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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 negative.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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 (http://www.oecd-ilibrary.org). 

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 effects.

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 mutation frequencies.

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 decision.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Mutagenicity 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. 

References:

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, 351-356.

 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., 49, 396–399.

 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.


Justification for classification or non-classification

Based on 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.