The Health Effects Institute


Program Summary:
Research on Oxygenates Added to Gasoline


ADDING OXYGENATES TO FUEL

The Clean Air Act of 1970 initiated a commitment by the U.S. government to reduce the public's exposure to air pollutants that may adversely affect health. Approaches to reduce emissions from mobile sources have focused on setting increasingly more stringent exhaust emission standards for new vehicles (which have in turn driven improvements in technology), in-use inspection, and maintenance of vehicles. These measures have substantially reduced emissions per vehicle mile traveled and the levels of air pollutants; however, these benefits have been largely offset by increases in the number of vehicle miles driven per year. The Clean Air Act Amendments of 1990 took further steps toward reducing motor vehicle emissions by mandating the use of two types of fuel reformulation that involve increasing the oxygen content of fuel by adding compounds containing oxygen (called oxygenates): oxygenated gasoline (oxyfuel) and reformulated gasoline (RFG).

Oxygenates used in oxyfuel and RFG include ethers such as methyl tert-butyl ether (MTBE, the most frequently used oxygenate at the present time) and alcohols such as ethanol. Because the oxygenates vary in molecular weight, a different amount of each oxygenate is needed to meet the oxygen requirements. For example, 15% MTBE (by volume) or 7.8% ethanol (by volume) would provide 2.7% oxygen (by weight), as required for oxyfuel. Other ethers that can be added to gasoline include ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and diisopropyl ether (DIPE).


THE HEI REVIEW OF THE OXYGENATES LITERATURE

Before 1992, MTBE was used as an octane enhancer in premium gasoline in amounts up to 9% (by volume). The introduction of oxyfuel (and later RFG) containing MTBE in higher concentrations elicited a number of complaints from workers and the general public in some areas of the United States, including reports of unpleasant odor, headaches, burning of the eyes and throat, and other symptoms of discomfort. At the same time, some consumers raised questions about the cost of oxyfuel, the performance of engines using it, and its effect on fuel economy. In response to continuing health questions, in the spring of 1995 the U.S. Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention asked HEI to (1) review the existing literature and conduct a qualitative assessment of the health effects of exposure to oxygenates, (2) compare the health effects of oxygenates with those of other pollutants whose levels in emissions would change when oxygenated fuels are used, and (3) discuss how the assessment of the health effects of oxygenates by HEI might differ from the risk evaluations that had been done by the EPA (U.S. Environmental Protection Agency 1993, 1994).

In response to this request, HEI assembled a committee of scientific experts (the Oxygenates Evaluation Committee) to work with HEI's scientific staff in reviewing what was known about exposure to and health effects of oxygenates. The Committee's report, The Potential Health Effects of Oxygenates Added to Gasoline: A Review of the Current Literature, issued in April 1996, focused on MTBE and ethanol, and briefly addressed other ethers whose use might increase in the future (Health Effects Institute 1996). This review was incorporated into a broader assessment of oxygenated fuels conducted by the White House Office of Science and Technology Policy (OSTP). The HEI review of the current literature and the draft OSTP assessment were reviewed by the National Research Council's Committee on Toxicological and Performance Aspects of Oxygenated and Reformulated Motor Vehicle Fuels (National Research Council 1996). The final version of the OSTP assessment was published in 1997 (National Science and Technology Council 1997). We report below the major conclusions of the HEI review on MTBE and other ethers.

Exposure Assessment
Exposure to oxygenates from their use in gasoline can occur by inhalation, ingestion, or skin contact, of which inhalation is the most common route. However, drinking water contaminated with oxygenates, primarily by gasoline leaking from underground storage tanks, is also of concern. Recently, finding lakes and reservoirs contaminated with MTBE from gasoline used in power boats and jet skis has suggested that exposure by ingestion and skin contact may be more widespread than previously thought.

Air concentrations have been measured primarily for MTBE; few data are available for other oxygenates. The highest exposures are experienced by workers involved in the manufacture and transport of neat oxygenates and fuels containing oxygenates, and by gas station attendants or others associated with the repair or maintenance of motor vehicles. Overall, the general public is exposed to low concentrations of oxygenates. Higher exposure levels are usually associated with activities of shorter duration. The highest exposure concentrations are encountered during vehicle refueling, for which median levels range from 0.3 to 6 ppm for 1- to 2-minute durations and peaks occasionally exceed 10 ppm. Commuters in automobiles are exposed to considerably lower MTBE concentrations (at levels of parts per billion rather than parts per million), but for longer time periods. However, the level of exposure can vary widely depending on the individual vehicle.

Metabolism
A number of studies conducted in vitro, in animals, and in humans have investigated the metabolism and disposition of MTBE, but limited information is available for other ethers. These studies have shown that MTBE (inhaled or ingested) is taken up into the blood stream and distributed to body water, and that within the range of concentrations relevant to human exposure, the level of MTBE in the blood is proportional to the MTBE concentration in the air. Following uptake, MTBE is either exhaled as such or metabolized. The extent to which MTBE is metabolized depends on the route of exposure and the dose administered. At comparable MTBE doses, a higher proportion of MTBE is exhaled after oral and intravenous exposure than after exposure by inhalation.

The half-life of MTBE in blood has been found to range from 0.5 to 1.5 hours in different species. It has been fairly well established that the first step in metabolizing MTBE is oxidative demethylation, which yields formaldehyde and tert-butyl alcohol (TBA); this reaction is catalized by cytochrome P-450–dependent enzymes. These enzymes are found in the microsomal fraction of eukaryotic cells and participate in the metabolism (oxidation) of foreign compounds; they occur in different forms (or isozymes) with different substrate specificities. Previous work conducted by Brady and coworkers (1990) in rat liver microsomes implicated the P-450 2E1 isozyme in the metabolism of MTBE. Formaldehyde is metabolized very rapidly, and its level in blood has not been followed. Tertiary butyl alcohol appears to be metabolized more slowly than MTBE, with glucuronidation being an important pathway for its elimination. Metabolism of TBA leads to 2-methyl-1,2-propanediol, which is further oxidized to either 1-hydroxybutyrate or to formaldehyde and acetone. Levels of TBA in blood are similar to or higher than the corresponding levels of MTBE. Although both MTBE and TBA appear to be reliable indicators of MTBE dose, further studies are needed to establish the time course of their blood or urine concentration as a function of the exposure concentration before either one can be used as a marker of exposure in human studies.

Health Effects
On the basis of its review of existing evidence, the HEI Oxygenates Evaluation Committee identified three areas of uncertainty and concern about potential health effects of MTBE for humans:

The Oxygenates Evaluation Committee noted that their conclusions were based on limited information, and that they still had questions about how to interpret the observed effects for humans; nevertheless, the Committee concluded that these effects point to potential human health risks. Not enough information was available on the toxicity of other ethers for the Committee to evaluate their potential health effects; further investigation is required if adding other ethers to fuels is to become widespread.

After qualitatively assessing the effects of gasoline and motor vehicle emissions with and without oxygenates, the Committee came to the following conclusions about gasoline containing oxygenates:

Research Needs
The HEI Oxygenates Evaluation Committee identified a number of research needs to address uncertainties about the health effects of oxygenates by themselves and as part of gasoline mixtures. The research needs of highest priority are listed below. A more detailed and longer list of research priorities can be found in the HEI Oxygenates Evaluation Committee's report (Health Effects Institute 1996), along with some information on work under way by other organizations.


THE HEI RESEARCH PROGRAM ON ETHERS

In August 1995, HEI issued RFA 95-1, Comparative Metabolism and Health Effects of Ethers Added to Gasoline to Increase Oxygen Content, which sought applications for research to investigate the uptake, metabolism, and disposition of various ethers (alone or in combination with components of gasoline) and their potential health effects in humans. HEI considered comparative research to be important because of the incomplete information about MTBE and the small amount of research on other ethers. Research to further characterize the enzymes responsible for the metabolism of ethers, particularly in humans, would help in extrapolating the animal toxicity data from one ether to another and from animals to humans, and may reveal interindividual metabolic differences that could be related to differences in susceptibility. In addition, studies on health effects could evaluate subtle adverse responses and identify mechanisms by which these effects occur. Research on how compounds present in gasoline vapor or combustion products may impact ether metabolism could identify interactions that might result in enhanced effects.

Studies Funded
From RFA 95-1, HEI funded three studies on the metabolism of various ethers. These studies were highly ranked among the applications submitted to HEI, and form a coherent program that, even though it does not address the health effects objective of the RFA, will contribute to a better understanding of ether metabolism across species. The goals of these studies are to:

Role of Human Cytochrome P-450 in the Metabolism and Health Effects of Gasoline Ethers
Jun-Yan Hong, Rutgers University
Previous work by Brady and coworkers (1990) has implicated the enzyme cytochrome P-450 2E1 in the metabolism of MTBE in rat liver microsomes. However, the metabolism of MTBE in human microsomes has not been studied. The goals of this study are to determine the metabolic pathways of MTBE, ETBE, and TAME in human liver microsomes, and identify the major cytochrome P-450 isozyme responsible for metabolizing these ethers. To accomplish this goal, human liver microsomes and human cells engineered to express different cytochrome P-450 isozymes are being used. The results so far indicate that isozyme 2A6 is more active than 2E1 in the metabolism of the three ethers (Hong et al. 1997).

Once the primary isozyme responsible for metabolizing MTBE has been identified, the investigators will test their hypothesis that genetic polymorphism may be linked to differences in sensitivity to MTBE among individuals. Polymorphic sites within a gene can be located either in the transcription regulatory region or in the coding region. Depending on its location, the polymorphism can affect either the level of expression of that gene (enzyme level) or the enzyme activity. The gene that codes for the P-450 2E1 isozyme is one of the genes most extensively studied for polymorphism. Large genetic differences have been documented in the frequency of appearance of various 2E1 polymorphic sites, and some of them have been associated with cancer susceptibility. The investigators will determine the impact of genetic polymorphism of the human ether-metabolizing enzyme by characterizing the genotypes of more than 300 individuals who have reported symptoms associated with exposure to MTBE, and comparing them with the genotypes of asymptomatic subjects. Finally, they will compare how rats and monkeys metabolize ethers in the liver and nasal mucosa, and determine how microsomes from rats exposed to MTBE in the presence of gasoline metabolize MTBE. These latter experiments complement the in vivo work in Dr. Dekant's study described below.

Comparative Biotransformation of MTBE, ETBE, and TAME in Rats and Humans
Wolfgang Dekant, University of Würzburg

This study is investigating the metabolism of three ethers in rats and humans exposed by inhalation in order to identify and compare the metabolites of the ethers and the pathways for their excretion, as a function of the time after exposure and the dose. An initial study was conducted to determine the structures of the metabolic products and to prepare the appropriate standard reference compounds: Three rats and one human subject were exposed to ethers labeled with 13C (a stable, nonradioactive isotope of carbon) on the central tertiary carbon of a tert-butyl group. Samples of urine were examined with 13C–nuclear magnetic resonance and gas chromatography/mass spectrometry to identify metabolic products. In the main study, six human subjects and six rats will be exposed to 4 or 40 ppm of each ether for 4 hours. Blood and urine will be collected before the exposure and at intervals during the 48-hour period after the end of exposure to analyze the metabolic products.

A minor component of this study involves identifying the P-450 isozymes responsible for oxidizing these ethers in human liver microsomes. This work complements the work being conducted by Dr. Hong described above.

The Toxicokinetics of MTBE Inhaled Alone and in Combination with Gasoline Vapor
Janet Benson, Lovelace Respiratory Research Institute

This study is designed to mimic human exposure to MTBE and gasoline vapor during refueling. It is investigating the effect of combined (single and repeated) exposure to MTBE and gasoline components on the uptake of MTBE; its distribution in blood, lung, liver, kidney, testes, heart, and fat; and its excretion in exhaled air, urine, and feces. Metabolites are being identified in selected tissues and excreta samples. The study design consists of:

The gasoline vapor mixture corresponds to the gasoline fraction present in an engine headspace, and is obtained by heating gasoline to 130°F. This mixture has been developed for the Fuel Testing Program, mandated under Section 211 of the Clean Air Act Amendments of 1990, to reflect the composition of evaporative emissions. When MTBE and the gasoline vapor are mixed, the resulting MTBE concentration in the mixture is 20% (by volume), which corresponds to the concentration of MTBE in evaporative emissions from gasoline that contains 15% MTBE (by volume) (R. Barter, personal communication).


SUMMARY

Taken together these three studies will provide information on the metabolism of MTBE alone and combined with gasoline evaporative emissions, and on the metabolism of other ethers in rats and humans. The concentrations of MTBE to be used in the two inhalation studies have been selected to represent occupational exposures at relatively low and high levels. These studies were initiated between August and November of 1996 and are scheduled to be completed by the end of 1998.

The results will (1) characterize possible interspecies differences in the biotransformation of ethers and facilitate interspecies extrapolation of health effects data, (2) provide information on whether (single or repeated) exposure to MTBE in combination with gasoline vapor affects the metabolism of MTBE, and (3) help determine whether sensitivity to MTBE exposure is related, at least in part, to differences in the DNA sequence of the gene for the P-450 enzyme responsible for its metabolism. The work supported by HEI complements other research currently under way on the mechanisms by which MTBE may cause tumors in rats and mice and on the health effects of TAME and ETBE (in conformity with requirements mandated in the Toxic Substances Control Act [Federal Register 1995]), and research to be initiated soon on the health effects of evaporative emissions containing MTBE (in conformity with the regulation on fuel registration [Federal Register 1994, 1997]).


REFERENCES

Brady JF, Xiao F, Ning SM, Yang CS. 1990. Metabolism of methyl tertiary-butyl ether by rat hepatic microsomes. Arch Toxicol 64:157–160.

Federal Register. 1994. Fuels and fuel additives registration regulation, June 27, 1994. Fed Regist 59:33042–33142.

Federal Register. 1995. Testing consent order for tertiary amyl methyl ether, March 21, 1995. Fed Regist 54:14910–14912.

Federal Register.1997. Proposed alternative tier 2 requirements for baseline gasoline and the oxygenated gasoline categories of methyl tertiary butyl ether, ethyl tertiary butyl ether, ethyl alcohol, tertiary amyl methyl ether, diisopropylether, and tertiary butyl alcohol, September 9, 1997. Fed Regist 62:47400–47401.

Hong J-Y, Yang CS, Lee M, Wang Y-Y, Huang W-Q, Tan Y, Patten CJ, Bondoc FY. 1997. Role of cytochrome P450 in the metabolism of methyl tert-butyl ether in human livers. Arch Toxicol 71:266–269.

Health Effects Institute. 1996. The Potential Health Effects of Oxygenates Added to Gasoline: A Review of the Current Literature (A Special Report of the Institute's Oxygenates Evaluation Committee). Health Effects Institute, Cambridge, MA.

National Research Council. 1996. Toxicological and Performance Aspects of Oxygenated Motor Vehicle Fuels. National Academy Press, Washington, DC.

National Science and Technology Council. 1997. Interagency Assessment of Oxygenated Fuels. Executive Office of the President of the United States, Washington, DC.

U.S. Environmental Protection Agency. 1993. Assessment of Potential Health Risks of Gasoline Oxygenated with Methyl Tertiary Butyl Ether (MTBE). EPA/600/R-93/206. Office of Research and Development, Washington, DC.

U.S. Environmental Protection Agency. 1994. Health Risk Perspectives on Fuel Oxygenates. EPA 600/R-94/217. Office of Research and Development, Washington, DC.


ABBREVIATIONS

CO carbon monoxide
DIPE diisopropyl ether
EPA U.S. Environmental Protection Agency
ETBE ethyl tert-butyl ether
MTBE methyl tert-butyl ether
OSTP White House Office of Science and Technology Policy
oxyfuel gasoline with added oxygen
ppm parts per million
RFG reformulated gasoline
TAME tert-amyl methyl ether
TBA tert-butyl alcohol

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