JPEC COMMENTS ON PRELIMINARY 

MATERIALS FOR THE INTEGRATED RISK 

INFORMATION SYSTEM (IRIS) TOXICOLOGICAL

REVIEW OF ETHYL TERT-BUTYL ETHER (ETBE)

EPA IRIS Bimonthly Meeting

EPA Conference Center, Arlington, Virginia

December 12, 2013

Fukumi Nishimaki, Ph.D. 

Senior Researcher

Auto Oil and New Fuels Dept.

Japan Petroleum Energy Center (JPEC)

EPA-HQ-ORD-2009-0229 1

Docket ID NO. EPA-HQ-ORD-2009-0229
JPEC Comments

ETBE use as an oxygenated gasoline additive in Japan to 

reduce CO2 emissions and support the objectives of the 

Kyoto Protocol is increasing.

JPEC conducted many studies to evaluate the toxicity and 

potential health risks of ETBE between 2006 and 2013, 

commissioned by and under the guidance of the Japan 

Ministry of Economy, Trade and Industry (METI). 

The resulting risk assessment, performed under the 

guidance of an independent committee of academic and 

government experts, concluded that the use of 7% ETBEblended gasoline does not pose a human health risk by

either the oral or inhalation route of exposure.

JPEC Public Comments EPA-HQ-ORD-2009-0229 2
JPEC Comments

EPA is an important authoritative body and its IRIS 

Toxicological Review of ETBE is expected to have 

significant regulatory impact globally.

JPEC is not aware of any significant use of ETBE as a 

gasoline additive within the U.S. during the past 15 years.

The U.S. Geological Survey (2006; USGS Circular 1292) 

reported that ETBE was “detected infrequently” in 

domestic and public wells. It was found in less than 0.5%

of public wells at concentrations of 0.2 g/L or greater.

Detection in domestic wells was less frequent. 

JPEC Public Comments EPA-HQ-ORD-2009-0229 3
JPEC Comments

We request that EPA define and communicate the 

problem and scope that the ETBE IRIS 

toxicological assessment intends to address 

before proceeding with the ETBE IRIS assessment.

Considering that the ETBE IRIS toxicological 

assessment is likely to have greater regulatory 

impact outside the U.S. than within the U.S., we 

also request that EPA consider the following 

comments as it proceeds with the ETBE 

toxicological review. 

JPEC Public Comments EPA-HQ-ORD-2009-0229 4
JPEC Comments

We appreciate that most of the original papers and study

reports on ETBE conducted by JPEC are included in the 

preliminary draft literature search and evidence tables for 

ETBE.

The preliminary draft literature search and evidence tables 

for ETBE, however, are insufficient for evaluating the 

human relevance of rodent tumorigenicity data without the 

inclusion of key literature regarding the mode of action 

(MOA) of ETBE.

We request that EPA include three original publications and 

two study reports that provide key information on the MOA

for ETBE hepatotumorigenicity in rats in both the literature 

search and evidence tables.

JPEC Public Comments EPA-HQ-ORD-2009-0229 5
JPEC Comments

We submitted our comments with these three key papers and two key study 

reports:

1 The original paper entitled: Anna Kakehashi et al. “Mode of action of ethyl tertiary-butyl ether 

hepatotumorigenicity in the rat: Evidence for a role of oxidative stress via activation of CAR, PXR 

and PPARs signaling pathways.” Published on December 1, 2013 in Toxicology and Applied 

Pharmacology. Volume 273, Issue 2, p.390-400, 2013.

2 The original paper entitled: Arata Saito et al. ”Hepatotumorigenicity of ethyl tertiary-butyl ether 

with 2-year inhalation exposure in F344 rats.” Published on February 7, 2013 in Archives of 

Toxicology, Volume 87, Number 5, p.905-914, 2013.

3 Accepted manuscript of the original paper entitled: Tadashi Noguchi et al. “Lack of 

micronucleus induction activity of ethyl tertiary-butyl ether in the bone marrow of F344 rats by subchronic drinking-water treatment, inhalation exposure, or acute intraperitoneal injection” (in press) 

The Journal of Toxicological Sciences. Accepted on September 9, 2013. Manuscript No.:JTS-13141. 

4 The full study report entitled: “Investigation of the Mechanisms of Ethyl tertiary-Butyl Ether 

(ETBE) Carcinogenicity in the Liver of F344 Rats. ” [Study No.: 1132] 

Conducted by DIMS Institute of Medical Science, Inc. and Osaka City University Graduate School of 

Medicine, Department of Pathology. (October 2012) Sponsored by JPEC.

5 The full study report entitled: “Investigation of the Mechanisms of Ethyl tertiary-Butyl Ether 

(ETBE) Carcinogenicity in the Liver of F344 Rats – Transmission Electron Microscopic 

Examination. [StudyNo.: 12138]

Conducted by DIMS Institute of Medical Science, Inc. and Osaka City University Graduate School of 

Medicine, Department of Pathology. (October 2012) Sponsored by JPEC.

JPEC Public Comments EPA-HQ-ORD-2009-0229 6
ETBE Toxicological Effects and Carcinogenicity 

Results of a 2-Year Inhalation Carcinogenicity Study in F344 Rats

7

Organ Observation Dose NOAEL Human Relevance

Male Liver Eosinophilic/

Basophilic cell foci

5000 ppm 1500 ppm

Kidney Chronic progressive 

nephropathy (CPN)

5000 ppm 1500 ppm Not relevant to humans

(Increase in age-related lesion specific to rats)

Kidney Mineral deposition in 

the renal papilla area

5000 ppm 1500 ppm Not relevant to humans

(Involvement of α2u globulin; observed only in 

males)

Kidney Urothelial 

hyperplasia in the 

renal pelvis

>1500 ppm 500 ppm Not relevant to humans

(Involvement of α2u globulin; observed only in 

males)

Female Kidney Chronic progressive 

nephropathy (CPN)

5000 ppm 1500 ppm Not relevant to humans

(Increase in age-related lesion specific to rats)

 Toxicological changes

 JPEC results and conclusions on ETBE kidney effects in rats were confirmed by a

Pathological Working Group Review conducted in 2011.

• Cited as “Cohen, SM; Hard, GC, Regan, KS, Seely, JC, Bruner, RH (2011) Pathology working group review

of selected histological changes in the kidneys of assigned to toxicological and carcinogenicity studies 

of ethyl tertiary butyl ether (ETBE): Japan Bioassay Research Center studies no.: 0665 and 0691” in 

“Preliminary Materials for IRIS Toxicological Review of ETBE”.

JPEC Public Comments EPA-HQ-ORD-2009-0229 7
JPEC Public Comments EPA-HQ-ORD-2009-0229 8

Male Female

Concentration of 

ETBE (ppm) 0 500 1500 5000 0 500 1500 5000

No. of examined 

animals 50 50 49 50 50 50 50 50

Hepatocellular 

adenoma 0 2 1 9** 1 0 1 1

Hepatocellular 

carcinoma 0 0 0 1 0 0 0 0

 Incidence of liver tumors

**: p  0.01 by Fisher exact test 

JPEC implemented two studies on Mode of Action (MOA) of ETBE 

hepatotumorigenicity in rats.

ETBE Toxicological Effects and Carcinogenicity 

Results of a 2-Year Inhalation Carcinogenicity Study in F344 Rats; JPEC (2007)
JPEC Public Comments EPA-HQ-ORD-2009-0229 9

[Study No. 1132] 

Investigation of the Mechanisms of Ethyl tertiary-Butyl Ether (ETBE) Carcinogenicity in the Liver of 

F344 Rats. Opened at JPEC English home page: http://www.pecj.or.jp/english/news/pdf/C-1-1.pdf

 Main study of ETBE hepatotumorigenicity MOA in rats.

 Conducted according to GLP.

 Results indicated:

 activation of CAR and PXR nuclear receptors predominantly

 induction of oxidative stress, DNA damage

 subsequent cell cycle arrest and apoptosis, suggesting regenerative cell 

proliferation

 ETBE hepatotumorigenicity MOA is similar to that of Phenobarbital.

 Concluded that ETBE hepatotumorigenicity in rats is not relevant to humans.

ETBE Hepatotumorigenicity MOA Studies in Rats
JPEC Public Comments EPA-HQ-ORD-2009-0229 10

[Study No. 12138]

Investigation of the Mechanisms of Ethyl tertiary-Butyl Ether (ETBE) Carcinogenicity in the Liver of 

F344 Rats – Transmission Electron Microscopic Examination. 

Opened at JPEC English home page: http://www.pecj.or.jp/english/news/pdf/C-2-1.pdf

 Additional testing for Study No.1132.

 Electron microscopic examination of liver cells in rats.

 Conducted according to GLP.

 Suggested very slight activation of PPARs nuclear receptors in liver cells in ETBE treated rats.

Anna Kakehashi et al. 

“Mode of action of ethyl tertiary-butyl ether hepatotumorigenicity in the rat: Evidence for a role of 

oxidative stress via activation of CAR, PXR and PPARs signaling pathways” 

Published on December 1, 2013 in Toxicology and Applied Pharmacology. Volume 237, Issue 2, 

p.390-400, 2013. 

ETBE Hepatotumorigenicity MOA Studies in Rats
Table 1. Difference of the included data in the reports or original 

paper on ETBE hepatotumorigenicity MOA

JPEC Public Comments EPA-HQ-ORD-2009-0229 11

Included Data 

Study Reports Paper

#1132 #12138

Anna Kakehashi et al. 

Toxicology and Applied 

Pharmacology. (in press). 

DOI :10.1016/j.taap.2013.09.016 

Accepted on Sept. 20, 2013. 

 Nuclear receptor activation 

(CAR, PXR, PPAR etc.) ○ - ○

 P450 induction(mRNA, 

Protein) ○ - ○

 Oxidative stress ○ - ○

 DNA damage (8OHG) ○ - ○

 Cell cycle arrest ○ - ○

 Apoptosis ○ - ○

 Cell proliferation - - ○

 Peroxisome proliferation - ○ ○
JPEC Public Comments EPA-HQ-ORD-2009-0229 12

Anna Kakehashi et al. Toxicology and Applied Pharmacology, Volume 273 (2), p.390-400, 2013. 

Summary of the Proposed ETBE MOA for Hepatotumorigenicity in Rats 

Conclusion: ETBE hepatotumorigenicity in rats lacks human relevance. 
ETBE Inhalation Exposure Level and Health Risk in Humans 

(based on “ETBE risk assessment report” in ETBE Risk Assessment Study by JPEC (March 2008))

ETBE Exposure level and Health Risk 7% ETBE blended with gasoline 

Exposure level: ETBE concentration in environment estimated by simulation

 Cair = Maximum atmospheric ETBE concentration 

Cair = 38 μg/m3

 NOAEL in rats

 Centrilobular hypertrophy of liver cells, hyaline droplet in 

kidney( 90-day inhalation toxicity study)

• NOAEL=500ppm =2,090mg/m3 

 Kidney changes (2-year inhalation carcinogenicity study)

• NOAEL = 500ppm =2,090mg/m3 

 Liver adenoma (2-year inhalation carcinogenicity study)

• NOAEL =1500ppm =6,270mg/m3

NOAEL :

1 Kidney changes 

=2,090,000μg/m3

2 Liver adenoma

= 6,270,000μg/m3

 MOE (Margin of Exposure) = NOAEL* /Cair 1 MOE = 55,000

2 MOE =165,000

 Uncertainty factors (UF) 

 Interspecies 10 * Intraspecies 10 * Test period 1 (Based on 

2-year carcinogenicity study)

UF = 100

Risk is assessed on comparison of “MOE versus UF” 

• In case of ; MOE < UF → High Risk

• In case of ; MOE > UF → Low Risk

Extremely Low Risk

MOE>USFs 55,000 >>100

165,000 >>100

CONCLUSION : Inhalation exposure to ETBE does not pose a health risk to humans. 

Opened at JPEC English home page: http://www.pecj.or.jp/english/news/pdf/b-2-8.pdf

13

MOE > UF

JPEC Public Comments EPA-HQ-ORD-2009-0229
We thank EPA for the opportunity to present these comments.

We request that EPA define and communicate the problem 

and scope that the ETBE IRIS toxicological assessment 

intends to address before proceeding with the assessment.

After EPA has defined the problem and scope of this 

assessment, we strongly request that EPA include our papers 

and our studies of ETBE toxicity, carcinogenicity, and the 

hepatotumorigenicity mode of action as part of a sound 

scientific evaluation of the toxicological effects of ETBE. 

JPEC Public Comments EPA-HQ-ORD-2009-0229 14

JPEC Comments
Thank you very much for your attention. 

JPEC Public Comments EPA-HQ-ORD-2009-0229 15