tert-butyl methyl ether

Carcinogenicity

Description of key information

MTBE produces tumours in mice and rats at concentrations ≥ 3000 ppm (10710 mg/m3) after inhalation exposure. There is no evidence of a direct genotoxic mode of action as MTBE is not genotoxic in vitro or in vivo. Furthermore, the treatment relation of the occurred tumours is equivocal in some cases, other types are not relevant for humans and for some the human relevance was questionable. Moreover, the tumours appear mostly at high and systemically toxic concentrations.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well reported modern guideline study to GLP. Original study report available

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic (Germantown, NY)
- Age at study initiation: 7-8 weeks
- Housing: individually in polycarbonate cages.
- Diet: NTP2000 ad libitum.
- Water (ad libitum): reverse osmosis purified.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16h April 2007 To: 24th April 2009.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Fresh solutions were prepared weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Every fourth week the made up solutions were checked by GC headspace analysis.

Duration of treatment / exposure:

743 days

Frequency of treatment:

Continuous

Remarks:

Doses / Concentrations:
Males: 0.5, 3.0. 7.5mg/l in drinking water. Females: 0.5, 3.0. 15mg/l in drinking water
Basis:
nominal in water

Remarks:

Doses / Concentrations:
Males: 0.50 (0.04), 3.00 (0.29), 7.44 (0.45) mg/l in drinking water (SD in brackets). Females: 0.50 (0.04), 3.00 (0.29), 14.96 (0.79) mg/l in drinking water (SD in brackets)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
Males: 25 (11), 140 (63), 330 (139) mg/kg bw/day in drinking water (SD in brackets).
Females: 49 (14), 232 (66), 1042(280) mg/kg bw/day in drinking water (SD in brackets).
Basis:actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on 13 week range finder and results from other published cancer studies.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, including weekends and holidays.
- Cage side observations checked: mortality and overt signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at start, weekly for 17 weeks then biweekly and at study end.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: at start, weekly for 17 weeks then biweekly and at study end.
- Final data normalised to bodyweight.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Gravimetrically per animal on a weekly basis for 17 weeks then monthly.

OTHER: Cage air was sampled for MTBE to ensure that no significant inhalation exposure occured through evaporation from the drinking water.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: Heart, Spleen, Liver, Kidneys, Ovaries, Epididymides, Testes, Brain all weighed.
HISTOPATHOLOGY: Yes:
- Cardiovascular/hematopoetic tissue: Aorta, Heart, Representative Lymph nodes, Spleen, Bone marrow-femur.
- Respiratory tissue: Larynx, Lungs/Bronchi, Pharynx, Trachea, Nose.
- Digestive system: Cecum, Colon, Duodenum, Esophagus, Ileum, Jejenum, Liver, Pancreas, Rectum, Stomach (fore and glandular), Salivary glands, Gall Bladder.
- Glandular system: Adrenals, Thyroid, Parathyroid, Thymus, Extraorbital lacrimal gland, Zymbal's gland.
- Nervous system: Brain, Pituitary gland, Eyes (retina and optic nerve), Peripheral nerve, Spinal cord.
- Urogenital system: Epididymides, Kidneys, Ovaries, Prostate, Seminal vesicles, Testes, Vagina, Urinary bladder, Uterus, Mammary gland.
- Other tissues: Skeletal muscle, Skin, Mesenteric fat, Tongue, Any gross lesions/masses observed.

Processed tissues from all high dose and control animals were examined plus kidneys, target tissues from all dose animals plus all gross lesions were examined.

Statistics:

Fisher's Exact test used to compare tumour incidence between control and high dose animals if tumour incidence was more than one in at least one of the two high dose groups (male and female). For neoplasms where all dose groups were examined, the Cochran-Armitage trend test was used. A survival adjusted neoplasm rate for each group was also calculated for the astrocytoma in male rats using a procedure based on the poly-3 method that modifies the Cochran-Armitage test to take into account survival differences.

Clinical signs:

no effects observed

Description (incidence and severity):

No effects related to substance exposure.

Mortality:

no mortality observed

Description (incidence):

No effects related to substance exposure.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Only statistitcally significant findings in kidney

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Only statistically significant finding in brain

Details on results:

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Significant reduction that persisted throughout study. Consumption in females averaged 80%, 63% and 56% in low, medium and high dose groups respectively of control consumption. Consumption in males averaged 77%, 71% and 68% in low, medium and high dose groups respectively of control consumption. Whilst not reported in this study, a one year study reported by Bermudez (2012) - see oral repeat dose chapter 7.5.1 of this IUCLID- showed no impairment of kidney function associated with decreased water intake.

ORGAN WEIGHTS: Male and female: Absolute and relative increase in kidney weight in high dose group. Relative increase only in kidney weight in mid and low dose groups. No other significant changes seen.

HISTOPATHOLOGY: NON-NEOPLASTIC. Kidney: In male rats, significant increase in incidence of mineralisation of cortex and nephropathy. In females: increase in incidence of tubular hyperplasia of cortex plus mineralisation of papilla and pelvis based on p<0.05 (two sided) from Cochran-Armitage trend test. However, based on a pairwise comparison, the only significant effect was nephropathy in males.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) Astrocytomas in brain. 9/460 animals seen. The incidence rates are shown in the table below. In males, Cochran-Armitage trend test and Poly-3 survival adjusted variant test indicated result was statistically significant (p values 0.032 and 0.037 respectively). However, a Fisher Exact test of the males comparing the control group with each exposure group in a pairwise comparison was not statistically significant (lowest p=0.181 for comparison of controls versus high dose group.)

Relevance of carcinogenic effects / potential:

The low but significant incidence of astrocytomas seen in the high dose males is thought to be a chance finding and unrelated to MTBE exposure. The rate was statistically significant compared to the in study control but fel within the historical ranges published in the literature for Wistar rats. Data from other studies does not indicate that the brain is a target organ for MTBE toxicity.

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on findings deemed significant. No biologically significant findings seen at maximum tested dose.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

1 042 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant findings seen at maximum tested dose.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

MTBE concentration in air was measured in the animal rooms and in selected cages, once per hour for the first 24 hours of the study and every six months thereafter. Control cage exposures averaged less than 0.05ppm for males and 0.19ppm for females. The measured average air concentration in the animal cages was 0.23 ppm for males, 0.43ppm for females. Maximum reading 0.91ppm. Animals were overall exposed to <1ppm MTBE throughout the study and inhalation was therefore an insignificant route of exposure.

Stability analysis showed an average 13% loss of MTBE from the drinking water over a 7 day period.

In terms of administered dose, there was an apparent decline in the average daily dose with time that corresponded with the increase in animal body weight.

Incidence rate of astrocytomas

Dose (mg/l)	Males (N=50)	Females (N=50)
0	1	0
0.5	1	0
3	1	0
7.5 (male), 15 (female)	4	1

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

330 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Dose descriptor:

NOAEC

1 465 mg/m³

Study duration:

chronic

Species:

rat

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for carcinogenicity.

Additional information

The oral carcinogenicity studies with Sprague-Dawley rats (Belpoggi et al., 1995 and Belpoggi et al., 1998),  was reviewed by IARC and disregarded given incomplete reporting and questionable study methodology and data interpretation. This is supported by a report by an expert Pathology Working Group sponsored by the U.S. National Toxicology Program and U.S. Environmental Protection Agency. The Pathology Working Group considered it inappropriate to combine lymphoma types in statistical analysis and the background level of lymphomas in the strain of rat used in this oral carcinogenicity study (Gift et al., 2013). A two-year drinking water guideline carcinogenicity study is available conducted in Wistar rats by with exposure at 0, 0.5, 3, 7.5 mg/ml in males and females, 0, 0.5, 3, and 15  mg/ml (Dodd et al., 2010). The study showed body weights were unaffected and water consumption reduced in MTBE-exposed males and females. Wet weights of male kidneys were increased at the end of two years of exposure to 7.5 mg/ml MTBE. Chronic progressive nephropathy was observed in males and females, was more severe in males, and was exacerbated in the high MTBE exposure groups. In this drinking water carcinogenicity study, a statistically significant finding of neoplasms was seen in the brain (Dodd et al., 2010). One astrocytoma (1/50) was found in a female rat (15 mg/ml). The incidence of brain astrocytomas in male rats was 1/50, 1/50, 1/50 and 4/50 for the 0, 0.5, 3 and 7.5  mg/mlexposure groups, respectively. This was a marginally significant statistical trend, but not statistically significant when pairwise comparisons were made or when multiple comparisons were taken into account. The incidence of astrocytoma fell within historical control ranges for Wistar rats and the authors note that brain has not been identified as a target organ following chronic administration of MTBE or TBA to mice and rats. The study report concludes that the astrocytomas observed were not associated with exposure to MTBE. Two inhalation carcinogenicity studies, one with Fischer-344 rats exposed for 2 years (Bushy Run Research Center, 1992a) and one with CD-1 mice exposed for 72 weeks (Bushy Run research Center, 1992b), were available. The longest available study, performed under GLP and according to EPA OTS 798.3300 (Carcinogenicity), was chosen as a key study (Bushy Run Research Center, 1992a). The results from both studies are considered for assessment of carcinogenicity. In male Fischer-344 rats (Bushy Run Research Center, 1992a), the incidence of parathyroid gland adenomas was increased at the two highest exposure concentrations: the 3000 ppm (10710 mg/m3) group males had 3/50 and the 8000 ppm (28560 mg/m3) males 1/50 parathyroid adenomas compared to zero in the 400 ppm (1428 mg/m3) and control groups. The proliferative changes seen in the parathyroid are likely due to hyperparathyroidism, which is commonly seen in cases where the parathyroid compensates in hypocalcaemia caused by, e. g., chronic renal failure as is the case for MTBE. The adenomas in the two high dose groups are probably associated with the resulted hyperplasia seen in parathyroid cells. Renal tubular cell tumours were increased in Fischer-344 male rats from the 3000 ppm and 8000 ppm groups. The adenoma rates were 2% at 0 ppm, 0% at 400 ppm, 10% at 3000 ppm and 6% at 8000 ppm. The respective carcinoma rates were 0%, 0%, 6% and 0%, respectively. The evidence suggests that these neoplasms are a result of proliferation in response to α2u-globulin associated nephropathy. No increase of tumours was seen at 400 ppm. The renal histopathology from this 2-year carcinogenicity bioassay of MTBE was re-evaluated by Hard (2006). He concluded that the modest increase in renal tubule tumors, observed in the assay, meets the criteria for supporting a mode of action involving the exacerbation of chronic progressive nephropathy (CPN). As rat CPN has no strict counterpart in humans (Hard et al., 2009), this finding is considered to be a secondary mechanism of renal tumor causation with no relevance for hazard assessment. It is also possible that there is interplay between two separate modes of action, comprising both a weak α2u-globulin response and CPN exacerbation. This would not alter the conclusion regarding hazard assessment, as α2u-globulin nephropathy is a male rat-specific phenomenon not relevant for species extrapolation. A dose dependent, statistically significant increase of testicular interstitial cell (Leydig cells) tumours was also demonstrated in Fischer-344 male rats, with an incidence of 64% at 0 ppm, 70% at 400 ppm, 82% at 3000 ppm (p<0.05), and 94% at 8000 ppm (p<0.05). The proportion of adenomas graded as “moderate” also increased in the high-concentration group. Although there was a clear dose-response relationship, the tumour incidences stayed mostly below intra-laboratory historical control values. Disturbances of the hypothalamic-pituitary-testis axis have been suggested as a possible mode of action. Although there were changes in plasma corticosterone and testosterone levels in the exposed animals, due to differences between rats and humans in the regulation of gonadotropins it is questionable whether a similar effect would occur in humans. Regarding the study with CD-1 mice (Bushy Run Research Center, 1992b), hepatocellular hypertrophy was increased in the males from the 3000 ppm and 8000 ppm dose groups and in females at 8000 ppm. Additionally female mice from the high dose group showed a 20% incidence of hepatocellular adenomas compared with 4% incidence in controls; the latter value is within the historical range of 0 - 4%. In males, while adenoma frequency was similar across the groups the incidence of liver carcinoma was increased 4-fold in the high dose group relative to the controls (16% vs. 4%). However, the difference was not statistically significant. Moser et al. (1998) have shown that MTBE causes changes in oestrogen sensitive tissues without affecting serum oestrogen level. There may be a connection with these changes and the increased amount of liver adenomas seen in female mice at 8000 ppm but there is no evidence to corroborate such a theory. MTBE increased cell proliferation may have contributed to the generation of tumours seen at high doses. MTBE did not show promoter activity when tested in female mice after N-nitrosodiethylamine (DEN) initiation (Moser et al, 1996). There was no increase in tumours in male mice at 400 or 3000 ppm (Bushy Run Research Center, 1992b). As discussed in the Toxicokinetics section, simulated predictions using a PBPK model clearly show the change from linear to non-linear metabolism occurs between 1000-2000 ppm following inhalation exposure and between 400-600 mg/kg bw MTBE following oral administration. These high exposure/dose conditions of MTBE with respect to the human health have implications associated with high-exposure/dose and the development of tumours in rodents. All these exposure/dose levels that were associated with tumour increases were well above the onset of metabolic saturation and well above potential human exposures (based on compliance to recommended occupational exposure values of 50 ppm). 

It is now widely recognized, as reflected in the OECD test guidelines, that care should be taken in selecting dose levels in toxicity studies to prevent saturation of absorption, metabolism or excretion as adverse effects observed at such doses are not relevant for safety assessment. 

There have been a number of comprehensive reviews that have emphasized some rodent tumour responses are restricted to high-test doses and may be questionable to the relevance of human health hazard and risk. Thus, high-dose specific saturation of metabolic processes, including toxicokinetics may result in transition to novel modes of action unique to those high dose levels that are not related to modes of action that operate at lower animal doses and substantially lower real-world human exposures (Foran et al., 1997; Slikker et al., 2004a,b; Barton et al., 2006; Carmichael et al., 2006; Doe et al., 2006). 

 

In conclusion, MTBE produces tumours in rats and mice at concentrations ≥ 3000 ppm (10710 mg/m3) after chronic inhalation exposure. There is no evidence of a direct genotoxic mode of action as MTBE is not genotoxic in vitro or in vivo. Furthermore, the treatment relationship of the observed tumours is equivocal in some cases, or of no or of questionable relevance for humans in others. Moreover, the tumours appear mostly at high systemically toxic concentrations. Cruzan et al (2007) reviewed the available data on the carcinogenicity of MTBE using the EPA framework. Their conclusions are in agreement with the conclusions as drawn above: the weight of the evidence does not support a genotoxic mode of action. Non genotoxic modes of action have been demonstrated or suggested that correspond to the weak tumorigenic responses. These modes of action either do not occur in humans or humans are much less susceptible to these effects. It is, therefore, unlikely that humans would be exposed to sufficient levels of MTBE to cause these tumorigenic responses. The SCOEL (2006) has also evaluated the carcinogenicity of MTBE and concluded the following: neither the kidney tumours caused by alpha-2-microglobulin and, consequently, the parathyroid tumours seen in male rats, nor the liver tumours in female mice which were seen also in the control animals seem to be of relevance for human health. The same is true for the Leydig-cell tumours. There are neither epidemiological studies addressing a possible association of MTBE with human cancer, nor grounds for assuming there to be a concern. MTBE is not genotoxic. Therefore, a threshold for the carcinogenic potential of this compound is assumed by SCOEL also. Furthermore, the available evidence convinced the EU member states and the CMR group to conclude that MTBE is not carcinogenic. The Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE) agreed to this opinion. Since then no new information has been published that could shed another light on the observations made, and thus potentially could change these conclusions. Based on this, MTBE is concluded to be non-carcinogenic. References:

Barton HA, Pastoor TP, Baetcke K, Chambers JE, Diliberto J, Doerrer NG, Driver JH, Hastings CE, Iyengar S, Krieger R, Stahl B, Timchalk C (2006). The acquisition and application of absorption, distribution, metabolism, and excretion (ADME) data in agricultural chemical safety assessments.Crit. Rev. Toxicol.36:9-35.

 

Carmichael NG, Barton HA, Boobis AR, Cooper RL, Dellarco VL, Doerrer NG, Fenner-Crisp PA, Doe JE, Lamb JC 4^th, Pastoor TP (2006). Agricultural chemical safety assessment: A multi-sector approach to the modernization of human safety requirements. Crit. Rev. Toxicol.36:1–7.

 

Doe JE, Boobis AR, Blacker A, Dellarco VL, Doerrer NG, Franklin C, Goodman JI, Kronenberg JM, Lewis R, McConnell, EE, Mercier T, Moretto A, Nolan C, Padilla S, Phang W., Solecki R, Tilbury L, van Ravenzwaay B, Wolf DC (2006). A tiered approach to systemic toxicity testing for agricultural chemical safety assessment.Crit. Rev. Toxicol.36:37–68.

 

Foran, JA. and ILSI risk Science Working Group on Dose Selection (1997).Principles for the Selection of Doses in Chronic Rodent Bioassays. ILSI Risk Science Working Group on Dose Selection. Environ. Health Perspect. 105(1):18-20.

 

Slikker W, Jr., Andersen ME, Bogdanffy MS, Bus JS, Cohen, SD, Conolly RB, David RM, Doerrer NG, Dorman DC, Gaylor DW, Hattis D, Rogers JM, Sletzer, WR, Swenberg JA, Wallace K. (2004a). Dose-dependent transitions in mechanisms of toxicity.Toxicol. Appl. Pharmacol. 201:203–225.

 

Slikker W, Jr., Andersen ME, Bogdanffy MS, Bus JS, Cohen, SD, Conolly RB, David RM, Doerrer NG, Dorman DC, Gaylor DW, Hattis D, Rogers JM, Sletzer, WR, Swenberg JA, Wallace K. (2004b). Dose-dependent transitions in mechanisms of toxicity: Case studies.Toxicol. Appl. Pharmacol.201:226–294.

Justification for selection of carcinogenicity via oral route endpoint:
No biologically significant carcinogenic findings seen at maximum tested dose.

Carcinogenicity: via inhalation route (target organ): glandular: parathyroids; urogenital: kidneys; urogenital: testes