1. An Evaluation of the Scientific Peer-Reviewed Research and Literature on the Human Health Effects of MTBE, its Metabolites, Combustion Products and Substitute Compounds John R. Froines, Ph.D. Principal Investigator Michael Collins, Ph.D. Elinor Fanning, Ph.D. Rob McConnell, M.D. Wendie Robbins, Ph.D. Ken Silver, M.S. Heather Kun Rajan Mutialu Russell Okoji, MSPH Robert Taber, M.D., MPH Naureen Tareen, MPH Catherine Zandonella
My name is elinor Fanning, and I’ll present Volume 2 of the UC report on MTBE, entitled __. The project was directed by John Froines, who is unable to be here today. I’ll start by acknowledging the colleagues whose work i will be discussing today.
Mike collins and Wendie Robbins worked on the devptl and repro toxicology sections, Rob McConell and Bob Taber developed the sections on acute effects and asthma, Ken Silver was our toxickinetics expert, and I did the sections on genotoxicity and cancer.
We had enthusiastic help from a number of students at Berkeley and UCLA, and would also like to acknowledge reviewers of sections of our document, as well as the written public comment we’ve received to date. In particular, HEI submitted a careful and throrough review of our volume.

2. Outline of Report MTBE Substitutes Taste and Odor Toxicokinetics
Acute Effects and Asthma
Neurotoxicity
Developmental and Reproductive Toxicity
Genotoxicity
Carcinogenicity
Substitutes
Ethanol
Acetaldehyde
PAN
Ethers
This is the very short version of the contents of Volume II. Given that our document is some 260 pages, and I have 10 minutes to summarize, I’ve had to be rather selective in what to emphasize. I’ll focus on the items in bold, with just quick mention of some other sections.
Acute, because of the public interest and uncertainty around these effects.
Cancer because this is the most controversial aspect of MTBE toxicology, and because of recent consideration by other review bodies
Finally, the section of our report concerning the toxicology of ethanol by-products has received little attention, but in our estimation, contains some of the most important findings in terms of policy implications, so I’d like to take a minute to look at them after we get through MTBE.
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3. Taste and Odor of MTBE Range of thresholds for taste:
2 ppb to 190 ppb
Range of thresholds for odor:
1.4 ppb to 680 ppb
Conclusion:
Detection in the low ppb range by sensitive individuals will impact aesthetics of drinking water
July 24, 1998, the California Department of Health Services proposed a secondary maximum contaminant level (SMCL) for MTBE at 5 ppb
These thresholds are dependant on several factors:
individual tested,
population tested
chlorination of the water
temperature of the water tested.
These findings have not been controversial, to my knowledge, but will have an important impact on the regulation of drinking water.
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4. Reports of acute effects cannot be dismissed
Acute Effects of MTBE
Studies of workers and general population
Headache, dizziness, nausea, respiratory irritation, have been observed. Study limitations and confounding make interpretation difficult.
Positive findings:
Results of one chamber study (out of four reviewed) found statistically significant increase in “heavy headedness” (Riihimaki et al, 1997)
One occupational study found that 20% of tanker drivers compared to 1% of milk drivers experienced acute effects (dizziness, dyspnea, nausea, salivation) (Hakkola et al, 1997)
Therapeutic use of MTBE associated with affects in some patients
Formaldehyde and TBA are irritants
Conclusion:
Reports of acute effects cannot be dismissed
Study populations
General population from MTBE use areas
Occupational populations in AK, CT, NJ, NY, Finland
Chamber studies (4 studies reviewed, one positive)
Gallstone patients
Limitations to the epi studies: confounding by media exposure, selection biases, incomplete follow up, and others.
Chamber studies generally negative.
While there is substantial uncertainty in these findings, we think it important not to rule them out just because the larger population studies are very difficult to interpret.
Likely to be a very significant effect of interindividual variabiltiy in these kinds of responses, and our document proposed experimental studies on specific, potentially sensitive subgroups.
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5. MTBE and Asthma No epidemiological studies
Insurance claim study did not detect an increase in asthma reporting in first year of MTBE use
MTBE combustion produces formaldehyde and TBA, both of which have irritant effects.
Studies of FA and asthma yield mixed results
Conclusion:
There is a need for epidemiological studies to assess what effect, if any, MTBE has on population asthma rates
There are no studies to address whether MTBE plays a role in asthma, so at this time, no conclusions can be drawn one way or the other.
A study of health insurance claims among Alaska state workers did not report an increase in asthma claims for the year MTBE was introduced. However, was the only year studied, so any confounding factors such as climate or other air pollutants can not be assessed.
Formaldehyde exacerbates and may cause asthma in sensitive people, but there are both positive and negative studies out there.
We really should do some studies on asthma and there are some suggestions in our document. Obviously, controlling for exposure to gasoline and exhaust is a critical factor.
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6. Developmental Toxicity of MTBE
4 studies (2 in mice, 1 in rat, 1 in rabbit)
Mice are most sensitive species
No significant effects in rat or rabbit studies
Skeletal defects and cleft palate, dose-related
Some defects occur below significant maternal toxicity
Conclusion:
MTBE has limited potential to act as a developmental toxicant
4 industry studies have been conducted to assess the ability of MTBE to induce developmental defects when females are exposed during gestation.
Rats and rabbits were not affected, but there were some limited findings in both studies of CD-1 mice.
Because there were doses at which no effects were seen, my colleagues concluded that the likelihood of effects in humans at ambient or occupational exposure levels is low.
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7. Reproductive Toxicity of MTBE
2 Studies (SD rats, inhalation)
Two generation
One generation, two matings
Findings:
Decreased pup weights at day 21 in both studies
Maternal toxicity
Conclusion:
MTBE has low potential to act as a reproductive toxicant, except at maternally toxic doses
Both studies were conducted by inhalation exposure to SD rats prior and during mating, but at different exposure concentrations.
Effects were seen on weight of offspring in both studies, and that was the only consistent finding.
Pup viabiltiy also affected in one time interval in each study but the time intervals affected were different.
Our document concludes that there is only low potential for MTBE to act as a reproductive toxicant
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8. Metabolism of MTBE
After exposure to MTBE, humans and rodents excrete the following compounds to the urine:
Alpha-hydroxyisobutyric acid (HBA, major urinary metabolite)
2-methyl-1,2-propanediol (MPD)
Glucuronide and sulfate conjugates of TBA
Pathways by which the major metabolites are formed are unknown, and therefore potentially toxic intermediates have not been identified
Toxicology of HBA and MPD, and potential intermediate forms is not known
Which metabolite or metabolites are involved in carcinogenicity of MTBE is not known
Before I move on to the genotoxicity and carcinogenicity material, I’d like to say a word about metabolism.
Major products found in urine are derivatives of TBA, we do not find mtbe, nor TBA, nor formaldehyde. (some TBA conjugates)
Metabolic pathways, enzymes and tissue sites responsible for transformation of TBA unknown, thus whether there are potentially toxic intermediates formed is not known
Tissue-specfic metabolism is an important area of research
CYP2A6 most importantin primary metabolism, 2E1 also involved. There is high interindividual variability of these enzyme activities in humans, indicating that there may be wide variation in sensitivity to the various health effects of MTBE.
Fraction metabolized 52-68% of the amount absorbed (Nihlen et al, 1998c).
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9. Genotoxicity of MTBE Positive tests Negative tests: Conclusions:
Forward mutation in mouse lymphoma cells (2)
Comet assay in fresh lymphocytes (abstract only)
Negative tests:
Salmonella strains (3 reports)
Mouse micronucleus assay (3 reports, )
Chromosomal aberrations (4 reports)
Recessive lethal in Drosophila (2 reports)
Hprt mutation in spleen lymphocytes
Conclusions:
MTBE displays little genotoxic activity in testing done to date
Tests for clastogenicity in target tissuesare needed
Mtbe has generally tested negative for genotoxicity, in assays that include in vitro and invivo, clastogenicity and point mutation.
However, there are some positive findings.
These raise concern that there may be genotoxic effects which are not adequately measured by the assays used. In vivo assays are preferable, but these assays measure damage to the bone marrow.
Assays of tissues that are target organs for MTBE toxicity would be more helpful. Of two in vivo studies done in target tissues, the comet assay measuring clastogenicity was positive and the hprt assay measuring point mutation was negative.
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10. Carcinogenicity of MTBE
No human data available
Excess cancer found in 3 animal studies:
Sprague Dawley rats (oral exposure)
females leukemia/lymphoma
males leydig cell adenoma
Fischer rats (inhalation exposure)
females no significant increase
males renal tubular adenoma and carcinoma leydig cell adenoma
CD-1 mice (inhalation exposure)
females hepatocellular adenoma
males hepatocellular carcinoma
Three studies
Two routes of exposure
Two species
Two strains of rat
Statistically significant increases in tumors in all 3 studies, and 5/6 if males and females are counted separately.
Both rat strains got LCTs, and I’ll have a bit more to say about that.
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11. Oral Study in Sprague-Dawley Rats
Using data from the most recent publication, giving the updated cancer data after a pathology review.
Let me breifly address some of the issues that have been raised about this study, although given the time limit I’d be happy to talk more about this during the question period.
First, the statistical methods used in the publication correct for the time at which tumors developed. The Mantel-Cox test is a time dependent test, more commonly used in human epidemiology. There seems to be widespread misunderstanding of this point, perpetuated in quote after quote about not correcting for mortality. (even occurred in the IARC deliberations, according to a memo)
Second, there has been quite an odd discussion, in my opinion about the classification of the lymphatic tumors in the females. It is biologically appropriate, and is common practice not only in toxicology, but in human epidemiology to combine tumors of this sort.
Finally, the LCTs observed were confirmed in the 1998 report to be adenomas. If hyperplasias were also included, the numbers would be much higher.
* statistically significant relative to controls, by either FET or Mantel-Cox test
Exposure: oral gavage 4d/wk, 104 wks
References: Belpoggi et al., 1995; 1997; 1998
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12. Inhalation Study in Fischer Rats
High dose affected mortality, may have been >MTD
Relevance of the LCTs have been challenged on the basis that the historical background rates in F344 rats ranges from 64 to 98 percent, and thus includes the range observed here.
Clear dose-response, stat sig at higher two doses.
Concurrent controls better than historical, because of strain variations, differences in caging protocols, feed used, etc.
Quite a bit of research has been done on the hypothesis that the kidney tumors were produced by a mechanism that is limited to male rats. There has been some excellent work from CIIT on this.
MTBE meets some criteria and not others.
Our document concluded that there may be a role for interaction with the alpha2 u globulin protein, but MTBE interacts only weakly and there is likely more to the story than just alpha two. Until we know for sure, it would be imprudent to rule the kidney tumors irrelevant for a compound with such widespread exposure.
Exposure 6hr/d, 5d/wk, 104 wk
High dose males terminated at 82 weeks
Mid-dose males terminated at 96 weeks
Bird et al, 1997
Mortality corrected data from USEPA, 1995
*statistically significant by FET (P<0.05)
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13. Inhalation Study in CD-1 Mice
In the original report, the male carcinomas were not identified as signficantly increased, because mortality-corrected data were not used.
Survival was not affected in the females, and andthere were no signs of liver toxicity.
Note the abbreviated study period, which may make it less likely that tumors would be detected, or that adenomas would progress to carcinoma, especially in females, whose survival was not affected by MTBE.
Some kidney lesions were observed, but not dose related and not cancer.
Endometrial hyperplasia reduced in female mice, suggesting hormonal effect, and CIIT, and I think Ann de Peyster’s lab in sandiego have looked into whether MTBE could act though estrogen. They’ve shown that this does not appear to be the case.
Exposure 6hr/d, 5d/wk, 68 wk
Bird et al, 1997
*statistically significant by FET (P<0.05)
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14. Conclusions on Carcinogenicity
MTBE is an animal carcinogen
Concordance of one tumor site (LCT in testes)
Supporting evidence:
TBA caused renal tubular tumors in rats
FA is an IARC 2A carcinogen
Mechanisms suggested to date are not adequately supported by available data
In the absence of data clearly showing otherwise, all four tumor endpoints must be considered potentially relevant to humans.
To help sort this out:
Metabolism/metabolites present in kidney? Is MTBE really what we want to be looking at with respect to alpha2?
Mechs that have been investigated:
MTBE does not seem to act on testes through LH
MTBE does not cause liver tumors by acting as a classic antiestrogen
MTBE does not cause kidney tumors strictly through alpha2
No hypotheses have been put forth for leuk/lymph, but we do have one of the positive assays for genotoxicity in that target tissue.
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15. Other Reviews of MTBE carcinogenicity
Proposition 65 Carcinogen Identification Committee
Apparent 3 to 3 tie (resulted in not listing)
Discussion based on whether MTBE had been clearly shown to cause cancer
National Toxicology Program Report on Carcinogens
Report on Carcinogens
Voted 6 to 5 not to list
International Agency for Research on Cancer
Classified in group 3, “not classifiable”
(Inadequate evidence in humans; limited evidence in animals)
The transcripts from the CIC hearing show that the majority of debate centered around the wording of the law, requiring that a compound be clearly shown to be a carcinogen.
IARC: the statement from IARC that I’ve seen on this indicates that there were some problems with the material given to the workgroup to review, although I cannot be sure. First, IARC concludes there was no tumor concordance, which indicates they are overlooking the LCTs. To my mind, this tumor finding is very important when we note that it occurred in two strains of rat, with a clear dose-response in both.
Second, the IARC memo stated that the statistical significance of leuk/lymph and LCTs in the Belpoggi study was produced by a method that failed to account for survival. This is a misunderstanding, and I’m not sure how it happened.
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16. Other Reviews of MTBE carcinogenicity
National Science and Technology Council
“Experimental studies indicate that MTBE is carcinogenic in rats and mice at multiple organ sites after inhalation or oral-gavage exposure. There is sufficient evidence to indicate that MTBE is an animal carcinogen and to regard MTBE as having a human cancer potential.”
Health Effects Institute (HEI)
“Tumors have been observed at multiple sites in rats and mice after exposure to high levels of MTBE. The Oxygenates Evaluation Committee consider these findings to be cause for concern. The Committee noted that the mechanisms that caused these tumors and the likelihood that these or other tumors will occur in humans exposed at substantially lower levels are both unknown.”
The transcripts from the CIC hearing show that the majority of debate centered around the wording of the law, requiring that a compound be clearly shown to be a carcinogen.
IARC: the statement from IARC that I’ve seen on this indicates that there were some problems with the material given to the workgroup to review, although I cannot be sure. First, IARC concludes there was no tumor concordance, which indicates they are overlooking the LCTs. To my mind, this tumor finding is very important when we note that it occurred in two strains of rat, with a clear dose-response in both.
Second, the IARC memo stated that the statistical significance of leuk/lymph and LCTs in the Belpoggi study was produced by a method that failed to account for survival. This is a misunderstanding, and I’m not sure how it happened.
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17. Other Reviews of MTBE carcinogenicity
Office of Environmental Health Hazard Assessment
“There is evidence for the carcinogenicity of MTBE at multiple sites in both sexes of the rat and mouse, MTBE is a multi-species, multi-strain, multi-sex, and multi-route carcinogen. Positive animal carcinogenicity data for HCHO and TBA, metabolites of MTBE, provide support for this conclusion.”
The transcripts from the CIC hearing show that the majority of debate centered around the wording of the law, requiring that a compound be clearly shown to be a carcinogen.
IARC: the statement from IARC that I’ve seen on this indicates that there were some problems with the material given to the workgroup to review, although I cannot be sure. First, IARC concludes there was no tumor concordance, which indicates they are overlooking the LCTs. To my mind, this tumor finding is very important when we note that it occurred in two strains of rat, with a clear dose-response in both.
Second, the IARC memo stated that the statistical significance of leuk/lymph and LCTs in the Belpoggi study was produced by a method that failed to account for survival. This is a misunderstanding, and I’m not sure how it happened.
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18. Ethanol Acetaldehyde Peroxyacetylnitrate (PAN) Conclusions
IARC 2B, TAC, irritant, clastogen, anueploidogen
Peroxyacetylnitrate (PAN)
Respiratory and ocular irritation
Synergy with respiratory infection (mice)
Mutagenicity weak, but positive
Potential carcinogen
Conclusions
Oxygenation with EtOH should not be taken lightly
Critical Research Needs:
Carcinogenicity bioassay of PAN
Respiratory effects of acetaldehyde and PAN
Synergy in respiratory effects with other TACs
Exposure to ethanol would increase during fuel dispensing, but is unlikely to be a major health concern. We are concerned, however, about combustion by-products of ethanol.
Acetaldehyde is a TAC in California, and is an IARC 2B carcinogen. It causes respiratory irritation, and cancers of the nose, larynx and respiratory epithelium in rodents.
There is also positive genotox on acetaldehyde, clastogenicity in culture. Not much in the way of invivo testing.
PAN: studies of eye and respiratory irriation have been mostly negative, but concentrations tested were quite low (9 exptl, 1 epi). There were effects on forced expiratory volume, and some synergy with ozone noted. It is critical that PAN be tested in sensitive individuals in the lab, since the concentrations at which effects were seen in healthy voluntters are only marginally above ambient concentrations.
Gentox: mixed, weak bacterial mutagen, more data on mammalian systems needed.
Cancer: only one study, Strain A mice exposed at 15ppm for 6 months, respiratory metaplasias in 50% of animals, and some kind of lesion in all. “the extent, rapidity and uniformity with which lesions developed in these mice exposed to 15 ppm PAN suggested that PAN may affect health significantly and that further studies to this end are indicated.” I’ll note that average PAN concentrations in some areas are 1,000 fold less than the conc tested.
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19. Conclusions (short list)
Given widespread exposure to MTBE, the data should be treated conservatively.
MTBE is an animal carcinogen and could potentially cause cancer in humans.
Further research is needed on tissue-specific metabolism and genotoxicity of MTBE.
Further research is needed on the respiratory effects, including asthma, of MTBE, combustion by-products, acetaldehyde and PAN.
While ethanol may be relatively safe, combustion by-products of ethanol have been associated with respiratory effects. PAN needs to be tested for carcinogenicity.
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