Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 216-653-1
CAS number: 1634-04-4
More than ten bacterial gene mutation assays (tests) according to or
similar to the OECD/EU guideline (and under GLP) are available that gave
mostly negative results. Positive results were found in an Ames TA102
test system (Williams-Hill et al, 1999), however this positive finding
in TA102 was not replicated in GLP-compliant, OECD 471 guideline
investigations conducted by RBM (1996f) and by McGregor (2005).
Therefore, MTBE is not considered mutagenic in bacteria.
Regarding gene mutations in mammalian cells in vitro, the HPGRT test
performed by Life Science Research (1989b) according to OECD guideline
476 and under GLP was considered to be the key study. No mutagenicity
was observed up to the highest tested concentration (10,000 µg/ml) in
the absence and presence of metabolic activation in Chinese hamster lung
fibroblasts. However, positive results were observed in a mouse lymphoma
assay (ARCO Chemical Company, 1980) where an increased and
dose-dependent mutation frequency was reported in the presence of
metabolic activation. Cells cultivated without S9-mix exhibited no
meaningful changes in mutation frequency when compared to controls,
suggesting the possible involvement of a MTBE metabolite. Mackerer et
al. (1996) investigated the potential role of formaldehyde, a metabolite
of MTBE and a known mutagenic substance, in this process. They exposed
mouse lymphoma cells to 1-4 μl/ml MTBE for 3 hours, in the absence or
presence of added formaldehyde dehydrogenase (FDH) and NAD; the latter
enzyme and cofactor convert formaldehyde to non-mutagenic formic acid.
The results showed that the mutation frequency did not increase when FDH
and its coenzyme were present, while there was a five-fold increase when
FDH/NAD were absent. Furthermore, Casanova et al. (1997) and the
subsequent review by McGregor et al. (2006) demonstrated that
formaldehyde generated endogenously from MTBE metabolism at
concentrations of up to 6.5 mM (650 µg/mL, corresponding to plasma
levels resulting from inhalation levels of greater than 8,000 ppm) did
not produce an increase over background levels in DNA-protein
cross-links in primary cultures of mouse and rat hepatocytes. Therefore,
positive mouse lymphoma assay findings obtained with MTBE in the
presence of metabolic activation are not considered physiologically
relevant since formaldehyde (the likely mutagenic product) is rapidly
and efficiently detoxified by formaldehyde dehydrogenase in vivo.
A correlation of increasing liver UDS activity in vitro with increasing
MTBE concentration was found in liver cells from Sprague Dawley rats
(Zhou et al, 2000). Yang et al. (2005; Chinese with abstract in English)
reported results of several in vitro assays: comet assay, DNA cross-link
test and unscheduled DNA synthesis (UDS) assay in cultured rat type II
pneumocytes and hepatocytes. No external metabolic activation was
included in the experiments. The abstract does not report any
quantitative data other than probability statistics associated with a
purported effect. For example, the abstract states that MTBE at doses
above 0.05 mmol/L caused greater DNA migration in cultured rat type II
pneumocytes and rat hepatocytes in vitro (p<0.05) whereas in the UDS
assay it gave positive results at concentrations of 5 and 10 mM. Without
having access to the full report in English, it is difficult to
critically examine this paper either for the acceptability of the
experimental methodology or for strength and reliability of the data. It
is extremely important to examine whether there is cytotoxicity
associated with any of the exposures since it is now well recognized
that cytotoxicity alone could induce false-positive findings in the
comet assay (Speit et al., 2015). However, two other in vitro liver UDS
studies (Cinelli et al, 1992 and Life Science Research, 1989c) and an in
vivo UDS test conducted according to a method similar to OECD guideline
486 and under GLP with CD-1 mice (Bushy Run Research Center, 1994) were
Tang et al. (1997) claimed genotoxicity for MTBE and its metabolites in
a human cell line (HL-60) in vitro as measured by the comet assay; no
external metabolic activation was used in this assay. The concentrations
of the test substances evaluated ranged from 1-30 mM It is also
remarkable that Tang et al. reported identical levels of DNA damage
following treatment of the cells with MTBE and two of its metabolites
(TBA and α-hydroxyisobutyric acid). The molecular structures of MTBE and
the metabolites are very different in their possible reactive moieties
and it would be very unlikely that they would produce the same comet
response. The cell line used in this study is not commonly used in the
comet assays and is not known to be metabolically competent. It is
therefore unlikely that a common proximate metabolite would be
responsible for the observed damage. Chen et al. (2008) reported DNA
single and double strand breaks and oxidative base modifications in
human lymphocytes treated in vitro with MTBE at concentrations ranging
from 50-200 µM. This study suffers from the following methodological
limitations: no image analysis and no data on cytotoxicity. In addition,
the DNA strand break response (reported in arbitrary units) was very
weak with no apparent dose response.
Results of in vitro comet assays reported in the literature should be
viewed with caution. Currently, there is no standardized and
internationally accepted guideline for the conduct of the in vitro comet
assay. Most importantly, cytotoxicity is a significant confounder in
interpreting comet assay results and studies that do not control this
variable are of questionable value. An international expert working
group on the comet assay (IWGT, 2015) concluded that “ .. that
cytotoxicity could be a confounder of comet results. It is recommended
to look at multiple parameters such as histopathological observations,
organ-specific clinical chemistry as well as indicators of tissue
inflammation to decide whether compound-specific toxicity might
influence the result.” Unfortunately, there was no evidence for such a
rigor in the conduct of the in vitro comet assays (as well as in vivo
comet studies - see below) on MTBE calling into question whether the
available comet data are robust or reliable enough to override a vast
database demonstrating lack of genotoxicity for MTBE.
An in vitro micronucleus study in a mouse cell linereported by Zhou et
al (2000) is considered the key study for clastogenicity. The test was
performed using a method similar to OECD guideline 473 and no
clastogenicity was observed.
Three well-conducted in vivo studies performed in accordance to EPA
guidelines/similar to OECD guidelines are available: an UDS test (Bushy
Run Research Center, 1994), a chromosome aberration test (Bushy Run
Research Center, 1989b) and a micronucleus test (Bushy Run Research
Center, 1993b). These studies were all negative. The highest exposure
concentration used in these studies, i.e., 8000 ppm, met or exceeded the
maximum tolerated dose criteria. First and foremost as discussed in the
Toxicokinetic section, the 8000 ppm exposure exceeded the of metabolic
saturation (Miller et al., 1997). It is now widely recognized, as
reflected in the OECD test guidelines, that care should be taken in
selecting dose levels in genetic toxicology studies to prevent
saturation of absorption, metabolism or excretion as adverse effects
observed at such doses are not relevant for safety
assessment. Additionally, the 8000 ppm also represents a concentration
where a variety of sensorimotor effects indicative of CNS depression
were observed even with a single 6 h exposure (McGregor,
2006). Finally, the target tissues used in these studies (bone marrow
and liver) were adequately exposed to MTBE and its metabolites because
of their systemic availability (McGregor, 2006; Borghoff et al., 2010).,
Yang et al. (2005; Chinese with English abstract) conducted a comet
assay in hepatocytes, renal cells, and pneumocytes of mice exposed by
inhalation to 108, 1440 and 4968 mg/m3 MTBE for 20 days. The abstract
does not report any quantitative on the endpoint other than stating
positive responses were observed; furthermore, no information on
cytotoxicity was presented. A positive result was observed in a comet
assay in lymphocytes of rats administered gavage doses up to 800
mg/kg/day for 28 days (Lee et al, 1998; reported as an abstract
only). Schreiner et al. (2014) reported small increases (up to 1.2-fold)
in sister chromatid exchanges (SCE) in peripheral blood lymphocytes of
male and female rats exposed by inhalation to baseline gasoline
containing MTBE at concentrations ranging from 2000 to 20000 mg/m3; no
increases in bone marrow micronucleus frequencies were observed in this
study. This is not a useful study because the test substance evaluated
was a mixture and not MTBE alone. Furthermore, SCEs induction is no
longer considered to be a bonafide genotoxic effect as reflected in the
recent decision by the OECD to delete the test guidelines for this assay
Du et al. (2005) reported the formation of DNA adducts in male mouse
lung, liver and kidney tissue following administration of14C-labeled
MTBE by oral gavage at doses ranging from 0.95 to 75.59 µg/kg body
weight. The authors used the ultrasensitive accelerator mass
spectrometry (AMS) technique which has a sensitivity to identify 1
adduct in a trillion nucleotides. The authors did not, however, compare
the adducts with synthetic standards and hence it is not possible to
ascertain whether the radioactivity identified in the DNA samples was in
fact due to DNA adduct formation and not due to metabolic incorporation
of14C into DNA through cellular carbon pools fueled with14C originating
from 14C-MTBE metabolism (e.g. formation of14C-formate).
As mandated in an ECHA Substance Evaluation decision, a Transgenic
Rodent Somatic and Germ Cell Gene Mutation assay conducted by
BioReliance (2018) is also available. This study was performed in
accordance with OECD Test Guideline 488 for MTBE whole body vapor
inhalation exposures administered for 6 hours per day for 28 consecutive
days to Fischer 344 Big Blue® homozygous transgenic rats. In general the
MTBE vapor concentrations were well tolerated by rats up to a nominal
exposure of 3000 ppm with some evidence of minor treatment related
After the 28 day exposure period, the rats were sacrificed and selected
tissues (bone marrow, liver, kidney and nasal epithelium) were harvested
to obtain sufficient DNA to permit determination and calculations of
The treatment with MTBE did not cause statistically elevated mutant
frequencies at the cII gene in these tissues in the Big Blue®
rat. As the results in the somatic cells were negative, the mutation
frequencies in germ cells were not assessed as detailed in the ECHA
A positive control group was also included which produced statistically
significant increases in mutation frequencies which demonstrated the
ability of the test system to detect and quantify mutants following
exposure to a known mutagen.
Weng et al. (2011, 2012,2013) reported a series of studies on ethyl
tertiary butyl ether (ETBE), Although these studies are of questionable
relevance to MTBE, they are briefly reviewed here. There was some
evidence in these studies that Aldh2-/- mice are slightly more sensitive
to ETBE-exposure-related genotoxicity than Aldh2+/+ mice and males are
slightly more responsive than females. However, the magnitude of effects
observed was small, often less than 2-fold, with no remarkable dose
response.The blood analyses do not support internal acetaldehyde
exposure as the only or most important factor in the sensitivity
differences between genotypes. The presentation of data in these papers
is at times confusing and there are questions regarding the validity of
some of the comparisons (e.g., comparing responses to treatments in one
genotype with controls in another; the apparent combining of data in
experiments conducted two years apart). As mentioned earlier, the
genotoxic responses observed in Aldh2+/+ and Aldh2 -/- mice were minor
and are of questionable biological relevance.
of MTBE has been extensively evaluated in vitro and in vivo using
multiple endpoints and the weight of evidence strongly favors the
assessment that MTBE does not pose a mutagenic hazard.
Borghoff SJ, Parkinson H and Leavens TL (2010). Physiologically based
pharmacokinetic rat model for methyl tertiary-butyl ether; Comparison of
selected dose metrics following various MTBE exposure scenarios used for
toxicity and carcinogenicity evaluation. Toxicology, 275, 79-91.
Casanova M and d. A. Heck H (1997). Lack of Evidence for the Involvement
of Formaldehyde in the Hepatocarcinogenicity of Methyl-t-Butyl Ether
(MTBE). Chemico-Biological Interactions 105, 131-143.
Charles GD, Spencer PJ, Schisler MR, Cifone M, Budinsky RA and Gollapudi
BB (2005). Mode of mutagenic action for the biocide Bioban CS-1246 in
mouse lymphoma cells and implications for its in vivo mutagenic
potential. Toxicol Sci. 84, 73-80.
Chen CS, Hseu YC, Liang SH, Kuo JY and Chen SC (2008). Assessment of
genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene,
and xylene to human lymphocytes using comet assay. J Hazard Mater. 153,
Du HF, Xu LH, Wang HF, Liu YF, Tang XY, Liu KX and Peng SX (2005).
Formation of MTBE-DNA adducts in mice measured with accelerator mass
spectrometry. Environ Toxicol. 20, 397-401.
McGregor D (2006). Methyl tertiary-butyl ether: studies for potential
human health hazards. Crit. Rev. Toxicol. 36, 319-358.
Miller MJ, Ferdinandi ES, Klan M, Andrews LS, Douglas JF and Kneiss
JJ (1997). Pharmacokinetics and disposition of methyl t-butyl ether in
Fischer-344 rats. J Appl. Toxicol. 17(S1), S3-S12.
Schreiner CA, Hoffman GM, Gudi R and Clark CR (2014). Health assessment
of gasoline and fuel oxygenate vapors: micronucleus and sister chromatid
exchange evaluations. Regul Toxicol Pharmacol. 70, S29-34.
Speit G, Kojima H, Burlinson B, Collins AR, Kasper P, Plappert-Helbig
U, Uno Y, Vasquez M, Beevers C, De Boeck M, Escobar PA, Kitamoto S, Pant
K, Pfuhler S, Tanaka J and Levy DD (2015). Critical issues with the in
vivo comet assay: A report of the comet assay working group in the 6th
International Workshop on Genotoxicity Testing (IWGT). Mutat Res Genet
Toxicol Environ Mutagen. 783, 6-12.
Tang G, Wang J, Zhuang Z (1997). [Cytotoxicity and genotoxicity of
methyl tert-butyl ether and its metabolite to human leukemia cells].
Zhonghua Yu Fang Yi Xue Za Zhi. 31, 334-337. Chinese.
Ward JB, Dalker DH, Hastings DA, Ammenhauser MM and Legator MS. 1995.
Assessment of the Mutagenicity of Methyl Tertiary Butyl Ether at
thehprtGene in CD-1 Mice. Annual Meeting of the Society of Toxicology.
Abstract No. 417.
Weng Z, Suda M, Ohtani K, Mei N, Kawamoto T, Nakajima T and Wang R-S
(2011). Aldh2 knockout mice were more sensitive to DNA damage in
leukocytes due to ethyl tertiary butyl ether exposure. Indust. Hlth.,
Weng Z, Suda M, Ohtani K, Mei N, Kawamoto T, Nakajima T and Wang R-S
(2012). Differential genotoxic effects of subchronic exposure to ethyl
tertiary butyl ether in the livers of Aldh2 knockout and wild-type mice.
Arch. Toxicol. 86, 675-682.
Weng Z, Suda M, Ohtani K, Mei N, Kawamoto T, Nakajima T and Wang RS
(2013). Subchronic exposure to ethyl tertiary butyl ether resulting in
genetic damage in Aldh2 knockout mice. Toxicology 311, 107-114.
Yang H, Kong L and Zhao JS (2005). [DNA damage induced by methyl
tertiary-butyl ether in vivo and in vitro]. Zhonghua Lao Dong Wei Sheng
Zhi Ye Bing Za Zhi. 5, 362-365. Chinese.
the available information, MTBE is considered to be not mutagenic.
In accordance with EU Classification, Labelling and Packaging of
Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008,
classification as a mutagen is not necessary.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
Welcome to the ECHA website. This site is not fully supported in Internet Explorer 7 (and earlier versions). Please upgrade your Internet Explorer to a newer version.
Do not show this message again