# JPEC COMMENTS **ON PRELIMINAR**  **Y MA**  **TERIALS FOR THE INTEGRA TED RISK**  **INFORMA**

**TION SYSTEM (IRIS) TO** **XICOLOGICAL REVIEW OF ETHYL**  **TER T**  **- BUTYL**  **ETHER (ETBE) EP**  **A IRIS Bimonthly** 

**Meeting EP**  **A Conference**  **Center**

**, Arlington**  **, Vi**  **rginia December**  **12, 2013**  **Fukumi Nishimaki,**  **Ph.D. Senior Researcher**  **Auto Oil and New** **F uels Dept.**  **Japan Petroleum Energy Center**  **(JPEC)**

EP A- HQ - ORD - 2009 - 0229 **1** 

## Docket ID **NO. EP**

**A -**  **HQ -**  **ORD -** **2009 -**  **0229**

# JPEC Comments **ETBE use as an oxy genated**  **gasoline additive in Japan to reduce CO**  **2** 

# emissions and support the objectives of the **Kyoto Protocol**  **is increasing .**  **JPEC conducted many** **s tudies to evaluate the toxicity** **a nd**  **potential health risks of ETBE between 2006 and 2013,**  **commissioned by a**  **nd under the guidance of the Japan Ministry**  **of Economy**

**, Tr** **ade 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% E**  **TBE**   ** blended gasoline does not pose a human health risk by** **either the oral or inhalation route of exposure.**  JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **2**

# JPEC Comments **EP A**  **is an important authoritative**  **body and its IRIS**  **To xicological**  **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 y**  **ears. 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 EP A- HQ - ORD - 2009 - 0229 **3**

# JPEC Comments **We request that EP** 

**A define and**  **communicate the**  **problem and scope that the ETBE**  **IRIS toxicological**  **assessment intends to address**  **before proceeding wi**  **th the ETBE IRIS assessment.**  **Considering that**  **the ETBE IRIS toxicological**  **assessment is likely**  **to have greater regulatory impact outside the U.S. than w**  **ithin the U.S., w**  **e also request that EP** 

**A consider the follow**  **ing comments**  **as it proceeds wi**  **th the ETBE**  **toxicological review**  **.** JPEC Public Comments EP A- 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**  **p reliminary**  **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** **d ata without the**  **inclusion of key l**  **iterature regarding the mode**  **of action**  **(MOA) of**  **ETBE. We**  **request that EP**

**A include three original publications**  **and two study** **r eports**  **that provide key information**  **on the MOA for ETBE hepatotumorigenicity**  **in rats in both the literature search and evidence tables.**  JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **5**

# JPEC Comments **We submitted**  **our comments w**  **ith thes**  **e three**  **key papers**  **and tw**  **o key**  **study reports:**  1 **The original paper entitled:**  **An na**  **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 PP ARs signaling pathways.” Published on December 1, 2013 in To xicology and Applied Pharmacology. V

olume 273, Issue 2, p.390 - 400, 2013. 2 **The original**  **paper entitled: Ar** **ata 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 To xicology, V

olume 87, Number 5, p.905 - 914 , 2013. 3 **A ccepted**  **manuscript of the original**  **paper entitled: Ta**  **dashi Noguchi et al.**  “ Lack of micronucleus induction activity of ethyl tertiary - butyl ether in the bone marrow of F344 rats by sub chronic drinking -water treatment, inhalation exposure, or acute intraperitoneal injection ” (in press) The Journal of To xicological Sciences. Accepted on September 9, 2013. Manuscript No.:JTS - 13141. 4 **The full**  **study**

**report entitled : “**  **Investigation of the Mechanisms**  **of Ethy l**  **tertiary -**  **Buty l Ether**  **(ETBE ) Carcinogenicity**  **in the Liver of F344 Rats. ” [Study**  **N o.: 1**  **132 ]**  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 Ethy l**  **tertiary -**  **Buty l Ether**  **( ETBE)**  **Carcinogenicity in the Liver of F344 Rats**  **– Tr** **ansmission Electron**  **Microscopic Examination**  **. [**  **Study No**  **.: 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 EP A- HQ - ORD - 2009 - 0229 **6**

# ETBE T

**oxicological Effects and Carcinogenicity Results of a 2**  **-**

**Ye ar**  **Inhalation Carcinogenicity Study**  **in F344 Rats**

7 **Organ Observation**  **Dose NOA**  **EL 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 ) 

##  **To xicological**  **changes**

 **JPEC results**  **and conclusions**  **on ETBE**  **kidney effects**  **in rats w ere**  **confirmed by**  **a Pathological**  **Working Group**  **Rev iew** **conducted in**  **201 1.** 

• **Cited as “Cohen, SM; Hard, GC, Regan, KS, Seely**  **, JC, Bruner**

**, RH (201 1)**  **Pathology wo**  **rking group review**  **of selected histological changes in the kidney s of assigned**  **to toxicological and carcinogenicity studies**  **of ethy l tertiary** 

**buty l**  **ether (ETBE): Japan Bioassay Research Center studies no.: 0665**  **and 0691” in**  **“Preliminary**

**Materials for IRIS T** 

**oxicological Review of ETBE”.**  JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **7**JPEC Public Comments EP A- 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 liv er**  **tumors**

**: p  0.01 by Fisher exact test 

## JPEC implemented tw **o studies on Mode of**  **Ac tion**  **(MOA )**  **of ETBE hepatotumorigenicity in rats.**  

# ETBE T **oxicological Effects**  **and Carcinogenicity Results of a 2**  **- Ye**  **ar Inhalation Carcinogenicity Study in F344 Rats; JPEC (2007)** JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **9 [Study**  **N o. 1**  **132 ]**  **Investigation of the Mechanisms**  **of Ethy l tertiary**  **- Buty**  **l 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 **H epatotumorigenicity**  **M OA**  **Studies in Rats**JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **10**

**[Study N**  **o. 12138] Investigation**  **of the Mechanisms of Ethy**  **l tertiary**  **- Buty**  **l Ether (ETBE) Carcinogenicity in the Liver of**  **F344 Rats –**  **Tr ansmission**  **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.1 132.

 Electron microscopic examination of liver cells in rats.  Conducted according to GLP

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

**An na**  **Kakehashi et al**  **. “Mode**  **of action of ethy**  **l tertiary -**  **buty l ether hepatotumorigenicity**  **in the rat: Evidence for a role**  **of oxidative**  **stress via activation**  **of CA R,**  **PXR and PP AR** **s signaling**  **pathw ay** **s”** Published on December 1, 2013 in To xicology and Applied Pharmacology

. Vo lume 237, Issue 2, p.390 - 400, 2013. 

## ETBE **H epatotumorigenicity**  **M OA**  **Studies in Rats**

## Ta **ble 1. Difference of the**  **included data in the reports**  **or original**

**paper on ETBE**  **hepatotumorigenicity MOA**  JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **11**

**Included Data**  **Study R** **eports Paper** 

# **11 32**  # **12138** 

## An **na Kakehashi et al.**  **To xicology** 

**and Ap**  **plied Pharmacology** 

**. (in press).**  **DOI :10.1016/j.taap.2013.09.016**  **Ac cepted on Sept. 20, 2013.**   **Nuclear receptor**  **activation (CA**  **R, PXR, PP** 

**AR e**  **tc.)** ○ - ○  **P450 induction**  ( **mRNA ,**  **Protein)** ○ - ○  **Oxidative stress**  ○ - ○  **DNA damage (**  **8OHG)** ○ - ○  **Cell cy cle arrest**  ○ - ○  **Ap optosis**  ○ - ○  **Cell proliferation**  - - ○  **Peroxisome proliferation**  - ○ ○JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **12**



### An **na Kakehashi et al. T oxicology** 

**and Ap**  **plied Pharmacology , V**  **olume 273 (2), p.390 -** 

**400, 2013.** 

## Summary

**of the Proposed ETBE MOA**  **for H**  **epatotumorigenicity in R**  **ats**

**Conclusion: ETBE hepatotumorigenicity**  **in rats lacks**  **human relevance.**

# ETBE Inhalation Exposure Level and Health **R isk in Humans**  (based on “ETBE risk assessment report” in ETBE Risk Assessment Study by JPEC (March 2008)) 

## ETBE Exposure lev**el and Health Risk**  **7% E**  **TBE blended w**  **ith gasoline Exposure**  **level:** ETBE concentration in environment estimated by simulation  

## C **air** = Maximum atmospheric ETBE concentration 

## C **air** = **38 μg/m 3**   **NOA EL**  **in rats**  Centrilobular hypertrophy of liver cells, hyaline droplet in kidney( 90 - day inhalation toxicity study)

• **NOA EL=500ppm =2,090mg/m**  **3**  Kidney changes (2 - year inhalation carcinogenicity study)

• **NOA EL** **= 500ppm =2,090mg/m**  **3**  Liver adenoma (2 - year inhalation carcinogenicity study)

• **NOA EL** **= 1500ppm**  **=6,270mg/m 3**  **NOA EL** **:** 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 ***  Te st period **1** (Based on 2 - year carcinogenicity study) **UF** = **100** 

### Risk is assessed on comparison **of “MOE v ersus**  **UF”**

• In case of ; MOE < UF → High Risk

• **In case of ; MOE**  **> UF** → **Low R**  **isk** 

## Extremely **Low R**  **isk 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 EP A- HQ - ORD - 2009 - 0229

# We **thank EP**

**A for the opportunity** **t o present these comments.**  **We request that EP** 

**A define and communicate the problem**  **and scope that the ETBE IRIS toxicological assessment**  **intends to address before proceeding with the assessment.**  **After EP A**  **has defined the problem and scope of this assessment, we strongly** **r equest that EP**  **A 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 EP A- HQ - ORD - 2009 - 0229 **14** 

# JPEC **Comments**

# Thank you very**m uch for y**  **our attention.**  JPEC Public Comments EP A- HQ - ORD - 2009 - 0229 **15**