lundquist et al., 1962); that result was confirmed in another human study (Tygstrup et al., 1965).

The number of toxicity studies involving ethanol is enormous; however, the goal of this document is to provide a rationale for setting inhalation exposure limits. With this goal in mind, my focus will be on the few inhalation studies conducted with ethanol and on studies by other routes of administration if those studies provide insight into how inhaled ethanol could adversely affect living systems. It is beyond the scope of this document to provide a comprehensive review of ethanol's toxicity by all routes. For example, adverse effects of ethanol on skeletal, cardiac, and vascular smooth muscle (Altura and Altura, 1982; Urbano-Marquez et al., 1989) and on the kidney (Ponticelli and Montagnino, 1979) will not be considered in detail because such effects are seen only in chronic alcoholics, and there is no indication that inhalation exposure would injure these tissues. Also, reports of responses to ethanol vapor at 0.5 ppm (0.001 mg/L) by 3 of 47 chemically sensi-

tive patients are not considered evidence of toxicity in this report. The responses were not applicable to nonsensitive populations, and the effects are hardly adverse (e.g., increased pulse rate) (Rea et al., 1991).

Short-term exposures to ethanol are known to induce irritation of mucosal surfaces, central-nervous-system (CNS) depression, and possibly a flush response in certain individuals. The first two effects have been demonstrated by inhalation exposure; however, the flush reaction has only been demonstrated by oral ingestion. The first two effects are thought to be mediated by ethanol, whereas the flush reaction is almost certainly due to acetaldehyde, which accumulates to unusually high concentrations in susceptible individuals.

The mechanism of toxic action of ethanol on the nervous system is believed to depend on its ability to fluidize the bulk lipid in membranes. A correlation between the severity of intoxication and the magnitude of ethanol-induced fluidization of brain membranes has been demonstrated in vitro, and long-sleep strains of mice show greater ethanol-induced fluidization of membranes than short-sleep strains (Wood and Schroeder, 1988). In a refinement of the bulk lipid effect, some of the membrane effects may be explained by differential effects in domains of the membrane defined by their degree of hydrophobicity, orientation (lateral or vertical) or composition (e.g., protein and phospholipid). Changes in membrane fluidity and lipid composition might be related to ethanol sensitivity and tolerance, but that relation has not been convincingly demonstrated (Wood and Schroeder, 1988).

Ethanol causes CNS depression in animals and humans exposed to air concentrations on the order of 10 mg/L and above. Table 55.4 in Rowe and McCollister (1982) summarizes mouse, guinea pig, and rat exposures to ethanol at 6 to 94 mg/L for 1 to 24 h that induce drowsiness, ataxia, narcosis, and death. Most of the data are from Loewy and von der Heide (1918) and suggest that guinea pigs are somewhat less

susceptible than rats. In rats, the no-observed-adverse-effect level (NOAEL) for a 6-h exposure was 6.1 mg/L, whereas in guinea pigs, the NOAEL was 12 mg/L for an exposure of 8 h. The table shows that in mice, guinea pigs, and rats exposed for 7 to 10 h, the concentration × time needed to induce death is about 400 h·mg/L. Quantifying the effect of inhaled ethanol on the human nervous system depends on correlating inhalation exposures with blood alcohol levels (BALs) and then correlating those BALs with specific nervous system effects. This correlation is necessary because the assessment of nervous-system effects in the human inhalation studies is inadequate as compared with the extensive work that has been reported on the effects after oral ingestion. For example, Loewy and von der Heide (1918) reported headache after 33 min of exposure at 2.6 mg/L and a slight daze after leaving the chamber, whereas Lester and Greenberg (1951) reported no CNS effects during 3 to 6 h exposures at 15 mg/L, even in subjects breathing at 24 L/min. The authors of the latter paper, in which subjects were exposed with a head-only hood, attribute the differences in their findings and those of Loewy and von der Heide (1918) to fatigue caused by confinement in the exposure chamber. As discussed in the section Rationale for Acceptable Concentrations, the data from Loewy and von der Heide (1918) are not that different from Lester and Greenberg's (1951) data. The older study was conducted in a sealed static chamber of 8000-L capacity, and the apparent concentrations of ethanol decreased far more rapidly than the authors expected. They attributed the rapid decrease to wall condensation (Loewy and von der Heide, 1918). Both studies suffer from having few test subjects, subjective end points, and no control exposures. Fortunately, Lester and Greenberg (1951) measured BALs and found that, at a light load (15 L/min), the BAL reached 9 mg/dL (0.009%). They reported no CNS effects at that concentration, but their methods to detect such effects were subjective.

It appears that minimal to no effects are detectable in humans with BALs of 50 mg/dL (0.05%), which is compatible with the report of Lester and Greenberg (1951). Flury and Klimmer (1943) indicated that the ''beginning of uncertainty'' is 0.06%. Small performance decrements (< 10%) were demonstrated in grammatical reasoning, code substitution, choice reaction, and tapping in 18 subjects with BALs of 0.06% after ingestion of 95% ethanol mixed with juice (Kennedy et al., 1993a). In another study with the same group, 20 males with BALs of

0.05% were subjected to a microcomputer performance test and found to be indistinguishable from their controls (Kennedy et al., 1993b). A BAL of 0.075% was found to be an effect level in that study.

According to Lester and Greenberg (1951), humans experienced smarting of the eyes and nose and coughing when exposed to ethanol at 10 to 20 mg/L. Those symptoms disappeared in 5 to 10 min. Again those data do not fully agree with the older data from Loewy and von der Heide (1918), in which nasal itching was reported in an unaccustomed subject exposed at 6 mg/L after 10 min of a 50-min exposure. They did report that 17 mg/L was at first intolerable to an unaccustomed person, but became tolerable except for continuing eye irritation. Because the 1951 study appears to be more complete than the 1918 study, 10 mg/L is considered the threshold for ethanol-induced irritation. Tolerance develops rapidly, so it is effectively a NOAEL for mucosal irritation.

Symptoms experienced primarily by Asian individuals soon after ingestion of ethanol are called the "alcohol sensitivity syndrome." When these persons consume ethanol at 0.3 to 0.5 mL/kg of body weight, they experience facial flushing, elevation of skin temperature, and an increase in pulse rate (Shibuya et al., 1989). Vasodilation in the neck and chest areas, headache, nausea, hypotension, and extreme drowsiness are also commonly reported (Thomasson, et al., 1993). No data show explicitly that this response has been elicited by inhalation exposure to ethanol; however, the description of the symptoms in Loewy and von der Heide (1918) suggests that they might have observed some of the symptoms of flushing. In test subjects exposed to ethanol vapor, they reported an increased feeling of heat in the forehead and ears, warmth in the head and trunk, and fatigue. It is unlikely that any of the three subjects had flush responses, because only about 10% of Caucasians have them; however, because acetaldehyde mediates the response, it is plausible that during the high-concentration exposures, a

sufficient quantity of acetaldehyde was produced to elicit the response even in those without the flush response.

Even though long-term oral ingestion of ethanol is hepatotoxic in humans and laboratory animals, there have been no contemporary studies showing lasting hepatotoxicity after repeated or continuous inhalation exposures. There are scattered old reports of studies of workers that suggest that cirrhosis of the liver develops after prolonged exposure to ethanol vapor (see Browning, 1953, 1965), but the accumulated experience in workers exposed to ethanol suggests that liver injury due to ethanol inhalation is rare. In animals, some investigators reported fatty infiltration of the liver in mice repeatedly exposed to high concentrations of ethanol (Weese, 1928, as cited in Rowe and McCollister, 1982), and cirrhosis of the liver was observed by Mertens (1896), as cited by Rowe and McCollister (1982), in rabbits exposed for 25 to 365 d to air saturated with ethanol. In rats exposed to ethanol at 1.4 mg/L for up to 14 d continuously, French and Morris (1972) reported that liver biopsies examined by light and electron microscopy showed no liver damage, although minimal fat accumulation was detected. Finally, it should be noted that liver injury is found in many of the inhalation models used to produce withdrawal effects in rodents. Such models use doses of pyrazole to maintain constant high blood concentrations of ethanol; however, the confounding effects of pyrazole preclude the conclusion that inhaled ethanol alone could induce hepatotoxicity (Goldstein, 1980). In an inhalation study of tolerance in rats exposed at 20 mg/L (initial 8-h 10-mg/L adjustment period) for 26 d, plasma triglyceride concentrations remained normal even though liver triglyceride concentrations increased on d 3, 6, and 9 (Di Luzio and Stege, 1979). In addition, GPT (glutamic pyruvic transaminase or alanine transaminase) activity was above control activity on the same days but returned to the control range by d 26. The authors characterized these changes as mild transient effects observed before adaptation; they found that BAL peaked at about 125 mg/dL (0.125%) on d 9 and had fallen to about 30 mg/dL (0.030%) by d 26.

The effects of oral ingestion of ethanol on the livers of animals and humans has been extensively studied and reviewed by many investigators (Lieber et al., 1975; Forsander, 1979; Ainley et al., 1988; Weiner et al., 1988). Many studies of cell injury show that damage to hepatocytes is mediated by acetaldehyde, which reaches progressively higher plasma (and presumably intracellular) concentrations for a given acute dose in those chronically consuming alcohol. This increasing concentration of the injurious metabolite appears to be a result of increasing production of acetaldehyde (induction of cytochrome P450 in the smooth endoplasmic reticulum (SER)) and decreasing catabolism of acetaldehyde (decreased mitochondrial ALDH activity). Acetaldehyde is known to cause many adverse effects on biochemical processes and on organelles, including forming complexes with proteins, depleting glutathione, increasing collagen synthesis, increasing lipid peroxidation, inhibiting protein secretion (by interfering with microtubules), and injuring mitochondria and SER. These biochemical and subcellular changes eventually result in gross changes in the liver including fatty liver, hepatitis, and cirrhosis. Acetaldehyde also induces many genetic alterations, which might lead to adverse effects including cancer (IARC, 1988).

The carcinogenicity of ethanol has been reviewed by scientists from many disciplines, thereby increasing the understanding of the many mechanisms through which ethanol might influence the rates of human cancer (Lowenfels, 1975; Seitz and Simanowski, 1986; IARC, 1988; Garro and Lieber, 1990). In 1975, it was clear that alcoholism was linked to cancer at sites where the liquid made direct contact with tissue (oropharynx, larynx, and esophagus) and where it is heavily metabolized to injurious products (liver). The frequent use of tobacco in combination with ethanol tended to confound the contribution from ethanol alone (Lowenfels, 1975). More recent evidence suggests that ethanol acts as a cocarcinogen primarily by inducing cytochrome-P-450-dependent mixed-function oxidase systems that activate such carcinogens as benzo (a) pyrene, nitrosamines, and aflatoxins. It is important to note that the induction of microsomal enzymes by orally ingested ethanol occurs in the lung as well as the intestine and liver (Lieber et al.,

1987). In addition to enzyme induction, several mechanisms that might contribute to the carcinogenic properties of ethanol are mitogenic effects, depletion of hepatic vitamin A, glutathione depletion, inhibition of DNA repair, and suppression of the immune system (Garro and Lieber, 1990). Despite the extensive studies of ethanol carcinogenesis, the findings do not tell us whether inhaled ethanol can act as a carcinogen or a cocarcinogen in the lung. Aside from the lung, it is very unlikely that inhaled ethanol would pose a significant cancer risk unless other symptoms of toxicity were present.

The genotoxicity of ethanol has been studied in many test systems and found to be attributable to ethanol's metabolism to acetaldehyde, which increases the incidence of sister chromatid exchanges (SCE) in bone-marrow cells of rodents exposed in vivo, induces chromosomal abnormalities in embryos exposed in vivo, and causes DNA cross links, chromosomal abnormalities, and SCE in human cells exposed in vitro (IARC, 1988). The important role of acetaldehyde is supported by the observation that without a test system capable of converting ethanol to acetaldehyde, ethanol does not exhibit genotoxicity. It must be noted that alcoholic beverages or their nonethanol extracts are often found to be mutagenic even in the absence of a metabolic system; however, this is attributed to the nonethanol components, which are themselves mutagenic (IARC, 1988).

Most in vitro test systems capable of metabolizing ethanol have given a positive analysis for the genotoxicity of ethanol. In mammalian cells exposed in vitro, the frequency of SCEs was increased when a metabolizing system was present (de Raat et al., 1983; Takehisa and Kanaya, 1983). Human lymphocytes exposed in vitro showed no increased frequency of SCEs at 1% ethanol; however, the frequency of SCEs was increased by the addition of ADH, but reduced by the addition of ADH and ALDH (Obe et al., 1986). Many mammalian in vivo genotoxicity assays have been negative; however, those involving embryonal cells have tended to show SCEs and other genetic abnormalities (IARC, 1988). Dominant lethal mutations have been induced by oral exposure of Sprague-Dawley rats (Klassen and Persaud, 1978), Long-Evans rats (Mankes et al., 1982), and CBA mice (Badr and Badr, 1975). Ethanol

induces aneuploidy in fertilized mouse eggs by directly interfering with the spindle apparatus during the first and second meiotic divisions (Kaufman and O'Neill, 1988). No studies were found that showed genotoxicity induced by inhalation exposure.

Many experiments have demonstrated some degree of reproductive toxicity by ethanol (Gavaler and Van Thiel, 1987); however, the doses used are typically 5% or more of the calories consumed or the liquid volume ingested. Such experiments may be pertinent to the human population consuming large quantities of alcoholic beverages, but the findings have little bearing on inhaled ethanol vapor. Structural abnormalities have been produced in reproductive organs of rodents administered ethanol as a major fraction (> 10%) of their diet; however, these doses often do not result in serious adverse effects on reproductive performance (Thiessen et al., 1966; Oisund et al., 1978; Mankes et al., 1982). High-concentration exposures of rat offspring to ethanol and its metabolites in utero have resulted in retarded physical and developmental maturation and disturbances in sexual behavior and performance as adults (Leichter and Lee, 1979; Parker et al; 1984). Hormonal changes often accompany morphological changes in rodents given high doses of ethanol. Male Sprague-Dawley rats given ethanol for 5 w (6-10% of a liquid diet) showed adverse effects on testes and a decrease in serum testosterone (Klassen and Pesaud, 1978). Female Wistar rats given ethanol as 36% of calories for 49 d showed a 60% decrease in ovarian weight and hormonal changes (Van Thiel et al., 1978). Despite disturbances in the estrus cycles of Holtzman rats given 5% ethanol for 16 w, there were no adverse effects on fertility or litter size after mating with unexposed males (Krueger et al., 1982).

A few recent studies have addressed the potential for inhaled ethanol to affect reproductive capacity. Male Sprague-Dawley rats exposed up to 16,000 ppm (30 mg/L), 7 h/d, for 6 w were mated 2 d after the exposure to unexposed females; compared with controls, no differences were seen in maternal weight gain, feed intake, water consumption, and number of offspring (Nelson et al., 1985a). As part of this study, it was found that exposure concentrations greater than 11,000 ppm (21

mg/L) were necessary for ethanol to begin accumulating in the blood. Hence, given the high oral doses that do not induce functional reproductive effects, it is not surprising that inhalation of ethanol vapor at 16,000 ppm (30 mg/L) also does not cause reproductive toxicity.

There is no question that maternal consumption of alcohol can increase the risk of developmental toxicity in offspring. Historically, the capacity for alcohol consumption to cause excess infant mortality and feeble children has been known for centuries (Warren and Bast, 1988). Modern recognition and naming of the fetal alcohol syndrome (FAS) by Jones and Smith (1973) have stimulated research to define the concentrations of alcohol consumption that might cause the syndrome and to better characterize the many adverse effects that are associated with maternal alcohol consumption. Nearly all developmental toxicity studies of ethanol involve oral ingestion; however, a few inhalation studies of rodents have been reported in an effort to address concerns about occupational exposure to ethanol vapor.

The increased risk of fetal abnormalities associated with FAS has been summarized in terms of specific effects and maternal alcohol consumption (Pratt, 1982). Even in offspring of women ingesting less than 10 g of alcohol per day, the risk of congenital malformations, abnormal behavior, and small (body length and body weight) for gestational age is about 10%. At this consumption level, the risk of characteristic FAS was considered very low. In offspring from heavy drinkers (consuming more than 50 g of alcohol per day), the risk of congenital malformation averaged 25%, the risk of mental deficiency averaged 35%, and the risk of small (body length and body weight) for gestational age averaged about 20%. In offspring of heavy drinkers, the risk of FAS was estimated to be 2.5% to 25%. After the review by Pratt (1982), a large study (12,440 women) from two Boston hospitals showed that consumption rates up to 14 drinks per week (average about 20 g/d) did not result in any adverse effects and that only abruptio placenta was observed with increased frequency above that rate (Marbury et al., 1983). However, in a study of 1122 pregnancies in the United Kingdom, intake of more than 10 g of ethanol per day early in pregnancy was found to double the risk of a low-birth-weight baby (Wright et al., 1984). Low

birth weight appears to be one of the most sensitive of the effects associated with FAS.

A diagnosis of FAS requires abnormalities in three specific areas: growth retardation (< 10th percentile), CNS deficits or mental retardation, and facial abnormalities (Warren and Bast, 1988). Immune deficiencies have also been characterized as an important aspect of the clinical problems associated with FAS (Johnson et al., 1981). The prevalence of FAS is approximately 2/1000 live births among Australians, Europeans, and North Americans, with wide variations depending on study site (Abel and Sokol, 1987). It appears that the most critical period for maternal alcohol use in causing FAS is near the time of conception (Ernhart et al., 1987).

Oral studies using rodents have given mixed results on rodents' ability to respond to ethanol in the same way as humans (IARC, 1988). The response of the animal model depends on strain, dose of ethanol, and gestational age at which ethanol is administered. From rodent models, it appears that reduced body weights in offspring are one of the most sensitive indicators. The potential for inhaled ethanol to induce fetotoxicity has been evaluated in a series of rat studies (Nelson et al., 1985a, b, 1988). Using maternal exposures up to 16,000 ppm (7 h/d for 6 w), no effect on behavior of offspring 10 to 60 d old could be demonstrated (Nelson et al., 1985a). Even when exposures were up to 20,000 ppm (7 h/d, gestational days 1 to 19) and the dams were narcotized, no definite increase in structural malformations could be found (Nelson et al., 1985b). In the latter study, offspring from males and females exposed at up to 16,000 ppm (7 h/d for 6 w) were found to perform as well as controls in a battery of neuromotor coordination tests and learning tests; however, a few neurochemical differences between exposed and control offspring were detected (Nelson et al., 1988).

Despite the well-founded concerns about ethanol's ability to induce fetal abnormalities when the mother consumes even modest amounts of alcohol, there is no evidence that fetotoxicity can occur at non-narcotic exposures via the inhalation route.

Ethanol has been found to potentiate the toxicity of many chemicals;

however, all data obtained were by noninhalation routes of administration of ethanol. Typical studies involved oral ingestion of ethanol coupled in some way to inhalation exposures to industrial chemicals. The summary provided here will be limited to ethanol-mediated effects on the inhalation toxicity of chemicals found at least occasionally in the space-shuttle atmosphere.

A number of studies have described the effect of ethanol on inhaled chlorocarbons. In human volunteers given trichloroethylene (TCE) at 50 or 100 ppm and enough ethanol to produce a BAL of 0.4% to 0.7%, Müller et al. (1975) found that the plasma TCE concentration was approximately 2.5 times higher in persons receiving ethanol than in controls. This result was due to blockage of TCE metabolism to trichloroethanol and trichloroacetic acid, thus increasing the CNS effects of TCE. Acute ethanol intoxication (5 g/kg) increases the incidence of chloroform-induced liver-function abnormalities in mice (Kutob and Plaa, 1962). Ethanol also potentiated the hepatotoxicity of TCE, but not 1, 1, 1-trichloroethane, in rats exposed to high solvent concentrations 18 h after ingestion of ethanol at 5 g/kg (Cornish and Adefuin, 1966). Interactions of various chlorocarbons have been studied in conditions in which ethanol is administered to rats and the chlorocarbons are administered by inhalation (Ikatsu and Nakajima, 1992).

The toxicity of inhaled aromatic chemicals is affected by concomitant administration of ethanol. Rats exposed to toluene at 2000 ppm (8 h/d for 2 w) and given 6% ethanol in drinking water showed reduced weight gain and abnormal clinical chemistry changes compared with toluene-exposed controls (Pryor et al., 1985). Padilla et al. (1992) showed that p-xylene inhaled by rats (1600 ppm, 6 h/d for 8 d) caused a reduction in rapid axonal transport, which was completely preventable by ethanol drinking (10% in water). Ingested ethanol (8 g/kg) and inhaled m-xylene (4 h at 6 or 11.5 mmol/m³) interacted in human volunteers in such a way that metabolic clearance of xylene was decreased and blood acetaldehyde concentrations were transiently increased. That interaction probably caused the dizziness and nausea reported by the test subjects given combined exposures (Riihimäki et al., 1982). In mice inhaling benzene at 300 ppm (6 h/d, 5 d/w, for 9 w), excess erythropoietic disruption (circulating normoblasts) developed in those also given 5% ethanol in drinking water as compared with controls not given ethanol (Baarson and Snyder, 1991).

The toxic effects of ethanol are unusual in several respects. There is a clearly defined population of flush-response persons who are highly susceptible to oral ingestion of alcohol; however, this group has never been tested for inhalation susceptibility. Long-term ingestion of alcohol (or inhalation in animal models) can lead to physiological dependency, which is evident when the toxicant is withdrawn from the environment.

The more clearly defined effects for which acceptable concentrations can be developed are as follows: neurotoxicity, mucosal irritation, and hepatotoxicity. As explained in the Toxicity Summary, no data show that inhaled ethanol can produce cancer, reproductive effects, or fetotoxicity at concentrations that do not cause short-term effects (e.g., narcosis) in the mother. The guidelines promulgated by the National Research Council will be used to structure the rationale for setting SMACs for ethanol (NRC, 1992).

Estimating the inhaled concentration of ethanol that will not induce neurobehavioral or performance decrements involves the results of two studies. First, the observations of Loewy and von der Heide (1918), who suggested effects at concentrations as low as 2.6 mg/L (2600 mg/m³), are discounted in favor of the report by Lester and Greenberg (1951). In the older study, individual subjects exposed at 2.6 or 4.7 mg/L were reported to be slightly dazed after leaving the chamber; however, that effect was not reported in subjects exposed to higher concentrations. In fact, two subjects exposed at 9.5 to 11.5 mg/L reported no effects (except temple pressure and slight pain), a result consistent with Lester and Greenberg's (1951) result. In the later study, the BAL after 3 to 6 h of exposure at 15 mg/L was less than 10 mg/dL (0.01%), and no symptoms were reported by the three test subjects. The lack of symptoms is not sufficient to exclude performance decrements, but Kennedy et al. (1993b) observed no performance decrements at a BAL of 0.05% in 20 test subjects. Exposures in rodents show that, at a constant exposure concentration, the blood concentration actually decreases over long periods of exposure, so time effects are unimportant after the first few hours. Noting that the BAL from inhalation of 15 mg/L was less than one fifth the BAL in a study of 20 human subjects showing no performance decrements, the AC was calculated as follows:

Because performance decrements are not acceptable for even short exposures, this AC applies to both short-and long-term exposures.

Lester and Greenberg (1951) reported that transient (< 10 min) coughing and smarting of the eyes and nose occurred at exposure concentrations between 10 and 20 mg/L. Based on that observation in three test subjects, the investigators exposed their subjects to ethanol at 15 mg/L for several hours, which the subjects found easily tolerable at low ventilation rates. Eye pain was not reported by Loewy and von der Heide (1918) in subjects exposed at concentrations below 11.5 mg/L (average); however, it was reported in two subjects exposed at 19.7 mg/L (starting concentration). That result is reasonably consistent with the finding of Lester and Greenberg (1951) that 10 mg/L is the irritation threshold. For short-term exposures, mild irritation is accepted, so the 1-h and 24-h ACs for irritation were set at 10 mg/L. Because the irritation is transient, 10 mg/L might also be taken as a NOAEL for purposes of setting long-term ACs, but the fact that only five subjects (three from the 1951 study and two from the 1918 study) were tested must be considered. The long-term (7-d and 180-d) ACs were set as follows:

This concentration should preclude risk of irritation regardless of the length of exposure.

Di Luzio and Stege (1979) found that Sprague-Dawley rats exposed continuously at 20 mg/L for 26 d did not exhibit any lasting liver damage, although mild transient changes were seen at 3, 6, and 9 d into the study. From this result, the long-term (7-d, 30-d and 180-d) ACs for hepatotoxicity were set as follows:

AC = 20 mg/L × 1 ¸ 10 (species) = 2 mg/L.

Increases in time of exposure were not considered a factor because tolerance to ethanol develops and BALs decrease as the exposure time

is increased beyond a few days. To induce hepatotoxicity with exposures of less than 24 h, the concentrations would have to be in a range that would cause narcosis; hence, short-term (1-h and 24-h) ACs were not set for liver damage.

No studies have directly measured the dose-response or NOAEL in a population of people known to exhibit the flush response. A NOAEL for flushing was estimated as follows: Inspection of the data of Zeiner et al. (1979) showed that the response was not observed when the breath acetaldehyde concentration (BAC) was below 10 ng/mL. Among those studied, the highest BAC was 60 ng/mL, and all subjects were given ethanol at 0.7 mL/kg. A sixfold reduction (to about 0.1 mL/kg) in the amount of ethanol given can be estimated to reduce the BAC of all subjects to below 10 ng/mL, a concentration at which no flush response would occur. Then, the NOAEL for a 70-kg subject would be as follows:

NOAEL = 0.1 mL/kg × 0.8 g/mL × 70 kg = 5.6 g.

Note that a NOAEL of 0.1 mL/kg is consistent with the dose range of 0.3 to 0.5 mL/kg often given to elicit the flush response in susceptible persons (Shibuya et al., 1989). The BAC profiles (Zeiner et al., 1979) show that the BAC remains above 10 ng/mL for only 2 h after oral ingestion (0.7 mL/kg); hence, one may assume that an inhaled dose must be delivered within 2 h at a threshold concentration that could induce flushing. During that period, an inhalation rate of 15 L/min and 62% uptake would result in the following concentration needed to deliver 5.6 g:

AC = 5.6 g × 0.62/(0.015 m³/min × 120 min) = 2 mg/L

That concentration should be a NOAEL and applies to long-term exposures (7 to 180 d). For 1-h and 24-h exposures, some degree of headache and other symptoms associated with the flush response can be tolerated. Doubling the AC estimate to 4 mg/L would result in a 2-h delivered dose of 0.2 mL/kg (assuming a weight of 70 kg), which is

below the typical lowest dose of 0.3 mL/kg used to elicit a flush response. Hence, the 1-h and 24-h ACs were set at 4 mg/L.

Despite the general consensus that inhaled ethanol is not harmful except in very high concentrations, no toxicity studies appear to have been conducted according to modern protocols to substantiate this conclusion. Only two studies involving human exposure were found, and they involved very few subjects, were uncontrolled, and relied on subjective reporting of symptoms. A carefully structured human inhalation study, including both flush response and nonflush response subjects, would provide a valuable addition to the data base.

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