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* Skip to Article Content * Skip to Article Information Search withinThis JournalAnywhere * Search term Advanced Search Citation Search * Search term Advanced Search Citation Search Login / Register * Individual login * Institutional login * REGISTER Environmental and Molecular Mutagenesis Early View RESEARCH ARTICLE Open Access MUTAGENICITY EVALUATION OF METHYL TERTIARY- BUTYL ETHER IN MULTIPLE TISSUES OF TRANSGENIC RATS FOLLOWING WHOLE BODY INHALATION EXPOSURE B. Bhaskar Gollapudi, B. Bhaskar Gollapudi Toxicology Consulting, Midland, Michigan, USA Search for more papers by this author Erik K. Rushton, Corresponding Author Erik K. Rushton * erik.rushton@lyb.com LyondellBasell, Rotterdam, The Netherlands Correspondence Erik K. Rushton, LyondellBasell, Delftseplein 27E, 3013 AA Rotterdam, The Netherlands. Email: erik.rushton@lyb.com Search for more papers by this author B. Bhaskar Gollapudi, B. Bhaskar Gollapudi Toxicology Consulting, Midland, Michigan, USA Search for more papers by this author Erik K. Rushton, Corresponding Author Erik K. Rushton * erik.rushton@lyb.com LyondellBasell, Rotterdam, The Netherlands Correspondence Erik K. Rushton, LyondellBasell, Delftseplein 27E, 3013 AA Rotterdam, The Netherlands. Email: erik.rushton@lyb.com Search for more papers by this author First published: 16 July 2024 https://doi.org/10.1002/em.22616 Accepted by: B. Engelward About * REFERENCES * RELATED * INFORMATION * PDF Sections * Abstract * 1 INTRODUCTION * 2 MATERIALS AND METHODS * 3 RESULTS * 4 DISCUSSION * AUTHOR CONTRIBUTIONS * ACKNOWLEDGMENTS * CONFLICT OF INTEREST STATEMENT * Open Research * REFERENCES PDF Tools * Request permission * Export citation * Add to favorites * Track citation ShareShare Give access Share full text access Close modal Share full-text access Please review our Terms and Conditions of Use and check box below to share full-text version of article. I have read and accept the Wiley Online Library Terms and Conditions of Use -------------------------------------------------------------------------------- Shareable Link Use the link below to share a full-text version of this article with your friends and colleagues. Learn more. Copy URL Share a link Share on * Email * Facebook * Twitter * LinkedIn * Reddit * Wechat ABSTRACT Methyl tertiary-butyl ether (MTBE) is used as a component of motor vehicle fuel to enhance combustion efficiency and to reduce emissions of carbon monoxide and nitrogen oxides. Although MTBE was largely negative in the in vitro and in vivo genotoxicity studies, isolated reports of positive findings along with the observation of tumors in the rat cancer bioassays raised concern for its in vivo mutagenic potential. To investigate this, transgenic male Big Blue Fischer 344 rats were exposed to 0 (negative control), 400, 1000, and 3000 ppm MTBE via whole body inhalation for 28 consecutive days, 6 h/day. Mutant frequencies (MF) at the cII locus of the transgene in the nasal epithelium (portal of entry tissue), liver (site of primary metabolism), bone marrow (rapidly proliferating tissue), and kidney (tumor target) were analyzed (5 rats/exposure group) following a 3-day post-exposure manifestation period. MTBE did not induce a mutagenic response in any of the tissues investigated. The adequacy of the experimental conditions to detect induced mutations was confirmed by utilizing tissue samples from animals treated with the known mutagen ethyl nitrosourea. These data provide support to the conclusion that MTBE is not an in vivo mutagen and male rat kidney tumors are not likely the result of a mutagenic mode of action. 1 INTRODUCTION Methyl tertiary-butyl ether (MTBE; CAS: 1634-04-4) is primarily used as an oxygenated fuel component to enhance the combustion of motor vehicle fuels and reduce the emission of carbon monoxide and nitrogen oxides. Although using MTBE was largely eliminated in the USA, its use continues in Asia Pacific and Europe. The primary route of potential human exposure to MTBE is via inhalation of gasoline vapors. McGregor (2006), Bogen and Heilman (2015), and Bus et al. (2022) reviewed the genetic toxicology literature on MTBE. The available data suggests that MTBE is not likely to pose an in vivo genotoxic hazard/risk. MTBE did not induce mutations in bacteria or cytogenetic damage in mammalian cell cultures. No mutagenicity was observed at the Hprt locus of Chinese hamster V79 cells. In vivo, MTBE did not induce cytogenetic damage in rodent bone marrow cells, UDS in the mouse liver, or mutations at the Hprt locus of the mouse splenic lymphocytes. However, there have been isolated positive findings both in vitro and in vivo such as the increased DNA damage reported in a human cell line (Tang et al., 1997), mutagenicity in the in vitro mouse lymphoma assay (Mackerer et al., 1996), and DNA adducts in the mouse liver, lung, and kidneys following oral gavage administration of MTBE (Du et al., 2005). In inhalation cancer bioassay, MTBE has been shown to increase male rat kidney and testicular (Leydig cell) tumors and liver adenomas in female mice (Bird et al., 1997). The in vivo mutagenic potential for MTBE contributing to the tumors observed in the animal studies continued to be of interest given the isolated positive genotoxicity findings in short-term genotoxicity assays. In the studies reported here, the mutagenicity of MTBE was investigated in several tissues (including a tumor target tissue) of the transgenic Big Blue rats using a relevant route of exposure (i.e., inhalation). 2 MATERIALS AND METHODS 2.1 TEST SUBSTANCE MTBE (Source: Sigma-Aldrich, St. Lous, MO, USA; Batch: SHBH0119V) was characterized under GLP and found to be 99.99% pure. The test material was kept under inert gas (nitrogen) in a room set to maintain 18°C to 24°C. 2.2 TEST ANIMALS The study was conducted in an AAALAC-accredited animal facility and with a protocol approved by the laboratory's Institution Animal Care and Use Committee. Nine-week-old, male transgenic Fischer 344 Big Blue® rats (TgF344) were obtained from Taconic Biosciences (Germantown, NY, USA). Animals were individually identified using a subcutaneously implanted electronic chip and housed in rooms with target temperatures of 68 °F to 78 °F, relative humidity of 30% to 70%, ≥10 fresh air changes/hour, and a 12-h light/12-h dark cycle. The animals were provided Certified Rodent LabDiet 5002 (PMI Nutrition International, LLC) and reverse osmosis and ultraviolet irradiation treated municipal water ad libitum except during inhalation exposures. Animals (N = 6) were assigned to various groups by a stratified randomization scheme designed to achieve similar group mean body weights. 2.3 EXPOSURE LEVELS The highest concentration of 3000 ppm used in this study represented a concentration where significant increases in renal and testicular tumors were observed in male Fischer 344 rats in a 24-month inhalation study (6 h/day, 5 days/week; Bird et al., 1997). In the above study, the incidences of renal tubular cell tumors were 2%, 0%, 16%, and 6% and that of interstitial adenomas of the testes were 64%, 70%, 82%, and 94% at exposure concentrations of 0, 400, 3000, and 8000 ppm, respectively. The intermediate concentration of 1000 ppm used in the Big Blue® rat study represented an inflection point where a change from linear to non-linear metabolism was predicted to occur based on the PBPK modeling (Borghoff et al., 2010; Bus et al., 2022; Leavens & Borghoff, 2009). The lowest concentration of 400 ppm used in the study was the no effect level for tumors in the inhalation carcinogenicity study. Negative controls (0 ppm) were exposed to filtered air. DNA extracted from frozen tissues of a previously conducted study where Big Blue® rats were treated by oral gavage with ethyl nitrosourea (ENU; 20 mg/kg/day) on Days 1, 2, 3, 12, 19 and 26 with necropsy on Day 31 served as a concurrent positive control group. 2.4 EXPOSURE METHODS MTBE was administered via whole-body inhalation for 28 consecutive days, 6 h/day. Exposures were conducted using approximately 66-L polycarbonate whole-body exposure chambers. Air supplied to the chambers was breathing quality, HEPA- and charcoal-filtered, temperature- and humidity-controlled air source. In-house compressed nitrogen was used for exposure atmosphere generation. Food and water were withheld during the animal exposure periods. The mean temperature and relative humidity of the exposure atmospheres were maintained between 20°C to 26°C and 30% to 70%, respectively. The oxygen content of the exposure atmospheres was measured during the method development phase and found to be 20.9% for all groups. Compressed nitrogen was delivered to Chamber 1 (0 ppm) at a flow rate of approximately 460 mL/min to approximate the maximum amount of nitrogen added to Chamber 4 (3000 ppm). Nitrogen was mixed with air at the exposure chamber inlet prior to entering the chamber. MTBE vapors were generated using a single gas washing bottle (GWB) filled with an appropriate amount of liquid test substance for Chambers 2 (400 ppm), 3 (1000 ppm), and 4 (3000 ppm). Compressed nitrogen was delivered to the GWB using a regulator and controlled using a flowmeter. Bubbling action through the GWB produced concentrated test substance vapors. Test substance vapors flowed from the GWB to a stainless-steel manifold where vapors were distributed to individual chambers. The concentrated vapors of the test substance were diluted to the desired exposure concentrations by mixing with the air prior to entering each chamber. Exposure atmospheres were sampled and analyzed approximately every 45 min using a gas chromatograph (GC) with a flame ionization detector. The spatial homogeneity of the MTBE concentrations within each exposure chamber was evaluated and confirmed during the method development phase of the study. 2.5 ANIMAL OBSERVATIONS Study animals were examined for mortality/viability, detailed clinical observations, and cage-side observations. Body weight and food consumption data were collected prior to exposure and at approximately weekly intervals during the study. 2.6 TISSUE COLLECTION Animals were necropsied 3 days after the final exposure to collect: (a) nasal epithelium from the right and left nasal passages to represent the portal of entry tissue; (b) liver representing the site of primary metabolism; (c) right and left femoral bone marrow representing a rapidly proliferating tissue; and (d) right and left kidneys representing tumor target tissue. Tissue samples were flash-frozen in liquid nitrogen and stored at approximately −70°C until the extraction of DNA. 2.7 EXTRACTION AND PACKAGING OF GENOMIC DNA Details of the methods were described in Gollapudi and Rushton (2023) with the exception that DNA from nasal epithelium was extracted following the method described in Young et al. (2015). DNA was extracted from the frozen tissues of the first 5 animals in each group using the RecoverEase DNA isolation kit (Agilent Technologies, Santa Clara, CA; liver, bone marrow, and kidney) or phenol: chloroform extraction method (nasal epithelium). DNA was packaged (Packaging Reaction Mix, New York University, New York, NY), and Agilent instruction manuals for transgenic shuttle vector recovery. The packaged phage was adsorbed onto E. coli G1250 suspension cultures, molten top agar added, and plated onto bottom agar plates. The titers were determined after incubating the plates overnight at 37 ± 1.0°C and the cII mutant selection plates were scored following incubation for 2 days at 24 ± 0.5°C. 2.8 STATISTICAL EVALUATION Body weight, food consumption, and organ weight data were analyzed by parametric ANOVA followed by Dunnett's test for pair-wise comparisons. Log10 transformed mutant frequency data were compared by one-way ANOVA followed by Dunnett's test for pair-wise comparisons if the data were normally distributed with equal variance as confirmed by the Ryan-Joiner test. If the data were not normally distributed, the non-parametric Kruskal-Wallis test followed by the Mann–Whitney test for pair-wise comparisons was employed. The test substance was considered positive if it induced a statistically significant increase in the frequency of cII mutants at any dose level, and the frequency in the treated group was outside the 95% control limits of the historical background mutant frequency. 3 RESULTS 3.1 CHAMBER CONCENTRATIONS The rationale used for the selection of the exposure levels of MTBE used in the study is described in Section 2.3. The mean analytically determined concentrations of MTBE in the chambers over the 28-day exposure period were 0, 400 (±18.3), 992 (±27.3), and 2977 (±77.7) ppm for groups targeted to receive 0 (filtered air), 400, 1000, and 3000 ppm, respectively. 3.2 IN-LIFE OBSERVATIONS There were no mortalities during the study in any of the treatment groups. Test substance-related clinical observations were limited to the 3000-ppm group and included dried red material around the left and right eye, dried red nasal discharge, red discharge from left and right eyes, dried brown material around the anogenital area, partially closed right eye, and enophthalmos of the right eye. Most observations were noted in single animals on 1 day only with no pattern except for one male that had multiple observations of dried red material around the eyes and/or nose on multiple (5) days; however, similar observations were also reported in this animal during the pre-study acclimation period. Absolute body weights were not significantly affected by MTBE administration during the study. A single observation of significantly higher mean cumulative body weight gain in the 3000-ppm group from Day 1 to 22 (55 g vs. 45 g in the in-air control) was not considered test substance-related because no such change was observed in this group at other intervals (Day 1 to 8, 15, 18 or 31; data not shown). There was no significant effect of treatment on food consumption. 3.3 CII MUTANT FREQUENCIES Data on mutant frequencies in various tissues following exposure to MTBE and the positive control ENU are presented in Tables 1–4. Laboratory historical vehicle and positive control data are shown in Table 5. TABLE 1. Mutant frequency (MF) in nasal epithelium of Big Blue rats exposed to MTBE. Group Animal No. No. of Packagings No. of phage screened No. of mutants cII MF (x 10−6) Mean MF (±SD) (×10−6) 0 ppm MTBE 7721 3 317,122 15 47.3 49.5 ± 14.2 7731 4 379,014 17 44.9 7732 2 371,215 12 32.3 7735 2 193,733 10 51.6 7736 3 252,468 18 71.3 400 ppm MTBE 7706 2 209,854 10 47.7 34.7 ± 9.3 7708 2 370,756 14 37.8 7711 3 461,848 17 36.8 7712 2 353,253 9 25.5 7720 2 385,328 10 26.0 1000 ppm MTBE 7707 2 429,923 13 30.2 21.9 ± 6.7 7724 2 358,411 10 27.9 7725 2 431,030 8 18.6 7728 2 642,355 11 17.1 7733 2 320,527 5 15.6 3000 ppm MTBE 7718 2 348,195 9 25.8 34.2 ± 28.8 7722 2 217,516 6 27.6 7723 4 306,726 26 84.8 7726 2 262,636 5 19.0 7727 2 368,627 5 13.6 Positive Control (ENU) 9807 3 343,513 59 171.8 171.2 ± 29.9* 9808 2 360,114 60 166.6 9809 3 493,279 108 218.9 9810 2 395,444 54 136.6 9811 3 289,879 47 162.1 * * Statistically significant (1-Way ANOVA, p < .001) vs. 0 ppm. TABLE 2. Mutant frequency (MF) in bone marrow of Big Blue rats exposed to MTBE. Group Animal No. No. of Packagings No. of phage screened No. of mutants cII MF (x 10−6) Mean MF (±SD) (×10−6) 0 ppm MTBE 7721 2 476,583 8 16.8 34.7 ± 21.8 7731 2 445,132 11 24.7 7732 2 332,526 8 24.1 7735 2 153,499 11 71.7 7736 2 221,739 8 36.1 400 ppm MTBE 7706 2 567,628 14 24.7 31.9 ± 10.9 7708 2 266,473 7 26.3 7711 2 438,046 15 34.2 7712 3 461,044 23 49.9 7720 3 330,433 8 24.2 1000 ppm MTBE 7707 2 731,283 14 19.7 26.4 ± 9.2 7724 2 402,520 13 25.4 7725 2 571,899 7 15.5 7728 2 513,800 9 34.7 7733 2 364,798 12 36.6 3000 ppm MTBE 7718 3 714,521 17 23.8 34.0 ± 11.7 7722 3 964,889 23 23.8 7723 2 224,295 11 49.0 7726 2 337,848 10 29.6 7727 2 321,501 14 43.5 Positive Control (ENU) 9807 2 507,348 244 480.9 650.3 ± 261.4* 9808 2 310,805 154 495.5 9809 2 463,641 199 429.2 9810 2 136,816 141 1030.6 9811 2 184,014 150 815.2 * * Statistically significant (1-Way ANOVA, p < .001) vs. 0 ppm. TABLE 3. Mutant frequency (MF) in livers of Big Blue rats exposed to MTBE. Group Animal No. No. of Packagings No. of phage screened No. of mutants cII MF (×10−6) Mean MF (±SD) (×10−6) 0 ppm MTBE 7721 3 325,369 27 83.0 63.3 ± 28.4 7731 3 223,926 23 102.7 7732 3 251,297 12 47.8 7735 2 287,820 14 48.6 7736 2 232,651 8 34.4 400 ppm MTBE 7706 3 348,719 23 66.0 49.1 ± 11.7 7708 2 203,142 10 49.2 7711 2 205,023 11 53.7 7712 2 277,556 10 36.0 7720 2 296,373 12 40.5 1000 ppm MTBE 7707 2 509,351 22 43.2 37.7 ± 16.7 7724 3 246,507 14 56.8 7725 4 331,527 14 42.2 7728 2 431,516 15 34.8 7733 2 354,108 4 11.3 3000 ppm MTBE 7718 2 360,694 9 25.0 35.2 ± 9.1 7722 2 232,651 9 38.7 7723 2 233,506 9 38.5 7726 2 235,217 11 46.8 7727 2 259,166 7 27.0 Positive Control (ENU) 9807 3 316,987 47 148.3 139.5 ± 22.1* 9808 2 227,091 39 171.7 9809 2 180,903 24 132.7 9810 2 231,795 26 112.2 9811 2 211,267 28 132.5 * * Statistically significant (1-Way ANOVA, p < .001) vs. 0 ppm. TABLE 4. Mutant frequency (MF) in kidneys of Big Blue rats exposed to MTBE. Group Animal No. No. of Packagings No. of phage screened No. of mutants cII MF (×10−6) Mean MF (±SD) (×10−6) 0 ppm MTBE 7721 2 294,235 6 20.4 28.9 ± 21.5 7731 2 321,178 7 21.8 7732 2 409,705 10 24.4 7735 2 789,045 9 11.4 7736 2 783,058 52 66.4 400 ppm MTBE 7706 2 391,315 10 25.6 22.1 ± 8.0 7708 2 421,679 10 23.7 7711 2 434,509 14 32.2 7712 2 894,679 16 17.9 7720 2 546,130 6 11.0 1000 ppm MTBE 7707 2 241,204 4 16.6 24.8 ± 14.0 7724 2 471,716 7 14.8 7725 2 435,792 10 22.9 7728 2 386,097 19 49.2 7733 2 637,651 13 20.4 3000 ppm MTBE 7718 2 523,464 16 30.6 24.1 ± 8.7 7722 2 558,533 20 35.8 7723 2 460,169 7 15.2 7726 2 526,885 11 20.9 7727 2 941,294 17 18.1 Positive Control (ENU) 9807 2 812,567 117 144.0 126.4 ± 22.5* 9808 2 500,798 78 155.8 9809 2 951,131 114 119.9 9810 2 849,774 89 104.7 9811 2 965,671 104 107.7 * * Statistically significant (Kruskal-Wallis, p = .009) vs. 0 ppm. TABLE 5. Laboratory historical Big Blue rat cII mutant frequency (×10−6). Nasal epithelium Bone marrow Liver Kidney Individuals Studies Individuals Studies Individuals Studies Individuals Studies Vechicle controla Mean 24.8 24.7 30.0 29.9 48.3 47.8 30.1 30.8 SD 13.1 6.4 12.9 5.9 22.2 14.2 12.9 11.0 95% Control limits 0.0–51.0 11.9–37.5 4.2–55.8 18.1–41.7 3.9–92.7 19.4–76.2 4.3–55.9 8.8–52.8 Range 7.3–61.7 18.9–33.3 12.3–66.1 19.3–38.0 16.5–120.2 29.7–76.2 12.7–67.1 20.9–48.1 Positive control: 20 mg/kg/day N-ETHYL-N-NITROSOUREA (ENU)b Mean 24.8 24.7 30.0 29.9 48.3 47.8 30.1 30.8 SD 13.1 6.4 12.9 5.9 22.2 14.2 12.9 11.0 95% Control limits 0.0–51.0 11.9–37.5 4.2–55.8 18.1–41.7 3.9–92.7 19.4–76.2 4.3–55.9 8.8–52.8 Range 7.3–61.7 18.9–33.3 12.3–66.1 19.3–38.0 16.5–120.2 29.7–76.2 12.7–67.1 20.9–48.1 * a Vehicle control data; pooled vehicle controls; dosed for 28 days and necropsied on Day 31 (relative to Day 1, first exposure). * b Bone marrow and liver: pooled data from animals dosed on Days 1, 2 and 3 and on Days 1, 2, 3, 12, 19 and 26 and necropsied on Day 31; nasal epithelium and kidney: data from animals dosed on Days 1, 2 and 3 and necropsied on Day 31. Nasal Epithelium (Table 1): The mean mutant frequency of the negative control group (49.5 × 10−6) was slightly outside the upper 95% historical control limit of 37.5 × 10−6 for nasal epithelium. This was attributed to the relatively small historical database (N = 34) which may not reflect the full range of animal-to-animal variation as seen with the other somatic tissues with larger historical databases. Mean cII mutant frequencies in the nasal epithelium from MTBE-exposed groups (21.9 × 10−6 to 34.7 × 10−6) were not significantly different from the negative control group. One animal (# 7723) in the 3000-ppm group did have a mutant frequency value above the 95% control limit of the historical negative control distribution, but this was again attributable to the relatively small historical database. ENU treated group demonstrated significantly higher mutant frequency (171.2 ± 29.9 × 10−6) compared to the concurrent negative control value. Bone Marrow (Table 2): The mutant frequency in the air control of 34.7 ± 21.8 × 10−6 was comparable to the historical experience of 30.0 ± 12.9 × 10−6 for this tissue. Among the MTBE-exposed groups, the mutant frequencies were not significantly different from the air control and ranged from 26.4 ± 9.2 × 10−6 to 34.0 ± 11.7 × 10−6. Individual animal frequencies of MTBE-exposed animals were within the 95% control limits of the historical vehicle control animals, with a single exception in filtered air control animal 7735 with a mutant frequency that slightly exceeded historical limits. ENU treated animals demonstrated a mean mutant frequency of 650.3 ± 261.4 × 10−6 which is significantly higher than the filtered air group. Liver (Table 3): For the liver, the mutant frequencies in the filtered-air control group of 63.3 ± 28.4 × 10−6 was within the 95% control limit for group mean mutant frequency for the liver. Mean cII mutant frequencies in MTBE-exposed groups (35.2 ± 9.1 × 10−6 to 49.1 ± 11.7 × 10−6) were not significantly different from the control value. Individual animal mutant frequencies were within the 95% control limit except for a filtered air animal having a frequency slightly above the 95% control limit. Results from liver DNA of ENU treated animals demonstrated a mean mutant frequency of 139.5 ± 22.1 × 10−6 which was significantly different from the corresponding control group. Kidney (Table 4): The filtered-air control mutant frequency of 28.9 ± 21.5 × 10−6 was consistent with the historical experience of 30.1 ± 12.9 × 10−6 for kidney. Mean cII mutant frequencies in MTBE-exposed rats of 22.1 ± 8.0 × 10−6, 24.8 ± 14.0 × 10−6, and 24.1 ± 8.7 × 10−6 in groups exposed to 400, 1000, and 3000 ppm, respectively, were not significantly different from the control mean. Individual animal mutant frequencies in the MTBE-exposed groups were within the historical control limits apart from a filtered air control animal (#7736) that had a frequency slightly above the 95% Control Limit. ENU-treated animals demonstrated a mean mutant frequency of 126.4 ± 22.5 × 10−6 which was significantly higher than the concurrent negative control value. 4 DISCUSSION In this study, MTBE was investigated for its potential to induce gene mutations in multiple tissues of transgenic male Big Blue Fischer 344 rats following whole body inhalation exposure for 28 consecutive days. The tissues investigated included the portal of entry tissue (nasal epithelium), the primary site of metabolism (liver), a rapidly proliferating tissue (bone marrow), and a tumor target tissue (kidney). The bioavailability of inhaled MTBE was assured in this study based on previous metabolism and kinetic studies conducted in Fischer 344 (Miller et al., 1997) and Wistar rats (Savolainen et al., 1985) demonstrating rapid (within 2 h) increase in steady-state plasma concentrations of MTBE following inhalation exposure. The highest concentration of MTBE used in the study (3000 ppm) was anchored to the exposure level that induced the maximum incidence of renal tumors in the inhalation cancer bioassay. This concentration was also substantially higher than the exposures that caused saturation of MTBE metabolism whereas the intermediate exposure level of 1000 ppm represented the predicted inflection point for metabolic saturation based on the physiologically based pharmacokinetic (PBPK) models (Borghoff et al., 2010; Leavens & Borghoff, 2009). The exposure levels evaluated in the study and the resulting expected blood concentrations are conservatively 3-5 orders of magnitude higher than those encountered in various human exposure scenarios (Bus et al., 2022). Results from this study showed that MTBE was not mutagenic in any of the examined tissues. In vitro, MTBE is not a bacterial mutagen in the Ames test, did not induce gene conversion in Saccharomyces cerevisiae D4, negative for the induction of UDS in rat hepatocytes, non-mutagenic at the Hprt locus of V79 cells, and did not induce micronuclei (tested only without S9) in mouse NIH/3 T3 cells (McGregor, 2006). In vivo, MTBE did not induce sex-linked recessive lethal mutations in Drosophila melanogaster, UDS in mouse hepatocytes, gene mutations at the Hprt locus of mouse splenic lymphocytes, micronuclei in mouse bone marrow or chromosomal aberrations in the rat bone marrow (McGregor, 2006). Recently, Bus et al. (2022) critically examined the positive findings reported by Du et al. (2005) for DNA adduct formation in various lung tissues following oral gavage administration of 14C-labeled MTBE. The accelerated mass spectroscopy methodology used to detect the adducts in the above study has certain limitations in distinguishing labeled DNA adducts from metabolic incorporation of the label into DNA through cellular carbon pools since comparisons with synthetic standards were not performed by Du et al. Results from the transgenic mutation assays reported here contribute important data indicating that MTBE is not an in vivo mutagen in any of the somatic tissues interrogated. Results from this study also support the conclusion that the male rat kidney tumors observed in the inhalation carcinogenicity study (Bird et al., 1997) are not mediated through a mode of action involving mutagenicity as an early key event. Several investigators suggested that these tumors are mediated through male rat specific α2u-globulin nephropathy, which is further exacerbated by rodent-specific chronic progressive nephropathy (reviewed in Bus et al., 2022). This mode of action for male rat-specific kidney tumors is not considered to be qualitatively relevant to humans (Hard et al., 2009, 2013). In conclusion, results from this investigation add support to the overall assessment that MTBE is not an in vivo mutagen, and thus, mutagenicity can be reasonably excluded as the initiating event in the etiology of the rodent tumors observed in the cancer bioassays on this substance. A potential limitation of the current study design may relate to the use of relatively younger rats (a standard practice for these types of studies) and thus any age-related susceptibility differences to mutagenic effects, if they exist, are not addressed by this study. AUTHOR CONTRIBUTIONS BG designed the study; BG and ER prepared the manuscript. ACKNOWLEDGMENTS The authors acknowledge the technical contributions of J. T. Weinberg of CRL Laboratories, Ashland, OH (USA) for the in-life portion of the study and Mr. R. R. Young and Joan Huynh of BioReliance Corporation, Rockville, MD (USA) for the analysis of tissues for mutations. The authors also acknowledge the contribution of Neslihan Aygun Kocabas in reviewing the document. CONFLICT OF INTEREST STATEMENT The study was sponsored and funded by ReachCentrum SA on behalf of the Fuel Ether REACH Consortium(FERC), Avenue E. van Nieuwenhuyse 6, 1160 Brussels, Belgium. Funding for the preparation of the manuscript was provided by LyondellBasell, Delftseplein 27E, 3013 AA Rotterdam, The Netherlands. ER is employed by LyondellBasell, a manufacturer of MTBE. 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Environmental and Molecular Mutagenesis, 56(7), 629–636. 10.1002/em.21951 CASPubMedWeb of Science®Google Scholar Early View Online Version of Record before inclusion in an issue * REFERENCES * RELATED * INFORMATION RECOMMENDED * Investigation of the potential mutagenicity of ethyl tertiary‐butyl ether in the tumor target tissue using transgenic Big Blue Fischer 344 rats following whole body inhalation exposure B. Bhaskar Gollapudi, Erik K. Rushton, Environmental and Molecular Mutagenesis * Toxicity of methyl tertiary‐butyl ether (MTBE) following exposure of Wistar Rats for 13 weeks or one year via drinking water Edilberto Bermudez, Gabrielle Willson, Horace Parkinson, Darol Dodd, Journal of Applied Toxicology * Methyl tert Butyl Ether Systemic Toxicity John J. Clary, Risk Analysis * Assessing the genotoxicity of N-nitrosodiethylamine with three in vivo endpoints in male Big Blue® transgenic and wild-type C57BL/6N mice Shaofei Zhang, Stephanie L. Coffing, William C. Gunther, Michael L. Homiski, Richard A. Spellman, Phu Van, Maik Schuler, Environmental and Molecular Mutagenesis * Within‐laboratory reproducibility of Ames test results: Are repeat tests necessary? Errol Zeiger, Constance A. Mitchell, Stefan Pfuhler, Yang Liao, Kristine L. Witt, Environmental and Molecular Mutagenesis METRICS Full text views:138 Full text views and downloads on Wiley Online Library. More metric information DETAILS © 2024 The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society. 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