vycrM
EPA /635/R-20/106b
Agency/Interagency Review Draft
www.epa.gov/iris
Toxicological Review of Ethyl Tertiary Butyl Ether
[CASRN 637-92-3]
Supplemental Information
August 2020
NOTICE
This document is a Final Agency and Interagency Draft. This information is distributed solely for
the purpose of predissemination peer review under applicable information quality guidelines. It
has not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Supplemental Information—ETBE
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of predissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
CONTENTS
APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS A-l
APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-REPONSE
ANALYSIS B-l
B.l.TOXICOKINETICS B-l
B.l.l. Absorption B-l
B.1.2. Distribution B-6
B.1.3. Metabolism B-8
B.1.4. Excretion B-17
B.1.5. Physiologically Based Pharmacokinetic Models B-22
B.1.6. Physiologically Based Pharmacokinetic (PBPK) Model Code B-26
B.1.7. Physiologically Based Pharmacokinetic (PBPK) Model Evaluation B-26
B.1.8. Toxicokinetic Data Extraction and Selected Model Outputs B-36
B.2.OTHER PERTINENT TOXICITY INFORMATION B-48
B.2.1. Other Toxicological Effects B-48
B.2.2. Genotoxicity Studies B-66
B.3.SUPPLEMENTAL ORGAN-WEIGHT DATA B-73
B.3.1. Relative Kidney-Weight Data B-73
B.3.2. Absolute Liver-Weight Data B-76
APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK ESTIMATES C-l
C.l.BENCHMARK DOSE MODELING SUMMARY C-l
C.1.1. Noncancer Endpoints C-l
C.l.2. Cancer Endpoints C-50
APPENDIX D. PATHOLOGY CONSULT FOR ETHYL TERTIARY BUTYL ETHER (ETBE) AND
TERT-BUTANOL D-l
APPENDIX E. SUMMARY OF EXTERNAL PEER-REVIEW COMMENTS AND EPA'S DISPOSITION E-l
APPENDIX F. QUALITY ASSURANCE (QA) FOR THE IRIS TOXICOLOGICAL REVIEW OF ETHYL
TERTIARY BUTYL ETHER F-l
REFERENCES FOR APPENDICES 1
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Supplemental Information—ETBE
TABLES
Table A-l. Health assessments and regulatory limits by other national and international
health agencies A-l
Table B-l. Radioactivity in blood and kidney of rats and blood and liver of mice, following 6
hours of [14C]ethyl tertiary butyl ether (ETBE) inhalation exposure B-4
Table B-2. Plasma radioactivity after a single oral or intravenous dose of [14C]ethyl tertiary
butyl ether (ETBE) to male Crl:CD(SD) rats B-6
Table B-3. Blood:tissue partition coefficients for ethyl tertiary butyl ether (ETBE) and tert-
butanol B-7
Table B-4. Unchanged ethyl tertiary butyl ether (ETBE) and its metabolites in plasma 8
hours after a single oral dose or repeated (7 or 14) daily oral dosing of [14C]ETBE
to male Crl:CD(SD) rats B-14
Table B-5. Unchanged ethyl tertiary butyl ether (ETBE) and its metabolites in the urine
(measured 0-24 hours) after a single oral dose or repeated (7 or 14) daily oral
dosing of [14C]ETBE to male Crl:CD(SD) rats B-15
Table B-6. Excretion of [14C]ethyl tertiary butyl ether (ETBE)-derived radioactivity from rats
and mice within 96 hours following a single 6-hour inhalation exposure B-20
Table B-7. Physiologically based pharmacokinetic (PBPK) model physiologic parameters
and partition coefficients3 B-33
Table B-8. Physiologically based pharmacokinetic (PBPK) model rate constants B-35
Table B-9. Summary of pharmacokinetic data used for model calibration and evaluation B-38
Table B-10. Conversion of ARCO (1983) total ferf-butanol (TBA) equivalents and serum
fraction data to TBA concentrations B-39
Table B-ll. Ratio of 14C activity in blood vs. plasma after [14C] ethyl tertiary butyl ether
(ETBE) exposures in rats (JPEC 2008a,b) B-40
Table B-12. Evidence pertaining to absolute kidney-weight effects in animals exposed to
ethyl tertiary butyl ether (ETBE) B-49
Table B-13. Evidence pertaining to body-weight effects in animals exposed to ethyl tertiary
butyl ether (ETBE) B-54
Table B-14. Evidence pertaining to adrenal effects in animals exposed to ethyl tertiary butyl
ether (ETBE) B-58
Table B-15. Evidence pertaining to immune effects in animals exposed to ethyl tertiary butyl
ether (ETBE) B-59
Table B-16. Evidence pertaining to mortality in animals exposed to ethyl tertiary butyl ether
(ETBE) B-63
Table B-17. Summary of genotoxicity (both in vitro and in vivo) studies of ethyl tertiary butyl
ether (ETBE) B-67
Table B-18. Evidence pertaining to relative kidney-weight effects in animals exposed to ethyl
tertiary butyl ether (ETBE) B-73
Table B-19. Evidence pertaining to absolute liver-weight effects in animals exposed to ethyl
tertiary butyl ether (ETBE) B-76
Table C-l. Noncancer endpoints selected for dose-response modeling for ethyl tertiary
butyl ether (ETBE) C-3
Table C-2. Summary of benchmark dose (BMD) modeling results for urothelial hyperplasia
of the renal pelvis in male F344 rats exposed to ethyl tertiary butyl ether (ETBE)
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in drinking water for 104 weeks (JPEC, 2010a) modeled with doses as mg/kg-day
(calculated by the study authors); benchmark response (BMR) = 10% extra risk C-7
Table C-3. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in female F344 rats exposed to ethyl tertiary butyl ether (ETBE) in
drinking water for 104 weeks (JPEC 2010); benchmark response (BMR) = 10%
relative deviation from the mean C-9
Table C-4. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in male Sprague-Dawley (S-D) rats exposed to ethyl tertiary butyl
ether (ETBE) by daily gavage for 26 weeks (Miyata et al., 2013; JPEC, 2008d);
benchmark response (BMR) = 10% relative deviation from the mean C-12
Table C-5. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in female Sprague-Dawley (S-D) rats exposed to ethyl tertiary
butyl ether (ETBE) by daily gavage for 26 weeks (Miyata et al., 2013; JPEC,
2008d); benchmark response (BMR) = 10% relative deviation from the mean C-15
Table C-6. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in P0 male Sprague-Dawley (S-D) rats exposed to ethyl tertiary
butyl ether (ETBE) by daily gavage for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups (Gaoua, 2004a); benchmark
response (BMR) = 10% relative deviation from the mean C-18
Table C-7. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in P0 female Sprague-Dawley (S-D) rats exposed to ethyl tertiary
butyl ether (ETBE) by daily gavage for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups (Gaoua, 2004a); benchmark
response (BMR) = 10% relative deviation from the mean C-21
Table C-8. Summary of benchmark dose (BMD) modeling results for absolute kidney
weight in F1 male Sprague-Dawley rats exposed to ethyl tertiary butyl ether
(ETBE) by gavage in a two-generation study (Gaoua, 2004b); benchmark
response (BMR) = 10% relative deviation from the mean C-24
Table C-9. Summary of benchmark dose (BMD) modeling results for absolute kidney
weight in F1 female Sprague-Dawley rats exposed to ethyl tertiary butyl ether
(ETBE) by gavage in a two-generation study (Gaoua, 2004b); benchmark
response (BMR) = 10% relative deviation C-26
Table C-10. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in P0 male Sprague-Dawley (S-D) rats exposed to ethyl tertiary
butyl ether (ETBE) by daily gavage for 16 weeks beginning 10 weeks prior to
mating (Fujii et al., 2010); benchmark response (BMR) = 10% relative deviation
from the mean C-29
Table C-ll. Summary of benchmark dose (BMD) modeling results for increased absolute
kidney weight in P0 female Sprague-Dawley (S-D) rats exposed to ethyl tertiary
butyl ether (ETBE) by daily gavage for 17 weeks beginning 10 weeks prior to
mating until Lactation Day 21 (Fujii et al., 2010); benchmark response
(BMR) = 10% relative deviation from the mean C-32
Table C-12. Summary of benchmark concentration (BMC) modeling results for urothelial
hyperplasia of the renal pelvis in male F344 rats exposed to ethyl tertiary butyl
ether (ETBE) by whole-body inhalation for 6 hours/day, 5 days/week, for 104
weeks (JPEC, 2010b); benchmark response (BMR) = 10% extra risk C-35
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Table C-13. Summary of benchmark concentration (BMC) modeling results for increased
absolute kidney weight in female F344 rats exposed to ethyl tertiary butyl ether
(ETBE) by whole-body inhalation for 6 hours/day, 5 days/week, for 104 weeks
(JPEC, 2010b); benchmark response (BMR) = 10% relative deviation from the
mean C-38
Table C-14. Summary of benchmark concentration (BMC) modeling results for increased
absolute kidney weight in male Sprague-Dawley (S-D) rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week for 13 weeks (JPEC, 2008b); benchmark response (BMR) = 10%
relative deviation from the mean C-39
Table C-15. Summary of benchmark concentration (BMC) modeling results for increased
absolute kidney weight in female Sprague-Dawley (S-D) rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day, 5
days/week for 13 weeks (JPEC, 2008b); benchmark response (BMR) = 10%
relative deviation from the mean C-42
Table C-16. Summary of benchmark concentration (BMC) modeling results for increased
absolute kidney weight in male F344 rats exposed to ethyl tertiary butyl ether
(ETBE) by whole-body inhalation for 6 hours/day, 5 days/week, for 13 weeks
(Medinsky et al., 1999; US EPA, 1997); benchmark response (BMR) = 10%
relative deviation from the mean C-45
Table C-17. Summary of benchmark concentration (BMC) modeling results for increased
absolute kidney weight in female F344 rats exposed to ethyl tertiary butyl ether
(ETBE) by whole-body inhalation for 6 hours/day, 5 days/week, for 13 weeks
(Medinsky et al., 1999; US EPA, 1997); benchmark response (BMR) = 10%
relative deviation from the mean C-48
Table C-18. Cancer endpoints selected for dose-response modeling for ethyl tertiary butyl
ether (ETBE) C-51
Table C-19. Summary of benchmark concentration (BMC) modeling results for
hepatocellular adenomas and carcinomas in male F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 104 weeks; modeled with concentrations as administered
exposure concentration in ppm (JPEC, 2010b); benchmark response
(BMR) = 10% extra risk C-51
Table C-20. Summary of benchmark concentration (BMC) modeling results for
hepatocellular adenomas and carcinomas in male F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 104 weeks; modeled with concentrations as mg/m3 (JPEC,
2010b); benchmark response (BMR) = 10% extra risk C-53
FIGURES
Figure B-l. Proposed metabolism of ethyl tertiary butyl ether (ETBE) B-9
Figure B-2. Example oral ingestion pattern for rats exposed via drinking water B-24
Figure B-3. Comparisons of liver tumors in male rats following 2-year oral or inhalation
exposure to ethyl tertiary butyl ether (ETBE) or ferf-butanol with internal dose
metrics calculated from the physiologically based pharmacokinetic (PBPK) mode B-25
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Figure B-4. Comparison of the ferf-butanol portions of existing methyl tertiary butyl ether
models with ferf-butanol blood concentrations from i.v. exposure by Poet et al.
(1997) B-28
Figure B-5. Schematic of the Salazar et al. (2015) physiologically based pharmacokinetic
(PBPK) model for ethyl tertiary butyl ether (ETBE) and its major metabolite
ferf-butanol in rats B-29
Figure B-6. Schematic of the Borghoff et al. (2016) physiologically based pharmacokinetic
(PBPK) model for ethyl tertiary butyl ether (ETBE) and its major metabolite
ferf-butanol in rats B-32
Figure B-7. ferf-Butanol PK data for 1 and 500 mg/kg oral exposures from ARCO (1983 B-37
Figure B-8. Comparison of the Borghoff et al. (2016) model predictions with measured ferf-
butanol blood concentrations for i.v., inhalation, and gavage exposure to ferf-
butanol B-43
Figure B-9. Comparison of Borghoff et al. (2016) model predictions with measured amounts
of ferf-butanol after gavage of ethyl tertiary butyl ether (ETBE) B-44
Figure B-10. Comparison of Borghoff et al. (2016) model predictions with measured amounts
after a 4-hour inhalation exposure to 4 and 40 ppm ethyl tertiary butyl ether
(ETBE B-45
Figure B-ll. Comparison of Borghoff et al. (2016) model predictions with measured amounts
of (A) ethyl tertiary butyl ether (ETBE) and (B) ferf-butanol in exhaled breath
after a 6-hour inhalation exposure to 500, 1,750, and 5,000 ppm ETBE B-46
Figure B-12. Comparison of the Borghoff et al. (2016) model predictions with measured
amounts of ferf-butanol in blood after repeated inhalation exposure to ferf-
butanol B-47
Figure B-13. Comparison of EPA model predictions with measured amounts of ferf-butanol in
blood after 5 mg/kg-day ethyl tertiary butyl ether (ETBE) gavage for up to
14 days in male rats B-48
Figure B-14. Exposure-response array of body-weight effects following oral exposure to ethyl
tertiary butyl ether (ETBE) B-64
Figure B-15. Exposure-response array of body-weight effects following inhalation exposure
to ethyl tertiary butyl ether (ETBE) B-65
Figure C-l. Plot of incidence rate by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-8
Figure C-2. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-10
Figure C-3. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-13
Figure C-4. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-16
Figure C-5. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-19
Figure C-6. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-22
Figure C-7. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-24
Figure C-8. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-27
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Figure C-9. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-30
Figure C-10. Plot of mean response by dose, with fitted curve for selected model; dose
shown in mg/kg-day C-33
Figure C-ll. Plot of incidence rate by concentration, with fitted curve for selected models-
concentration shown in mg/m3 C-36
Figure C-12. Plot of mean response by concentration, with fitted curve for selected models-
concentration shown in ppm C-40
Figure C-13. Plot of mean response by concentration, with fitted curve for selected models-
concentration shown in ppm C-43
Figure C-14. Plot of mean response by concentration, with fitted curve for selected models-
concentration shown in ppm C-46
Figure C-15. Plot of mean response by concentration, with fitted curve for selected models-
concentration shown in ppm C-49
Figure C-16. Plot of incidence rate by concentration, with fitted curve for selected models-
concentration shown in ppm C-52
Figure C-17. Plot of incidence rate by concentration, with fitted curve for selected models-
concentration shown in mg/m3 C-54
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Supplemental Information—ETBE
ABBREVIATIONS
ADAF
age-dependent adjustment factor
HBA
hydroxyisobutyric acid
ADJ
adjusting experimental exposure
HEC
human equivalent concentration
concentrations to a value reflecting
HED
human equivalent dose
continuous exposure duration
HERO
Health and Environment Research on
ADME
absorption, distribution, metabolism,
Online
excretion
HGPRT
hypoxanthine-guanine
AIC
Akaike's information criterion
phosphoribosyltransferase
ALDH
aldehyde dehydrogenase
HIBA
2-hydroxyisobutyrate
atm
atmosphere
HT
heterogeneous
ATSDR
Agency for Toxic Substances and
IARC
International Agency for Research on
Disease Registry
Cancer
ALP
alkaline phosphatase
IRIS
Integrated Risk Information System
ALT
alanine
i.v.
intravenous
aminotransferase/transaminase
JPEC
Japan Petroleum Energy Center
AST
aspartate
Km
Michaelis-Menten constant
aminotransferase/transaminase
KO
knockout
AUC
area-under-the-curve
LD
lactation day
BBN
/V-butyl-/V-(hydroxybutyl]nitrosamine
LOAEL
lowest-observed-adverse-effect level
BMC
benchmark concentration
MN
micronucleus, micronucleated
BMCL
benchmark concentration lower
MNPCE
micronucleated polychromatic
confidence limit
erythrocytes
BMD
benchmark dose
MNRET
micronucleated reticulocyte
BMDL
benchmark dose lower confidence limit
MNU
iV-methyl-iV-nitrosourea
BMDS
Benchmark Dose Software
MOA
mode of action
BMDU
benchmark dose upper confidence limit
MPD
2 -methyl-1,2 -propanediol
BMR
benchmark response
MTBE
methyl tert-utyl ether
BUN
blood urea nitrogen
MTD
maximum tolerated dose
BW
body weight
N.D.
not detected
CAAC
Chemical Assessment Advisory
NADPH
reduced form of nicotinamide adenine
Committee
dinucleotide phosphate
CAR
constitutive androstane receptor
No.
number
CASRN
Chemical Abstracts Service registry
NOAEL
no-observed-adverse-effect level
number
NR
not reported
Cmax
maximum plasma concentration
NTP
National Toxicology Program
CPHEA
Center for Public Health and
OECD
Organisation for Economic
Environmental Assessment
Co-operation and Development
CPN
chronic progressive nephropathy
ORD
Office of Research and Development
CSL
continuous simulation language
OSF
oral slope factor
CYP450
cytochrome P450
PBPK
physiologically based pharmacokinetic
DAF
dosimetric adjustment factor
PCE
polychromatic erythrocyte
DEN
diethylnitrosamine
PND
postnatal day
df
degrees of freedom
PNW
postnatal week
DHPN
N- bis (2-hydroxypropyl]nitrosamine
POD
point of departure
DMH
1,2-dimethylhydrazine dihydrochloride
PPARa
peroxisome proliferator-activated
DNA
deoxyribonucleic acid
receptor a
EHEN
N- ethyl -iV-hy dr oxy ethylni tr o s amin e
PXR
pregnane X receptor
EPA
Environmental Protection Agency
QA
quality assurance
ETBE
ethyl tertiary butyl ether
QSAR
quantitative structure-activity
GD
gestation day
relationship
GGT
y-glutamyl transferase
RD
relative deviation
GLP
good laboratory practice
RfC
inhalation reference concentration
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RfD
oral reference dose
UF
uncertainty factor
rho
Spearman's rank coefficient
UFa
animal-to-human uncertainty factor
RNA
ribonucleic acid
UFc
composite uncertainty factor
S-D
Sprague-Dawley
UFd
database deficiencies uncertainty factor
SAB
Science Advisory Board
UFh
human variation uncertainty factor
SD
standard deviation
UFl
LOAEL-to-NOAEL uncertainty factor
SE
standard error
UFs
subchronic-to-chronic uncertainty
SRBC
sheep red blood cell
factor
SS IICA
Stoddard Solvent IICA
USGS
U.S. Geological Survey
TBA
tert-butyl alcohol, tert-butanol
Vmax
maximum substrate turnover velocity
TSCATS
Toxic Substances Control Act Test
voc
volatile organic compounds
Submissions
WT
wild type
TWA
time-weighted average
wt
weight
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APPENDIX A. OTHER AGENCY AND
INTERNATIONAL ASSESSMENTS
Table A-l. Health assessments and regulatory limits by other national and
international health agencies
Organization
Toxicity value
National Institute for Public
Health and the Environment
(Bilthoven, The Netherlands)
Oral noncancer tolerable daily intake: 0.25 mg/kg-d
Inhalation noncancer tolerable concentration in air: 1.9 mg/m3
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APPENDIX B. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-REPONSE
ANALYSIS
B.l. TOXICOKINETICS
B.l.l. Absorption
Absorption in Humans
Most of the available human data on the uptake of ethyl tertiary butyl ether (ETBE) were
obtained from volunteers. Nihlen etal. (19981 exposed eight healthy male volunteers (average age:
29 years) to 5, 25, or 50 ppm (20.9,104, or 210 mg/m3) ETBE by inhalation for 2 hours. Each
volunteer was exposed at each concentration in sequence with 2-week intervals between
exposures. The study was performed according to the Declaration of Helsinki after approval by the
Regional Ethical Committee of the institution where the study was performed, and written
informed consent was obtained from the volunteers. The volunteers performed light physical
exercise (50 watts) on a bicycle ergometer during exposure. Exhaled air was collected before
exposure, every 30 minutes during exposure, and 6 times after exposure. The concentrations of
ETBE and one of its primary metabolites, tert-butanol (TBA), were determined in exhaled air
samples. Blood was drawn before exposure, approximately every 10 minutes during exposure,
approximately every 30 minutes from 1 to 4 hours after exposure, and an additional 4 times up to
48 hours after exposure. Urine was collected before exposure, at 0 and 2 hours, and at
approximately 4, 7,11, 20, 22, and 46 hours after exposure. ETBE, tert-butanol, and acetone (an
ETBE metabolite) concentrations were determined in blood and urine. The blood profiles of the
parent compound and metabolites were similar at all three exposure levels and reflected exposure
concentrations, as judged by linear increases in blood area-under-the-curve (AUC) values for the
concentration-time curve calculated (but only reported in graphical form by the authors).
Acetone levels were highly variable and seemed to reflect not only ETBE exposure, but the
physical activity of the volunteers. Nihlen etal. (1998) calculated the ETBE doses to the volunteers
to be 0.58, 2.9, and 5.8 mmol for the 20.9-, 104-, and 210-mg/m3 exposure levels, respectively. The
concentrations of ETBE in blood rose sharply during the first 30 minutes of exposure and kept
rising at a lower rate until the end of exposure, reaching peak concentrations of about 10, 5.4, and
1.1 [J.M at 210,104, and 20.9 mg/m3, respectively. By 6 hours, the concentrations of ETBE had
fallen to very low levels (<1 |j.M), even after the 210-mg/m3 exposure. Based on blood AUC values
for ETBE, the authors calculated two types of respiratory uptake: (1) net respiratory
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uptake = (concentration in inhaled air - concentration in exhaled air) multiplied by the pulmonary
ventilation and (2) respiratory uptake = net respiratory uptake + amount exhaled during the
exposure. During the 2 hours of exposure, the authors calculated that 32-34% of each dose was
retained by the volunteers (respiratory uptake), and the net respiratory uptake was calculated to
be 26% of the dose at all three exposure levels. Over 24 hours, the respiratory expiration was
calculated as 45-50% of the respiratory uptake, and because the net respiratory uptake and
expiration do not consider the amount of ETBE cleared during exposure, the net respiratory
excretion was lower, at 30-31% of the net respiratory uptake. These authors determined that the
ETBE blood:air partition coefficient in humans was 11.7.
Ambergetal. f20001 exposed six volunteers (three males and three females, average age
28 ± 2 years) to 4.5 ppm (18.8 mg/m3) and 40.6 ppm (170 mg/m3) ETBE. The exposures lasted
4 hours, and the two concentrations were administered to the same volunteers 4 weeks apart.
These volunteers were healthy nonsmokers and were asked to refrain from alcohol and medication
intake from 2 days before the experiment until its end. The study was performed according to the
Declaration of Helsinki after approval by the Regional Ethical Committee of the institution where
the study was performed, and written informed consent was obtained from the volunteers. Urine
was collected at 6-hour intervals for 72 hours. Blood was drawn immediately after exposure and
thereafter every 6 hours for 48 hours. Levels of ETBE and its primary metabolite, tert-butanol,
were determined in blood. The same two substances, plus additional metabolites of tert-butanol,
were assessed in urine. The authors estimated the retained doses to be 1,090 [imol following
170-mg/m3 ETBE exposure and 121 [imol following 18.8-mg/m3 exposure. These estimates were
derived using a resting human respiratory rate of 9 L/minute (13 m3/day) and a retention factor
for ETBE of 0.3, which was based on data reported by Nihlen etal. f 19981. These estimates of
retained dose are lower than those reported during light exercise fNihlen et al.. 19981.
Absorption in Animals
Ambergetal. (2000) exposed F344 NH rats (5/sex/dose group) concurrent with the human
volunteers in the same exposure chamber. Blood was taken from the tail vein of each rat at the end
of the exposure period, and urine was collected for 72 hours at 6-hour intervals following exposure.
The authors reported that immediately after the 4-hour exposure period blood levels of ETBE were
lower in the rats than in humans, although the exact values were not reported. The authors
estimated that the rats received doses of 20.5 and 2.3 [imol at the 170- and 18.8-mg/m3 exposures,
respectively, using an alveolar ventilation rate of 0.169 L/minute and a retention factor of 0.3 for
rats.
No published oral dosing studies of the absorption of ETBE in humans were identified. The
Japan Petroleum Energy Center (JPEC), however, conducted an oral dosing study of the absorption
of ETBE in rats after single and repeated dosing for 14 days flPEC. 2008e. f). Seven-week-old
Crl:CD(SD) male rats (4/dose group) were administered either a single oral dose of 5, 50, or
400 mg/kg [14C]ETBE via gavage or 5 mg/kg-day [14C]ETBE daily for 14 days. In the single-dose
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study by TPEC (2008f). plasma levels were compared with those observed after a single intravenous
dose of 5 mg/kg-day [14C]ETBE. There is no indication that a similar comparison was conducted in
the repeated-dose study flPEC. 2008el Plasma radioactivity was measured in rats at 1, 2, 4, 6, 8,
10, and 24 hours after the first exposure in the repeated-dose study; 8 and 24 hours after the
second to 13th exposures; and at 1, 2, 4, 6, 8,10,12, 24, 32, 48, 72, 96,120,144, and 168 hours after
the last exposure in the repeated-dose study and after the single-dose study.
Plasma radioactivity levels increased following a single dose of [14C]ETBE; this increase was
not proportional as the dose increased, especially at the high dose (i.e., the peak plasma
radioactivity levels were 2,800, 22,100, and 89,900 ng equivalents of ETBE/mL [ng equivalent
ETBE/mL] in the 5-, 50-, and 400-mg/kg dose groups, respectively). Maximum plasma [14C]ETBE
levels (Cmax) were estimated to be reached at 9.0,11.5, and 8.0 hours after administration in the 5-,
50-, and 400-mg/kg dose groups, respectively. The [14C]ETBE levels in the plasma were higher
following oral exposure than after intravenous exposure (see Table B-2). The estimated
elimination plasma half-lives were 17.5,19.8, and 9.9 hours for the 5-, 50-, and 400-mg/kg dose
groups, respectively. With repeated dosing of 5 mg/kg-day [14C]ETBE flPEC. 2008e], the Cmax was
achieved 6 hours after the first exposure and increased until it reached a steady state around the
fifth day of exposure. After the last exposure on Day 14, the Cmax of 6,660 ± 407 ng equivalent
ETBE/mL was achieved 10 hours after administration of [14C]ETBE, and plasma radioactivity
steadily decreased after this point. The elimination plasma half-life from Cmax to 24 hours was
17.9 hours after the first dose and 14.2 hours after the final dose. The elimination half-life from
Cmax to 168 hours after the final dose following repeated dosing was 24.7 hours. Based on
radioactivity levels measured in urine and exhalation, more than 90% of the administered dose was
absorbed.
In two parallel studies, the pharmacokinetics of ETBE was studied in mice fSun and Beskitt.
1995a) and male Fischer 344 (F344) rats fSun and Beskitt. 1995bl. The study authors investigated
the pharmacokinetics of [14C]ETBE in mice and rats (3/sex/dose) exposed by nose-only inhalation
at target concentrations of 500, 750,1,000,1,750, 2,500, and 5,000 ppm (2,090, 3,130, 4,180, 7,310,
10,450, and 20,900 mg/m3) for a single 6-hour period (the true doses differed by less than 10%
from the targets). Specific activity of the administered [14C]ETBE and localization of the label were
not reported. Note, that in the absence of the specific activity and localization of the label, it is not
clear how the "mg ETBE equivalents" were calculated in the Sun and Beskitt f!995a. 1995bl report
or for the specific tissues. Of the three animals per sex exposed concurrently, two were used to
determine blood and tissue concentrations of radiolabel, and the third was kept in a metabolism
cage for up to 118 hours to quantify radiolabel excretion in urine, feces, exhaled carbon dioxide
(CO2), and as volatiles in expired air. Exhaled organic volatiles were trapped in charcoal filters.
Exhaled C02 was trapped in aqueous 1 M KOH. Samples from the 20,900-mg/m3 treated animals
were collected at 3, 6,12,18, 24, 48, 72, 96, and 118 hours after termination of exposure. At the
lower exposure concentrations listed above, the samples were collected at fewer time points,
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generally at full-day intervals up to 96 hours. Animals were euthanized either immediately after
exposure or after being removed from the metabolic cages, and blood and kidneys were collected.
The cages were washed, and the wash fluid collected. Charcoal traps were eluted with methanol.
Urine, cage wash, trapped 14CC>2, and charcoal filter eluates were measured directly by liquid
scintillation spectrometry. Blood and kidney tissue from rats and blood and liver tissue from mice
were combusted in a sample oxidizer and analyzed by liquid scintillation spectrometry.
Immediately upon cessation of exposure, radiolabel was quantified in the blood and kidneys
of two rats and in the blood and liver of two mice. Results in Table B-l demonstrate the absorption
of radiolabel expressed as mg equivalents of ETBE into blood. Because the ETBE carbon(s) bearing
the radiolabel was not identified, further speciation is not possible. The concentration of radiolabel
in rat blood is proportionate with exposure concentration to the highest concentration
(20,894 mg/m3), although in mice, such proportionality is absent at concentrations of
10,447 mg/m3 and above. These data indicate that ETBE is well absorbed following inhalation
exposure, but that higher concentrations (e.g., 10,447 mg/m3 and above) could result in reduced
respiration rates or otherwise affect mechanisms of inhalation uptake. Additional support for
reduction of absorption is presented in Table B-l, demonstrating the excretion of the radiolabel
from rats and mice in these studies fSun and Beskitt. 1995a. b).
Table B-l. Radioactivity in blood and kidney of rats and blood and liver of
mice, following 6 hours of [14C]ethyl tertiary butyl ether (ETBE) inhalation
exposure
Exposure level
(mg/m3)
F344 rat3
CD-I mouse3
Bloodb
Kidney0
Bloodb
Liver0
2,089
0.037
0.074
0.154
0.208
3,134
0.062
0.094
0.340
0.348
4,179
0.080
0.116
0.336
0.540
7,313
0.124
0.152
0.481
0.724
10,447
0.156
0.185
0.474
0.628
20,894
0.114
0.182
0.408
0.592
aMean values of one male and one female per rat/mouse.
bln mg [14C]ETBE equivalents per gram blood.
cln mg [14C]ETBE equivalents.
Sources: Sun and Beskitt (1995a) and Sun and Beskitt (1995b).
In contrast, Borghoff and Asgharian f 19961 evaluated the disposition of 14C radiolabel in
F344 rats and CD-I mice after whole-body and nose-only inhalation exposure to 500,1,750, or
5,000 ppm [14C]ETBE. Besides recovery of total radioactivity in urine, feces, and expired air, air and
urine samples were analyzed for ETBE and tert-butanol. Urine samples were also analyzed for
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tert-butanol metabolites hydroxyisobutyric acid (HBA) and 2-methyl-l,2-propanediol (MPD), and
14C02 was measured in exhaled air. Results obtained after both a single 6-hour exposure or after
13 days of preexposure to 0, 500, or 5,000 ppm ETBE indicate that total inhalation uptake increases
linearly with exposure concentration over this range, although there are dose- and
preexposure-related shifts in the form and route of excretion. Because the later study used four
rats per sex and exposure level, rather than just two, it should be given higher weight
No studies investigating dermal absorption of ETBE were identified, but because dermal
absorption of homologous organic substances is thought to be a function of the octanol:water
partition coefficient, ETBE might be assumed to penetrate rat skin relatively well. For humans,
Potts RO T19921 proposed an equation to calculate the dermal permeability coefficient, Kv\
log Kp (cm/sec) = -6.3 + 0.71 x logKow - 0.0061 x (molecular weight) (B-l)
Using the log Kow [identified as Koct in Potts RO (1992)] values for ETBE [0.95-2.2; Drogos
and Diaz f20011] and converting cm/second values to cm/hour, the estimated Kv values are
0.0020-0.016 cm/hour for ETBE.
ETBE is moderately absorbed following inhalation exposure in rats and humans, and blood
levels of ETBE approached—but did not reach—steady-state concentrations within 2 hours. Nihlen
etal. (19981 calculated the net respiratory uptake of ETBE in humans to be 26%. The AUC for the
concentration-time curve was linearly related to the ETBE exposure level, suggesting linear kinetics
up to 209 mg/m3. The JPEC studies (TPEC. 2008e. f) demonstrated that ETBE is readily absorbed
following oral exposure in rats with >90% of a single dose (5-400 mg/kg-day) or repeated doses
(5 mg/kg-day) estimated to be absorbed. In the repeated-dose study, peak plasma [14C]ETBE levels
were reached 6 hours after the first dose and 10 hours after the final (14th) dose, and the maximum
plasma concentration reached a steady state on Day 5. No data are available on dermal absorption
of ETBE.
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Supplemental Information—ETBE
Table B-2. Plasma radioactivity after a single oral or intravenous dose of
[14C]ethyl tertiary butyl ether (ETBE) to male Crl:CD(SD) rats
Time (h)
Radioactive concentration (ng eq of ETBE/mL)
Oral
Intravenous
Dose administered
5 mg/kg
50 mg/kg
400 mg/kg
5 mg/kg
0.083
-
-
-
918 ± 188a
0.25
-
-
-
822 ±165
0.5
-
-
-
914±156
1
2,150 ± 281
11,100 ± 1,007
47,000 ± 11,900
907 ±143
2
2,400 ±151
12,100 ± 883
58,200 ± 7,340
923 ±158
4
2,620 ± 109
14,800 ± 659
73,300 ± 6,800
929 ±193
6
2,750 ± 146
18,700 ± 1,550
82,900 ± 12,500
981 ±216
8
2,760 ± 265
19,900 ± 2,430
89,900 ± 16,300
973 ±196
10
2,710 ± 303
21,400 ±2,830
87,300 ± 15,300
943 ± 203
12
2,660 ± 426
22,000 ± 3,060
78,500 ± 18,100
862 ± 205
24
1,330 ± 419
10,800 ± 2,820
17,200 ± 6,460
383 ±184
32
1,170 ± 424
9,310 ± 2,510
13,100 ± 6,580
334 ±190
48
443 ±271
3,900 ± 1,480
3,180 ± 1,480
144 ± 93.8
72
204 ±165
1,660 ± 845
2,000 ± 1,820
65.2 ±34.0
96
81.3 ±70.3
792 ± 338
N.D.
31.3 ± 11.4
120
35.9 ±44.0
385 ±110
N.D.
16.1 ±3.8
144
19.6 ±26.0
179 ±129
N.D.
11.9 ± 13.8
168
N.D.
85.4 ± 103
N.D.
N.D.
- = not measured; N.D. = not detected.
aMean ± standard deviation; n = 4.
Source: JPEC(2008e).
1 B.1.2. Distribution
2 There are no in vivo data on the tissue distribution of ETBE in humans. Nihlen etal. (1995)
3 measured the partitioning of ETBE and tert-butanol in air into human blood from 10 donors
4 (5 males, 5 females), saline, or oil inside of sealed vials. Also, human tissue-to-blood partitioning
5 coefficients were estimated in brain, fat, liver, kidney, lung, and muscle based on their relative
6 water and fat contents. Kaneko etal. (2000) conducted a similar series of in vitro studies to
7 measure the partitioning of ETBE and tert-butanol in air to various rat tissues (5 male Wistar rats),
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including blood, brain, fat, liver, kidney, lung, muscle, and testis. The blood:air partition coefficients
for ETBE were much lower than for tert-butanol. Both studies reported efficient uptake of these
substances from air into blood, with blood:air partition coefficients of 11.7 and 11.6 for ETBE and
462 and 531 for tert-butanol in humans and rats, respectively. Nihlen etal. f 19951 also estimated
oil:water partition (log Kow) coefficients and obtained values of 0.278 for tert-butanol and 22.7 for
ETBE. These values have a similar ranking, but are not identical, to those listed in a report by
Drogos and Diaz (20011 (namely, 0.35 for tert-butanol and 1.48-1.74 for ETBE). Nihlen etal.
(19951 used the coefficients of tissue:air and blood:air partition coefficients to calculate human
tissue:blood partition coefficients. These values are listed in Table B-3.
Table B-3. Bloochtissue partition coefficients for ethyl tertiary butyl ether
(ETBE) and tert-butanol
Partition coefficient
tert-Butanol
ETBE
Blood:air
462
11.7
Brain:blood
1.05
2.34
Muscle:blood
1.06
1.78
Fat:blood
0.646
11.6
Lung:blood
1.02
0.835
Kidney:blood
1.06
1.42
Liver:blood
1.05
1.44
Source: Nihlen et al. (1998).
The TPEC (2008e. 2008f) study examined the distribution of radioactivity in 7-week-old
Crl:CD(SD) male rats (4/dose group) following either a single oral dose of 5 or 400 mg/kg
[14C]ETBE via gavage or a repeated dose of 5 mg/kg-day for 7 or 14 days. Tissue samples were
collected at 8, 24, 72, and 168 hours after a single dose; 8 and 24 hours after 7 days of repeated
dosing; and 8, 24, 72, and 168 hours after 14 days of repeated dosing. Although the highest
radioactivity levels were generally detected in plasma, [14C]ETBE was also detected in all tissues
examined (brain, peripheral nerve, eyes, submaxillary gland, thyroid gland, thymus, lungs, kidneys,
heart, liver, adrenal glands, spleen, pancreas, bone marrow, mesenteric lymph node, prostate,
epididymis, testes, muscle, skin, adipose tissue, stomach, large intestines, and small intestines).
Tissue concentrations after a single 400 mg/kg dose of [14C]ETBE were higher than after a single
5 mg/kg dose; however, the percentage distribution of radioactivity in tissues was lower with the
higher dose. Tissue radioactivity levels reached a maximum at 8 hours after a single dose of either
5 or 400 mg/kg [14C]ETBE and rapidly decreased by 72 hours. In the repeated-dose study, the
radioactivity was the same 8 hours after the seventh administration when compared to 8 hours
after the 14th administration. The levels of [14C]ETBE in the tissues declined steadily from 8 hours
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through 168 hours after the last exposure except in adipose tissue. In adipose tissue, there was a
rapid decline between 8 and 24 hours, but the levels remained consistent between the 24- and
168-hour time points. The percentage of radioactivity found in red blood cells was estimated to be
20-27% within 72 hours of administration, and little was found to be bound to plasma proteins.
Sun and Beskitt f!995al and Sun and Beskitt f!995bl studied the distribution of radiolabel
derived from [14C]ETBE in rats and mice, respectively. The animals were subjected to a single
nose-only inhalation exposure to [14C]ETBE for 6 hours. Immediately upon cessation of exposure,
radiolabel was quantified in the blood and kidneys of two rats and in the blood and liver of two
mice. Results in Table B-l (shown earlier) demonstrate the distribution of radiolabel expressed as
mg equivalents of ETBE from blood to kidney (rats) and liver (mice) during exposure. The
concentration of radiolabel in rat kidney and mouse liver parallels the concentration of radiolabel
in blood of the respective species, leading to an expectation of the proportionate distribution of 14C
from ETBE to rat kidney and mouse liver up to exposure concentrations of 7,313 mg/m3 in rats and
10,447 mg/m3 in mice. Because radiolabel levels do not distinguish between parent ETBE and its
metabolites, these results need to be interpreted with some caution, as the distribution of
individual chemical species may differ.
Leavens and Borghoff f20091 evaluated the distribution of the structurally similar
compound, methyl tert-butyl ether (MTBE), and the common metabolite, tert-butanol, after
inhalation exposure to those two compounds, specifically in the brain, kidney, and liver of male and
female rats and testis of male rats. Concentrations of MTBE and tert-butanol were similar in the
female rat brain, kidney, and liver, and concentrations in the male rat brain, liver, and testes, were
similar for exposure level and across time points, indicating an even distribution of MTBE and
tert-butanol in those tissues/sexes. While total concentrations of MTBE and tert-butanol were
higher in male rat kidneys than other tissues, consistent with the mechanism of binding to alpha
2u-globulin for those two compounds fLeavens and Borghoff. 20091. the overall observations are
consistent with the conclusion that unbound ETBE and tert-butanol distribute rapidly and evenly
through the body, although additional accumulation of material bound to alpha 2u-globulin occurs
for tert-butanol and may occur for ETBE in the male rat kidney.
B.1.3. Metabolism
The metabolism of ETBE has been studied in rats and humans using both in vivo and in
vitro methods. A schematic of the proposed metabolism of ETBE is presented in Figure B-l. Based
on elucidated structures of urinary metabolites from rats that were exposed to ETBE by inhalation,
ETBE is initially metabolized by cytochrome P450 (CYP450) enzymes via oxidative deethylation by
the addition of a hydroxyl group to the a-carbon of the ethyl ether group (Bernauer et al.. 1998).
The resulting hemiacetal is unstable and decomposes spontaneously into tert-butanol and
acetaldehyde. In human liver microsome preparations, this step is catalyzed mainly by CYP2A6,
with some contribution from CYP3A4 and CYP2B6 and possible contribution from CYP2E1 fLe Gal
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etal.. 2001: Hong etal.. 1999a). Using data from rat hepatic microsome preparations, Turini etal.
f!9981 suggested that CYP2B1 is the primary enzyme responsible for this step in rats but that
CYP2A1 may also have an important role. Acetaldehyde is oxidized to acetic acid by aldehyde
dehydrogenase enzymes (some of which are polymorphically expressed) and eventually to CO2.
tert-Butanol can be sulfated, glucuronidated, and excreted into urine, or it can undergo further
oxidation by the CYP enzymes (but not by alcohol dehydrogenases) to form MPD, and
2-hydroxyisobutyrate (HIBA), acetone, and formaldehyde (Bernauer et al.. 1998). Note also that
these metabolites have been identified in studies using liver preparations from human or rat
studies using ETBE, MTBE, or tert-butanol fBernauer et al.. 1998: Cederbaum and Cohen. 19801:
however, all the enzymes that perform these metabolic steps have not been fully described.
Excretion studies indicate that final metabolism to CO2 plays only a minor role (see Section B.1.4).
CYP2A6
CYP3A4
O | CH3-
CH3
ETBE
glucuronide-0 ——CH3
CH3
t-butyl glucuronide
CH,
0X0,
H3c—( ch3
OH
CH, OH
CYP450
ch3 H,C-
ETBE—hemi-acetal
CH3
t-butanol
y°
rats,
humans
A
CH3 oh
2-methyl-1,2-propanediol
r
H3C—^OH
CH,
/
h3c—V v5' ch|3
o \
acetaldehyde 0
ch3
t-butyl sulfate
H°_°
[O]
2-hydroxyisobutyric acid
h2c=o
formaldehyde
h3c" "ch3
acetone
Figure B-l. Proposed metabolism of ethyl tertiary butyl ether (ETBE).
Source: Adapted from Dekant et al. (2001), NSF International (2003), ATSDR (1996), Bernauer et al. (1998), Amberg
et al. (1999), and Cederbaum and Cohen (1980).
Zhang etal. f!9971 used computer models to predict the metabolites of ETBE. The
metabolism model correctly predicted cleavage into tert-butanol and acetaldehyde and that
tert-butanol would undergo glucuronidation and sulfation. For the further metabolism of
tert-butanol, however, the computer model predicted reductive steps leading to metabolites that
have not been identified in vivo or in vitro. The software did not predict the formation of MPD or
HIBA, which have been found in vivo.
Metabolism in Humans
Metabolism of ethyl tertiary butyl ether fETBEl in humans in vivo
Nihlen etal. (1998) exposed eight healthy male volunteers (average age: 29 years) to 0,
20.9,104, or 209 mg/m3 ETBE by inhalation for 2 hours. Profiles of ETBE, tert-butanol, and acetone
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were established for blood throughout exposure and for up to 22 hours thereafter. The blood
profiles of parent compounds and metabolites were similar at all three exposure levels and
reflected exposure concentrations, as judged by linear increases in concentration-time AUC values
calculated by the authors (only reported graphically). Acetone levels were highly variable before,
during, and after the exposure period, and the variation could likely be due to variations in
endogenous acetone production due to diet or physical activity.
The concentration of ETBE in blood rose sharply during the first 3 0 minutes of exposure
and kept rising at a lower rate until the end of exposure to reach peak concentrations of about 10, 5,
and 1 [J.M at 209,104, and 20.9 mg/m3, respectively. By 6 hours, ETBE concentrations had fallen to
low levels even after exposure to 209 mg/m3. The blood concentration of tert-butanol continued to
rise for the full 2-hour exposure period, with peak values of about 12 and 7 |j.M at 209 and
104 mg/m3, respectively. Blood concentrations leveled off for 3-4 hours and then began a slow
decline to less than one-half maximum levels by 24 hours [tert-butanol levels could not be
determined following 20.9 mg/m3 exposure). Acetone blood levels began to increase after about
1 hour of exposure and continued to increase after the end of exposure (high dose) or leveled off for
about 1.5 hours after exposure (lower doses and controls). Blood acetone levels fell rapidly during
the next half hour but remained slightly above normal for the exposed volunteers until 4 hours
after exposure when measurements were terminated.
Ambergetal. (2000) exposed six volunteers (three males and three females; average age:
28 ± 2 years) to 18.8 and 170 mg/m3 of ETBE. The exposures lasted 4 hours, and the two
concentrations were administered to the same volunteers 4 weeks apart Urine was collected at
6-hour intervals for 72 hours. Blood was drawn immediately, at 4 or 6 hours after exposure, and
thereafter every 6 hours for 48 hours. Levels of parent ETBE and its primary metabolite,
tert-butanol, were determined in blood and urine. In urine, two further metabolites of tert-butanol,
MPD and HIBA, were also assayed.
At 170 mg/m3, the mean peak blood concentration of ETBE was 12.1 ± 4.0 |j.M, although for
tert-butanol it was 13.9 ± 2.2 [J.M. The corresponding values at 18.8 mg/m3 were 1.3 ± 0.7 and
1.8 ± 0.2 [J.M, respectively. The time courses of metabolite appearance in urine after 170 and
18.8 mg/m3 were similar, but relative urinary levels of metabolites after 18.8 mg/m3 differed from
those after 170 mg/m3. Using parent ETBE as the reference, molar ratios for total urinary excretion
(ETBE:tert-butanol:MPD:HIBA) were 1:25:107:580 after 170 mg/m3, and 1:17:45:435 after
18.8 mg/m3. Individual variations were large, but the authors did not report any sex differences in
the metabolism of ETBE based on data from only three subjects of each sex.
In vitro metabolism of ethyl tertiary butyl ether (ETBE) using human enzyme preparations
The metabolism of ETBE has been studied in vitro using microsomal protein derived from
human liver and from genetically engineered cells expressing individual human CYP isozymes.
Hong etal. f!997bl coexpressed human CYP2A6 or CYP2E1 with human CYP reductase in insect SF9
cells. In this heterologous expression system, in the presence of 1 mM ETBE, tert-butanol was
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formed at rates of 13.6 nmol/min-nmol CYP2A6 and 0.8 nmol/min-nmol CYP2E1, indicating a
greater capacity for ETBE metabolism by CYP2A6 than by CYP2E1 at high (e.g., 1 mM)
concentrations of ETBE.
Hong etal. f!999al obtained hepatic microsomal protein preparations from 15 human
donor liver microsomal samples and used them to evaluate the contributions of several CYP
enzymes to ETBE metabolism. The 15 samples displayed very large interindividual variations in
metabolic activities towards ETBE ranging from 179 to 3,130 pmol/minute-mg protein.
Michaelis-Menten constant (Km) values, estimated in three human liver microsomal samples using
MTBE, ranged from 28 to 89 |iM, with maximum substrate turnover velocity (Vmax) values ranging
from 215 to 783 pmol/minute-mg protein. The Vmax:Km ratios, however, varied only between 7.7
and 8.8. After evaluating the activities of multiple different CYP forms in the 15 donor samples, the
study authors demonstrated that the metabolism of ETBE was highly correlated with certain CYP
forms. The highest degree of correlation was found for CYP2A6, which also displayed the highest
metabolic capacity for ETBE.
As part of CYP inhibition studies in the same paper, human liver microsomes were
coincubated with ETBE in the presence of chemical inhibitors or specific antibodies against either
CYP2A6 or CYP2E1. For chemical inhibition, coumarin was added to the liver microsomes prior to
initiation of the reaction. For antibody inhibition, monoclonal antibodies against human CPY2A6 or
CYP2E1 were preincubated with liver microsomes prior to incubation with the rest of the reaction
mixture. Methanol alone caused approximately 20% inhibition of the metabolism of ETBE, and
coumarin, a CYP2A6 substrate, caused a significant dose-dependent inhibition of ETBE metabolism,
which reached a maximal inhibition of 99% at 100-|j.M coumarin. The antibody against CYP2A6
inhibited metabolism by greater than 75% but did not do so against CYP2E1.
In the same paper, several specific human CYPs were expressed into human
P-lymphoblastoid cells which were used to evaluate ETBE metabolism. Based on the ETBE
metabolizing activities in the 15 human liver microsomes and the enzyme activity profiles towards
known CYP specific substrates, correlation coefficients (ranging from 0.94 for CYP2A6 to 0 for
CYP2D6) were calculated for each CYP enzyme. The correlation ranking for ETBE metabolism by
nine human CYP isozymes was as follows:
2A6 > 3A4 « 2B6 « 3A4/5 » 2C9 > 2E1 « 2C19 » 1A2 « 2D6. The reported direct enzyme
activities towards ETBE by the heterologous expression systems (in pmol tert-butanol formed per
minute per pmol CYP enzyme) were 1.61 for CYP2A6; 0.34 for CYP2E1; 0.18 for CYP2B6; and 0.13
for CYP1A2. CYPs 1B1, 2C8, 2C9, 2C19, and 2D6 were not investigated. CYP3A4 and 1A1 did not
metabolize ETBE. The authors concluded that CYP2A6 is the major enzyme responsible for the
oxidative metabolism of ETBE in human livers. Furthermore, they concluded that the results of the
correlation analysis and antibody inhibition study strongly suggest that CYP2E1 is nota major
enzyme responsible for metabolism of ETBE. Le Gal etal. (2001) used similar human cytochrome
preparations as Hong etal. f!999al (i.e., from human donors) or used genetically modified human
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P-lymphoblastoid cell lines transfected with CYP2A6, CYP2B6, CYP3A4, or CYP2E1 and human CYP
reductase to elucidate the metabolism of ETBE, MTBE, and tertiary amyl methyl ether. They
identified acetaldehyde and tert-butanol as primary metabolites from ETBE.
Metabolism in Animals
Metabolism of ethyl tertiary butyl ether (ETBE) in animals in vivo
Bernauer et al. (19981 studied the metabolism and excretion of [13C]ETBE and tert-butanol
in rats. F344 rats, 2/sex, were exposed via inhalation to 2,000 ppm (8,400 mg/m3) ETBE; three
male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours. The
excretion profile for ETBE metabolites was
MPD > HIBA > tert-butanol-sulfate > tert-butanol-glucuronide. Oral administration of tert-butanol
produced a similar metabolite profile, with
HIBA > tert-butanol-sulfate > MPD » tert-butanol-glucuronide ~ tert-butanol. tert-Butanol could
not be detected in urine following inhalation exposure to ETBE. Traces of acetone were also
detected in urine. Ambergetal. f20001 exposed F344 NH rats, 5/sex/dose, to ETBE in the same
exposure chamber described earlier for the human volunteers. Urine was collected for 72 hours
following exposure. Blood samples were drawn from the tail vein every 6 hours up to 48 hours.
Peak blood levels of ETBE and tert-butanol were 5.3 ± 1.2 and 21.7 ± 4.9 |j.M at 170 mg/m3 and
1.0 ± 0.7 and 5.7 ± 0.8 |j.M at 18.8 mg/m3, respectively. Peak levels of tert-butanol were higher in
rats than in humans. Like humans, rats excreted mostly HIBA in urine, followed by MPD and
tert-butanol. The molar ratios for total urinary excretion of tert-butanol:MPD:HIBA were 1:2.3:15
after exposure to 170 mg/m3 and 1:1.5:11 after exposure to 18.8 mg/m3. Parent ETBE was not
identified in rat urine in this study.
In a review covering mostly their own work on fuel oxygenate metabolism, Dekant et al.
f20011 focused on aspects of ETBE metabolism that were considered quantitatively similar in
humans and rats, with no sex-dependent differences and no likely accumulation of metabolites or
parent compound. They reported that at a high exposure level (8,400 mg/m3 ETBE), rats
predominantly excreted the glucuronide of tert-butanol in urine; however, at low exposure levels
(16.7 or 167.1 mg/m3 ETBE), the relative concentration of tert-butanol to the received dose was
much smaller. This result seems to indicate that at high exposure levels, the normally rapid
metabolism of tert-butanol to MPD and HIBA became saturated, forcing more of the tert-butanol
through the glucuronidation pathway. The apparent final metabolite of ETBE was HIBA, which can
undergo further metabolism to acetone. The latter process appeared to play a minor role in the
overall metabolism of ETBE. Dekant etal. (2001) also noted that many metabolites of the fuel
oxygenate ethers, such as formaldehyde, acetaldehyde, tert-butanol, HIBA, or acetone, occur
naturally in normal mammalian physiology, providing a highly variable background that needs to
be accounted for in metabolic experiments.
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TPEC (2008e. 2008f) measured metabolite distribution in the plasma and urine of
7-week-old Crl:CD(SD) male rats (4/dose group) following either a single oral dose of 5 or
400 mg/kg [14C]ETBE via gavage or a repeated dose of 5 mg/kg-day for 7 or 14 days. The
metabolites were measured in the plasma 8 hours after single or repeated dosing and measured in
urine collected on Days 1, 7, and 14 after repeated dosing or during a 24-hour period after
administration of the single dose. The number of doses did not appear to affect the metabolic
pattern. The study authors determined the identities of five metabolites, and the results in plasma
and urine are summarized in Table B-4 and Table B-5, respectively. When combined with what is
known of the metabolic pathway for ETBE, these data indicate that ETBE is efficiently metabolized
to tert-butanol, which is then metabolized to tert-butanol glucuronide, 2-methyl-l,2-propanediol,
and finally to 2-hydroxyisobutyrate.
Although Sun and Beskitt f!995al did not identify the radiolabel excreted, their
investigations do yield information pertinent to determining whether metabolic saturation might
occur under bioassay conditions. In their single-exposure protocol, rats and mice were exposed via
inhalation to ETBE. These investigators reported the fraction of absorbed dose that was excreted in
urine and feces, as expired volatiles, and as expired CO2 from one rat and one mouse. At inhaled
concentrations between 4,180 and 7,310 mg/m3 a shift in the primary route of excretion was
observed, as demonstrated by a marked decrease in the fraction of radiolabel excreted in urine and
a marked increase in the fraction of radiolabel eliminated as volatiles in expired air, and (in rats) a
doubling of the fraction eliminated as exhaled CO2. Given the different solubilities, molecular size,
and other characteristics of ETBE and its multiple metabolites, it is thought that this shift in the
excretion pattern of radiolabel is indicative of a shift in metabolism at these exposure levels.
Considering the potential shift in metabolic pattern relative to the pattern of toxicity can be
informative, especially related to species and dose extrapolation. These data might still be
considered preliminary because they are from one animal of each species, have not been replicated
by other study authors, and the radiolabel has not been speciated as to chemical form. The
unfortunate limitation of the application of the physiologically based pharmacokinetic (PBPK)
model for human inhalation precludes its combination with rat PBPK models to complete species
extrapolation. The inhalation toxicity study by Saito etal. (2013). however, demonstrated an
increased incidence of urothelial hyperplasia at an exposure concentration of 6,270 mg/m3 and
higher, and an increased incidence of hepatocellular adenoma or carcinoma only at an exposure
concentration of 20,900 mg/m3. Additional data are required to determine whether increases in
incidence could be related to pharmacokinetic effects (e.g., metabolic saturation).
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Supplemental Information—ETBE
Table B-4. Unchanged ethyl tertiary butyl ether (ETBE) and its metabolites in
plasma 8 hours after a single oral dose or repeated (7 or 14) daily oral dosing
of [14C]ETBE to male Crl:CD(SD) rats
Compound
Metabolite
Percentage of dose
1 Dose
7 Doses
14 Doses
5 mg/kg-d
400 mg/kg-d
5 mg/kg-d
5 mg/kg-d
Unchanged ETBE
ETBE
N.D.
N.D.
N.D.
N.D.
P-l
2-Hydroxyisobutyrate
75.4 ± 8. la
35.7 ±2.5
71.4 ±4.7
69.8 ±7.3
P-2
tert-Butanol glucuronide
N.D.
N.D.
N.D.
N.D.
P-3
Not enough to
determine
N.D.
N.D.
N.D.
N.D.
P-4
2-Methyl-l,2-
propanediol
9.7 ±2.4
9.328 ±0.9
9.1 ±0.8
8.1 ± 1.4
P-5
tert-Butanol
12.9 ±3.1
55.0 ±2.9
18.2 ±3.8
22.2 ±6.0
N.D. = not detected.
aMean ± standard deviation; n = 4.
Source: JPEC (2008e, 2008f) unpublished reports.
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Table B-5. Unchanged ethyl tertiary butyl ether (ETBE) and its metabolites in
the urine (measured 0-24 hours) after a single oral dose or repeated (7 or 14)
daily oral dosing of [14C]ETBE to male Crl:CD(SD) rats
Compound
Metabolite
Percentage of dose
1 Dose
7 Doses
14 Doses
5 mg/kg-d
400 mg/kg-d
5 mg/kg-d
5 mg/kg-d
Unchanged ETBE
ETBE
0.7 ±0.5a
N.D.
0.9 ±0.6
1.4 ±0.4
P-l
2-Hydroxyisobutyrate
53.0 ±3.4
55.4 ±4.7
58.9 ±4.2
56.0 ±5.2
P-2
te/t-Butanol glucuronide
29.2 ±3.0
25.9 ±4.6
22.8 ±3.2
25.2 ±5.8
P-3
Not enough to determine
2.5 ±0.2
1.7 ±0.4
2.2 ±0.3
1.7 ±0.4
P-4
2-Methyl-l,2-
propanediol
13.1 ±0.6
13.3 ±2.5
13.4 ± 1.5
13.9 ±2.3
P-5
te/t-Butanol
1.5 ±0.5
3.7 ±0.6
1.9 ±0.2
1.8 ±0.0
N.D. = not detected.
aMean ± standard deviation; n = 4.
Source: JPEC (2008e, 2008f) unpublished reports.
Borghoff and Asgharian T19961 evaluated the disposition of a 14C radiolabel in F344 rats and
CD-I mice after whole-body and nose-only inhalation exposure to 500,1,750, or 5,000 ppm
[14C]ETBE. Besides recovery of total radioactivity in urine, feces, and expired air, the air and urine
samples were analyzed for ETBE and tert-butanol. Urine samples were also analyzed for
tert-butanol metabolites, HBA and MPD. Results obtained after both a single 6-hour exposure or
after 13 days of preexposure to 0, 500, or 5,000 ppm ETBE indicated dose- and preexposure-related
shifts in the form and route, likely due to metabolic factors. Excretion shifted from being primarily
in the urine after 500 ppm exposure to primarily by exhalation at 5,000 ppm in naive rats,
indicating a saturation of metabolism of ETBE to TBA. This shift was greater in female rats than in
males. However, in rats preexposed to 5,000 ppm ETBE for 13 days, most of the excretion was in
the urine even at 5,000 ppm. Rats preexposed to 500 ppm ETBE also showed a shift from
exhalation to urinary excretion in comparison to naive rats, but to a smaller degree than elicited by
5,000 ppm preexposure. The changes in excretion after preexposure indicated an induction of the
metabolism of ETBE to tert-butanol. As with rats, the fraction of radiolabel in exhaled volatiles in
mice increased with exposure level, while the fraction excreted in urine decreased. The exhalation
pattern observed in rats showed levels of ETBE falling ~90% in the first 8 hours postexposure,
whereas levels of TBA exhaled actually rose between 0 and 3 hours postexposure and then fell
more slowly between 3 and 16 hours, particularly after 5,000 ppm ETBE exposure. The increase in
TBA between 0 and 3 hours postexposure can be explained by the continued metabolism of ETBE
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during that period. The slower decline after 3 hours can be explained as a result of the generally
slower clearance of TBA, which is saturated by the higher ETBE exposure levels.
Metabolism of ethyl tertiary butyl ether fETBEl in animal tissues in vitro
Using microsomal protein isolated from the olfactory epithelium from male
Sprague-Dawley (S-D) rats, Hongetal. (1997a) measured ETBE metabolism as the formation of
TBA. They found that metabolism occurred only in microsomal protein (not in cytosol) and only in
the presence of an nicotinamide adenine dinucleotide phosphate (NADP)-regenerating system. The
metabolic activity was inhibited by 80% after treating the microsomal preparation with carbon
monoxide and by 87% in the presence of coumarin (a CYP2A6 inhibitor), which indicates CYP
involvement. Using an in vitro concentration of 1 mM ETBE, metabolic activity could not be
detected in microsomal protein from the olfactory bulb, lungs, or kidneys. Activity toward ETBE
was 8.78, 0.95, and 0.24 nmol/minute-mg microsomal protein in olfactory mucosa, respiratory
mucosa, and liver, respectively. In olfactory mucosa, the study authors reported a Km value of
125 for ETBE.
Hong etal. f!999bl used hepatic microsomal protein derived from Cyp2el knockout mice to
investigate whether this enzyme plays a major role in ETBE metabolism. They compared the
metabolizing activity of liver microsomes (incubated for 30 minutes at 37°C and with 0.1 mM
ETBE) between the Cyp2el knockout mice and their parental lineage strains using four or five
female mice (7 weeks of age) per group. The ETBE-metabolizing activities were not significantly
different between the Cyp2el knockout strain (0.51 ± 0.24 nmol/minute-mg protein) compared
with those observed in the Cyp2el wild-type parental strains (0.70 ± 0.12 for C57BL/6N mice, and
0.66 ± 0.14 for 129/Sv mice). Therefore, microsomal protein from mice thatdid notexpress any
CYP2E1 did not differ from microsomal protein derived from wild-type animals in their ability to
metabolize ETBE in vitro, suggesting that CYP2E1 might contribute only little to ETBE metabolism
in vivo. Furthermore, these authors evaluated potential sex- and age-dependent differences for the
metabolism of 1 mM concentrations of ETBE by hepatic microsomal protein. Although activities in
female knockout mice were approximately 60% of those in male knockout mice, the difference did
not reach the level of statistical significance. Finally, observed rates of ETBE metabolism
(approximately 0.5 to 0.9 nmol/min/mg microsomal protein) did not seem to differ when assayed
at 0.1 or 1 mM, indicating that for mouse hepatic microsomal ETBE metabolism, saturation can
occur at concentrations no higher than 0.1 mM in vitro, and that Km values would be expected to be
lower than 0.1 mM in vitro.
Turini etal. (1998) investigated the effects of ETBE exposure on P450 content and
activities, and characteristics of ETBE metabolism in hepatic microsomal protein from male
Sprague-Dawley rats in an attempt to elucidate the role of CYP2E1 in ETBE metabolism.
Administration of ETBE at 200 or 400 mg/kg for 4 days did not alter hepatic CYP profiles, but the
administration of 2 mL ETBE/kg resulted in significant increases of metabolic activities toward
substrates characteristic for CYP2B and CYP2E1 (p-NPH) forms, but not of activities catalyzed by
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CYP3A or 1A forms. Studies of ETBE metabolism were based on high-performance liquid
chromatography detection of the acetaldehyde ETBE metabolite. Induction of CYP2B forms in vivo
via the administration of phenobarbital slightly reduced the Km value and produced a significant,
approximate threefold increase in Vmax; in these preparations; chemical inhibition of CYP2B forms
resulted in significant inhibition of ETBE metabolism. Studies with CYP enzymes purified from rats
confirmed metabolic competency of several CYP forms, with the activity of purified rat CYP forms
2B1 > 2E1 > 1A1 > 2C11. Chemical inhibition of CYP2E1 did not reduce ETBE metabolic activity;
CYP2A forms were not evaluated. In microsomal preparations from rats treated with phenobarbital
(a CYP2B inducer), incubation with chemical inhibitors of CYP2B forms produced a significant
decrease in ETBE metabolism. Pretreatment of rats with chemicals known as inducers of CYP2E1,
CYP3A, and CYP1A forms did not result in significant changes in Km or Vmax values for ETBE
metabolism, as measured in vitro. The results of these investigations indicate that, in rats, CYP2E1
is apparently minimally involved in ETBE metabolism, and that under some conditions, CYP2B
forms can contribute to ETBE metabolism. The role of CYP2A forms was not studied in this
investigation. This study also investigated the kinetic constants for ETBE metabolism in control rat
hepatic microsomal protein, indicating a Km value of 6.3 mM and a Vmax value of 0.93 nmol/min/mg
microsomal protein. When compared with the kinetic constants indicated by the results of Hong et
al. f 1999bl. the rate of ETBE metabolism at in vitro concentrations below 1 mM are expected to be
higher in mouse than in rat microsomal preparations.
The enzymes that metabolize tert-butanol to MPD, HIBA, and even acetone, have not been
fully characterized; however, tert-butanol is not subject to metabolism by alcohol dehydrogenases
(Dekant etal.. 20011.
B.1.4. Excretion
Excretion in Humans
Nihlen etal. (1998) exposed eight healthy male volunteers (average age 29 years) to 20.9,
104, and 209 mg/m3 ETBE by inhalation for 2 hours. ETBE, and two metabolites [tert-butanol and
acetone) were measured in urine for up to 22 hours after exposure. The blood profiles of the
parent compound and metabolites were similar at all three exposure levels and reflected exposure
concentrations. The study authors estimated the inhaled amount of ETBE in the volunteers to be
0.58, 2.9, and 5.8 mmol for the 20.9-, 104-, and 209-mg/m3 exposure levels, respectively. Based on
blood AUC values for ETBE and metabolites, the authors calculated respiratory uptake to be
32-34% in humans and the net uptake (which excludes ETBE exhaled during exposure) to be 26%
of the dose at all three exposure levels. During the 24 hours following the start of inhalation
exposure, respiratory expiration was calculated at 45-50% of the inhaled ETBE (respiratory
uptake), and net respiratory expiration was 31% (of the net respiratory uptake), of which
tert-butanol accounted for only 1.4-3.8%. Urinary excretion of parent ETBE (as percentage of the
respiratory uptake of ETBE) accounted for even less: 0.12, 0.061, and 0.056% after the exposures to
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20.9,104, and 209 mg/m3, respectively. The authors identified four phases of excretion of ETBE
from blood, with half-lives of about 2 and 20 minutes and 1.7 and 28 hours. Only one phase for
excretion of tert-butanol from blood was identified with a half-life of 12 hours compared with
10 hours in another study with volunteers flohanson etal.. 19951. In urine, ETBE displayed two
phases of excretion, with half-lives of about 8 minutes and 8.6 hours. The half-life of tert-butanol in
urine was determined to be 8 hours (lohanson et al.. 1995).
ETBE displayed a multiphasic excretion from blood. The first phase likely indicates uptake
into highly perfused tissues. The other phases could indicate uptake into less perfused tissues and
fat, or result from metabolism events. The apparent total body clearance of ETBE (based on the net
respiratory uptake) was 0.57 L/hour-kg (average of the three exposure levels). The metabolic
clearance was calculated as 0.39 L/hour-kg and the exhalation clearance as 0.35 L/hour-kg. These
authors reported that the kinetics of ETBE in humans was linear over the range of concentrations
studied (Nihlen etal.. 1998).
In the study by Amberg etal. (2000) that was described earlier, two excretion half-lives
were found for ETBE (1.1 ± 0.1 and 6.2 ± 3.3 hours) at the high exposure concentration
(170 mg/m3), although tert-butanol displayed only one half-life (9.8 ± 1.4 hours). Atthe low
exposure concentration (18.8 mg/m3), only the short half-life for ETBE could be measured at
1.1 ± 0.2 hours, although that for tert-butanol was 8.2 ± 2.2 hours. The predominant urinary
metabolite, identified was HIBA, was excreted in urine at 5-10 times the amount of MPD and
12-18 times the amount of tert-butanol (note: urine samples had been treated with acid before
analysis to cleave conjugates). Excretion of unchanged ETBE in urine was minimal. The time
courses of urinary excretion for 170 and 18.8 mg/m3 were similar, but relative urinary levels of
HIBA after 18.8 mg/m3 were higher, although those for MPD were lower, as compared to
170 mg/m3. HIBA in urine showed a broad maximum at 12-30 hours after exposure to both
concentrations, with a slow decline thereafter. MPD in urine peaked at 12 and 18 hours after 170
and 18.8 mg/m3, respectively, although tert-butanol peaked at 6 hours after both concentrations.
The time to peak of the three metabolites reflected the sequence of their formation and
interconversion as ETBE is metabolized. Interindividual variations were large, but the authors did
not report differences in the toxicokinetics of ETBE by sex. Amberg etal. (2000) calculated that
43 ± 12% of the 170 mg/m3 dose and 50 ± 20% of the 18.8 mg/m3 dose had been excreted in urine
by 72 hours. Respiratory elimination was not monitored.
Excretion in Animals
Amberg etal. (2000) exposed F344 NH rats, 5/sex/dose, concurrent with the human
volunteers in the same exposure chamber. Urine was collected for 72 hours following exposure.
Like humans, rats excreted mostly HIBA in urine, followed by MPD and tert-butanol. Parent ETBE
was not identified in rat urine. The half-life for tert-butanol in rat urine was 4.6 ± 1.4 hours at
170 mg/m3 but could not be calculated at 18.8 mg/m3. Corresponding half-lives were 2.6 ± 0.5 and
4.0 ± 0.9 hours for MPD, and 3.0 ± 1.0 and 4.7 ± 2.6 hours for HIBA. The authors concluded that rats
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excreted ETBE considerably faster than humans. Urinary excretion accounted for 53 ± 15 and
50 ± 30% of the estimated dose at 170- and 18.8-mg/m3 exposures, respectively, with the
remainder of the dose being eliminated via exhalation, as suggested by the study authors.
Bernauer et al. T19981 studied the excretion of [13C]ETBE and MTBE in rats. F344 rats,
2/sex, were exposed via inhalation to 8,400 mg/m3 ETBE or 7,200 mg/m3 MTBE for 6 hours, or
3 male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours, and
the ETBE metabolite prevalence in urine was
MPD > HIBA > tert-butanol-sulfate > tert-butanol-glucuronide. Oral administration of tert-butanol
produced a similar metabolite profile, with relative amounts of
HIBA > tert-butanol-sulfate > MPD » tert-butanol-glucuronide ~ tert-butanol.
Although there are several unpublished reports relevant to the elimination of ETBE
following inhalation exposure, no additional peer-reviewed publications were identified.
Unpublished reports have not gone through the public peer-review process and are of unknown
quality. They are included here as additional information only.
During 96 hours in metabolic cages, rats excreted approximately 60% of the radioactivity in
urine, approximately 38% was recovered as exhaled organic volatiles, and approximately 1% as
exhaled CO2. This pattern was maintained at an exposure concentration of 4,180 mg/m3; above
that, urinary excretion of radioactivity decreased to 34% of the recovered radioactivity, although
exhalation of organic volatiles increased to 63%. A shift in the excretion profile of radiolabel was
seen at concentrations of 7,310 mg/m3 and above, which remained fairly constant to the highest
exposure of 20,900 mg/m3. In this range of concentrations, approximately 39% of the excreted
radiolabel was found in urine, approximately 58% was exhaled as organic volatiles, and 2% was
eliminated as exhaled CO2.
A review of the data demonstrating the percentage of recovered radiolabel via various
routes of elimination demonstrate, in the rat and mouse, a pattern indicative of metabolic
saturation occurring at inhaled concentrations above 4,180 mg/m3.
In rats, the time course of excretion indicated that exhalation of organic volatiles was
essentially complete by 24 hours, although urinary excretion of ETBE-derived radioactivity
displayed a broad peak at 12-48 hours. The bulk of each dose was excreted within 48 hours after
the end of exposure. At 20,900 mg/m3,14CC>2 exhalation and fecal excretion of radioactivity
remained rather constant from 12 to 118 hours. In comparing the total radiolabel excreted to the
inhaled concentrations (see Table B-6), a proportionate relationship is observed in rats at all
concentrations, but less than proportionate excretion of total radiolabel at the highest
concentration in mice. The complete data set led the study authors to conclude that saturation of
the inhalation absorptive processes might have occurred at concentrations of approximately
7,310 mg/m3. The findings of Sun and Beskitt (1995a) in mice at 20,900 mg/m3 were essentially
confirmed by Borghoff T19961 (unpublished report, a pilot study) and Borghoff and Asgharian
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1 (1996) (unpublished report, final study), which used the identical species, experimental protocol,
2 materials, and methods but which were conducted later at a different laboratory (see Table B-6).
Table B-6. Excretion of [14C]ethyl tertiary butyl ether (ETBE)-derived
radioactivity from rats and mice within 96 hours following a single 6-hour
inhalation exposure
Exposure level
(mg/m3)
Volatile
organics3
Exhaled C02a
Urine3
Feces3
Total"
F344 ratc
2,090d
37
[28, 32]
1
[1.2, 1.3]
60
[59, 59]
2
[2.8,1.0]
9.9
[16.1, 13.6]
3,130
36
1
62
2
17.5
4,180
42
1
56
2
22.1
7,310d
58
[41, 52]
2
[1.5, 1.7]
38
[53, 41]
3
[0.7,0.5]
56.9
[45, 31]
10,400
52
2
45
2
56.2
20,900de
63
(51)
[51, 64]
2
(1)
[1.6, 2.0]
34
(44)
[45, 30]
1
(3)
[0.2,0.2]
97.5
(116)
[143, 94]
CD-I mouse?
2,090d
10
[12.7, 26.8]
1
[1.2, 1.2]
74
[81.3, 67.2]
16
[3.2,2.3]
6.38
[10.4, 6.8]
3,130
28
2
60
10
7.9
4,180
29
2
64
6
12.8
7,310d
42
[23, 36]
2
[2.2, 1.9]
46
[71, 61]
10
[1.1,0.6]
13.7
[22.4, 17.3]
10,400
42
2
47
10
22.7
20,900de
44
(37)
[40, 47]
5
(2)
[2.9, 3.3]
39
(57)
[53, 47]
12
(2)
[0.6,0.8]
18.9
(28)
[37.1, 25.2]
Percentage of total excreted radioactivity; mean of one male and one female.
bln mg [14C]ETBE equivalents.
cSun and Beskitt (1995b).
dValues in brackets: [males, females], nose-only exposures, excretion up to 48 h Borghoff and Asgharian (1996).
eValues in parentheses: Borghoff (1996).
fSun and Beskitt (1995b).
3 Similarities between rats (Sun and Beskitt. 1995b) and mice (Sun and Beskitt. 1995a) are
4 evident. Both species demonstrate similar excretion pathways and present evidence of saturation
5 of metabolic pathways at concentrations lower than those that demonstrate saturation of
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absorptive pathways. Metabolic saturation (evidenced as a shift from urine as the predominant
excretion pathway and an increase in the fraction of dose eliminated via exhalation) occurred in
both species at concentrations approximating 7,310 mg/m3. Noteworthy differences between the
two species were that, in general, mice excreted a smaller percentage of the dose in the form of
volatile organics and a higher amount in urine, at least up to 4,180 mg/m3 (see Table B-6),
compared with rats and that mice excreted about five times as much [14C]ETBE-derived
radioactivity via feces than did rats. The total amounts of excreted radioactivity (mg equivalents)
were considerably higher in rats than in mice; however, the values in the respective columns of
Table B-6 are not corrected for body weight (BW). When normalized to body weight, it is apparent
that mice absorbed a higher dose than rats; however, the total excreted radioactivity at
20,900 mg/m3 showed no further increase over the values at 10,450 mg/m3, indicating that the
absorptive capacities of mice had become saturated; however, this analysis conducted in rats does
not indicate a saturation of absorptive capacities over the range of concentrations studied.
Borghoff (1996). in an unpublished report, conducted studies to establish experimental
conditions for future bioassays of ETBE, based on the two studies previously conducted by Sun and
Beskitt f!995a. 1995b! The experimental protocol and materials were identical to the ones used
by Sun and Beskitt f!995a. 1995bl: however, in this pilot study, only three male F344 rats and
three male CD-I mice were used per experiment, with only one exposure level at 20,900 mg/m3.
Also, only blood was collected from the animals, while the whole carcasses were liquefied and
assayed for retained radioactivity immediately after exposure and after the end of the animals' stay
in metabolic cages. The carbon at "the central position of the tert-butyl group" was radiolabeled.
Radioactive ETBE was obtained by mixing [14C]ETBE with unlabeled material in the gas phase for a
specific activity of 2.74 |j.Ci/mmol. The rats, when assayed immediately after exposure, were found
to have absorbed 2.57 ± 0.14 |iCi radioactivity, although the balance of radioactivity after 96 hours
in metabolic cages from other animals accounted for 3.17 ± 0.08 |j.Ci (mean ± standard deviation
[SD], n = 3). The authors could not make any suggestion as to the origin of this discrepancy.
Absorbed doses in mice were 0.85 ± 0.08 |j.Ci immediately after exposure and 0.77 ± 0.16 |j.Ci for
other mice placed in metabolism cages. Excretion values detected in these rats and mice are shown
in parentheses in Table B-6; the percentage values shown in this table were based on the total body
burden of the individual animals from which the excretion data were obtained, not on group means.
Mice had excreted most of the dose within 12 hours after exposure, rats within 24 hours.
Organic volatiles collected on charcoal filters were analyzed for ETBE and tert-butanol contents.
Rats exhaled 22% of the absorbed ETBE within 1 hour after exposure, 12% during the following
2 hours, and only another 3% during the next 3 hours, tert-Butanol exhalation accounted for 1% of
the total during the first hour, 3% during the following 2 hours, and 4% during the last 3 hours of
the experimental period. Mice, on the other hand, exhaled 16% of the unmetabolized ETBE within
1 hour after exposure and 1% during the following 2 hours, with immeasurable amounts thereafter.
tert-Butanol exhalation made up 6% of total during the first hour, 8% in the next 2 hours, and 4%
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during the final 3 hours. Excretion of ETBE, tert-butanol, HIBA, and MPD in urine were assayed.
During 24 hours of collection, the rats excreted about 7 times as much tert-butanol as ETBE in
urine; in mice, the ratio was >60. HIBA was detected in urine of both species but could not be
quantified. MPD was not detected. These results could be interpreted as suggesting that mice
metabolize, and hence excrete, ETBE faster than rats.
A subsequent larger study by Borghoff and Asgharian (1996) (see previous details)
essentially confirmed the results of the pilot study (Borghoff. 1996). F344 rats and CD-I mice were
exposed by inhalation to 500,1,750, or 5,000 ppm [14C]ETBE. Concentrations of ETBE and
tert-butanol were measured in exhaled breath up to 16 hours postexposure. The exhalation pattern
observed in rats showed levels of ETBE falling ~90% in the first 8 hours postexposure, while levels
of TBA exhaled actually rose between 0 and 3 hours postexposure and then fell more slowly
between 3 and 16 hours, particularly after 5,000 ppm ETBE exposure. The increase in TBA
between 0 and 3 hours postexposure can be explained by the continued metabolism of ETBE during
that period. The slower decline after 3 hours can be explained by the generally slower clearance of
TBA, which is saturated by the higher ETBE exposure levels. Exhaled breath levels declined much
more rapidly in mice than in rats.
Unpublished reports by TPEC f2008el determined that following oral exposure of
7-week-old Crl:CD(SD) male rats to [14C]ETBE, the largest amount of radioactivity was recovered in
expired air, followed by urinary excretion, with very little excretion occurring via the feces. With
increasing dose, increasing proportions of radioactivity were found in expired air. The total
radioactivity recovered by 168 hours after a single dose of 5 mg/kg [14C]ETBE was 39.16% in the
urine, 0.58% in the feces, and 58.32% in expired air, and, after a single dose of 400 mg/kg, 18.7% in
the urine, 0.15% in the feces, and 78.2% in expired air. With repeated dosing, the recovery of
radioactivity through excretion increased through Day 6 when a steady state was achieved;
however, the radioactivity level in the feces increased throughout the 14 days, but the level was too
low to affect the total recovery. After 14 days, 36.3% of the administered dose was recovered in the
urine, 2.33% was recovered in the feces, and 56.7% was recovered in expired air.
B.1.5. Physiologically Based Pharmacokinetic Models
Two PBPK models have been developed specifically for describing the absorption,
distribution, metabolism, and excretion of ETBE in rats fBorghoffetal.. 2016: Salazar etal.. 20151.
A detailed summary of these and other toxicokinetic models is provided in U.S. EPA f2017I The
PBPK model described in Borghoff et al. (2016) and in U.S. EPA (2017) was considered to conduct a
route-to-route extrapolation based on an equivalent internal dose (the rate of ETBE metabolism in
the liver), but was not ultimately used for this purpose because of feedback from external peer
review by EPA's Science Advisory Board (SAB). The SAB recommended that in the absence of a
consistent dose-response relationship for ETBE when combining oral and inhalation studies to
assess liver tumors, extrapolation between inhalation and oral routes of exposure not be
This document is a draft for review purposes only and does not constitute Agency policy.
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performed. Regarding the extrapolation from animals to humans, the existing human PBPK model
was not considered adequate (see below); therefore, default methodologies were applied to
extrapolate toxicologically equivalent exposures from adult laboratory animals to adult humans.
The PBPK model described in Borghoff et al. f20161 and in U.S. EPA f20171 includes a
possible adjustment for induction of tert-butanol metabolism; however, this induction has only
been observed in mice exposed directly to tert-butanol (McComb and Goldstein. 1979).
Furthermore, implementing metabolic induction does not allow for dependence on exposure or
dose, nor for any de-induction that might occur during periods without exposure, such as weekends
during 5 days/week exposures. Finally, because induction is expected to have an equal impact on
oral and inhalation exposures—and only if tert-butanol levels or metabolism is used as a dose
metric—induction's potential impact on risk evaluation for ETBE is considered minimal. Therefore,
this adjustment was not turned off in the model; instead, the maximum induction level was set to
zero.
While model simulations accounted for variations during the day and week (e.g.,
6 hours/day, 5 days/week inhalation exposure), simulations reached a condition of "periodicity" by
the second week, such that the time course of internal doses were identical between the second
week and subsequent weeks of exposure with metabolic induction turned off. However, to ensure
applicability should metabolic induction is considered (predicted to take 2-3 weeks), simulations
were generally run for 7 weeks, with results for the last 1-2 weeks used to estimate average tissue
or blood concentrations or metabolic rates.
For simulating exposure to drinking water, the water consumption was modeled as
episodic, based on the pattern of drinking observed in rats (Spiteri. 1982). In particular, rats were
assumed to ingest water in pulses or "bouts," which were treated as periods of continuous
ingestion, interspersed with periods of no ingestion. During the active dark period (12 hours/day),
80% of total daily ingestion is assumed to occur in 45-minute bouts alternating with 45 minutes of
other activity. During the relatively inactive light period (12 hours/day), the remaining 20% of
daily ingestion is assumed to occur; the bouts are only assumed to last 30 minutes, with 2.5 hours
between. This pattern is thought to be more realistic than assuming continuous 24 hours/day
ingestion. The resulting ingestion rate for one exposure is shown in Figure B-2.
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15 ,
12
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3
o 11II 11II ii—u—U—U—IIIII1111IIII111II—u—u—U—
0 6 12 18 24 30 36 42 48
Time (h)
Figure B-2. Example oral ingestion pattern for rats exposed via drinking
water.
PBPK modeling was also used to evaluate a variety of internal dose metrics (daily average
TBA blood concentration, daily amount of TBA metabolized in liver, daily average of ETBE blood
concentration, and daily amount of ETBE metabolized in liver) to assess the correlation with
different endpoints following exposure to ETBE or TBA (Salazar etal.. 20151. Administering ETBE
either orally or via inhalation achieved similar or higher levels of TBA blood concentrations or TBA
metabolic rates as those induced by direct TBA administration. Altogether, the PBPK model-based
analysis by Salazar etal. f20151 [which applied a model structurally similar to Borghoff et al.
£2016}] indicated a consistent dose-response relationship between kidney weight, urothelial
hyperplasia, and chronic progressive nephropathy (CPN) and TBA blood concentration (as the dose
metric for both ETBE and TBA). Kidney and liver tumors, however, were not consistently
correlated with any dose metric. These data are consistent with TBA mediating the noncancer
kidney effects following ETBE administration, but additional factors besides internal dose are
necessary to explain the induction of liver and kidney tumors.
Oi
E
<1)
4-)
c
o
¦+J
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Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
terf-butanol-oral
CO
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.10
-0.15
A. rho= 0.099 (all datasets)
rho= 0.15 (ETBE only)
B. rho= 0.33 (ETBE only)
0 1 2 3 0 1 2 3 4
ferf-butanol metabolized (mg/hr) ETBE metabolized (mg/hr)
Salazaretal. (2015)
•
ETBE-oral
¦
ETBE-inhalation
O
ferf-butanol-oral
CO
0.25
0.20 -
0.15 -
0.10 -
0.05 -
0.00 -
-0.05 -
-0.10 -
-0.15
A rho= 0.14 (all datasets)
" rho= 0.33 (ETBE only)
¦
o
II
o
.c
cri
(ETBE only)
¦
¦
¦
• • O
o
o
¦
•
¦
•
• • •
• •
•
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3
fert-butanol metabolized (mg/hr) ETBE metabolized (mg/hr)
Borghoff et al. (2016)
Figure B-3. Comparisons of liver tumors in male rats following 2-year oral or
inhalation exposure to ethyl tertiary butyl ether (ETBE) or tert-butanol with
internal dose metrics calculated from the physiologically based
pharmacokinetic (PBPK) model. Results applying the model of Salazar et al.
(2015) (top) and Borghoff etal. (2016) (bottom).
Dose metrics expressed are metabolism rate of tert-butanol (A) and metabolism rate of
ETBE (B). Liver tumor incidences following ETBE oral or inhalation exposure did not present a
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consistent dose-response relationship using either the ETBE or tert-butanol metabolism rate dose
metric, and the correlation coefficients was not statistically significant. These data indicate that
internal dose is inadequate to explain differences in tumor response across these studies.
B.1.6. Physiologically Based Pharmacokinetic (PBPK) Model Code
The PBPK acslX model code is available electronically through EPA's Health and
Environmental Research Online (HERO) database. All model files may be downloaded in a zipped
workspace from HERO (U.S. EPA. 20161.
B.1.7. Physiologically Based Pharmacokinetic (PBPK) Model Evaluation
All available PBPK models of ETBE and its principal metabolite tert-butanol were evaluated
for potential use in the assessments.
Overview of Available Models
A PBPK model of ETBE and its principal metabolite tert-butanol has been developed for
humans exposed while performing physical work (Nihlen and Tohanson. 19991. The Nihlen and
Johanson model is based on measuring blood concentrations of 8 individuals exposed to 5, 25, and
50 ppm ETBE for 2 hours while physically active. This model differs from conventional PBPK
models in that the tissue volumes and blood flows are calculated from individual data on body
weight and height Additionally, to account for physical activity, blood flows to tissues are
expressed as a function of the workload. These differences from typical PBPK models preclude
allometric scaling of this model to other species for cross-species extrapolation. As there are no
oral exposure toxicokinetic data in humans, this model does not have a mechanism for simulating
oral exposures, which prevents its use in animal-to-human extrapolation for that route.
A number of PBPK models were developed previously for the related compound, MTBE and
the metabolite tert-butanol that is common to both MTBE and ETBE (Borghoffetal.. 2010: Leavens
andBorghoff. 2009: Blancato etal.. 2007: Kim etal.. 2007: Rao and Ginsberg. 1997: Borghoffetal..
19961. A PBPK model for ETBE and tert-butanol in rats was then developed by the EPA (Salazar et
al.. 20151 by integrating information from across these earlier models. Another model for ETBE
and tert- butanol was published by Borghoffetal. (20161. adapted with modest structural
differences from the Leavens and Borghoff f20091 MTBE/tert-butanol model. Brief descriptions
below highlight the similarities and differences between the MTBE/tert-butanol models of Blancato
etal. (2007) and Leavens and Borghoff (2009). and the ETBE/tert-butanol models of Salazar et al.
(2015). and Borghoffetal. (2016).
The Models of Blancato etal. (2007) and Leavens and Borahoff (2009)
The Blancato etal. f20071 model is an update of the earlier Rao and Ginsberg T19971 model,
and the Leavens and Borghoff f20091 model is an update of the Borghoffetal. f 19961 model. Both
the Blancato etal. f20071 and Leavens and Borghoff f20091 models are flow-limited models that
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predict amounts and concentrations of MTBE and its metabolite tert-butanol in blood and six tissue
compartments: liver, kidney, fat, brain, and rapidly and slowly perfused tissues. These tissue
compartments are linked through blood flow, following an anatomically accurate, typical,
physiologically based description f Andersen. 19911. The parent (MTBE) and metabolite
(tert-butanol) models are linked by the metabolism of MTBE to tert-butanol in the liver. Oral and
inhalation routes of exposure are included in the models for MTBE; Leavens and Borghoff (2009)
also included oral and inhalation exposure to tert-butanol. Oral doses are assumed to be 100%
bioavailable and 100% absorbed from the gastrointestinal tract represented with a first-order rate
constant Following inhalation of MTBE or tert-butanol, the chemical is assumed to enter the
systemic blood supply directly, and the respiratory tract is assumed to be at pseudo-steady state.
Metabolism of MTBE by CYP450s to formaldehyde and tert-butanol in the liver is described with
two Michaelis-Menten equations representing high- and low-affinity enzymes, tert-Butanol is
either conjugated with glucuronide or sulfate or further metabolized to acetone through MPD and
HBA; the total metabolic clearance of tert-butanol by both processes is described by a single
Michaelis-Menten equation in the models. All model assumptions are considered valid for MTBE
and tert-butanol.
In addition to differences in fixed parameter values between the two models and the
addition of exposure routes for tert-butanol, the Leavens and Borghoff f20091 model has three
features not included in the Blancato etal. (2007) model: (1) the alveolar ventilation was reduced
during exposure, (2) the rate of tert-butanol metabolism was increased over time to account for
induction of CYP enzymes, and (3) binding of MTBE and tert-butanol to alpha 2u-globulin was
simulated in the kidney of male rats. The Blancato etal. (2007) model was configured through
EPA's PBPK modeling framework, Exposure-Related Dose Estimating Model, which includes explicit
pulmonary compartments. The modeling assumptions related to alveolar ventilation, explicit
pulmonary compartments, and induction of metabolism of tert-butanol are discussed in the model
evaluation section below.
MTBE and tert-butanol binding to alpha 2u-globulin in the kidneys of male rats were
incorporated in the PBPK model of MTBE by Leavens and Borghoff f20091. Binding to alpha
2u-globulin is one hypothesized mode of action for the observed kidney effects in MTBE-exposed
animals. For a detailed description of the role of alpha 2u-globulin and other modes of action in
kidney effects, see the kidney mode-of-action section of the Toxicological Review. In the Leavens
and Borghoff f20091 model, binding of MTBE to alpha 2u-globulin was applied to describe sex
differences in kidney concentrations of MTBE and tert-butanol, but acceptable estimates of MTBE
and tert-butanol pharmacokinetics in the blood are predicted in other models that did not consider
alpha 2u-globulin binding. Moreover, as discussed below, the EPA's implementation of the Leavens
and Borghoff (2009) model did not adequately fit the available tert-butanol i.v. dosing data, adding
uncertainty to the parameters they estimated.
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The Blancato etal. (2007) and Leavens and Borghoff (2009) PBPK models for MTBE were
specifically evaluated by comparing predictions from the tert-butanol portions of the models with
the tert- butanol i.v. data of Poet etal. f19971 (see Figure B-4). Neither model adequately
represented the tert-butanol blood concentrations. Modifications of model assumptions for
alveolar ventilation, explicit pulmonary compartments, and induction of metabolism of tert-butanol
did not significantly improve model fits to the data.
300 mg/kg ~ male ® female
— 150 mg/kg ¦ male o female
75 mg/kg • male o female
- • -37.5 mg/kg a male a female
0 2 4 6 8 10 12 14 16 18 20 22 24
time (hours)
10000
10000
1000
1000
300 mg/kg
-- 150 mg/kg
75 mg/kg
~ male
¦ male
• male
< male
° female
~ female
o female
a female
0 2 4 6 8 10 12 14 16 18 20 22 24
time (hours)
Figure B-4. Comparison of the tert-butanol portions of existing methyl
tertiary butyl ether models with tert-butanol blood concentrations from i.v.
exposure by Poet etal. (1997). Neither the (A) Blancato et al. (2007) nor the (B)
Leavens and Borghoff f20091 model adequately represents the measured tert-
butanol blood concentrations.
The Model of Salazar et al. (2015)
To better account for the tert-butanol blood concentrations after intravenous tert-butanol
exposure, the model by Leavens and Borghoff (2009) was modified by adding a pathway for
reversible sequestration of tert-butanol in the blood (Salazar et al.. 2015). Sequestration of
tert-butanol was modeled using an additional blood compartment, into which tert-butanol can
enter reversibly, represented by a differential mass balance (see Figure B-5). Other differences in
model structure are that the brain was included in the other richly perfused tissues compartment
and that binding to alpha 2u-globulin was not included. Binding to alpha 2u-globulin was neglected
because it was assumed to not significantly affect the blood concentration or metabolic rate of
ETBE to TBA, the two dose metrics being used for route-to-route extrapolation. This model
improved the fit to tert-butanol blood concentrations after tert-butanol i.v. exposures [see Salazar
etal. f20151]. Additionally, the model adequately estimated the tert- butanol blood concentrations
after inhalation and gavage exposures. The ETBE submodel was based on the MTBE component of
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Supplemental Information—ETBE
1 the Leavens and Borghoff (2009) model. The model assumed two-pathways for metabolism of
2 ETBE to TBA, and the metabolic parameters were optimized to fit toxicokinetic data. Partition
3 coefficients of ETBE were based on data of Nihlen and Tohanson f!9991.
ETBE TBA
Inhalation Exhalation Inhalation Exhalation
Dose
Figure B-5. Schematic of the Salazar etal. (2015) physiologically based
pharmacokinetic (PBPK) model for ethyl tertiary butyl ether (ETBE) and its
major metabolite tert-butanol in rats. Exposure can be via multiple routes
including inhalation, oral, or i.v. dosing. Metabolism of ETBE and tert-butanol occur
in the liver and are described by Michaelis-Menten equations with two pathways for
ETBE and one for tert-butanol. ETBE and tert-butanol are cleared via exhalation,
and tert-butanol is additionally cleared via urinary excretion.
4 The Model of Borahoff et al. f2016)
5 The Borghoff et al. (20161 models for ETBE and tert-butanol were based on Leavens and
6 Borghoff f20091. including binding of ETBE and TBA to alpha 2u-globulin and induction of
7 tert-butanol metabolism, with some structural changes. The revised model lumped gastrointestinal
8 tract tissue and brain tissue into the richly perfused compartment [Leavens and Borghoff (2009)
9 modeled these compartments separately], Borghoff et al. (2016) assumed that urinary clearance
10 was a function of central venous blood concentration and effectively occurs from that compartment,
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as opposed to clearance from the kidney venous blood assumed by Leavens and Borghoff (2009).
Using the new structure, urinary clearance was reparameterized to fit the intravenous data by Poet
etal. f!9971. The model assumed a single oxidative metabolic pathway for metabolism of ETBE to
tert- butanol using parameters from Rao and Ginsberg f!9971. instead of the two-pathway models
assumed by Leavens and Borghoff f20091 (for MTBE) and Salazar etal. f20151. The model did not
incorporate the tert-butanol blood sequestration kinetics included in the tert-butanol model. It did,
however, incorporate the oral absorption rate of tert- butanol estimated by Salazar etal. (20151.
Partition coefficients for ETBE were obtained from Kaneko etal. (20001 and from metabolic
parameters. Rate constants for binding of ETBE to alpha 2u-globulin and its dissociation were
assumed to be the same as estimated for MTBE by Leavens and Borghoff f20091. Finally, unlike
Leavens and Borghoff f20091. Borghoff et al. f20161 assumed a lower-bound alveolar ventilation for
all times and exposures, not just during periods of inhalation exposure.
To simulate induction of tert-butanol metabolism, the default metabolic rate of tert-butanol
clearance is multiplied by an exponential function of the form [1 + A[1 - e-fct)], where A is the
maximum fold increase above baseline metabolism, k is the rate constant for the ascent to
maximum induction, and t is time. Because metabolic induction does not occur instantaneously, but
involves a delay for induction of ribonucleic acid transcription and translation, Borghoff et al.
f20161 assumed that induction did not begin until 24 hours after the beginning of exposure. But
the computational implementation then treated the effect as if the enzyme activity suddenly
jumped each 24 hours to the level indicated by the time-dependent equation shown in the paper.
This stepwise increase in activity was not considered realistic. Therefore, in evaluating the impact
of induction, the EPA treated the induction as occurring continuously with time but beginning at
12 hours after the start of exposure. This change would not affect long-term steady-state or
periodic simulations, in particular those used to characterize bioassay conditions, but has a modest
effect on simulations between 12 and 24 hours, which are compared to experimental data below
for the purpose of model validation. However, with further review of the existing data on liver
histology (which would also reflect metabolic induction if it occurs, as detailed below), the EPA
determined that it is likely to only occur at the very highest exposure levels and hence not at levels
where the model is applied for route-to-route extrapolation. Therefore, the maximal induction was
set to zero unless otherwise noted.
The form of the equations for hepatic metabolism in the Borghoff et al. f20161 model was
revised to be a function of the free liver concentration, specifically the concentration in the venous
blood leaving the liver, rather than the concentration in the liver tissue. To maintain the integrity of
all prior model simulations and parameter estimations, EPA updated the Michaelis-Menten
constants (Km's) for ETBE and TBA by scaling them by the liver:blood partition coefficients. As a
result, the model produces identical results as before without reestimating a fitted parameter.
Finally, a discrepancy between the pulmonary ventilation value as described by Borghoff et
al. f20161. in particular as the lower limit of values reported by Brown etal. f!9971. should be
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noted. Borghoff et al. (2016) claimed that an allometric coefficient of 18.9 L/hour/kg°75 (allometric
coefficient provided here reflects actual use in model code) is the lower limit For a 0.25 kg rat, this
value yields an absolute ventilation rate of 6.6822 L/hour or 111.37 mL/min. In Table 31 of Brown
etal. f!9971. the mean and range of values given for the rat are 52.9 and
31.5-137.6 mL/min/(100 g BW). From the text immediately following this table, it is clear that
these mean and range values are not scaled to BW0-75, but exactly as indicated. Hence for a 250 g rat
they correspond to 132.25 and 78.75-344 mL/min. Use of 18.9 L/hour/kg°75 corresponds to a
ventilation rate 61% of the way between the lower limit and the mean for a 0.25 kg rat Note that
31.5 mL/min/100 g BW, the actual lower limit, equals 18.9 L/hour/kg10 (i.e., the respiration per kg
BW, not per kg BW0-75). Thus, the discrepancy appears due to a mistaken translation in allometric
scaling.
The fact that Borghoff et al. f20161 and Leavens and Borghoff f20091 used a ventilation rate
closer to the mean than the lower limit may explain why it was also necessary to incorporate a
fraction of TBA available for alveolar absorption of 0.6. From considering the plots of model
simulations versus data below, it appears that model fits to the data would be improved by further
decreasing ventilation, which could now be justified. But EPA has chosen to keep the value of
alveolar ventilation (QPC) and absorption fraction as published by Borghoff et al. f2016I
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Supplemental Information—ETBE
ETBE TBA
Inhalation Exhalation Inhalation Exhalation
Dose
Figure B-6. Schematic of the Borghoff etal. (2016) physiologically based
pharmacokinetic (PBPK) model for ethyl tertiary butyl ether (ETBE) and its
major metabolite tert-butanol in rats.
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Supplemental Information—ETBE
Table B-7. Physiologically based pharmacokinetic (PBPK) model physiologic
parameters and partition coefficients3
Parameter
Value
Source or reference
Body weight and organ volumes as fraction of body weight
Body weight (kg)
0.25
Brown et al. (1997)
Liver
0.037
Brown et al. (1997)
Kidney
0.0073
Brown et al. (1997)
Fat
0.35 x BW + 0.00205
Brown et al. (1997)
Richly perfused (total)
0.136
Brown et al. (1997)
Richly perfused
0.0177
b
Poorly perfused (total)
0.757
Brown et al. (1997)
Poorly perfused
0.75495-0.35 x BW
Blood
0.074
Brown et al. (1997)
Rest of body (not perfused)
0.107
Brown et al. (1997)
Cardiac output and organ blood flows as fraction of cardiac output
Cardiac output (L/h-kg)
18.9
Brown et al. (1997)°
Alveolar ventilation (L/h-kg)
18.9
Brown et al. (1997)°
Liver
0.174
Brown et al. (1997)d
Kidney
0.141
Brown et al. (1997)
Fat
0.07
Brown et al. (1997)
Richly perfused (total)
0.47
e
Richly perfused
0.155
f
Poorly perfused (total)
0.53
Brown et al. (1997)
Poorly perfused
0.46
g
Partition coefficients for ETBE
Blood:air
11.6
Kaneko et al. (2000)
Liver:blood
2.9
Kaneko et al. (2000)
Fat:blood
11.7
Kaneko et al. (2000)
Richly perfused:blood
2.9
Kaneko et al. (2000)
Poorly perfused:blood
1.9
h
Kidney:blood
2.9
1
Partition coefficients for tert-butanol
Blood:air
481
Borghoff et al. (1996)
Liver:blood
0.83
Borghoff et al. (1996)
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Supplemental Information—ETBE
Table B-7. Physiologically based pharmacokinetic (PBPK) model physiologic
parameters and partition coefficients3 (continued)
Parameter
Value
Source or reference
Fat:blood
0.4
Borghoff et al. (1996)
Richly perfused:blood
0.83
Borghoff et al. (1996)
Poorly perfused:blood
1.0
Borghoff et al. (1996)
Kidney:blood
0.83
Borghoff et al. (2001)
aValues have been updated to incorporate corrections from a quality assurance review and to include values to
the number of digits used in the model code.
b0.165 -Z(kidney, liver, blood).
cLower limit of alveolar ventilation for rat reported in Brown et al. (1997); alveolar ventilation is set equal to
cardiac output.
dSum of liver and gastrointestinal blood flows.
eBrown et al. (1997) only accounts for 94% of the blood flow. This assumes unaccounted 6% is richly perfused.
f0.47 - Z(kidney, liver).
g0.53 - fat.
hSet equal to muscle tissue (Borghoff et al., 2016).
'Set equal to richly perfused tissue (Borghoff et al., 2016).
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Supplemental Information—ETBE
Table B-8. Physiologically based pharmacokinetic (PBPK) model rate
constants
Parameter
Value
Source or reference
tert-Butanol rate constants
TBA first-order absorption constant (1/h)
5.0
Salazar et al. (2015)
Fraction of TBA absorbed in alveolar region
0.6
Medinsky et al. (1993)
Urinary clearance of TBA (L/h/kg075)
0.015
Borghoff et al. (2016)
Scaled maximum metabolic rate of TBA (nmol/h/kg)
54
Borghoff et al. (1996), Rao and Ginsberg
(1997)
Michaelis-Menten constant (nmol/L)
457a
Borghoff et al. (1996), Rao and Ginsberg
(1997)
Maximum percentage increase in metabolic rate
0.0
124.9 used bv Leavens and Borghoff (2009)
Rate constant for ascent to maximum (l/d)b
0.3977
Leavens and Borghoff (2009)
ETBE rate constants
ETBE first-order absorption constant (1/h)
1.6
Leavens and Borghoff (2009)
Scaled maximum metabolic rate of ETBE
(nmol/h/kg0 75)
499
Rao and Ginsberg (1997)
Michaelis-Menten constant for ETBE (nmol/L)
430a
Rao and Ginsberg (1997)
Alpha 2u-globulin binding parameters
Steady-state free kidney alpha 2u-globulin (nmol/L)
550°
Leavens and Borghoff (2009)
First-order constant for hydrolysis of free alpha
2u-globulin (1/h)
0.31
Leavens and Borghoff (2009)
First-order constant for hydrolysis of bound alpha
2u-globulin (1/h)
0.11
Leavens and Borghoff (2009)
Second-order binding constant for TBA to alpha
2u-globulin (L/nmol/h)
1.3
Leavens and Borghoff (2009)
Alpha 2u-globulin dissociation constant for TBA
(nmol/L)
120
Leavens and Borghoff (2009)
First-order constant for unbinding of TBA from alpha
2u-globulin (1/h)
Calculatedd
Second-order binding constant for ETBE to alpha
2u-globulin (L/nmol/h)
0.15
Leavens and Borghoff (2009)
Alpha 2u-globulin dissociation constant for ETBE
(nmol/L)
1
Leavens and Borghoff (2009)
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Supplemental Information—ETBE
Table B-8. Physiologically based pharmacokinetic (PBPK) model rate
constants (continued)
Parameter
Value
Source or reference
First-order constant for unbinding of ETBE from alpha
2u-globulin (1/h)
Calculated6
aBased on dividing the original values in Borghoff et al. (1996) and Rao and Ginsberg (1997) [used by Borghoff et
al. (2016)1 by the corresponding liver partition coefficients: 379/0.83 = 457 for te/t-butanol kinetics, and
1,248/2.9 = 430 for ETBE kinetic pathway 1.
bNote: model revised from a daily stepwise induction change to a continuous change (with a 12-h time lag), while
still maintaining the default parameters.
cBased on values ranging from ~160 to 1,000 nmol/L (Carruthers et al., 1987; Charbonneau et al., 1987; Olson et
al., 1987; Stonard et al., 1986).
dProduct of alpha 2u-globulin dissociation constant for te/t-butanol and second-order binding constant for
te/t-butanol to alpha 2u-globulin.
eProduct of alpha 2u-globulin dissociation constant for ETBE and second-order binding constant for ETBE to alpha
2u-globulin.
B.1.8. Toxicokinetic Data Extraction and Selected Model Outputs
Data Extraction and Adjustments
A study by ARCO (19831 reported tert-butanol blood levels after gavage exposure, primarily
as tert-butanol equivalents based on total 14C activity, which does not distinguish between
tert-butanol and its metabolites. However, for oral doses of 1 and 500 mg/kg, the fraction of
activity identifiable as tert-butanol was also reported, although not at identical time points. To
estimate total equivalents at other times, the study authors used empirical bi-exponential curves
(see Figure B-7) to interpolate between the time points at which total tert-butanol equivalents were
measured. The total equivalents calculated this way were then multiplied by the fraction of
tert-butanol reported at 0.5, 3, 6, and 12 hours for 1 mg/kg [ARCO (19831. Table 24] and 500 mg/kg
[ARCO (19831. Table 25] to obtain the data used for PBPK modeling (see Table B-10).
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Time(h) Time(h)
Figure B-7. tert-Butanol PK data for 1 and 500 me/ke oral exposures from
ARCO fl9831.
Time-course data and empirical regressions for te/t-butanol equivalents in rats following oral exposure to 1 or
500 mg/kg [14C]TBA (ARCO, 1983). For 1 mg/kg, the single exponential regression reported by ARCO (1983) was
1.73 x exp(-0.0946t) (dashed line), but it did not appear to adequately fit the data. A bi-exponential regression
(solid line) was found by minimizing the sum of square errors between the regression and data in Excel:
0.4874 x exp(-0.7055t) + 1.404 x exp(-0.06983t). For 500 mg/kg, the bi-exponential regression reported by ARCO
(1983) appeared sufficient: 554 x exp(-0.0748t) - 426 x exp(-3.51t).
The single-dose data from TPEC (2008f) were taken from Appendix Table 12 of that report.
The values for the P-5 component were converted from ETBE equivalents to mg/L tert-butanol. For
example, at 5 mg/kg-day, 416 ng ETBE eq/mL is reported for P-5 in animal #17. The
corresponding concentration in mg/L for tert-butanol are then calculated as (416 ng
ETBE eq/mL) x (1,000 mL/L) x (10~6 mg/ng) x (74.12 [MW tert-butanol])/(102.17 [MW
ETBE]) = 0.302 mg tert-butanol eq/L, where MW represents molecular weight. Likewise the data
for the repeated-dose study flPEC. 2008el. Days 7 and 14, were converted from the P-5 values in
Appendix Table 7, p. 53 of that report (The data from the single-dose study were combined with the
Day 7 and 14 data from the multiple dose study for comparison with model simulations of 14-day
dosing.).
The TPEC (2008a) TPEC (2008b) studies measured tert-butanol in plasma only, unlike the
Poetetal. (1997) and Leavens and Borghoff (2009) studies, which measured tert-butanol in whole
blood. Based on the measurements of plasma and whole blood by JPEC (2008a,b), the
concentration of tert-butanol in plasma is approximately 130% of the concentration in whole blood
(see Table B-ll). The tert-butanol plasma concentrations measured by JPEC were therefore
divided by 1.3 to obtain the expected concentration in whole blood for comparison with the PBPK
model.
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Supplemental Information—ETBE
Table B-9. Summary of pharmacokinetic data used for model calibration and evaluation
Exposure
Measured
Data source
Figure no. in
Salazar et al.
(2015)
Conversion
Notes
Chemical
Route
Chemical
Medium
TBA
i.v.
TBA
Blood
Poet et al. (1997) Figure 1
and 2
3A
HM to mg/L
Digitized from the figure
Inhalation
TBA
Blood
Leavens and Borghoff (2009)
Figure 8A-B
3B
HM to mg/L
Digitized from the figure, showing
only 1 d of exposure
Gavage
TBA
Blood
ARCO (1983),% total TBA,
Tables 24-25; TBA
equivalents, Figure 6
3C
TBA equivalents to
TBA concentration
ETBE
Gavage
TBA
Blood
JPEC (2008f) Appendix 12
4A
ETBE equivalents to
mg/L TBA
"P5" is TBA
TBA
Urine
JPEC (2008f) Appendix 13
4B
ETBE equivalents to
mg/L TBA
"P5" is TBA
ETBE
Inhalation
ETBE
Blood
Amberg et al. (2000) Table 5
4C
HM to mg/L
TBA
Blood
Amberg et al. (2000)Table 5
4D
HM to mg/L
TBA
Urine
Amberg et al. (2000) Table 6
and Figure 4
4E
HM to mg/L
ETBE
Exhaled air
Borghoff (1996)
4F
Hmoles to mg
Cumulative mass
TBA
Exhaled air
Borghoff (1996)
4G
Hmoles to mg
Cumulative mass
TBA
Inhalation
TBA
Blood
Leavens and Borghoff (2009)
Figure 8B
5A-B
HM to mg/L
Digitized from the figure
TBA
Blood
Leavens and Borghoff (2009)
Figure 8A
5C-D
HM to mg/L
Digitized from the figure
ETBE
Gavage
TBA
Blood
JPEC (2008f) Appendix 12
5E
ETBE equivalents to
mg/L TBA
"P5" is TBA
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Supplemental Information—ETBE
Table B-10. Conversion of ARCO T19831 total tert-butanol (TBA) equivalents and serum fraction data to TBA
concentrations
Time (h)
% TBAa
Total TBA equivalents
interpolated (ng/mL)b
TBA concentration using
interpolated equivalents
(Hg/mL = mg/L)c
Total TBA equivalents measured at
nearest time point
(time measured)d
TBA concentration using
nearest time point (mg/L)e
1 mg/kg data
0.5
57.3
1.6982
0.9731
1.69 (0.5 h)
0.9684
3
25
1.1972
0.2993
1.26 (2.67 h)
0.3150
6
18.1
0.9304
0.1684
0.97 (5.33 h)
0.1756
12
1
0.6074
0.006074
0.68 (10.67 h)
0.006800
500 mg/kg data
0.5
22.9
460.0
105.34
445 (0.5 h)
101.91
3
20.4
442.6
90.30
438 (2.67 h)
89.35
6
18.7
353.7
66.14
393 (5.33 h)
73.49
12
18.5
225.8
41.77
269 (10.67 h)
49.77
aFrom Table 24, p. 48 of ARCO (1983) (1 mg/kg) and Table 25, p. 49 of ARCO (1983) (500 mg/kg).
bUsing bi-exponential functions.
"Values used in PBPK modeling; %TBA x total TBA equivalents interpolated.
dFrom Table 14, p. 32 of ARCO (1983) (1 mg/kg) and Table 11, p. 27 of ARCO (1983) (500 mg/kg).
e%TBA x total TBA equivalents at nearest time point.
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Supplemental Information—ETBE
Table B-ll. Ratio of 14C activity in blood vs. plasma after [14C] ethyl tertiary
butyl ether (ETBE) exposures in rats IPEC (2008a) IPEC (2008b)
Time (h)
Animal no.
Plasma
(ng 14C eq/mL)
Blood
(ng 14C eq/mL)
Plasma/blood (%)
Sinale dose. JPEC (2008f) Appendix Table 5. p. 94
8
97
78,133
40,667
192.1
98
95,533
80,000
119.4
99
89,367
64,667
138.2
100
72,400
62,333
116.2
24
37
10,900
8,800
123.9
38
19,133
14,433
132.6
39
19,433
15,400
126.2
40
30,767
22,967
134.0
72
41
2,133
1,600
133.3
42
2,833
3,033
93.4
43
4,033
3,200
126.0
44
3,167
2,333
135.7
Mean ± SD
130.9 ± 22.8
Sinale dose. JPEC I2008f) Appendix Table 3. p. 91
8
17
2,853
1,784
159.9
18
2,850
1,802
158.2
19
2,629
1,568
167.7
20
3,918
2,718
144.2
24
21
1,692
1,255
134.8
22
846.7
642.9
131.7
23
1,048
785
133.5
24
761.7
591.3
128.8
72
25
49.6
40
124.0
26
34.2
29.2
117.1
27
79.2
60.8
130.3
28
107.9
84.6
127.5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-ll. Ratio of 14C activity in blood vs. plasma after [14C] ethyl tertiary
butyl ether (ETBE) exposures in rats IPEC (2008a) IPEC (2008b) (continued)
Time (h)
Animal no.
Plasma
(ng 14C eq/mL)
Blood
(ng 14C eq/mL)
Plasma/blood (%)
168
29
12.9
13.3
97.0
30
17.5
13.8
126.8
31
26.7
24.2
110.3
32
40
35.8
111.7
Mean ± SD
131.5 ± 18.9
Repeated dose. JPEC (2008e). Appendix Table 3. p. 49
8 (7 d dosing)
3,789
3,029
125.1
5,041
3,988
126.4
4,914
3,938
124.8
5,608
4,638
120.9
24 (7 d dosing)
2,740
1,908
143.6
3,433
2,575
133.3
2,488
1,888
131.8
963.3
812.5
118.6
8 (14 d dosing)
5,665
4,546
124.6
5,175
4,075
127.0
3,889
3,058
127.2
5,090
3,858
131.9
24 (14 d dosing)
2,003
1,508
132.8
2,121
1,692
125.4
1,948
1,354
143.9
1,037
804.2
128.9
72 (14 d dosing)
1,378
1,138
121.1
301.3
245.8
122.6
110
N.D.
421.3
337.5
124.8
Mean ± SD
128.1 ± 6.85
N.D. = not detected.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Selected Model Comparisons Applying the Borah off etal. (20161 Model
2 The modeling code was obtained by the authors of Borghoff et al. f20161. The modeling
3 language and platforms is acslX (Advanced Continuous Simulation Language, Aegis, Inc., Huntsville,
4 AL).
5 The following modifications were made:
6 1) Periodic drinking water pathway was incorporated into the continuous simulation language
7 (CSL) file, and the continuous oral dose rate function was modified slightly to improve
8 flexibility of the model.
9 2) For simulations showing the effect of including enzyme induction, the code was modified
10 slightly in the CSL file to improve continuity. Daily step functions in metabolic chances were
11 replaced with a continuous function but delayed by 12 hours.
12 3) Otherwise, enzyme induction was not used (set to zero).
13 4) In the PBPK model code, the changes to the Michaelis-Menten constants described as
14 footnotes in Table B-8 above were not made in the PBPK parameter script (MTBEparam.m).
15 Instead, parameters were redefined in the core model *CSL file as scaling calculations in the
16 parameters section of the INITIAL bloc:
17 a. Kmlvetbe = Kmletbe/Pletbe
18 b. Km2vetbe = Km2etbe/Pletbe
19 c. Kmvtba = Kmtba/Pltba
20 5) Tissue volumes and the rate of hydrolysis of free alpha 2u-globulin were corrected (slightly)
21 to values shown in Table B-7.
22 6) All model scripts previously used to evaluate model fits of the Salazar etal. (2015) model
23 were adapted to run the Borghoff et al. f20161 model. Model parameters were set to
24 uniform values for all simulations highlighted in this section, unless otherwise noted.
25 7) Digitized data from Ambergetal. (2000) were updated after a quality assurance (QA)
26 review.
27 8) Tabulated data from Borghoff and Asgharian f!9961 were updated subsequent to a QA
28 review.
29 The PBPK acslX model code is available electronically through EPA's HERO database. All
30 model files may be downloaded in a zipped workspace from HERO fU.S. EPA. 20161. The model
31 contains workspaces for EPA's implementation of the Salazar etal. (2015) model, the unchanged
32 version of the of Borghoff et al. f20161 model, and the EPA implementation of the Borghoff etal.
33 f20161 model.
34 Selected model outputs compared with experimental data sets are provided below.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
(A)
CB)
1,000 -
10 -
TBA iv exposure
r— 300 mg/kg ~
male
O
female ^
— 150 mg/kg ¦
male
~
female
75 mg/kg •
male
O
female
lk™¦¦ 37.5 mg/kg A
male
A
female j
Turn
400-
<
Qj
300-
200
_o
CQ
100
Time (h)
12
Time (h)
-
1726 ppm
~
males
444 ppm
¦
males
239 ppm
*
males
1914 ppm
O
females
444 ppm
~
females
256 ppm
I O
females ^
(C)
1,000
100
10-
1 -
0.1
0.01 -
0.001 -
TBA gavage exposures
500 mg/kg
1 mg/kg
A 500 mg/kg data
• 1 mg/kg data
—i—
10
12
Time (h)
Figure B-8. Comparison of the Borghoff etal. f20161 model predictions with
measured tert-butanol blood concentrations for i.v., inhalation, and gavage
exposure to tert-butanol.
Source: (A) i.v. data from Poet et al. (1997); (B) inhalation data from Leavens and Borghoff (2009); and (C) gavage
data from ARCQ (1983).
1 The model results for the i.v. data are significantly improved from the Blancato etal. f20071
2 and Leavens and Borghoff f2 009) model results presented previously. As evident here and in the
3 Borghoff et al. (20161 study, the Borghoff etal. (2016) model generally overpredicts TBA blood and
4 urine concentrations. Some attempts were made to improve model fit in the EPA model
5 implementation (such as adjusting inhalation, urinary, and induction parameter values); however,
6 the default values were maintained in the final model.
This document is a draft for review purposes only and does not constitute Agency policy,
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3
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5
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Supplemental Information—ETBE
j 100
C
O
¦J 10
fO
L.
4J
£
V
(J
C
S i
o
o
<
as
0.1
lOeO
1 ~10e-l
5 rng/kg, model
A 5 mg/kg, data
¦¦¦¦ 400 mg/kg, model
• 400 mg/kg data
a> 10e-2
_E
L
3
c10e-3
<
ca
10e-4 -I
10e-5
4 6
Time (h)
10
i
6 12 18 24
Time (h)
Figure B-9. Comparison of Borghoff etai. (2016) model predictions with
measured amounts of tert-butanol after gavage of ethyl tertiary butyl ether
[ETBE).
The data points show the measurements from the four individual rats in the J PEC (2008f) study. The
concentrations of tert-butanol in blood are shown in (A). The amount of tert-butanol in urine is shown in (B).
Note that the overprediction of tert-butanol in urine (B) is by a factor of 3- to 10-fold.
The predictions of the model are compared with amounts measured by Amberg et al.
f2000! after ETBE inhalation in Figure B-10A. The predicted tert-butanol blood concentrations are
slightly higher than the measured ones. The tert-butanol blood concentration would be reduced if
the exposed animals were reducing their breathing rate or other breathing parameters, but the
exposure concentration of ETBE are unlikely to be high enough to cause a change in breathing
parameters, because at the much higher ETBE concentration in the ARCO (19831 study
(5,000 ppm], changes in breathing were not noted. The model already uses a lower bound estimate
of respiration rate and cardiac output for all simulations, and the model predictions fit most
measured concentrations well. However, the urinary excretion of tert-butanol is significantly
overestimated (~3- to 10-fold] by the tert-butanol submodel (see Figure B-10B).
This document is a draft for review purposes only and does not constitute Agency policy,
B-44 ' DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
i—
6
0.09
0.08
» 0.07
B 0.06
v
c 0,05
= 0.04
~ 0.03
<
£ 0.02
0,01
0
iii
6 12 18 24
Time (h)
Figure B-l 0. Comparison of Borghoff etal. f2016) model predictions with
measured amounts after a 4-hour inhalation exposure to 4 and 40 ppm ethyl
tertiary butyl ether (ETBE).
Concentrations in blood are shown in (A) for ETBE and (B) for te/t-butanol. The amount of te/t-butanol in urine is
shown in (C) for the 40-ppm exposure. The data are from Amberg et al. (2000).
This document is a draft for review purposes only and does not constitute Agency policy,
B-45 ' DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Time after exposure (h) Time after exposure (h)
Figure B-ll. Comparison of Borghoff et al. f20161 model predictions with
measured amounts of ethyl tertiary butyl ether (ETBE) and tert-butanol in
exhaled breath after a 6-hour inhalation exposure to 500,1,750, and
5,000 ppm ETBE.
The data points are from the Borghoff and Asgharian (1996) study. The model significantly overpredicted the
concentrations of both ETBE and tert-butanol in the exhaled breath of male rats and of tert-butanol in female rats
following ETBE inhalation exposure. The model currently assumes that 100% of inhaled ETBE, though only 60% of
inhaled tert-butanol, is available for alveolar absorption. The inhalation availability may have a significant impact
on estimated exhaled breath amounts but was not adjusted to fit this data set.
The increased tert-butanol metabolism better estimates the measured tert-butanol blood
concentrations as shown in a comparison of the model predictions and experimental
This document is a draft for review purposes only and does not constitute Agency policy,
B-46 ' DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 measurements in Figure B-12. The male rats have lower tert-butanol blood concentrations after
2 repeated exposures than female rats, and this difference could indicate greater induction of
3 tert-butanol metabolism in males or other physiologic changes such as ventilation or urinary
4 excretion.
Male rats, no induction
Male rats with induction
C
Q
500 -
400 -
<0
£ 300
V
u
O 200
o
-2 100
<
CQ
I- 0
48 96
Time (h)
Female rats, no induction 3
¦ 1 1 I ( l l l
96 144
Time (h)
Female rats with induction
-r-
48
1
96
Time (h)
144
192
96
Time (h)
192
Figure B-12. Comparison of the Borghoff etal. f20161 model predictions with
measured amounts of tert-butanol in blood after repeated inhalation
exposure to tert-butanol.
Male rats were exposed to 239,444, or 1,726 ppm and female rats were exposed to 256, 444, or 1,914 ppm
te/t-butanol for up to 8 consecutive days (Borghoff et al., 2001). te/t-Butanol blood concentrations are better
predicted by the model after 8 days of exposure with enzyme induction (right panels) than without enzyme
induction (left panels).
This document is a draft for review purposes only and does not constitute Agency policy,
B-47 ' DRAFT-DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
Supplemental Information—ETBE
Time (h)
Figure B-13. Comparison of EPA model predictions with measured amounts of
tert-butanol in blood after 5 mg/kg-day ethyl tertiary butyl ether (ETBE)
gavage for up to 14 days in male rats.
The data show the individual measurements of the four rats in the JPEC (2008e, 2008fi study. Adding enzyme
induction to the model has a small effect on the predicted tert-butanol blood concentrations and the model
predictions are closer to measured data when induction is not included.
B.2. OTHER PERTINENT TOXICITY INFORMATION
B.2.1. Other Toxicological Effects
Syn thesis of Other Effects
The database for effects other than kidney, liver, reproduction, and cancer contain only 11
rodent studies. These effects included decreased body weight, increased adrenal weights, altered
spleen weights, and increased mortality. All selected studies used inhalation, gavage, or drinking
water exposures for >90 days. Shorter duration, multiple-exposure studies that examined
immunological endpoints were also included. No studies were removed for methodological
concerns.
Kidnev effects
Absolute kidney-weight data are presented in Table B-12.
This document is a draft for review purposes only and does not constitute Agency policy,
B-48 ' DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-12. Evidence pertaining to absolute kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Fuiiietal. (2010); JPEC (2008d)
Rat, Sprague-Dawley
Oral—gavage
PO, male (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 16 wk beginning 10 wk prior to
mating
P0, female (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 17 wk beginning 10 wk prior to
mating to LD 21
P0, Male
P0, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
100
5
100
-2
300
8
300
0
1,000
18a
1,000
7a
Gaoua (2004b)
Rat, Sprague-Dawley
Oral—gavage
P0, male (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until after weaning of the
pups
P0, female (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
and lactation, then Fl doses beginning
PND 22 until weaning of the F2 pups
P0, Male
P0, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
250
ir
250
-1
500
15a
500
2
1,000
21a
1,000
5
Fl, Male
Fl, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
250
10
250
4
500
22a
500
3
1,000
58a
1,000
ir
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0 or 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
1,000
19a
This document is a draft for review purposes only and does not constitute Agency policy.
B-49 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-12. Evidence pertaining to absolute kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Mivata et al. (2013); JPEC (2008b)
Rat, Crl:CD(SD)
Oral—gavage
Male (15/group): 0, 5, 25,100, or
400 mg/kg-d; female (15/group): 0, 5, 25,
100, or 400 mg/kg-d
Daily for 26 wk
Male
Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
5
1
5
1
25
6
25
0
100
5
100
7
400
25a
400
10a
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (34-37/group):
Female (36-38/group): 0, 625, 2,500, or
10,000 ppm (0, 46,171, or 560 mg/kg-d)
Daily for 104 wk
(Organ weights measured for animals
surviving to study termination)
Male
Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
28
-4
46
3
121
5
171
_Q
o
1
542
_Q
00
1
560
14b
Saito et al. (2013); JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (30-44/group):
Female (29-39/group): 0, 500,1,500, or
5,000 ppm (0, 2,090, 6,270, or
20,900 mg/m3)b
Dynamic whole-body inhalation; 6 h/d,
5 d/wk for 104 wk; generation method,
analytical concentration reported
(Organ weights measured for animals
surviving to study termination)
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
5
2,090
5
6,270
8
6,270
6a
20,900
18a
20,900
_Q
00
1
This document is a draft for review purposes only and does not constitute Agency policy.
B-50 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-12. Evidence pertaining to absolute kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (NR): 0,150, 500,1,500, or 5,000 ppm
(0, 627, 2,090, 6,270, or 20,900 mg/m3);
female (NR): 0,150, 500,1,500, or
5,000 ppm (0, 627, 2,090, 6,270, or
20,900 mg/m3);
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
627
10
627
1
2,090
11
2,090
-1
6,270
18a
6,270
4
20,900
16a
20,900
7
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (6/group): 0 or 5,000 ppm (0 or
20,900 mg/m3);c female (6/group): 0 or
5,000 ppm (0 or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk followed by a 28 d
recovery period; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
20,900
19
20,900
8
Medinskv et al. (1999); US EPA (1997)
Rat, F344
Inhalation—vapor
Male (48/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3);c female (48/group): 0, 500,
1,750, or 5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
7
2,090
4
7,320
10a
7,320
12a
20,900
19a
20,900
21a
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Supplemental Information—ETBE
Table B-12. Evidence pertaining to absolute kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Medinskv et al. (1999); Bond et al. (1996)
Mice, CD-I
Inhalation—vapor
Male (40/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3);b female (40/group): 0, 500,
1,750, or 5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3)b
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
9
2,090
0
7,320
10
7,320
6
20,900
5
20,900
4
- = for controls, no response relevant; for other doses, no quantitative response reported; LD = lactation day;
n = number evaluated from group; NR = not reported; PND = postnatal day.
aResult is statistically significant (p < 0.05) based on analysis of data by study authors.
b4.18 mg/m3 = 1 ppm.
Body weight
As presented in Table B-12, body weights were significantly reduced compared with vehicle
controls following 2-year oral and inhalation exposures to ETBE fSaito etal.. 2013: Suzuki etal..
2012: TPEC. 2010a. b). Reductions were also reported in studies of exposure durations shorter than
2 years (Banton etal.. 2011: Hagiwara etal.. 2011: Fujii etal.. 2010: TPEC. 2008a. b; Gaoua. 2004b:
Medinskv etal.. 19991: however, these effects were frequently not statistically significant Food
consumption did not correlate well with body weight fSaito etal.. 2013: Suzuki etal.. 2012: TPEC.
2010a. b). Water consumption was reduced in the 2-year oral exposure study flPEC. 2010a],
Reduced water consumption due to ETBE exposure and the chemical's unpalatability might
contribute to the reduced body weight, particularly for dietary or drinking water exposures.
Hypersalivation, which is frequently observed with unpalatable chemicals following gavage, was
observed in rats gavaged for 18 weeks (Gaoua. 2004b). Body-weight changes are poor indicators of
systemic toxicity but are important when evaluating relative organ-weight changes.
Adrenal weight
Adrenal weights were increased in the 13- and 23-week studies (see Table B-13). For
instance, a 13-week inhalation study found that absolute adrenal weights were increased in male
and female rats (Medinskv etal.. 1999). In another study, absolute and relative adrenal weights
were increased in male rats (Hagiwara etal.. 2011). None of the observed organ-weight changes
This document is a draft for review purposes only and does not constitute Agency policy.
B-52 DRAFT-DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Supplemental Information—ETBE
corresponded with functional or histopathological changes; thus, adrenal effect data are inadequate
to draw conclusions as a human hazard of ETBE exposure.
Immune system
Functional immune assays represent clear evidence of immunotoxicity and generally
outweigh immune organ weight and cell population effects when establishing hazard [WHO (2012):
see Table B-15], The single published functional assay available reported that the number of IgM+
sheep red blood cell (SRBC)-specific antibody forming cells was not significantly affected after a
28-day oral exposure to ETBE fBanton etal.. 20111. Relative spleen weights were inconsistently
affected in male and female rats following oral and inhalation >13-week exposures to ETBE
(see Table B-15). The only dose-responsive changes in spleen weights were increased relative
weights in male rats and decreased absolute weights in female rats following 2-year inhalation
exposure (Saito etal.. 2013: TPEC. 2010b) and increased relative weights in female rats following
2-year oral exposure (Suzuki etal.. 2012: TPEC. 2010a). Spleen weights are heavily influenced by
the proportion of red blood cells, which do not impact immune function of the organ (Elmore.
20061. Thus, spleen-weight changes must be correlated with histopathological and functional
changes for evidence of immunotoxicity (Elmore. 2006). none of which are observed for ETBE.
CD3+, CD4+, and CD8+ T cells were modestly reduced in male mice after 6 or 13 weeks of ETBE
exposure via inhalation but are not correlated with any change in T cell function as indicated by the
SRBC assay (Li etal.. 2011). No other indicators of histopathological or functional changes were
reported with a single chemical exposure. The ETBE database contains no evidence of altered
immune function that correlate with modest T cell population reductions and altered splenic organ
weights; thus, the immune effect data are inadequate to draw conclusions as a human hazard of
ETBE exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
B-53 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-13. Evidence pertaining to body-weight effects in animals exposed to
ethyl tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Banton et al. (2011)
Rat, Sprague-Dawley
Oral—gavage
Female (10/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for 28 consecutive d
Female
Dose
(mg/kg-d)
Body weight
(%)
0
-
250
3
500
5
1,000
-1
Fuiiietal. (2010); JPEC (2008d)
Rat, Sprague-Dawley
Oral—gavage
P0, male (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 16 wk beginning 10 wk prior to
mating; P0, female (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 17 wk beginning 10 wk before mating
to LD 21
P0, Male
P0, Female
Dose
(mg/kg-d)
Body weight
(%)
Dose
(mg/kg-d)
Body weight
(%)
0
-
0
-
100
-4
100
1
300
-4
300
1
1,000
-7
1,000
5
Gaoua (2004b)
Rat, Sprague-Dawley
Oral—gavage
P0, male (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until after weaning of the pups
P0, female (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until PND 21
Fl, male (25/group): 0, 250, 500, or
1,000 mg/kg-d
Dams dosed daily through gestation and
lactation, then Fl doses beginning PND 22 until
weaning of the F2 pups
Fl, female (24-25/group): 0, 250, 500, or
1,000 mg/kg-d
P0 dams dosed daily through gestation and
lactation, then Fl dosed beginning PND 22
until weaning of the F2 pups
P0, Male
P0, Female
Dose
(mg/kg-d)
Final body
weight (%)
Dose
(mg/kg-d)
Final body
weight (%)
0
-
0
-
250
-1
250
-7
500
-3
500
-2
1,000
-5a
1,000
0
Fl, Male
Fl, Female
Dose
(mg/kg-d)
Final body
weight (%)
Dose
(mg/kg-d)
Final body
weight (%)
0
-
0
-
250
0
250
-2
500
3
500
-3
1,000
1
1,000
2
This document is a draft for review purposes only and does not constitute Agency policy.
B-54 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-13. Evidence pertaining to body-weight effects in animals exposed to
ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0 or 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Final body
weight (%)
0
-
1,000
-5a
Mivata et al. (2013);JPEC (2008b)
Rat, Crl:CD(SD)
Oral—gavage
Male (15/group): 0, 5, 25,100, or 400 mg/kg-d;
female (15/group): 0, 5, 25,100, or
400 mg/kg-d
Daily for 26 wk
Male
Female
Dose
(mg/kg-d)
Body weight
(%)
Dose
(mg/kg-d)
Body weight
(%)
0
-
0
-
5
-6
5
-5
25
0
25
-2
100
-5
100
-2
400
2
400
-3
Maltoni et al. (1999)
Rat, Sprague-Dawley
Oral—gavage
Male (60/group): 0, 250, or 1,000 mg/kg-d;
female (60/group): 0, 250, or 1,000 mg/kg-d;
4 d/wk for 104 wk; observed until natural
death
Male
No significant difference at any dose
Female
No significant difference at any dose
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (50/group): 0, 625, 2,500, or 10,000 ppm
(0, 28,121, or 542 mg/kg-d);b female
(50/group): 0, 625, 2,500, or 10,000 ppm (0,
46,171, or 560 mg/kg-d)b
Daily for 104 wk
Male
Female
Dose
(mg/kg-d)
Terminal body
weight (%)
Dose
(mg/kg-d)
Terminal body
weight (%)
0
-
0
-
28
-4
46
-10a
121
-T
171
-ir
542
-9a
560
-17a
This document is a draft for review purposes only and does not constitute Agency policy.
B-55 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-13. Evidence pertaining to body-weight effects in animals exposed to
ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (NR): 0,150, 500,1,500, or 5,000 ppm (0,
627, 2,090, 6,270, or 20,900 mg/m3);c female
(NR): 0, 150, 500, 1,500, or 5,000 ppm (0, 627,
2,090, 6,270, or 20,900 mg/m3)
Dynamic whole-body chamber; 6 h/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male
Female
Dose
(mg/m3)
Body weight
(%)
Dose
(mg/m3)
Body weight
(%)
0
-
0
-
627
0
627
-6
2,090
1
2,090
-7
6,270
-1
6,270
-7
20,900
-7
20,900
-11
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (6/group): 0 or 5,000 ppm (0 or
20,900 mg/m3);c female (6/group): 0 or
5,000 ppm (0 or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk
for 13 wk followed by a 28 d recovery period;
generation method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Body weight
(%)
Dose
(mg/m3)
Body weight
(%)
0
-
0
-
20,900
3
20,900
4
Medinskv et al. (1999); US EPA (1997)
Rat, F344
Inhalation—vapor
Male (48/group): 0, 500,1,750, or 5,000 ppm
(0, 2,090, 7,320, or 20,900 mg/m3);c female
(48/group): 0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male
Female
Dose
(mg/m3)
Body weight
(%)
Dose
(mg/m3)
Body weight
(%)
0
-
0
-
2,090
2
2,090
-3
7,320
4
7,320
3
20,900
2
20,900
6a
Medinskv et al. (1999); US EPA (1997)
Mice, CD-I
Inhalation—vapor
Male (40/group): 0, 500,1,750, or 5,000 ppm
(0, 2,090, 7,320, or 20,900 mg/m3);c female
(40/group): 0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male
Female
Dose
(mg/m3)
Body weight
(%)
Dose
(mg/m3)
Body weight
(%)
0
-
0
-
2,090
0
2,090
-2
7,320
-1
7,320
-1
20,900
-3
20,900
2
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-13. Evidence pertaining to body-weight effects in animals exposed to
ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Saito et al. (2013);JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (50/group): 0, 500,1,500, or 5,000 ppm
(0, 2,090, 6,270, or 20,900 mg/m3);c female
(50/group): 0, 500,1,500, or 5,000 ppm (0,
2,090, 6,270, or 20,900 mg/m3)c
Dynamic whole-body inhalation; 6 h/d, 5 d/wk
for 104 wk; generation method, analytical
concentration, and method were reported
Male
Female
Dose
(mg/m3)
Body weight
(%)
Dose
(mg/m3)
Body weight
(%)
0
-
0
-
2,090
-7a
2,090
-6a
6,270
-7a
6,270
-10a
20,900
-26a
20,900
-23a
- = for controls, no response relevant; for other doses, no quantitative response reported; NR = not reported;
PND = postnatal day.
aResult is statistically significant (p < 0.05) based on analysis of data by study authors.
Conversion performed by study authors.
c4.18 mg/m3 = 1 ppm.
Percentage change compared to controls calculated as 100 x [(treated value - control value) -f control value].
This document is a draft for review purposes only and does not constitute Agency policy.
B-57 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-14. Evidence pertaining to adrenal effects in animals exposed to ethyl
tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Adrenal weight
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0, 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Absolute
weight (%)
Relative weight
(%)
0
-
-
1,000
16a
19a
Medinskv et al. (1999); US EPA (1997)
Rat, F344
Inhalation—vapor
Male (48/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3);b female
(48/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b
dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
11
2,090
7
7,320
9
7,320
7
20,900
34a
20,900
18a
Medinskv et al. (1999); Bond et al. (1996)
Mice, CD-I
Inhalation—vapor
Male (40/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3);b female
(40/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
0
2,090
-8
7,320
50
7,320
8
20,900
0
20,900
-8
- = for controls, no response relevant; for other doses, no quantitative response reported; n = number evaluated
from group.
aResult is statistically significant (p < 0.05) based on analysis of data by study authors.
b4.18 mg/m3 = 1 ppm.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-15. Evidence pertaining to immune effects in animals exposed to
ethyl tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Functional immune effects
Banton et al. (2011)
Rat, Sprague-Dawley
Oral—gavage
Female (10/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for 28 consecutive d
Immunized i.v. 4 d prior to sacrifice
with sheep red blood cells
Female
Dose
(mg/kg-d)
IgM antibody forming
cells/106 spleen cells
(%)
IgM antibody forming
cells/spleen (%)
0
-
-
250
-21
-20
500
42
36
1,000
8
8
Immune cell populations
Li et al. (2011)
Mice, 129/SV
Inhalation—vapor
Male (6/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3)a
Whole body, 6 h/d for 5 d/wk over
6 wk; generation method not
reported; analytical concentration
and method were reported
Male
Dose (mg/m3)
Number of CD3+
T cells (%)
Number of CD4+
T cells (%)
Number of CD8+T
cells (%)
0
-
-
-
2,090
_Q
00
1
1
-16
-13
7,320
-16
-11
-14
20,900
-21b
-17b
-25
Li et al. (2011)
Mice, C57BL/6
Inhalation—vapor
Male (6/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3)a
Whole body, 6 h/d for 5 d/wk over
6 wk; generation method not
reported; analytical concentration
and method were reported
Male
Dose (mg/m3)
Number of CD3+
T cells (%)
Number of CD4+
T cells (%)
Number of CD8+T
cells (%)
0
-
-
-
2,090
-14
-15
-12
7,320
-13
-11
-13b
20,900
-24b
-23b
-23b
Li et al. (2011)
Mice, C57BL/6
Inhalation—vapor
Male (5/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3)a
Whole body, 6 h/d for 5 d/wk over
13 wk; generation method not
reported; analytical concentration
and method were reported
Male
Dose (mg/m3)
Number of CD3+
T cells (%)
Number of CD4+
T cells (%)
Number of CD8+T
cells (%)
0
-
-
-
2,090
-9
-11
-8
7,320
-17b
-28b
-12
20,900
-24b
-37b
-20
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-15. Evidence pertaining to immune effects in animals exposed to ethyl
tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Spleen weight
Banton et al. (2011)
Rat, Sprague-Dawley
Oral—gavage
Female (10/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for 28 consecutive d
Female
Dose
(mg/kg-d)
Absolute
weight (%)
Relative
weight (%)
0
-
-
250
-3
0
500
-15
-18
1,000
-9
0
Fuiiietal. (2010); JPEC (2008d)
Rat, Sprague-Dawley
Oral—gavage
P0, male (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 16 wk beginning 10 wk prior
to mating
P0, female (24/group): 0,100, 300,
or 1,000 mg/kg-d
Daily for 17 wk beginning 10 wk prior
to mating to LD 21
P0, Male
P0, Female
Dose
(mg/kg-d)
Absolute
weight
(%)
Relative
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
Relative
weight (%)
0
-
-
0
-
-
100
-4
-1
100
0
-2
300
-2
2
300
-2
-3
1,000
0
8
1,000
-1
-5
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0 or 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Absolute
weight
(%)
Relative
weight (%)
0
-
-
1,000
-5
0
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (50/group): 0, 625, 2,500, or
10,000 ppm (0, 28,121, or
542 mg/kg-d);a female (50/group): 0,
625, 2,500, or 10,000 ppm (0, 46,
171, or 560 mg/kg-d)a
Daily for 104 wk
Male
Female
Dose
(mg/kg-d)
Absolute
weight
(%)
Relative
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
Relative
weight (%)
0
-
-
0
-
-
628
-3
-35
46
-35
2
121
19
3b
171
-1
28
542
39
-45
560
_Q
o
LO
1
55b
This document is a draft for review purposes only and does not constitute Agency policy.
B-60 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-15. Evidence pertaining to immune effects in animals exposed to ethyl
tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (NR): 0,150, 500,1,500, or
5,000 ppm (0, 627, 2,090, 6,270, or
20,900 mg/m3);c female (NR): 0,150,
500,1,500, or 5,000 ppm (0, 627,
2,090, 6,270, or 20,900 mg/m3)
Dynamic whole-body chamber;
6 h/d, 5 d/wk for 13 wk; generation
method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight
(%)
Relative
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
Relative
weight (%)
0
-
-
0
-
-
627
0
0
627
-9
-3
2,090
7
5
2,090
-2
5
6,270
-1
1
6,270
-5
1
20,900
-9
-2
20,900
1
12
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (6/group): 0 or 5,000 ppm (0 or
20,900 mg/m3);c female (6/group): 0
or 5,000 ppm (0 or 20,900 mg/m3)c
Dynamic whole-body chamber;
6 h/d, 5 d/wk for 13 wk followed by
a 28-d recovery period; generation
method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight
(%)
Relative
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
Relative
weight (%)
0
-
-
0
-
-
20,900
10
6
20,900
6
0
Saito et al. (2013); JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (50/group): 0, 500,1,500, or
5,000 ppm (0, 2,090, 6,270, or
20,900 mg/m3);c female (50/group):
0, 500,1,500, or 5,000 ppm (0,
2,090, 6,270, or 20,900 mg/m3)c
Dynamic whole-body inhalation;
6 h/d, 5 d/wk for 104 wk; generation
method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight
(%)
Relative
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
Relative
weight (%)
0
-
-
0
-
-
2,090
4
15
2,090
5
30
6,270
32
43b
6,270
-39
-31
20,900
17
66b
20,900
-43b
-25
This document is a draft for review purposes only and does not constitute Agency policy.
B-61 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-15. Evidence pertaining to immune effects in animals exposed to ethyl
tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Medinskv et al. (1999); US EPA
(1997)
Rat, F344
Inhalation—vapor
Male (48/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3);c female (48/group):
0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber;
6 h/d, 5 d/wk for 13 wk; generation
method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight
(%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
6
2,090
-3
7,320
3
7,320
3
20,900
5
20,900
0
Medinskv et al. (1999); Bond et al.
(1996)
Mice, CD-I
Inhalation—vapor
Male (40/group): 0, 500,1,750, or
5,000 ppm (0, 2,090, 7,320, or
20,900 mg/m3);c female (40/group):
0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber;
6 h/d, 5 d/wk for 13 wk; generation
method, analytical concentration
and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight
(%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
-5
2,090
-11
7,320
0
7,320
-2
20,900
-15
20,900
-11
- = for controls, no response relevant; for other doses, no quantitative response reported; LD = lactation day;
n = number evaluated from group; NR = not reported.
Conversion performed by the study authors.
bResult is statistically significant (p < 0.05) based on analysis of data by the study authors.
c4.18 mg/m3 = 1 ppm.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-16. Evidence pertaining to mortality in animals exposed to ethyl
tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Maltoni et al. (1999)
Rat, Sprague-Dawley
Oral—gavage
Male (60/group): 0, 250, or
1,000 mg/kg-d; female (60/group): 0, 250,
or 1,000 mg/kg-d
4 d/wk for 104 wk; observed until natural
death
Male
Female
Dose (mg/m3)
Survival at
104 wk (%)
Dose (mg/m3)
Survival at
104 wk (%)
0
-
0
-
250
-8
250
-8
1,000
-54
1,000
18
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (50/group): 0, 625, 2,500, or
10,000 ppm (0, 28,121, or 542 mg/kg-d);a
female (50/group): 0, 625, 2,500, or
10,000 ppm (0, 46,171, or 560 mg/kg-d)a
Daily for 104 wk
Male
Female
Dose
(mg/kg-d)
Percentage
survival (%)
Dose
(mg/kg-d)
Percentage
survival (%)
0
-
0
-
628
-3
46
3
121
-11
171
6
542
-11
560
6
Saito et al. (2013);JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (50/group): 0, 500,1,500, or
5,000 ppm (0, 2,090, 6,270, or
20,900 mg/m3);b female (50/group): 0,
500,1,500, or 5,000 ppm (0, 2,090, 6,270,
or 20,900 mg/m3)b
Dynamic whole-body inhalation; 6 h/d,
5 d/wk for 104 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose (mg/m3)
Survival at
104 wk (%)
Dose (mg/m3)
Survival at
104 wk (%)
0
-
0
-
2,090
-14
2,090
3
6,270
-9
6,270
-21°
20,900
-32°
20,900
-21°
- = for controls, no response relevant; for other doses, no quantitative response reported; n = number evaluated
from group.
Conversion performed by the study authors.
b4.18 mg/m3 = 1 ppm.
cResult is statistically significant (p < 0.05) based on analysis of data by the study authors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Decreased Body
Weight
subchronic/
reproductive
Female rat; 28d (A)
PO Female rat; 16wks (B)
PO Male rat; 16wks (B)
PO Male rat; 18wks (C)
PO Female rat; 18wks (Q
F1 Male rat; GD 0-adult (C)
F1 Female rat; CD 0-adult (CJ
Male rat; 23wks (D)
Female rat; 26wks (F)
Male rat; 26wks (FJ
0-
~ B B
~ B B
B—B—B
B—B-
B—B—B
B—B—B
B—B—B
a b b
a b 0
chronic
Female rat; 104wks (G)
Male rat; 104wks (G)
Female rat; 104wks (E)
Male rat; 104wks (E)
~ 0
Q 0
1 10 100 1,000 10,000
Dose (mg/kg-day)
Sources: (A) Banton et al, 2011 (B) Fujii et al., 2010; JPEC, 2008e [C] Gaoua, 2004b (D) Hagiwara et al., 2011
(E) Maltoni et al., 1999 (F) Miyata et al., 2013; JPEC, 2008c (G) Suzuki et al., 2012; JPEC, 2010a
Figure B-14. Exposure-response array of body-weight effects following oral
exposure to ethyl tertiary butyl ether (ETBE).
This document is a draft for review purposes only and does not constitute Agency policy,
B-64 ' DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Decreased Body
Weight
Female rats; 13wks (A]
Male rats; 13wks (A)
Female rats; 13wks, 28d recovery (A]
Male rats; 13wks, 28d recovery (A)
subchronic
Female rats; 13 wks (B)
Male rats; 13 wks (B)
Female mice; 13 wks (B)
Male mice; 13 wks (B)
~ B B-
~ B B-
~ B
B B
B B
B B
~
-B
-B
chronic
Female rats; 104wks (C)
Male rats; 104wks (C)
1 10 100 1,000 10,000 100,000
Exposure Concentration (mg/mJ)
¦"significantly increased body weight
Sources: (A] JPEC, 2008b (B) Medinsky et al., 1999; Bond et al., 1996 [C] Saito et al, 2013; JPEC,2010b
Figure B-15. Exposure-response array of body-weight effects following
inhalation exposure to ethyl tertiary butyl ether (ETBE).
This document is a draft for review purposes only and does not constitute Agency policy,
B-65 ' DRAFT-DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Supplemental Information—ETBE
Mortality
Mortality was significantly increased in male and female rats following a 2-year ETBE
inhalation exposure fSaito etal.. 2013: TPEC. 2010bl but not significantly affected following a 2-year
drinking water exposure f Suzuki etal.. 2012: IPEC. 2010a], Increased mortality in male rats
correlated with increased CPN severity in the kidney. The study authors attributed increased
mortality in females to pituitary tumors; however, pituitary tumors were not dose responsively
increased by ETBE exposure. Survival was also reduced in a lifetime gavage study at the highest
exposure in males and females after 72 weeks (data not shown), and after 104 weeks, survival was
reduced 54% in males at the highest dose fMaltoni et al.. 19991. After 104 weeks, however, survival
in the controls was approximately 25% in males and 28% in females, percentages that are much
lower than expected for a 2-year study fMaltoni etal.. 19991. The survival data in this study was
likely confounded by chronic respiratory infections, which could have contributed to the reduced
survival (Malarkev and Bucher. 20111. These data do not suggest that mortality was increased in
these studies due to excessively high exposure concentrations of ETBE; thus, the mortality data are
inadequate to draw conclusions as a human hazard of ETBE exposure.
Mechanistic Evidence
No relevant mechanistic data are available for these endpoints.
Summary of Other Toxicity Data
EPA concluded that the evidence does not support body-weight changes, adrenal and
immunological effects, and mortality as potential human hazards of ETBE exposure based on
confounding factors, lack of progression, and study quality concerns.
B.2.2. Genotoxicity Studies
Bacterial Systems
The mutagenic potential of ETBE has been tested by Zeiger etal. (19921 using different
Salmonella typhimurium strains for 311 chemicals, including ETBE, both in the absence and
presence of metabolic activation (S9). Preincubation protocol was followed and precaution was
exercised to account for the volatility of the compound. Five doses ranging from 100 to
10,000 |ig/plate were tested using different Salmonella strains, including TA97, TA98, TA100, and
TA1535. The results showed that the ETBE did not cause mutations in any of the Salmonella strains
tested. It should be noted that TA102, a sensitive strain for oxidative metabolite, was not used in
this study. The available genotoxicity data for ETBE are discussed below, and the summary of the
data is provided in Table B-17.
This document is a draft for review purposes only and does not constitute Agency policy.
B-66 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table B-17. Summary of genotoxicity (both in vitro and in vivo) studies of
ethyl tertiary butyl ether (ETBE)
Species
Test system
Dose/
concentration
Results3
Comments
Reference
-S9
+S9
Bacterial systems
Salmonella
typhimurium
(TA97, TA98,
TA100,
TA1535)
Mutation
assay
100, 333, 1,000,
3,333,
10,000 ng/plate
Preincubation
procedure was
followed. Experiment
was conducted in
capped tubes to
control for volatility
Zeiger et al. (1992)
In vitro systems
Chinese
hamster
ovary cells
(hgprt locus)
Gene
mutation
assay
100, 300, 1,000,
3,000,
5,000 Hg/mL
Experiments
conducted both with
and without metabolic
activation
Vergnes and Kubena
(1995b)
(unpublished
report)
Chinese
hamster
ovary cells
Chromosomal
aberration
assay
100, 300, 1,000,
3,000,
5,000 Hg/mL
Experiments
conducted both with
and without metabolic
activation
Vergnes (1995)
(unpublished
report)
In vivo animal studies
CD-I mice
(male and
female)
Bone marrow
micronucleus
test
0, 400, 2,000,
5,000 ppm (0,
1,670, 8,360,
20,900 mg/m3)b
Whole-body
inhalation, 6 h/d, 5 d,
5/sex/group
Vergnes and Kubena
(1995a)
(unpublished
report)
B6C3Fi mice
(male)
Bone marrow
micronucleus
test
0,1,300, 1,700,
2,100,
2,500 mg/kg
Intraperitoneal
injection 3x, 72 h;
5/group, 3 animals in
dose 1,700 mg/kg
dose. Surviving
animals were not
scored at doses of
2,100 and
2,500 mg/kg
NTP(1996a)
F344 rats
(male)
Bone marrow
micronucleus
test
0, 625,1,250,
2,500 mg/kg
Intraperitoneal
injection 3x, 72 h;
5/group, 3 animals in
2,500 mg/kg dose
group
NTP(1996b)
F344 rats
(male and
female)
Bone marrow
micronucleus
test
0, 500,1,000,
2,000 mg/kg-d
Gavage, 24 h apart,
2 d, 5/sex/group
JPEC (2007b)
(unpublished
report)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-17. Summary of genotoxicity (both in vitro and in vivo) studies of
ethyl tertiary butyl ether (ETBE) (continued)
Species
Test system
Dose/
concentration
Results3
Comments
Reference
-S9 +S9
F344 rats
(male and
female)
Bone marrow
micronucleus
test
0, 250, 500,
1,000,
2,000 mg/kg-d
Intraperitoneal
injection, 24 h apart,
2 d, 5/sex/group
Noguchi et al.
(2013); JPEC
(2007b),
unpublished report
F344 rats
(male and
female)
Bone marrow
micronucleus
test
0, 1,600, 4,000,
10,000 ppm (0,
101, 259,
626 mg/kg-d in
males; 0,120,
267,
629 mg/kg-d in
females)0
Drinking water, 13 wk,
10/sex/group
Noguchi et al.
(2013); JPEC
(2007d),
unpublished report
F344 rats
(male and
female)
Bone marrow
micronucleus
test
0, 500,1,500,
5,000 ppm (0,
2,090, 6,270,
20,900 mg/m3)b
Whole-body
inhalation, 6 h/d,
5 d/wk, 13 wk.
10/sex/group
Noguchi et al.
(2013); JPEC
(2007d),
unpublished report
C57BL/6 WT
and Aldh2
KO mice
DNA strand
breaks
(alkaline
comet assay);
leukocytes
0, 500, 1,750,
5,000 ppm
Male
WT/KO
+d/+
Whole-body
inhalation, 6 h/d,
5 d/wk, 13 wk
Weng et al. (2011)
Female
WT/KO
-/+d
C57BL/6 WT
and Aldh2
KO mice
DNA strand
breaks
(alkaline
comet assay)
0, 500, 1,750,
5,000 ppm
Male
WT/KO
+d/+
Whole-body
inhalation, 6 h/d,
5 d/wk, 13 wk
Weng et al. (2012)
Female
WT/KO
-/+d
C57BL/6 WT
and Aldh2
KO mice
Micronucleus
assay;
erythrocytes
0, 500, 1,750,
5,000 ppm
Male6
WT/KO
+d/+
Whole-body
inhalation, 6 h/d,
5 d/wk, 13 wk
Weng et al. (2013)
Female6
WT/KO
-/+
C57BL/6 WT
and Aldh2
KO mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 50, 200,
500 ppm
WT/HT/KO
-/+/+
Whole-body
inhalation, 6 h/d,
5 d/wk, 9 wk
Weng et al. (2014)
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Table B-17. Summary of genotoxicity (both in vitro and in vivo) studies of
ethyl tertiary butyl ether (ETBE) (continued)
Species
Test system
Dose/
concentration
Results3
Comments
Reference
-S9 +S9
C57BL/6 WT
and Aldh2
KO mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 500, 1,750,
5,000 ppm
WT/KO
+/+
Whole-body
inhalation, 6 h/d,
5 d/wk, 13 wk
Weng et al. (2014)
KO = knockout; WT = wild type.
a+ = positive; - = negative; (+) = equivocal.
b4.18 mg/m3 = 1 ppm.
Conversions performed by study authors.
dPositive in highest dose tested.
eWhen the data of ETBE-induced MNRETs were normalized with corresponding control, the effect disappeared.
In Vitro Mammalian Studies
The two available studies in in vitro mammalian systems were unpublished reports.
Vergnes and Kubena (1995b) evaluated the mutagenicity of ETBE using the hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) forward mutation assay in Chinese hamster ovary K1-BH4
cells. Duplicate cultures were treated with five concentrations of ETBE (>98% purity; containing
13 ppm A022, an antioxidant stabilizer) ranging from 100 to 5,000 |ig/mL, both in the presence and
absence of S9 activation. No statistically significant or concentration-related increase in the HGPRT
mutation frequencies were observed at any of the ETBE concentrations tested, either in the absence
or in the presence of metabolic (S9) activation.
The same authors [Vergnes and Kubena (1995b) unpublished report] studied the
clastogenic potential of ETBE in vitro using a chromosome aberration assay in Chinese hamster
ovary cells. The cells were exposed from 100 to 5,000 ng/mL of ETBE in culture medium, both in
the presence and absence of S9 metabolic activation system. No statistically significant or
concentration-related increase in the frequency of chromosomal aberrations, in the presence or
absence of the S9 metabolic activation system, was observed. Neither the effect of the antioxidant
stabilizer used in ETBE nor control for volatility of the compound was described for both studies
although capped glass bottles were used in the experiments.
In Vivo Animal Studies
In vivo studies were conducted by the same authors that tested ETBE for in vitro
genotoxicity. Vergnes and Kubena f!995al. in an unpublished report, performed an in vivo bone
marrow micronucleus (MN) test in mice in response to ETBE exposure. Male and female CD-I mice
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(5/sex/group) were exposed to ETBE by inhalation at target concentrations of 0, 400, 2,000, or
5,000 ppm (0,1,671, 8,357, or 20,894 mg/m3) for 6 hours/day, for 5 days. Following treatment,
polychromatic erythrocytes (PCE) from bone marrow were analyzed for micronucleus formation.
The results showed that no statistically significant increases in the mean percentages of
micronucleated polychromatic erythrocytes (MNPCE) were observed in mice (male or female)
when exposed to ETBE.
In addition to Vergnes and Kubena (1995a). four animal studies were conducted by JPEC in
rats using different routes of exposure (oral, inhalation, intraperitoneal, or drinking water) to
detect micronucleus as a result of exposure to ETBE [TPEC f2007a. 2007b. 2007c. 2007dl published
as Noguchi etal. f20131].
The first two studies (oral and intraperitoneal injection) were part of an acute (2-day)
exposure. In the first study, both male and female F344 rats (5/sex/dose group) were
administered ETBE (99.3% pure) via gavage in olive oil at doses of 0, 500,1,000, or
2,000 mg/kg-day every 24 hours [TPEC (2007a). unpublished report]. The animals were sacrificed,
and bone marrow smears were collected and stained 24 hours after the final administration.
Following treatment, polychromatic erythrocytes from bone marrow were analyzed for MN
formation. The results were expressed as the ratio of PCE to total erythrocytes. No
treatment-related effects on the number of MNPCE or the ratio of PCE to total erythrocytes were
found. ETBE was determined to be negative for micronuclei induction in rat bone marrow cells
after acute oral exposure.
In the second study (intraperitoneal injection), male and female F344 rats (5/sex/dose
group) were administered two ETBE intraperitoneal injections separated by 24 hours at doses of 0,
250, 500,1,000, or 2,000 mg/kg-day in olive oil fNoguchi etal.. 2013: TPEC. 2007bl. The animals
were sacrificed, and bone marrow smears were collected and stained 24 hours after the final
injection. All animals in the 2,000 mg/kg-day group died on the first day of treatment. There were
no treatment-related effects on either the number of MNPCEs or the ratio of polychromatic
erythrocytes to total erythrocytes. In addition, no dose-dependent tendencies for the increase in
the MNPCE to PCE ratio or alterations in the ratios of PCE to total erythrocytes were noted in either
sex of the treated groups. ETBE was determined to be negative for micronuclei induction in rats
after acute intraperitoneal exposure.
The next two studies (drinking water and inhalation) were part of 13-week toxicity studies
in rats in which the effects of ETBE on the micronuclei in PCE were examined at the end of the
study. In the first 13-week study, male and female F344 rats (10/sex/dose group) were given
drinking water containing 0,1,600, 4,000, or 10,000 ppm ETBE for 13 weeks (Noguchi etal.. 2013:
TPEC. 2007c). The concentrations were stated to be equivalent to 0,101, 259, and 626 mg/kg-day
in males and 0,120, 267, and 629 mg/kg-day in females. Following treatment, polychromatic
erythrocytes from bone marrow were analyzed for MN formation. The results were expressed as
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the ratio of PCE to total erythrocytes. There were no treatment-related effects on the number of
MNPCEs or the ratio of PCE to total erythrocytes.
In the second 13-week study (inhalation), male and female F344 rats (10/sex/dose group)
were exposed to ETBE (99.2-99.3% pure) through whole-body inhalation exposure at 0, 500,
1,500, or 5,000 ppm (0, 2,089, 6,268, or 20,894 mg/m3) for 6 hours/day, 5 days/week fNoguchi et
al.. 2013: TPEC. 2007b). Normochromatic and polychromatic erythrocytes and micronuclei were
counted as in the previous study. There were no treatment-related effects on the number of
MNPCE or the ratio of PCE to total erythrocytes. ETBE was determined to be negative for
micronuclei induction in rat bone marrow cells after a 13-week inhalation exposure.
Furthermore, NTP f!996a. 1996bl performed an in vivo bone marrow micronucleus test in
both B6C3Fi mice and Fischer rats. The animals were exposed through intraperitoneal injection
3 times in a period of 72 hours (n = 5). Doses for the mice were 0,1,300,1,700, 2,100, or
2,500 mg/kg, and the doses for rats were 0, 625,1,250, or 2,500 mg/kg. No increase in
micronucleated PCEs were observed in either mice or rats. Two of five mice died in the
1,700 mg/kg dose group, while 3 of 5 and 4 of 5 animals died in the 2,100 and 2,500 mg/kg dose
groups, respectively, and the surviving animals in the two highest dose groups were not scored. In
the rat study, 2 of 5 animals died in the highest dose group.
Wengetal. f20111 conducted several studies evaluating the differences in genotoxicity of
ETBE in various tissues or systems (i.e., erythrocytes, leukocytes, liver, and sperm) in C57BL/6 wild
type and Aldh2 knockout mice after subchronic inhalation exposure. All studies used the same
exposures (i.e., 0, 500,1,750, or 5,000 ppm ETBE for 6 hours/day, 5 days/week for 13 weeks).
Deoxyribonucleic acid (DNA) strand breaks were observed in leukocytes of male (all
concentrations) and female (high dose only) Aldh2 knockout mice and with the high dose in
wild-type male mice fWeng etal.. 20111.
Wengetal. f20121 studied the differential genotoxic effects of subchronic exposure to ETBE
in the liver of C57BL/6 wild-type and Aldh2 knockout mice. DNA strand breaks in the hepatocytes
of male and female with different Aldh2 genotypes were determined using the alkaline comet assay.
In addition, 8-hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification, and
8-hydroxydeoxyguanosine were determined as endpoints for genetic damage. There was
significant increase in damage in all three exposure groups in the knockout male mice, although the
increase was only found in the 5,000-ppm exposure group for the knockout female mice. In the
wild type, significant DNA damage was seen only in males in the 5,000-ppm group, but not in
females. This indicates the sensitivity of sex differences both in knockout and wild-type mice.
Another study by the same authors performed in vivo micronucleus tests (on what appear
to be the same set of animals), in addition to the DNA strand breaks, 8-hydroxyguanine DNA
glycosylase 1 (8-hOGGl)-modified oxidative base modification, and 8-hydroxydeoxyguanosine
fWeng etal.. 20131. The mice (wild type and knockout, males and females) were exposed to 0, 500,
1,750, or 5,000 ppm ETBE for 6 hours/day, 5 days/week for 13 weeks. Peripheral blood samples
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were obtained and processed to detect micronucleated reticulocytes (MNRETs) and micronuclei in
the mature normochromatic erythrocyte population. The results indicate that ETBE significantly
affected frequencies of MNRETs in male and female mice. In knockout male mice, the frequencies of
MNRETs of the 1,750- and 5,000-ppm exposure groups were significantly increased when
compared with the control group. In the wild-type male mice, however, only the 5,000-ppm group
had a higher frequency of MNRETs than that of the control group. In female wild-type mice, there
was no difference in the frequencies of MNRETs between exposure groups and the control group.
In the same exposure group (5,000 ppm), the knockout mice had a higher frequency of MNRETs
than the wild type. These results inform the influence of AIdh2 and sex difference on genotoxicity
as a result of exposure to ETBE.
In yet another study by the same authors fWeng etal.. 20141. DNA strand breaks and
8-hydroxyguanine DNA glycosylase 1 (8-hOGGl)-modified oxidative base modification were
measured in sperm collected from the left cauda epididymis. In addition to the 13-week protocol
used in the other studies, Weng etal. (20141 included a 9-week study in which the male mice (wild
type, knockout, and heterogeneous [HT]) were exposed to 0, 50, 200, or 500 ppm ETBE for
6 hours/day, 5 days/week. In the 13-week study, there were significant increases in damage in all
three exposure groups in the knockout male mice, but only in the two highest dose groups in the
wild-type males. In the 9-week study, there was no change in the wild-type mice, but both the
heterogeneous and the knockout mice had significant increases in the two highest doses.
Summary
Limited studies have been conducted to understand the genotoxic potential of ETBE. Most
studies indicate that ETBE does not induce genotoxicity in the systems tested. More recently, Weng
and coauthors illustrated the influence of AIdh2 on the genotoxic effects of ETBE. With respect to
overall existing database, it should be noted that the array of genotoxic tests conducted are limited.
The inadequacy of the database is two dimensional: (1) the coverage of the studies across the
genotoxicity tests needed for proper interpretation of the weight of evidence of the data is sparse
and (2) the quality of the available data is questionable. With respect to the array of types of
genotoxicity tests available, ETBE has only been tested in one bacterial assay. Only two in vitro
studies are available. The existing in vivo studies have tested only for the micronucleus assay, DNA
strand breaks, or both. Key studies on chromosomal aberrations and DNA adducts are missing.
Additionally, the few existing studies are unpublished reports lacking peer review. Given the above
limitations (i.e., the significant deficiencies and sparse database both in terms of quality and
quantity), the database is insufficient to draw a definitive conclusion on the genotoxic effects of
ETBE.
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Supplemental Information—ETBE
1 B.3. SUPPLEMENTAL ORGAN-WEIGHT DATA
2 B.3.1. Relative Kidney-Weight Data
Table B-18. Evidence pertaining to relative kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Fuiiietal. (2010); JPEC (2008d)
Rat, S-D
Oral—gavage
PO, male (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 16 wk beginning 10 wk prior to
mating
P0, female (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 17 wk beginning 10 wk prior to
mating to LD 21
P0, Male
P0, Female
Dose
(mg/kg-d)
Relative weight
(%)
Dose
(mg/kg-d)
Relative weight
(%)
0
-
0
-
100
8a
100
-3
300
12a
300
-1
1,000
26a
1,000
2
Gaoua (2004b)
Rat, S-D
Oral—gavage
P0, male (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until after weaning of the
pups
P0, female (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
and lactation, then Fl doses beginning
PND 22 until weaning of the F2 pups
P0, Male
P0, Female
Dose
(mg/kg-d)
Relative weight
(%)
Dose
(mg/kg-d)
Relative weight
(%)
0
-
0
-
250
ir
250
9
500
18a
500
5
1,000
28a
1,000
3
Fl, Male
Fl, Female
Dose
(mg/kg-d)
Relative weight
(%)
Dose
(mg/kg-d)
Relative weight
(%)
0
-
0
-
250
10a
250
6
500
19a
500
6
1,000
58a
1,000
10a
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0 or 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Relative weight
(%)
0
-
1,000
25a
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Supplemental Information—ETBE
Table B-18. Evidence pertaining to relative kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Mivata et al. (2013);JPEC (2008b)
Rat, Crl:CD(SD)
Oral—gavage
Male (15/group): 0, 5, 25,100, or
400 mg/kg-d; female (15/group): 0, 5, 25,
100, or 400 mg/kg-d
Daily for 26 wk
Male
Female
Dose
(mg/kg-d)
Relative weight
(%)
Dose
(mg/kg-d)
Relative weight
(%)
0
-
0
-
5
8
5
7
25
6
25
4
100
12a
100
ir
400
21a
400
15a
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (50/group): 0, 625, 2,500, or
10,000 ppm (0, 28,121, or 542 mg/kg-d);b
female (50/group): 0, 625, 2,500, or
10,000 ppm (0, 46,171, or 560 mg/kg-d)b
Daily for 104 wk
Male
Female
Dose
(mg/kg-d)
Relative weight
(%)
Dose
(mg/kg-d)
Relative weight
(%)
0
-
0
-
28
0
46
13a
121
12a
171
22a
542
31a
560
37a
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (NR): 0,150, 500,1,500, or 5,000 ppm
(0, 627, 2,090, 6,270, or 20,900 mg/m3);c
female (NR): 0,150, 500,1,500, or
5,000 ppm (0, 627, 2,090, 6,270, or
20,900 mg/m3)
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose (mg/m3)
Relative weight
(%)
Dose (mg/m3)
Relative weight
(%)
0
-
0
-
627
10
627
8
2,090
9
2,090
7
6,270
20a
6,270
12a
20,900
24a
20,900
20a
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (6/group): 0 or 5,000 ppm (0 or
20,900 mg/m3);c female (6/group): 0 or
5,000 ppm (0 or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d,
5 d/wk for 13 wk followed by a 28 d
recovery period; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Relative weight
(%)
Dose
(mg/m3)
Relative weight
(%)
0
-
0
-
20,900
15a
20,900
5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-18. Evidence pertaining to relative kidney-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Saito et al. (2013); JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (50/group): 0, 500,1,500, or
5,000 ppm (0, 2,090, 6,270, or
20,900 mg/m3);c female (50/group): 0, 500,
1,500, or 5,000 ppm (0, 2,090, 6,270, or
20,900 mg/m3)c
Dynamic whole-body inhalation; 6 h/d,
5 d/wk for 104 wk; generation method,
analytical concentration and method were
reported
Male
Female
Dose
(mg/m3)
Relative weight
(%)
Dose
(mg/m3)
Relative weight
(%)
0
-
0
-
2,090
19a
2,090
ir
6,270
26a
6,270
16a
20,900
66a
20,900
51a
- = for controls, no response relevant; for other doses, no quantitative response reported; LD = lactation day;
NR = not reported; PND = postnatal day.
aResult is statistically significant (p < 0.05) based on analysis of data by the study authors.
Conversion performed by the study authors.
c4.18 mg/m3 = 1 ppm.
Percentage change compared to controls calculated as 100 x [(treated value - control value) -f control value].
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 B.3.2. Absolute Liver-Weight Data
Table B-19. Evidence pertaining to absolute liver-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE)
Reference and study design
Results (percentage change compared to control)
Fuiiietal. (2010); JPEC (2008d)
Rat, S-D
Oral—gavage
P0, male (24/group): 0,100, 300, or 1,000 mg/kg-d
daily for 16 wk beginning 10 wk before mating
P0, female (24/group): 0,100, 300, or
1,000 mg/kg-d
Daily for 17 wk beginning 10 wk before mating to
LD 21
P0, Male
P0, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
100
-3
100
-1
300
-1
300
3
1,000
13a
1,000
14a
Gaoua (2004b)
Rat, S-D
Oral—gavage
P0, male (25/group): 0, 250, 500, or 1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups
P0, female (25/group): 0, 250, 500, or
1,000 mg/kg-d
Daily for a total of 18 wk beginning 10 wk before
mating until PND 21
Fl, male (25/group): 0, 250, 500, or 1,000 mg/kg-d
P0 dams dosed daily through gestation and
lactation, then Fl doses beginning PND 22 until
weaning of the F2 pups
Fl, female (24-25/group): 0, 250, 500, or
1,000 mg/kg-d
P0 dams dosed daily through gestation and
lactation, then Fl dosed beginning PND 22 until
weaning of the F2 pups
P0, Male
P0, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
250
2
250
-1
500
2
500
4
1,000
17a
1,000
6
Fl, Male
Fl, Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
250
0
250
1
500
14a
500
3
1,000
27a
1,000
10a
Hagiwara et al. (2011); JPEC (2008c)
Rat, F344
Oral—gavage
Male (12/group): 0 or 1,000 mg/kg-d
Daily for 23 wk
Male
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
1,000
21a
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-19. Evidence pertaining to absolute liver-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Mivata et al. (2013); JPEC (2008b)
Rat, Crl:CD(SD)
Oral—gavage
Male (15/group): 0, 5, 25,100, or 400 mg/kg-d;
female (15/group): 0, 5, 25,100, or 400 mg/kg-d
Daily for 26 wk
Male
Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
5
-2
5
-4
25
7
25
-1
100
4
100
2
400
19
400
9
Suzuki et al. (2012); JPEC (2010a)
Rat, F344
Oral—water
Male (50/group): 0, 625, 2,500, or 10,000 ppm (0,
28,121, or 542 mg/kg-d);b female (50/group): 0,
625, 2,500, or 10,000 ppm (0, 46, 171, or
560 mg/kg-d)b
Daily for 104 wk
Male
Female
Dose
(mg/kg-d)
Absolute
weight (%)
Dose
(mg/kg-d)
Absolute
weight (%)
0
-
0
-
28
_ir
46
-5
121
-4
171
-2
542
2
560
-10
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (NR): 0,150, 500,1,500, or 5,000 ppm (0,
627, 2,090, 6,270, or 20,900 mg/m3);c female (NR):
0, 150, 500, 1,500, or 5,000 ppm (0, 627, 2,090,
6,270, or 20,900 mg/m3)
Dynamic whole-body chamber; 6 h/d, 5 d/wk for
13 wk; generation method, analytical
concentration, and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
627
5
627
-3
2,090
6
2,090
-8
6,270
4
6,270
-2
20,900
2
20,900
5
JPEC (2008a)
Rat, Crl:CD(SD)
Inhalation—vapor
Male (6/group): 0 or 5,000 ppm (0 or
20,900 mg/m3);c female (6/group): 0 or 5,000 ppm
(0 or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk for
13 wk followed by a 28 d recovery period;
generation method, analytical concentration, and
method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
20,900
13
20,900
11
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-19. Evidence pertaining to absolute liver-weight effects in animals
exposed to ethyl tertiary butyl ether (ETBE) (continued)
Reference and study design
Results (percentage change compared to control)
Saito et al. (2013); JPEC (2010b)
Rat, F344
Inhalation—vapor
Male (50/group): 0, 500,1,500, or 5,000 ppm (0,
2,090, 6,270, or 20,900 mg/m3);c female
(50/group): 0, 500,1,500, or 5,000 ppm (0, 2,090,
6,270, or 20,900 mg/m3)c
Dynamic whole-body inhalation; 6 h/d, 5 d/wk for
104 wk; generation method, analytical
concentration, and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
1
2,090
-3
6,270
ir
6,270
-8
20,900
10
20,900
1
Medinskv et al. (1999); US EPA (1997)
Rat, F344
Inhalation—vapor
Male (48/group): 0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3);c female
(48/group): 0, 500,1,750, or 5,000 ppm (0, 2,090,
7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk for
13 wk; generation method, analytical
concentration, and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
6
2,090
2
7,320
14a
7,320
9
20,900
32a
20,900
26a
Medinskv et al. (1999); Bond et al. (1996)
Mice, CD-I
Inhalation—vapor
Male (40/group): 0, 500,1,750, or 5,000 ppm (0,
2,090, 7,320, or 20,900 mg/m3);c female
(40/group): 0, 500,1,750, or 5,000 ppm(0, 2,090,
7,320, or 20,900 mg/m3)c
Dynamic whole-body chamber; 6 h/d, 5 d/wk for
13 wk; generation method, analytical
concentration, and method were reported
Male
Female
Dose
(mg/m3)
Absolute
weight (%)
Dose
(mg/m3)
Absolute
weight (%)
0
-
0
-
2,090
4
2,090
2
7,320
13a
7,320
19a
20,900
18a
20,900
33a
- = for controls, no response relevant; for other doses, no quantitative response reported; NR = not reported;
PND = postnatal day.
aResult is statistically significant (p < 0.05) based on analysis of data by study authors.
Conversion performed by study authors.
c4.18 mg/m3 = 1 ppm.
Percentage change compared to controls calculated as 100 x [(treated value - control value) -f control value].
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
APPENDIX C. DOSE-RESPONSE MODELING FOR
THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE
DERIVATION OF CANCER RISK ESTIMATES
C.l. BENCHMARK DOSE MODELING SUMMARY
This appendix provides technical detail on dose-response evaluation and determination of
points of departure (PODs) for relevant toxicological endpoints. The endpoints were modeled using
EPA's Benchmark Dose Software (BMDS, version 2.2). Section C.l.l (noncancer) and Section C.l.2
(cancer) describe the common practices used in evaluating the model fit and selecting the
appropriate model for determining the POD, as outlined in the Benchmark Dose Technical Guidance
Document fU.S. EPA. 20121. In some cases, it might be appropriate to use alternative methods
based on statistical judgment; exceptions are noted as necessary in the summary of the modeling
results.
C.l.l. Noncancer Endpoints
Evaluation of Model Fit
For each dichotomous endpoint, BMDS dichotomous models1 were fitted to the data using
the maximum likelihood method. Each model was tested for goodness of fit using a chi-square
goodness-of-fit test (x2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess
model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the
vicinity of the benchmark response (BMR).
For each continuous endpoint, BMDS continuous models2 were fitted to the data using the
maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model,
the homogeneity of the variances was tested first using a likelihood ratio test (BMDS Test 2). If
Test 2 was not rejected (x2 p-value > 0.10), the model was fitted to the data assuming constant
variance. If Test 2 was rejected (x2 p-value < 0.10), the variance was modeled as a power function
'Unless otherwise specified, all available BMDS dichotomous models besides the alternative and nested
dichotomous models were fitted. The following parameter restrictions were applied: for the Log-Logistic
model, restrict slope >1; for the Gamma and Weibull models, restrict power >1.
2Unless otherwise specified, all available BMDS continuous models were fitted. The following parameter
restrictions were applied: for the Polynomial models, restrict the coefficients bl and higher to be nonnegative
or nonpositive if the direction of the adverse effect is upward or downward, respectively; for the Hill, Power,
and Exponential models, restrict power >1.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
of the mean, and the variance model was tested for adequacy of fit using a likelihood ratio test
(BMDS Test 3). For fitting models using either constant variance or modeled variance, models for
the mean response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with x2
p-value < 0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such
as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the
BMR.
Model Selection
For each endpoint, the lower confidence limit of the benchmark dose or concentration
(BMDL/BMCL), as estimated by the profile likelihood method and Akaike's information criterion
(AIC) value, were used to select a best-fit model from among the models exhibiting adequate fit If
the BMDL/BMCL estimates were "sufficiently close," that is, differed by at most threefold, the model
selected was the one that yielded the lowest AIC value. If the BMDL/BMCL estimates were not
sufficiently close, the lowest BMDLBMCL was selected as the POD.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose-response modeling for ethyl tertiary butyl ether (ETBE)
Endpoint, study
Sex, strain,
species
Doses/concentrations and effect data
Oral
Urothelial hyperplasia of
the renal pelvis
Suzuki etal. (2012); JPEC
(2010a)
Male F344 rats
Dose (mg/kg-d)
0
28
121
542
Incidence/total
0/50
0/50
10/50
25/50
Increased absolute
kidney weight
Suzuki etal. (2012); JPEC
(2010a)
Female F344 rats
Dose (mg/kg-d)
0
46
171
560
No. of animals
36
37
38
38
Mean ± SD
1.81 ±0.12
1.863 ±0.14
1.988 ±0.19
2.057 ±0.26
Increased absolute
kidney weight
Miyata et al. (2013);
JPEC (2008b)
Male
Sprague-Dawley rats
Dose (mg/kg-d)
0
5
25
100
400
No. of animals
15
15
14
15
13
Mean ± SD
3.27 ±0.34
3.29 ±0.3
3.47 ±0.32
3.42 ± 0.48
4.09 ± 0.86
Increased absolute
kidney weight
Mivata et al. (2013);
JPEC (2008b)
Female
Sprague-Dawley rats
Dose (mg/kg-d)
0
5
25
100
400
No. of animals
15
15
15
15
15
Mean ± SD
1.88 ±0.2
1.89 ±0.16
1.88 ±0.15
2.02 ±0.21
2.07 ±0.23
Increased absolute
kidney weight
Gaoua (2004b)
PO male
Sprague-Dawley rats
Dose (mg/kg-d)
0
250
500
1,000
No. of animals
25
25
25
25
Mean ± SD
3.58 ±0.413
3.96 ±0.446
4.12 ±0.624
4.34 ± 0.434
Increased absolute
kidney weight
Gaoua (2004b)
PO female
Sprague-Dawley rats
Dose (mg/kg-d)
0
250
500
1,000
No. of animals
25
24
22
25
Mean ± SD
2.24 ±0.185
2.22 ±0.16
2.29 ±0.207
2.35 ±0.224
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose-response modeling for ethyl tertiary butyl ether (ETBE)
(continued)
Endpoint, study
Sex, strain,
species
Doses/concentrations and effect data
Increased absolute
kidney weight
Gaoua (2004b)
F1 male
Sprague-Dawley rats
Dose (mg/kg-d)
0
250
500
1,000
No. of animals
24
25
24
25
Mean ± SD
3.38 ±0.341
3.73 ±0.449
4.13 ±0.64
5.34 ±5.39
Increased absolute
kidney weight
Gaoua (2004b)
F1 female
Sprague-Dawley rats
Dose (mg/kg-d)
0
250
500
1,000
No. of animals
25
24
25
23
Mean ± SD
2.24 ±0.178
2.34 ±0.242
2.3 ±0.226
2.49 ±0.284
Increased absolute
kidney weight
Fuiiietal. (2010); JPEC
(2008d)
Male
Sprague-Dawley rats
Dose (mg/kg-d)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
3.46 ±0.57
3.62 ±0.45
3.72 ±0.35
4.07 ±0.53
Increased relative kidney
weight
Fuiiietal. (2010); JPEC
(2008d)
Male
Sprague-Dawley rats
Dose (mg/kg-d)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
0.546 ± 0.059
0.592 ±0.06
0.609 ± 0.042
0.689 ± 0.049
Increased absolute
kidney weight
Fuiiietal. (2010); JPEC
(2008d)
Female
Sprague-Dawley rats
Dose (mg/kg-d)
0
100
300
1,000
No. of animals
21
22
23
19
Mean ± SD
2.17 ±0.18
2.13 ±0.14
2.17 ±0.17
2.33 ±0.24
Increased relative kidney
weight
Fuiiietal. (2010); JPEC
(2008d)
Female
Sprague-Dawley rats
Dose (mg/kg-d)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
0.674 ±0.053
0.656 ± 0.048
0.668 ±0.057
0.687 ± 0.045
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose-response modeling for ethyl tertiary butyl ether (ETBE)
(continued)
Endpoint, study
Sex, strain,
species
Doses/concentrations and effect data
Inhalation
Urothelial hyperplasia of
the renal pelvis
Saito etal. (2013); JPEC
(2010b)
Male F344 rats
Exposure concentration
(mg/m3)
0
2,090
6,270
20,900
Incidence/total
2/50
5/50
16/49
41/50
Increased absolute
kidney weight
Saito etal. (2013); JPEC
(2010b)
Female
Sprague-Dawley rats
Exposure concentration
(ppm)
0
2,090
6,270
20,900
No. of animals
37
39
29
30
Mean ± SD
1.81 ±0.18
1.90 ±0.20
1.92 ±0.13
2.13 ±0.28
Increased absolute
kidney weight
JPEC (2008a)
Male
Sprague-Dawley rats
Exposure concentration
(ppm)
0
150
500
1,500
5,000
No. of animals
10
10
10
10
10
Mean ± SD
3.15 ±0.243
3.45 ±0.385
3.49 ±0.314
3.72 ±0.365
3.64 ±0.353
Increased absolute
kidney weight
JPEC (2008a)
Female
Sprague-Dawley rats
Exposure concentration
(ppm)
0
150
500
1,500
5,000
No. of animals
10
10
10
10
10
Mean ± SD
1.84 ±0.129
1.85 ±0.18
1.83 ±0.118
1.92 ±0.173
1.97 ±0.16
Increased absolute
kidney weight
Medinsky et al. (1999);
US EPA (1997)
Male F344 rats
Exposure concentration
(ppm)
0
500
1,750
5,000
No. of animals
11
11
11
11
Mean ± SD
1.73 ±0.155
1.85 ±0.137
1.903 ±0.1
2.067 ±0.124
Increased absolute
kidney weight
Female F344 rats
Exposure concentration
(ppm)
0
500
1,750
5,000
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose-response modeling for ethyl tertiary butyl ether (ETBE)
(continued)
Endpoint, study
Sex, strain,
species
Doses/concentrations and effect data
Medinskv et al. (1999);
US EPA (1997)
No. of animals
10
11
11
11
Mean ± SD
1.077 ± 0.069
1.125 ±0.048
1.208 ± 0.076
1.306 ± 0.055
No. = number; SD = standard deviation.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Modeling Results
2 Below are tables summarizing the modeling results for the noncancer endpoints modeled.
3 Oral exposure endpoints
Table C-2. Summary of benchmark dose (BMD) modeling results for urothelial
hyperplasia of the renal pelvis in male F344 rats exposed to ethyl tertiary
butyl ether (ETBE) in drinking water for 104 weeks (IPEC. 2010a) modeled
with doses as mg/kg-day (calculated by the study authors); benchmark
response (BMR) = 10% extra risk
Model3
Goodness of fit
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Basis for model selection
p-value
AIC
Gamma
0.196
127.93
88.1
60.9
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Quantal-Linear
model was selected based on
lowest AIC.
Logistic
1.00 x 10"3
139.54
217
177
Log-Logistic
0.264
127.28
85.3
49.5
Probit
0.0015
138.30
197
162
Log-Pro bit
0.374
126.14
85.8
51.3
Weibull
0.202
128.00
85.7
60.7
Multistage
(3 degree)b
Multistage
(2 degree)0
0.395
126.07
79.3
60.5
Quantal-linear0
0.395
126.07
79.3
60.5
aSelected model in bold; scaled residuals for selected model for doses 0, 28,121, and 542 mg/kg-d were 0.000,
-1.377,1.024, and -0.187, respectively.
bFor the Multistage (3 degree) model, the beta coefficient estimates were 0 (boundary of parameters space), and
the model reduced to the Multistage (2 degree) model.
cThe Multistage (2 degree) model and Quantal-Linear models appear equivalent; however, differences exist in
digits not displayed in the table.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Quantal Linear Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for the
1 3:1 O 09/1 O 2014
Figure C-l. Plot of incidence rate by dose, with fitted curve for selected model;
dose shown in mg/kg-day.
Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013)
The form of the probability function is:
P[response] = background + (1 - background) x [1 - exp(- slope x dose)]
Benchmark Dose Computation.
BMR = 10% extra risk
Benchmark dose (BMD) = 79.3147
BMDL at the 95% confidence level = 60.5163
Parameter Estimates
Variable
Estimate
Default initial parameter values
Background
0
0.0192308
Slope
0.00132839
0.00124304
Power
N/A
1
Analysis of Deviance Table
Model
Log(likelihood)
# Parameters
Deviance
Test df
p-value
Full model
-59.6775
4
Fitted model
-62.0369
1
4.71891
3
0.1936
Reduced model
-92.7453
1
66.1356
3
<0.0001
AIC = 126.074
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Goodness-of-Fit Table
Dose
Estimated
probability
Expected
Observed
Size
Scaled residual
0
0
0
0
50
0
28
0.0365
1.826
0
50
-1.377
121
0.1485
7.424
10
50
1.024
542
0.5132
25.662
25
50
-0.187
2 x2 = 2.98; degrees of freedom (df) = 3; p-value = 0.3948
Table C-3. Summary of benchmark dose (BMD) modeling results for increased
absolute kidney weight in female F344 rats exposed to ethyl tertiary butyl
ether (ETBE) in drinking water for 104 weeks (IPEC. 2010a): benchmark
response (BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.00752
-339.97
385
290
The Exponential (M4) model was
selected as the only model with a
goodness-of-fit p-value > 0.1.
Exponential (M4)
0.621
-347.50
204
120
Exponential (M5)
N/A
-345.75
192
116
Hill
N/A
-345.75
195
107
Power0
Polynomial
(3 degree)d
Polynomial
(2 degree)6
Linear
0.0115
-340.82
367
272
aModeled variance case presented (BMDS Test 3 p-value = <0.8167), selected model in bold; scaled residuals for
selected model for doses 0, 46,171, and 560 mg/kg-d were 0.0259, -0.19,0.474, and -0.289, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Power model, the power parameter estimate was 1 (boundary), and the model reduced to the Linear
model.
dFor the Polynomial (3 degree) model, the b3 and b2 coefficient estimates were 0 (boundary), and the model
reduced to the Linear model.
eFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary), and the model reduced to
the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
16:55 06/18 2019
Figure C-2. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Exponential 4 Model. (BMDS Version 1.10; Date: 01/12/2015)
2 The form of the response function is: P[dose] = a*[c-(c-l)x exp(-b x dose)]
3 A modeled variance is fit: Var[i] = exp(log-alpha + log[mean(i)] x rho)
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 204 mg/kg-day
7 BMDL at the 95% confidence level = 120 mg/kg-day
8 Parameter Estimates
Variable
Model 4
Standard error
Inalpha
-11.0816
1.89029
rho
11.431
2.93477
a
1.80851
0.0173746
b
0.00518165
0.00207201
c
1.15314
0.0322089
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
37
1.809
1.809
0.122
0.116
0.02585
46
39
1.863
1.867
0.135
0.1392
-0.1903
171
29
1.988
1.971
0.189
0.1898
0.4744
560
30
2.057
2.07
0.261
0.2511
-0.2889
2 Likelihoods of Interest
Model
Log(likelihood)
df
AIC
A1
166.6724
5
-323.3449
A2
179.0769
8
-342.1539
A3
178.8744
6
-345.7488
R
148.74
2
-293.4799
4
178.7521
5
-347.5042
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
60.67
6
<0.0001
Test 2
24.81
3
<0.0001
Test 3
0.4051
2
0.8167
Test 6a
0.2446
1
0.6209
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-4. Summary of benchmark dose (BMD) modeling results for increased
absolute kidney weight in male Sprague-Dawley (S-D) rats exposed to ethyl
tertiary butyl ether (ETBE) by daily gavage for 26 weeks (Mivata et al.. 2013:
IPEC. 2008d): benchmark response (BMR) = 10% relative deviation from the
mean
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Model3
p-value
AIC
Basis for model selection
Exponential (M2)
Exponential (M3)b
0.752
-47.963
186
126
The linear model was selected
based on lowest AIC.
Exponential (M4)
Exponential (M5)c
0.603
-46.156
157
67.7
Hill
0.605
-46.161
156
63.6
Powerd
Polynomial
(2 degree)®
Linear'
0.774
-48.055
176
115
Polynomial
(3 degree)8
0.774
-48.055
176
115
aModeled variance case presented (BMDS Test 3 p-value = <0), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-d were -0.421, -0.288,1.29, -0.669, and 0.15,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
the Linear and Polynomial (3 degree) models appear equivalent; however, differences exist in digits not
displayed in the table.
gThe Linear model, Polynomial (2 degree and 3 degree) models and the Power models appear equivalent;
however, differences exist in digits not displayed in the table.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
15:56 05/15 2014
0 50 100 150 200
dose
Figure C-3. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMD
1 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = beta_0 + beta_l x dose
3 A modeled variance is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 176.354
7 BMDL at the 95% confidence level = 114.829
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
-13.8218
-1.41289
rho
9.65704
0
beta_0
3.30477
3.30246
beta_l
0.00187393
0.00193902
This document is a draft for review purposes only and does not constitute Agency policy.
C-13 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
15
3.27
3.3
0.34
0.32
-0.421
5
15
3.29
3.31
0.3
0.325
-0.288
25
14
3.47
3.35
0.32
0.343
1.29
100
15
3.42
3.49
0.48
0.418
-0.669
400
13
4.09
4.05
0.86
0.859
0.15
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
17.455074
6
-22.910149
A2
29.755425
10
-39.51085
A3
28.583571
7
-43.167142
fitted
28.027315
4
-48.05463
R
6.041664
2
-8.083328
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
47.4275
8
<0.0001
Test 2
24.6007
4
<0.0001
Test 3
2.34371
3
0.5042
Test 4
1.11251
3
0.7741
This document is a draft for review purposes only and does not constitute Agency policy.
C-14 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-5. Summary of benchmark dose (BMD) modeling results for increased
absolute kidney weight in female Sprague-Dawley (S-D) rats exposed to ethyl
tertiary butyl ether (ETBE) by daily gavage for 26 weeks (Mivata et al.. 2013:
IPEC. 2008d): benchmark response (BMR) = 10% relative deviation from the
mean
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Model3
p-value
AIC
Basis for model selection
Exponential (M2)
Exponential (M3)b
0.369
-168.25
406
271
The Exponential (M4) model was
selected based on lowest BMDL.
Exponential (M4)
0.670
-168.60
224
56.9
Exponential (M5)
0.865
-167.37
Error0
0
Hill
0.986
-169.37
Error0
Error0
Powerd
Polynomial
(3 degree)6
Polynomial
(2 degree)'
Linear
0.382
-168.34
402
263
aConstant variance case presented (BMDS Test 2 p-value = 0.425), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-d were 0.2257, 0.2206, -0.737,0.3806, and -0.08999,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
CBMD or BMDL computation failed for this model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
fFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
16:35 05/15 2014
Figure C-4. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a*[c-(c-l)x exp(-b x dose)]
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 223.57
7 BMDL at the 95% confidence level = 56.8917
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
-3.35462
-3.36529
rho(S)
N/A
0
a
1.86911
1.786
b
0.0100557
0.00368689
c
1.11181
1.21697
d
1
1
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
15
1.88
1.869
0.2
0.1869
0.2257
5
15
1.89
1.879
0.16
0.1869
0.2206
25
15
1.88
1.916
0.15
0.1869
-0.737
100
15
2.02
2.002
0.21
0.1869
0.3806
400
15
2.07
2.074
0.23
0.1869
-0.08999
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
88.69837
6
-165.3967
A2
90.62918
10
-161.2584
A3
88.69837
6
-165.3967
R
82.20147
2
-160.4029
4
88.29837
4
-168.5967
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
16.86
8
0.03165
Test 2
3.862
4
0.4251
Test 3
3.862
4
0.4251
Test 6a
0.8
2
0.6703
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-6. Summary of benchmark dose (BMD) modeling results for increased
absolute kidney weight in PO male Sprague-Dawley (S-D) rats exposed to ethyl
tertiary butyl ether (ETBE) by daily gavage for a total of 18 weeks beginning
10 weeks before mating until after weaning of the pups (Gaoua. 2004a):
benchmark response (BMR) = 10% relative deviation from the mean
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Model3
p-value
AIC
Basis for model selection
Exponential (M2)
Exponential (M3)b
0.155
-38.410
551
423
The Hill model is selected based
on lowest BMDL
Exponential (M4)c
0.727
-40.012
255
123
Exponential (M5)c
0.727
-40.012
255
123
Hill
0.811
-40.077
244
94.0
Powerd
Polynomial
(3 degree)6
Polynomial
(2 degree)'
Linear
0.199
-38.902
517
386
aConstant variance case presented (BMDS Test 2 p-value = 0.119), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-d were -0.0247, 0.14, -0.181, and 0.0657, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
cThe Exponential (M4) model and the Exponential (M5) model appear equivalent; however, differences exist in
digits not displayed in the table.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
fFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
14:47 05/15 2014
Figure C-5. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v x dosen/(kn + dosen)
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 243.968
7 BMDL at the 95% confidence level = 93.9617
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.227462
0.236804
rho
N/A
0
intercept
3.58236
3.58
V
1.16337
0.76
n
1
0.647728
k
548.322
250
This document is a draft for review purposes only and does not constitute Agency policy.
C-19 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
25
3.58
3.58
0.413
0.477
-0.0247
250
25
3.96
3.95
0.446
0.477
0.14
500
25
4.12
4.14
0.624
0.477
-0.181
1,000
25
4.34
4.33
0.434
0.477
0.0657
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
24.067171
5
-38.134342
A2
26.992591
8
-37.985183
A3
24.067171
5
-38.134342
fitted
24.038627
4
-40.077253
R
9.48179
2
-14.963581
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
35.0216
6
<0.0001
Test 2
5.85084
3
0.1191
Test 3
5.85084
3
0.1191
Test 4
0.057089
1
0.8112
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-7. Summary of benchmark dose (BMD) modeling results for increased
absolute kidney weight in PO female Sprague-Dawley (S-D) rats exposed to
ethyl tertiary butyl ether (ETBE) by daily gavage for a total of 18 weeks
beginning 10 weeks before mating until after weaning of the pups (Gaoua.
2004a); benchmark response (BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
0.625
-214.58
1,734
1,030
Exponential (M2) model is
selected based on lowest AIC;
however, BMDL is higher than
the maximum dose.
Exponential (M3)
0.416
-212.86
1,458
1,040
Exponential (M4)
0.327
-212.56
1,774
1,032
Exponential (M5)
N/Ab
-211.39
Error0
0
Hill
0.715
-213.39
Error0
Error0
Power
0.418
-212.87
1,470
1,041
Polynomial
(3 degree)
0.400
-212.81
1,409
1,035
Polynomial
(2 degree)
0.400
-212.81
1,409
1,037
Linear
0.619
-214.56
1,774
1,032
aConstant variance case presented (BMDS Test 2 p-value = 0.391), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-d were 0.5052, -0.7974, 0.1844, and 0.1033,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
CBMD or BMDL computation failed for this model.
This document is a draft for review purposes only and does not constitute Agency policy.
C-21 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bl
15:14 05/15 2014
Figure C-6. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a x exp(sign x b x dose)
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 1,734.24
7 BMDL at the 95% confidence level = 1,030.08
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
-3.29773
-3.30752
rho(S)
N/A
0
a
2.22057
2.22078
b
0.0000549578
0.0000546688
c
0
0
d
1
1
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
25
2.24
2.221
0.185
0.1923
0.5052
250
24
2.22
2.251
0.16
0.1923
-0.7974
500
22
2.29
2.282
0.207
0.1923
0.1844
1,000
25
2.35
2.346
0.224
0.1923
0.1033
2 Likelihoods of Interest
Model
Log(likelihood)
# Parameters
AIC
A1
110.761
5
-211.522
A2
112.2635
8
-208.5269
A3
110.761
5
-211.522
R
107.4777
2
-210.9553
2
110.2909
3
-214.5817
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
9.572
6
0.1439
Test 2
3.005
3
0.3909
Test 3
3.005
3
0.3909
Test 4
0.9403
2
0.6249
This document is a draft for review purposes only and does not constitute Agency policy.
C-23 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-8. Summary of benchmark dose (BMD) modeling results for absolute
kidney weight in F1 male Sprague-Dawley rats exposed to ethyl tertiary butyl
ether (ETBE) by gavage in a two-generation study (Gaoua. 2004b): benchmark
response (BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
6.30 x 10"4
89.912
232
175
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Polynomial (3
degree) model was selected
based on lowest AIC.
Exponential (M3)
0.129
79.474
335
256
Exponential (M4)
<0.0001
98.039
263
179
Exponential (M5)
N/Ab
82.504
347
267
Hill
N/Ab
82.509
347
267
Power
0.0680
80.504
347
267
Polynomial
(3 degree)
0.374
77.965
318
235
Polynomial
(2 degree)
0.0943
79.973
330
251
Linear
<0.0001
96.039
263
179
aModeled variance case presented (BMDS Test 3 p-value = <0), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-d were -0.584,0.717,0.225, and -0.837, respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
dose
13:43 09/12 2014
Figure C-7. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
C-24 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Polynomial Model. (Version: 2.19; Date: 06/25/2014)
2 The form of the response function is: Y[dose] = beta_0 + beta_l x dose + beta_2 x dose2 + ...
3 A modeled variance is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 318.084
7 BMDL at the 95% confidence level = 235.491
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
-13.8779
2.02785
rho
9.40248
0
beta_0
3.41732
3.38
beta_l
0.000881597
0.00138667
beta_2
2.232 x 10"28
0
beta_3
1.90507 x 10"9
6.93333 x 10"9
9 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
24
3.38
3.42
0.341
0.313
-0.584
250
25
3.73
3.67
0.449
0.436
0.717
500
24
4.13
4.1
0.64
0.734
0.225
1,000
25
5.34
6.2
5.39
5.16
-0.837
10 Likelihoods of Interest
Model
Log(likelihood)
# Parameters
AIC
A1
-146.32249
5
302.644981
A2
-32.521507
8
81.043013
A3
-33.58656
6
79.17312
Fitted
-33.982384
5
77.964768
R
-149.897277
2
303.794554
This document is a draft for review purposes only and does not constitute Agency policy.
C-25 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
234.752
6
<0.0001
Test 2
227.602
3
<0.0001
Test 3
2.13011
2
0.3447
Test 4
0.791648
1
0.3736
Table C-9. Summary of benchmark dose (BMD) modeling results for absolute
kidney weight in F1 female Sprague-Dawley rats exposed to ethyl tertiary
butyl ether (ETBE) by gavage in a two-generation study (Gaoua. 2004bl:
benchmark response (BMR) = 10% relative deviation
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
0.311
-180.23
978
670
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Exponential (M2)
model was selected based on
lowest AIC.
Exponential (M3)
0.147
-178.46
1,016
679
Exponential (M4)
0.121
-178.16
980
654
Exponential (M5)
N/Ab
-176.44
1,019
613
Hill
N/Ab
-176.44
1,019
611
Power
0.145
-178.44
1,019
666
Polynomial
(3 degree)
0.184
-178.80
1,001
684
Polynomial
(2 degree)
0.159
-178.58
1,002
673
Linear
0.301
-180.16
980
654
aConstant variance case presented (BMDS Test 2 p-value = 0.159), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-d were -0.05426, 0.8898, -1.173, and 0.3711,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
This document is a draft for review purposes only and does not constitute Agency policy.
C-26 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
13:47 09/12 2014
Figure C-8. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a x exp(sign x b x dose)
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 978.157
7 BMDL at the 95% confidence level = 669.643
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
-2.91989
-2.94397
rho(S)
N/A
0
a
2.24252
2.24321
b
0.0000974385
0.000096475
c
0
0
d
1
1
This document is a draft for review purposes only and does not constitute Agency policy.
C-27 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
25
2.24
2.243
0.178
0.2322
-0.05426
250
24
2.34
2.298
0.242
0.2322
0.8898
500
25
2.3
2.354
0.226
0.2322
-1.173
1,000
23
2.49
2.472
0.284
0.2322
0.3711
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
94.28268
5
-178.5654
A2
96.87585
8
-177.7517
A3
94.28268
5
-178.5654
R
87.16418
2
-170.3284
2
93.11474
3
-180.2295
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
19.42
6
0.003505
Test 2
5.186
3
0.1587
Test 3
5.186
3
0.1587
Test 4
2.336
2
0.311
This document is a draft for review purposes only and does not constitute Agency policy.
C-28 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-10. Summary of benchmark dose (BMD) modeling results for
increased absolute kidney weight in PO male Sprague-Dawley (S-D) rats
exposed to ethyl tertiary butyl ether (ETBE) by daily gavage for 16 weeks
beginning 10 weeks prior to mating (Fujii etal.. 2010): benchmark response
(BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.668
-41.247
648
479
The Hill model was selected
based on lowest BMDL (BMDLs
were greater than threefold
difference.)
Exponential (M4)
Exponential (M5)c
0.600
-39.779
438
163
Hill
0.613
-39.799
435
139
Powerd
Polynomial
(3 degree)6
Polynomial
(2 degree)'
Linear
0.700
-41.342
625
448
aConstant variance case presented (BMDS Test 2 p-value = 0.102), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-d were -0.202,0.399, -0.232, and 0.0344, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model. For the Polynomial (3 degree) model, the b3 and b2
coefficient estimates were 0 (boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
13:13 05/15 2014
0 200 400
dose
Figure C-9. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
4.4
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v x dosen/(kn + dosen)
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 434.715
7 BMDL at the 95% confidence level = 139.178
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.223598
0.2327
rho
N/A
0
intercept
3.47949
3.46
V
1.24601
0.61
n
1
0.27452
k
1,122
1,610
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
24
3.46
3.48
0.57
0.473
-0.202
100
24
3.62
3.58
0.45
0.473
0.399
300
24
3.72
3.74
0.35
0.473
-0.232
1,000
24
4.07
4.07
0.53
0.473
0.0344
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
24.027112
5
-38.054223
A2
27.13071
8
-38.26142
A3
24.027112
5
-38.054223
Fitted
23.899392
4
-39.798783
R
14.313578
2
-24.627156
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
25.6343
6
0.0002604
Test 2
6.2072
3
0.102
Test 3
6.2072
3
0.102
Test 4
0.25544
1
0.6133
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-ll. Summary of benchmark dose (BMD) modeling results for
increased absolute kidney weight in PO female Sprague-Dawley (S-D) rats
exposed to ethyl tertiary butyl ether (ETBE) by daily gavage for 17 weeks
beginning 10 weeks prior to mating until Lactation Day 21 (Fujii etal.. 2010):
benchmark response (BMR) = 10% relative deviation from the mean
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Model3
p-value
AIC
Basis for model selection
Exponential (M2)
0.483
-199.73
1,135
781
Polynomial (2 degree) is selected
Exponential (M3)
0.441
-198.60
1,089
826
based on most parsimonious
model with lowest AIC.
Exponential (M4)
0.217
-197.67
1,144
771
Exponential (M5)
N/Ab
-196.66
Error0
0
Hill
N/Ab
-196.66
Error0
Error0
Power
0.441
-198.60
1,092
823
Polynomial
(3 degree)d
Polynomial
(2 degree)
0.743
-200.60
1,094
905
Linear
0.467
-199.67
1,144
771
aConstant variance case presented (BMDS Test 2 p-value = 0.103), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-d were 0.499, -0.579,0.0914, and -0.00282,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
CBMD or BMDL computation failed for this model.
dFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
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Supplemental Information—ETBE
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the B
14:09 05/15 2014
Figure C-10. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
1 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = beta_0 + beta_l x dose + beta_2 x dose2 + ...
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% relative deviation
6 BMD = 1,093.86
7 BMDL at the 95% confidence level = 905.267
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.0323691
0.0337309
rho
N/A
0
beta_0
2.1504
2.15624
beta_l
7.16226 x 10"28
0
beta_2
1.79719 x 10"6
0
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
21
2.17
2.15
0.18
0.18
0.499
100
22
2.13
2.15
0.14
0.18
-0.579
300
23
2.17
2.17
0.17
0.18
0.0914
1,000
19
2.33
2.33
0.24
0.18
-0.00282
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
103.595625
5
-197.191249
A2
106.684319
8
-197.368637
A3
103.595625
5
-197.191249
fitted
103.298361
3
-200.596722
R
96.89324
2
-189.78648
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
19.5822
6
0.003286
Test 2
6.17739
3
0.1033
Test 3
6.17739
3
0.1033
Test 4
0.594528
2
0.7428
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Supplemental Information—ETBE
1 Inhalation exposure endpoints
Table C-12. Summary of benchmark concentration (BMC) modeling results for
urothelial hyperplasia of the renal pelvis in male F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 104 weeks (IPEC. 201 Obi: benchmark response (BMR) = 10%
extra risk
Model3
Goodness of fit
BMCio
(mg/m3)
BMCLio
(mg/m3)
Basis for model selection
p-value
AIC
Gamma
0.874
164.37
2,734
1,498
Of the models that provided an
adequate fit and a valid BMCL
estimate, the Gamma model was
selected based on lowest AIC.
Logistic
0.146
166.30
4,329
3,522
Log-Logistic
0.814
164.40
3,010
1,831
Probit
0.202
165.59
4,059
3,365
Log-Pro bit
0.633
164.57
3,050
1,896
Weibull
0.758
164.44
2,623
1,478
Multistage
(3 degree)
0.565
164.69
2,386
1,412
Multistage
(2 degree)
0.565
164.69
2,386
1,422
Quantal-Linear
0.269
165.16
1,541
1,227
BMC = benchmark concentration; BMCL = benchmark concentration lower confidence level.
aSelected model in bold; scaled residuals for selected model for concentrations 0, 2,089, 6,268, and
20,893 mg/m3 were 0.036, -0.107,0.104, and -0.040, respectively. Exposure concentrations were converted
from 0, 500,1,500, and 5,000 ppm to mg/m3 using the calculation mg/m3 = (102.17, molecular weight of
ETBE) x ppm t- 24.45.
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1
2
3
4
5
6
7
8
9
10
Supplemental Information—ETBE
Gamma Multi-Hit Model, with BMR of 1 0% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for the
1
o
CD
Gamma Multi-Hit
1 3:40 09/1 O 201 4
10000
dose
Figure C-ll. Plot of incidence rate by concentration, with fitted curve for
selected model; concentration shown in mg/m3.
Gamma Model. (Version: 2.16; Date: 2/28/2013)
The form of the probability function is:
P [response] = background + (1 - background) x CumGamma[slope x concentration,power], where
CumGamma(.) is the cumulative Gamma distribution function.
Power parameter is restricted as power >1.
Benchmark Concentration Computation.
BMR = 10% extra risk
BMC = 2,734.41
BMCL at the 95% confidence level = 1,497.7
Parameter Estimates
Variable
Estimate
Default initial parameter values
Background
0.0390054
0.0576923
Slope
0.000121504
0.000132454
Power
1.59019
1.84876
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Supplemental Information—ETBE
1 Analysis of Deviance Table
Model
Log(likelihood)
# Parameters
Deviance
Test df
p-value
Full model
-79.1741
4
Fitted model
-79.1867
3
0.0253512
l
0.8735
Reduced model
-124.987
1
91.626
3
<0.0001
2 AIC = 164.373
3 Goodness-of-Fit Table
Concentration
Estimated
probability
Expected
Observed
Size
Scaled residual
0
0.039
1.95
2
50
0.036
2,089
0.1046
5.231
5
50
-0.107
6,268
0.3196
15.659
16
49
0.104
20,893
0.8222
41.109
41
50
-0.04
4 x2 = 0.03; df = 1; p-value = 0.8737
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-13. Summary of benchmark concentration (BMC) modeling results for
increased absolute kidney weight in female F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day, 5
days/week, for 104 weeks (IPEC. 2010b). benchmark response (BMR) = 10%
relative deviation from the mean
Model3
Goodness of fit
BMCiord
(mg/m3)
BMCLiord
(mg/m3)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.1482
-293.77
13,422.9
10,431.3
Of the models that provided an
adequate fit and a valid BMCL
estimate, the Exponential (M2)
model was selected based on
lowest AIC.
Exponential (M4)
0.04944
-291.73
13,028.1
7,023.54
Exponential (M5)
0.04944
-291.73
13,027.3
7,023.54
Hill
0.04939
-291.73
13,027.3
9,893.86
Polynomial
(2 degree)0
0.05124
-291.79
13,959.9
9,936.46
Polynomial
(3 degree)d
0.05454
-291.89
14,857.4
9,985.31
Power6
Linear
0.1451
-293.73
13,029.1
9,909.08
BMC = benchmark concentration; BMCL = benchmark concentration lower confidence level.
aSelected model in bold; modeled variance case presented. For this data set variance was modeled as a power
function of the mean, but the p-value (BMDS Test 3 p-value = <0.018) is below the threshold criteria for variance
testing of 0.1.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
cFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
dFor the Polynomial (3 degree) model, the b3 and b2 coefficient estimates were 0 (boundary), and the model
reduced to the Linear model.
eFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-14. Summary of benchmark concentration (BMC) modeling results for
increased absolute kidney weight in male Sprague-Dawley (S-D) rats exposed
to ethyl tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week for 13 weeks (IPEC. 2008b): benchmark response (BMR) = 10%
relative deviation from the mean
Model3
Goodness of fit
BMCiord
(ppm)
BMCLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.168
-43.014
1,105
750
Of the models that provided an
adequate fit and a valid BMCL
estimate, the Hill model was
selected based on lowest BMCL
(BMCLs differed by more than 3).
Exponential (M4)
0.200
-42.943
380
1.73
Exponential (M5)
0.200
-42.943
380
2.61
Hill
0.294
-43.484
264
15.4
Power0
Polynomial
(3 degree)d
Polynomial
(2 degree)6
Linear
0.178
-43.133
1,071
703
aConstant variance case presented (BMDS Test 2 p-value = 0.506), selected model in bold; scaled residuals for
selected model for concentrations 0,150, 500, and 1,500 ppm were -0.13, 0.54, -0.8, 0.38, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
dFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model. For the Polynomial (3 degree) model, the b3 and b2
coefficient estimates were 0 (boundary of parameters space), and the model reduced to the Linear model.
eFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
4
3.8
3.2
3
800
dose
18:02 05/28 2015
Figure C-12. Plot of mean response by concentration, with fitted curve for
selected model; concentration shown in ppm.
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is:
3 Y[concentration] = intercept + v x concentration11/(kn + concentration11)
4 A constant variance model is fit
5 Benchmark Concentration Computation.
6 BMR = 10% relative deviation
7 BMC = 264.371
8 BMCL at the 95% confidence level = 15.4115
9 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.101559
0.109774
rho
N/A
0
intercept
3.16295
3.15
V
0.600878
0.57
n
1
0.169179
k
237.864
157.5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Concentration
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
10
3.15
3.16
0.24
0.32
-0.129
150
10
3.45
3.4
0.38
0.32
0.542
500
10
3.49
3.57
0.31
0.32
-0.795
1,500
10
3.72
3.68
0.36
0.32
0.381
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
26.293887
5
-42.587775
A2
27.46147
8
-38.922941
A3
26.293887
5
-42.587775
Fitted
25.742228
4
-43.484456
R
19.334386
2
-34.668772
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
16.2542
6
0.01245
Test 2
2.33517
3
0.5058
Test 3
2.33517
3
0.5058
Test 4
1.10332
1
0.2935
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-15. Summary of benchmark concentration (BMC) modeling results for
increased absolute kidney weight in female Sprague-Dawley (S-D) rats
exposed to ethyl tertiary butyl ether (ETBE) by whole-body inhalation for
6 hours/day, 5 days/week for 13 weeks (IPEC. 2008b): benchmark response
(BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMCiord
(ppm)
BMCLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.8
-135.38
6,790
4,046
The Linear model is selected
based on lowest AIC; however,
the BMC is higher than the
maximum concentration.
Exponential (M4)
0.731
-133.76
Error0
0
Exponential (M5)
0.760
-132.29
Error0
0
Hill
0.760
-132.29
Error0
Error0
Powerd
Polynomial
(3 degree)6
Polynomial
(2 degree)'
Linear
0.806
-135.40
6,840
3,978
aConstant variance case presented (BMDS Test 2 p-value = 0.623), selected model in bold; scaled residuals for
selected model for concentrationsO, 150, 500,1,500, and 5,000 ppm were -0.0742,0.0535, -0.578, 0.774, and
-0.176, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
CBMC or BMCL computation failed for this model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
fFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
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Supplemental Information—ETBE
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMC
13:40 05/16 2014
Figure C-13. Plot of mean response by concentration, with fitted curve for
selected model; concentration shown in ppm.
1 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[concentration] = beta_0 + beta_l x concentration
3 A constant variance model is fit
4 Benchmark Concentration Computation.
5 BMR = 10% relative deviation
6 BMC = 6,840.02
7 BMCL at the 95% confidence level = 3,978.09
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.021752
0.0236988
rho
N/A
0
beta_0
1.84346
1.84346
beta_l
0.0000269511
0.0000269511
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Concentration
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
10
1.84
1.84
0.129
0.147
-0.0742
150
10
1.85
1.85
0.18
0.147
0.0535
500
10
1.83
1.86
0.118
0.147
-0.578
1,500
10
1.92
1.88
0.173
0.147
0.774
5,000
10
1.97
1.98
0.16
0.147
-0.176
2 Likelihoods of Interest
Model
Log(likelihood)
# Parameters
AIC
A1
71.192285
6
-130.384569
A2
72.502584
10
-125.005168
A3
71.192285
6
-130.384569
Fitted
70.701239
3
-135.402478
R
67.96809
2
-131.93618
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
9.06899
8
0.3365
Test 2
2.6206
4
0.6232
Test 3
2.6206
4
0.6232
Test 4
0.982091
3
0.8056
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-16. Summary of benchmark concentration (BMC) modeling results for
increased absolute kidney weight in male F344 rats exposed to ethyl tertiary
butyl ether (ETBE) by whole-body inhalation for 6 hours/day, 5 days/week,
for 13 weeks (Medinskv et al.. 1999: US EPA. 1997): benchmark response
(BMR) = 10% relative deviation from the mean
Goodness of fit
BMCiord
(ppm)
BMCLiord
(ppm)
Model3
p-value
AIC
Basis for model selection
Exponential (M2)
Exponential (M3)b
0.184
-129.97
3,107
2,439
The Hill model was selected
based on lowest BMCL.
Exponential (M4)
Exponential (M5)c
0.199
-129.71
1,798
808
Hill
0.224
-129.89
1,667
603
Powerd
Polynomial
(3 degree)6
Polynomial
(2 degree)'
Linear
0.208
-130.22
2,980
2,288
BMC = benchmark concentration; BMCL = benchmark concentration lower confidence level.
aConstant variance case presented (BMDS Test 2 p-value = 0.540), selected model in bold; scaled residuals for
selected model for concentrationsO, 500,1,750, and 5,000 ppm were -0.441, 0.91, -0.635, and 0.166,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
fFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
14:00 05/16 2014
Figure C-14. Plot of mean response by concentration, with fitted curve for
selected model; concentration shown in ppm.
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is:
3 Y[concentration] = intercept + v x concentration11/(kn + concentration11)
4 A constant variance model is fit
5 Benchmark Concentration Computation.
6 BMR = 10% relative deviation
7 BMC = 1,666.92
8 BMCL at the 95% confidence level = 603.113
9 Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
0.0160221
0.0170425
rho
N/A
0
intercept
1.74684
1.73
V
0.521534
0.337
n
1
0.225826
k
3,309.8
1,856.13
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Concentration
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
11
1.73
1.75
0.155
0.127
-0.441
500
11
1.85
1.82
0.137
0.127
0.91
1,750
11
1.9
1.93
0.1
0.127
-0.635
5,000
11
2.07
2.06
0.124
0.127
0.166
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
69.681815
5
-129.36363
A2
70.76062
8
-125.521241
A3
69.681815
5
-129.36363
Fitted
68.943332
4
-129.886663
R
55.026208
2
-106.052416
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
31.4688
6
<0.0001
Test 2
2.15761
3
0.5403
Test 3
2.15761
3
0.5403
Test 4
1.47697
1
0.2242
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Supplemental Information—ETBE
Table C-17. Summary of benchmark concentration (BMC) modeling results for
increased absolute kidney weight in female F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 13 weeks (Medinskv et al.. 1999: US EPA. 1997): benchmark
response (BMR) = 10% relative deviation from the mean
Model3
Goodness of fit
BMCiord
(ppm)
BMCLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0630
-187.67
2,706
2,275
The Exponential (M4) model was
selected as the most
parsimonious model of adequate
fit.
Exponential (M4)
Exponential (M5)c
0.956
-191.20
1,342
816
Hill
N/Ad
-189.20
1,325
741
Power6
Polynomial
(3 degree)'
Polynomial
(2 degree)8
Linear
0.0928
-188.45
2,552
2,111
BMC = benchmark concentration; BMCL = benchmark concentration lower confidence level.
aConstant variance case presented (BMDS Test 2 p-value = 0.428), selected model in bold; scaled residuals for
selected model for concentrations 0, 500,1,750, and 5,000 ppm were -0.0252, 0.043, -0.02385, and 0.004872,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dNo available degrees of freedom to calculate a goodness-of-fit value.
eFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
fFor the Polynomial (3 degree) model, the b3 coefficient estimates was 0 (boundary of parameters space), and
the model reduced to the Polynomial (2 degree) model.
gFor the Polynomial (2 degree) model, the b2 coefficient estimate was 0 (boundary of parameters space), and the
model reduced to the Linear model.
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Supplemental Information—ETBE
14:13 05/16 2014
Figure C-15. Plot of mean response by concentration, with fitted curve for
selected model; concentration shown in ppm.
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[concentration] = a*[c-(c-l)* exp(-b x concentration)]
3 A constant variance model is fit
4 Benchmark Concentration Computation.
5 BMR = 10% relative deviation
6 BMC = 1,341.66
7 BMCL at the 95% confidence level = 815.742
8 Parameter Estimates
Variable
Estimate
Default initial parameter values
Inalpha
-5.63259
-5.63266
rho(S)
N/A
0
a
1.07748
1.02315
b
0.000383798
0.000348471
c
1.24847
1.34027
d
1
1
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Concentration
N
Observed
mean
Estimated
mean
Observed
standard
deviation
Estimated
standard
deviation
Scaled residual
0
10
1.077
1.077
0.069
0.05983
-0.0252
500
11
1.125
1.124
0.048
0.05983
0.043
1,750
11
1.208
1.208
0.076
0.05983
-0.02385
5,000
11
1.306
1.306
0.055
0.05983
0.004872
2 Likelihoods of Interest
Model
Log(likelihood)
No. parameters
AIC
A1
99.60217
5
-189.2043
A2
100.9899
8
-185.9798
A3
99.60217
5
-189.2043
R
75.30605
2
-146.6121
4
99.60063
4
-191.2013
3 Tests of Interest
Test
-2 x log(likelihood ratio)
Test df
p-value
Test 1
51.37
6
<0.0001
Test 2
2.775
3
0.4276
Test 3
2.775
3
0.4276
Test 6a
0.003077
1
0.9558
4 C.1.2. Cancer Endpoints
5 For the Multistage Cancer models, the coefficients were restricted to be nonnegative (beta
6 >0). For each endpoint, Multistage Cancer models were fitted to the data using the maximum
7 likelihood method. Each model was tested for goodness of fit using a chi-square goodness-of-fit test
8 (x2 p-value < 0.053 indicates lack of fit). Other factors were used to assess model fit, such as scaled
9 residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
10 For each endpoint, the BMDL/BMCL estimate (95% lower confidence limit on the
11 BMD/BMC, as estimated by the profile likelihood method) and AIC value were used to select a
3A significance level of 0.05 is used for selecting cancer models because the model family (Multistage) is
selected a priori; see Benchmark Dose Technical Guidance Document (U.S. EPA. 2012).
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Supplemental Information—ETBE
1 best-fit model from among the models exhibiting adequate fit. If the BMDL/BMCL estimates were
2 "sufficiently close," that is, differed by more than threefold, the model selected was the one that
3 yielded the lowest AIC value. If the BMDL/BMCL estimates were not sufficiently close, the lowest
4 BMDL/BMCL was selected as the POD.
5 The incidence of liver tumors in male F344 rats was found to be statistically significantly
6 increased following a 2-year inhalation exposure; hepatocellular adenomas and a single
7 hepatocellular carcinoma in the high-dose group were combined in modeling the data set The data
8 were modeled using two different exposure metrics: administered concentration as ppm as mg/m3.
Table C-18. Cancer endpoints selected for dose-response modeling for ethyl
tertiary butyl ether (ETBE)
Species/sex
endpoint
Concentrations and effect data
Hepatocellular
adenomas and
carcinomas in
male rats;
JPEC (2010b)
Exposure concentration
(ppm)
0
500
1,500
5,000
Exposure concentration
(mg/m3)
0
2,089
6,268
20,893
Incidence/total
0/50
2/50
1/49
10/50
9 Modeling Results
10 Below are tables summarizing the modeling results for the cancer endpoints modeled.
Table C-19. Summary of benchmark concentration (BMC) modeling results for
hepatocellular adenomas and carcinomas in male F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 104 weeks; modeled with concentrations as administered
exposure concentration in ppm (IPEC. 2010b): benchmark response
(BMR) = 10% extra risk
Model3
Goodness of fit
BMCio
(ppm)
BMCLio
(ppm)
Basis for model
selection
p-value
Scaled residuals
AIC
3 degree
0.0991
-0.030,1.382, -0.898, and 0.048
84.961
2,942
1,735
Multistage (1
degree) was
selected based
on lowest AIC.
2 degree
0.264
0.000, 1.284, -1.000, and 0.137
83.093
2,756
1,718
1 degree
0.490
0.000,1.009, -1.144, and 0.309
81.208
2,605
1,703
BMC = benchmark concentration; BMCL = benchmark concentration lower confidence level.
aSelected model in bold.
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
14:57 05/16 2014
Figure C-16. Plot of incidence rate by concentration, with fitted curve for
selected model; concentration shown in ppm.
Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is:
P[response] = background + (1 - background) x [1 - exp(-betal x concentration1-beta2
x concentration2...)]
The parameter betas are restricted to be positive.
Benchmark Concentration Computation.
BMR = 10% extra risk
BMC = 2,604.82
BMCL at the 95% confidence level = 1,703.47
Benchmark concentration upper confidence limit (BMCU) at the 95% confidence level = 4,634.52
Collectively, (1,703.47, 4,634.52) is a 90% two-sided confidence interval for the BMC.
Multistage cancer slope factor = error
Parameter Estimates
Variable
Estimate
Default initial parameter values
Background
0
0
Beta(l)
4.04483 x 10"4
4.38711 x 10"4
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Supplemental Information—ETBE
1 Analysis of Deviance Table
Model
Log(likelihood)
# Parameters
Deviance
Test df
p-value
Full model
-38.2989
4
Fitted model
-39.6042
1
2.61063
3
0.4556
Reduced model
-48.0344
1
19.4711
3
0.0002184
2 AIC = 81.2084
3 Goodness-of-Fit Table
Concentration
Estimated
probability
Expected
Observed
Size
Scaled residual
0
0
0
0
50
0
500
0.02
1.001
2
50
1.009
1,500
0.0589
2.885
1
49
-1.144
5,000
0.1831
9.155
10
50
0.309
4 x2 = 2.42; df = 3; p-value = 0.4898
Table C-20. Summary of benchmark concentration (BMC) modeling results for
hepatocellular adenomas and carcinomas in male F344 rats exposed to ethyl
tertiary butyl ether (ETBE) by whole-body inhalation for 6 hours/day,
5 days/week, for 104 weeks; modeled with concentrations as mg/m3 (IPEC.
2010b): benchmark response (BMR) = 10% extra risk
Model3
Goodness of fit
BMDio
(mg/m3)
BMDLio
(mg/m3)
Basis for model
selection
p-value
Scaled residuals
AIC
3
degree
0.0991
-0.040, 1.382, -0.897, and 0.048
84.961
12,300
7,251
Multistage (1
degree) was
selected based on
lowest AIC
2
degree
0.264
0.000, 1.284, -1.000, and 0.137
83.093
11,514
7,179
1
degree
0.490
0.000,1.009, -1.144, and 0.309
81.209
10,884
7,118
aSelected model in bold.
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
15:02 05/16 2014
Figure C-17. Plot of incidence rate by concentration, with fitted curve for
selected model; concentration shown in mg/m3.
Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is:
P[response] = background + (1 - background) x [1 - exp(-betal x concentration1- beta2
x concentration2...)]
The parameter betas are restricted to be positive.
Benchmark Concentration Computation.
BMR = 10% extra risk
BMC = 10,884.4
BMCL at the 95% confidence level = 7,118.08
BMCU at the 95% confidence level = 19,366.3
Collectively, (7,118.08,19,366.3) is a 90% two-sided confidence interval for the BMC.
Multistage cancer slope factor = error
Parameter Estimates
Variable
Estimate
Default initial parameter values
Background
0
0
Beta(l)
9.6799 x 10"6
1.04989 x 10"4
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Supplemental Information—ETBE
1 Analysis of Deviance Table
Model
Log(likelihood)
# Parameters
Deviance
Test df
p-value
Full model
-38.2989
4
Fitted model
-39.6044
1
2.61098
3
0.4556
Reduced model
-48.0344
1
19.4711
3
0.0002184
2 AIC = 81.2087
3 Goodness-of-Fit Table
Concentration
Estimated
probability
Expected
Observed
Size
Scaled residual
0
0
0
0
50
0
2,089
0.02
1.001
2
50
1.009
6,268
0.0589
2.885
1
49
-1.144
20,893
0.1831
9.155
10
50
0.309
4 x2 = 2.42; df = 3; p-value = 0.4897
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Supplemental Information—ETBE
APPENDIX D. PATHOLOGY CONSULT FOR ETHYL
TERTIARY BUTYL ETHER (ETBE) AND
TERT-BUTANOL
o
November 28. 20IS
To: John Buclier, NTP
From: Kristina Thayer, NCEA-IRIS
Subject: Pathology- consult for ETBE and tBA
Purpose
Hie purpose of this memo is to request a consult for pathology-related issues discussed in the
ethyl tertiary butyl ether (ETBE) and ten-butyl alcohol (tBA) draft IRIS assessments. Tim
request is being conducted under the existing MOU between EPA NCEA and the National
Toxicology Program (NTP) that covers cooperation and communication in the development of
human health toxicological assessments.
Background
The draft IRIS assessments identify kidney effects as a potential human hazard of ETBE and its
metabolite tBA. primarily based on evidence in rats (ETBE and tBA Sections 1.2.1. U.l). EPA
evaluated the evidence, including the role of a2u — globulin (in accordance with EPA guidance
[U.S. EPA. 1991]) and chronic progressive nephropathy (CPN: for which no formal guidance is
available). tBA was determined to induce a2u -globulin mediated nephrotoxicity, however, for
ETBE. although increased hyaline droplets of ct2u -globulin were observed, data were insufficient
to conclude that ETBE induces a2u-globulin nephropathy (only one of the five steps in the
pathological sequence, linear mineralization, was consistently observed). Both chemicals show
dose-related exacerbation of CPN (increased incidence and or severity), as well as lesions that are
not specifically defined as CPN (increased urothelial hyperplasia of the renal pelvis and
suppurative inflammation) but are reported to be associated with late stages of CPN (Frazier et
al.. 2012), Thus. EPA selected urothelial hyperplasia, transitional epithelial hyperplasia of the
renal pelvis as the basis for the reference values for both ETBE and tBA.
The SAB committee reviewing ETBE and tBA was unable to reach a consensus with respect to
how the EPA interpreted the ETBE and tBA databases for noncancer kidney effects. There was
disagreement within the SAB as to whether any noncancer kidney effects for ETBE and tBA
should be considered a hazard relevant to humans. Specifically, the difference in opinion was
related to the extent of confidence in the roles that CPN and or a2u-globulin-based mechanisms
played in the development of the renal effects seen with tBA and ETBE.
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
This document is a draft for review purposes only and does not constitute Agency policy,
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Supplemental Information—ETBE
Charge Questions
111 tins pathology consult. IRIS is seeking additional input on the role that «2u-globulin and CPN
play m the observed kidney toxicity, Please consider the following questions and provide
references, a* applicable, with yoiir responses, Please also comment on any sex-related aspects
that are pertinent to these questions,
• Is the etiology of CPX" in rats known?
• Are urothelial hyperplasia of the renal pelvis and transitional epithelial hyperplasia of the
renal pelvis considered to be the same lesion?
• Suppurative inflammation and urothelial hyperplasia have been reported to be associated
with advanced stages of CPN (Frazier et al 2012; Does XTP agree with this conclusion?
Are the-e lesions also associated with «2u -globulin nephropathy?
• CPX exacerbation has been reported m some chemicals that XT? identified as candidates
for acting via the o2«-giobulin pathway (Travlos et al. 2011 J, A theory has been
proposed that CPX" exacerbation seen m male animals with ETBE and tBA exposure is
caused by a2u-globulin related processes, Please comment on the strength of the above
proposition
• It has been hjpothesized that there is no analog to the CPX" process in the aging human
kidney, Does this position reflect the consensus in the field of pathology?
• Given what is known about the biology of CPX development in rodents, is it plausible a
chemical which exacerbates CPX m rats could also exacerbate disease processes in the
human kidney i e g, diabetic nephropathy, glomerulonepliritis. interstitial nephritis >l?
Refei ences
Frazier KS. Seely JC. Hard GC. Betton G. Burnett R. Xakatsuji S. Xishikawa A.
Durchfeld-Meyer B. Bube A 2012 Proliferative and nonproliferative lesions of the rat and mouse
miliary system, Toxicol Pathol, 40(4 Suppli:14S-S6S
Travlos GS. Hard GC. Betz U. Kisslnu GE 2011, Chionic progressive nephiopathy m male
F344 rats in 9D-day toxicity- studies its occurrence and association with renal tubule tumors m
subsequent 2-year bioassays Toxicol Pathol, 39l 2 i;38 1-9,
U.S. EPA 1991, Alpha-2u-globuim: Association with chemically induced renal toxicity and
neoplasia in the male rat, EPA 625 391 019F.
Attachments
JPEC {Japan Petroleum Energy Center J, ! 2010a} Carcinogenicity test of 2-Ethoxy-2-
methylpropnne m rats iDrinking water study). i Study Xo: 0691 i
JPEC s Japan Petroleum Energy Center i, ! 2010b I C arcinogenieity test of 2-Ethoxy-2-
methyipropane in rats Ihilialation study! i Study Xo: 06S6!
Kiistina Thayei'. Ph.D.
Dnecroi. XCEA-IRIS
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Supplemental Information—ETBE
*- era I theories have been postulated to be the etiology of CPN none have been
retogn z«-i) v the absolute cause of CPN Factors wh'ch have been suggested to be associated \v:th
the etiology of CPN include genetics, increased glomerular permeabihty and dysfunction due to
hyperfiltration and functional overload, high renal protein levels, arid hemodynamic changes. All ot
these may influence the progression of CPN but do not appear to initiate renal CPN disease {Baylis,
1984; Barthold, 1998; Abrass, 2000; Hard and Khan, 2004). CPN is a spontaneous and complex
degenerative/regenerative disease process influenced by age (incidence and severity increases
vCth age), sen (males affected more than females), and strain (in order of highest to lowest CPN
incidence- Sprague-Oawley -^Fischer 344 -^Wistar rats). It can be modified by d'et (increased
piote n and high caloric intake), hormones (testosterone, estrogen), and many other factors (Seely
et ->! , 20191
Are urothelial hyperplasia of the renal pelvis and transitional epithelial hypeiplasia of the renal
pelvis considered to be the same lesion?
Yes, the older terminology of "transitional epithelium hyperplasia, renal pelvis'' s be ng updated
and replaced by the newer terminology of "'urothelial hyperplasia, renal pelv:s', Urothel urn is
recognized as the collect terminology of the ep.theiiurn lining the tenal pelvis, ureter, urinary
bladder and a portion of the urethra (Fraziet and Seely, 2013). However, in advanced stage j ot CPN
a type of ep'thelial proliferation/hypeiplasia may be ob.eived along the epithelial lining ot the
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Supplemental Information—ETBE
Page 2 - Kristine Thayer, Ph.D.
renal papilla wh'ch n some older stud'es was designated as "urothelial hyperplas'Y'. Recently, the
epithelial lining of the renal papilla has been unequivocally demonstrated to represent a type of
epithelium different from the urothelium lining the renal pelv's. The difference between
urothelium (uroplakin pos;t've) and the ep:thelium lining the renal papilla (uroplahn negative! was
confirmed by irrmunostaining for uroplakin (a d'st'nct cell marker for urotheLum) (Souza et al ,
2018).
3, Suppurat ve 'nflamrnation and urothelial hyperplasia have been reported to be assoc'ated with
advanced stages of CRN (Frazier et al,, 2012). Does NTP agree with this conclus'on? Are these
lesions also assoc'ated w;th a2u-globul'n nephropathy?
Renal inflarr(nation is not uncommon in the laboratory rat and can be observed throughout all
portions of the kidney, Witlvn the pelvis, 'nflamrnation tends to result in a reactive hyperplasia of
the urothelium (Seely et at,, 2018). Most cases of suppurative inflammation and urothelial
hyperplasia are observed as spontaneous changes of undetermined origin, Interstitial mononuclear
cell "nfiltrates are commonly observed in advanced stages of CPN (Frazier and Seely, 2018),
However, suppurative inflammation and urothelial hyperplas:a are typically unrelated to CPN or, at
most, occasionally noted as an uncommon secondary change to CPN, Therefore, CPN does not
directly result in suppurative inflammation or urothelal hyperplas'a of the renal pelv's in its
advanced stages. Cases of suppurat've "nflamrnation and urothel-al hyperplasia are more I'kely to
be associated with the presence of renal pelv:c mineralization, pelvic calculi, or from an ascending
bacterial infection ot pyelonephritis (Seely et al., 2018), furthermore, mineral'zation has been
reported to be associated w'th an increased inc'dence and severity of spontaneous 'nflamrnation
and urothelial hyperplasia in the renal pelvis of female rats (Tomonar" et al , 2016), In addition,
there is no information that appears to support that suppurative inflammation and pelv'c urothelial
hyperplasia are directly assoc'ated with the spectrum of morphological changes associated v/th
ct2u-globulin nephropathy (Frazier et al,, 2012; Frazier and Seely, 2018).
4, CPN exacerbation has been reported in some cherrr'cals that NTP identified as candidates for acting
via the a2u-globu!in pathway |Travlos et al,, 2011). A theory has been proposed that CPN
exacerbation seen in male animals with ETBE and tBA exposure is caused by a2-globul;n related
processes. Please comment on the strength of the above proposition
According to the IARC Scientific Publication No. 147 (1999), chemicals which cause ci2u-gIobulin
nephropathy are often associated with an accelerated onset and severity (exacerbation) of the
cortical changes typical of chronic progressive nephropathy seen in older male rats iftlden et al.,
1984; Svvenberg and Lehman-McKeenan, 1999; Travlos et al,, 2011; Frazier et al,, 2012), However,
studies on 2-ethoxy-2 methylpropene (ethyl tertiary butyl ether; inhalation and drinking water
studies) confirmed the presence of exacerbated CPN in both male and female rats at the highest
dose levels (Japan Industrial Safety and Health Association/Japan Bioassay Research Center, 2010*
2010b), Because of ''urothelial hyperplasia" and linear pelvic (papillary! m'neralizat'on noted in the
male rats from these studies, it was proposed that u2u-globulin nephropathy contributed to the
exacerbation of CPN in the males although no pathogenesis of the exacerbated CPN in rem,ales was
given. Additionally, :n these studies, "urothelial hyperplasia' was apparently and according to its
description more likely to represent a proliferation of the papillary lining epithel'um and not
representative of true "urothelial hyperplasia". This proliferative epithelial finding is often observed
This document is a draft for review purposes only and does not constitute Agency policy.
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Page 3 - Kristina Thayer, Ph.D.
as part of advanced cases of rat CPN and has no similarity to any human renal papillary find'ng
fSeely et al,, 2018; Souza et al., 2018), Long term exposures to methvl tertiary -butyl ether also
resulted in an «2u-globulin nephropathy and exacerbated CPN in both male and female rats
(Cruzan et al,, 2007) The etiology of exacerbated CPN in females is not known since a2u-globulin
nephropathy is regarded as a male only condition. Therefore, although ct2u-globulin nephropathy
may account for cases of chemically exacerbated CPN, other undetermined factors contr buting to
CPN exacerbation cannot be discounted (Doi et al., 2007),
5. It has been hypothesized that there is no analog to the CPN process in the aging human kidney.
Does this posVon reflect the consensus in the field of pathology,
Yes, the publication by Hard, Johnson, and Cohen makes a very strong case that the renal
development, b'ological behavior, and morphological spectrum of CPN have no analog in the
human kidney and that CPN is a distinct entity in the rat, (Hard et al., 2009), Overall, CPN has
prominent protein filed dilated tubules, no vascular changes, no immunological or auto'mmune
bas s, and little inflammation which distinguishes CPN from most human nephropathies (Hard et
al., 2009). There appears to be nothing :n the literature that counters this assumption,
6, Given what is known about the biology of CPN development in rodents, is :t plausible a chemical
which exacerbates CPN in rats could also exacerbate disease processes in the human kidney (e.g.
diabetic nephropathy, glomerulonephritis, inter$t't>al nephritis)?
The etiology of CPN is unknown and represents a complex disease process in rats. Given the fact
that there is no definitive pathogenesis for this multifactorial disease process, it cannot be fully
ruled out that chemicals which exacerbate CPN in rats may have the potential to exacerbate
disease processes in the human kidney.
Please let me know if you have additional questions or wish further clarification of any of these
responses.
Sincerely,
John Bucher, Ph.D.
National Toxicology Program,
NIEH5
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References:
Abrass CK. 2000. The nature of chronic progressVe nephropathy in aging rats. Adv Renal
Replacement Therapy 7(1): 4-10.
Alden CL, Kanerva RL, Ridder G, Stone LC. 1984, The pathogenes's of the nephrotoxicity of volatile
hydrocarbons in the male. In: Renal Effects of Petroleum Hydrocarbons, Advances in Modern
Environmental Toxicology, Vol 7, MA Mehlrran, CP Hemstreet, JJ Thorpe, NK Weaver teds),
Princeton Sci Pub, Princeton, NJ, pp, 107-120.
Barthold SW. 1998. Chronic progressive nephropathy, rat. In: Urinary System. 2"" ed. TC Jones, GC
Hard, U Mohr (eels), pp.228-233, Sprnger-Verlag, Berlin.
Baylis C. 1994. Age-dependent glomerular damage in the rat: dissociation between glomerular
injury and both glomerular hypertension and hypertrophy. Male gender as a primary risk factor, J
Clin Invest 94:1823-1829.
Capen CC, et al. (eels) 1999. Spec'es Differences in Thyroid, Kidney and Urinary Bladder
Carcinogenesis. IARC Scient'fic Publications No. 147. international Agency for Research on Cancer,
Lyon, France,
Cruzan 6, Borghoff SJ, de Peyster A, Hard GC, McClain M, McGregor DB,Thomas MG, 2007. Reg
Toxicol Pharmacol 47: 156-165.
Doi AM, Hill G, Seely J, Hailey JR, Kissling G Bucher JR. 2007. a2.»-globulin nephropathy and renal
tumors in National Toxicology Program studies. Toxicol Pathol 35:533-540.
Frazier KS, Seely JC, Hard GC, Betton G, Burnet R, Nakatsuj' S, Nbhikaiva A, Durchfeld-Meyer B,
Bube A. 2012, Proliferative and nonproliferative lesions of the rat and mouse urinary system,
Toxicol Pathol 40 (4 Suppl): 14S-86S.
Frazier KS, Seely JC. 2018, Urinary system. In: Toxicologic Pathology: Nonclin'cal Safety
Assessment. 2nc' eel, PS Sahota, JA Popp, PR Bouchard, JF Hardisty, C Gopinath {eds}, CRC
press/Taylor and Francis, Boca Raton, pp. 569-638,
Hard GC, Khan KN. 2004, A contemporary overview of chrorrc progressive nephropathy 'n the
laboratory rat and its significance for human rsl. assessment. Tox'col Pathol 32:171-180.
Hard GC, Johnson KJ, Cohen SM. 2009, A comparison of rat chronic progressive nephropathy with
human renal disease-implications for human risk assessment, Crit Rev Toxicol, 39(4):332-346.
Japan Industrial Safety and Health Association/ Japan Bioassay Research Center, 2010",
Carcinogen'city Test of 2-ethoxy-2-methypropene 'n Rat (Dr'nking Water Study (Study 0691).
Japan Industrial Safety and Health Association/Japan Bioassay Research Center. 2010",
Carcinogenicity Study of 2-ethyi-2-methylpropene in F344 Rats (Study 0686).
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Peter CP, Burek JD, van Zwieten MJ, 1986. Spontaneous nephropathies in rats, Toxicol Pathol
14(1):91-100.
Seeiy it, Hard GC, Biankenship B. 2018. Kidney. In: doorman's Pathology of the Rat. AWSuttie, JR
Leininger, AE Bradley (eels), pp. 125-186, Elsevier, Academic Press.
Souza N, Hard G, Arnold L, Foster K, Pennington K, Cohen S, 2018, Epithelium lin:ng rat renal
papilla: nomenclature and association with chronic progressive nephropathy (CPN), Toxicol Pathol
46(3):266-272.
Swenoerg JA, Lehman-fvlcKeenan LP, l'1??. ok Urinary globulin-associated nephropathy as a
mechanism of renal tubule cell carc'nogenesls in male rats, 199S. In: Species Differences in
Thyroid, Kidney, and Urinary Bladder Carcinogenesis, CC Capen, E Dybing E, JM Rice, JD Eilbourn
feds'). IARC Scientific Publications No. 147, IARC, Lyon pp. 95-118.
Tomonari Y, Kurotaki T, Sato i, Doi T, Kokoshima H, Kan no T, Tsuchitani M, Seely JC 2016.
Spontaneous age-related lesions of the kidney forn'ces in Sprague-Dawley rats Toxicol Pathol 44:
226-232.
Travlos GS, Hard GC, Betz U, Kissing GE, 2011. Chronic progressive nephropathy in male F344 rats
in 90-day toxicity studies: its occurrence and associat on with renal tubule tumors in subsequent 2-
year bioassays. Toxicol Pathol 39:381-339,
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APPENDIX E. SUMMARY OF EXTERNAL
PEER-REVIEW COMMENTS AND EPA'S
DISPOSITION
The Toxicological Review of ethyl tertiary butyl ether (ETBE), dated June 2017, underwent
a formal external peer review in accordance with U.S. Environmental Protection Agency (EPA)
guidance on peer review (U.S. EPA, 2015). This peer review was conducted by the Chemical
Assessment Advisory Committee (CAAC) augmented for review of the draft Integrated Risk
Information System (IRIS) ETBE assessment (SAB-CAAC ETBE panel) of EPA's Science Advisory
Board (SAB). An external peer review workshop was held on August 15-17, 2017. Public
teleconferences of the SAB-CAAC ETBE panel were held on July 11, 2017, March 22, 2018, March
27, 2018, and June 6, 2018. The Chartered SAB held a public meeting on September 26, 2018 to
conduct a quality review of the draft SAB-CAAC peer review report4. The final report of the SAB
was released on February 27, 2019.
The SAB-CAAC was tasked with providing feedback in response to charge questions that
addressed scientific issues related to the hazard identification and dose-response assessment of
ETBE. Key recommendations of the SAB5 and EPA's responses to these recommendations,
organized by charge question, follow. Editorial changes and factual corrections offered by SAB were
incorporated in the document as appropriate and are not discussed further.
1. Literature Search/Study Selection and Evaluation
Charge Question 1. The section on Literature Search Strategy / Study Selection and Evaluation
describes the process for identifying and selecting pertinent studies. Please comment on
whether the literature search strategy, study selection considerations, including exclusion
criteria, and study evaluation considerations, are appropriate and clearly described. Please
identify additional peer-reviewed studies that the assessment should consider.
Key Recommendation: The SAB recommended EPA should provide clarification on the rationales
for several decisions that impacted how the literature search was conducted. This includes (1) the
4 During the quality review by the Chartered SAB, 2 of the 44 members provided dissenting comments related to
the cancer weight of evidence descriptors and the quantitative cancer risk estimates for ETBE and tBA. These
comments were included as an appendix to the final SAB report and are summarized and addressed following the
disposition of the SAB-CAAC recommendations below.
5 The SAB provided tiered recommendations: Tier 1 (key recommendations), Tier 2 (suggestions), and Tier 3 (future
considerations).
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rationale for the selection of some synonyms of ETBE as key search words and not others; (2) the
rationale for imposing limitations on sources in the first stage of the scientific literature search (i.e.,
PubMed, Web of Science); and (3) the rationale for limiting the search for additional citations to
only some of the publications available in peer-reviewed literature and secondary sources, but not
others.
Response: The literature search was developed and executed in consultation with information
specialists and librarians through EPA's Health and Environmental Research Online (HERO)
database. The process includes developing, testing, and implementing a comprehensive literature
search strategy in an iterative and collaborative manner. Responses to the above enumerated SAB
concerns follow. (1) The most common synonyms and trade names were used as the keywords in
the literature search. This included the preferred IUPAC name of 2-Methoxy-2-methylpropane. (2)
PubMed, Web of Science, and Toxline are the core sources that IRIS uses for published studies. Past
experience has demonstrated searching of PubMed, Web of Science and Toxline generally provides
sufficient coverage for literature pertinent to human health assessments. The TSCATS2 database
was included to capture submissions of health and safety data submitted to the EPA either as
required or voluntarily under certain sections of TSCA. Based on the attributes of the chemical,
along with input from HERO, EPA did not include supplemental databases (e.g., databases for
pesticides, U.S. Department of Agriculture -related compounds or inhalation values). (3) To ensure
no key studies were missed, a manual search of citations was performed on published reviews and
studies identified from public comments, as well as reviews previously conducted by other
international and federal agencies. Table LS-2 lists the approach and sources used in the manual
searching of citations.
Key Recommendation: The SAB recommended that the EPA address why not all databases were
updated through December 2016.
Response: All databases were updated through December 2016 in the external peer review draft
assessment; text has been edited to ensure clarity. In addition, a literature update following SAB
review was conducted through July 2019. Information on this literature update has been added to
the Literature Search Section of the document.
Key Recommendation: The SAB recommended that the EPA address the discrepancy in the number
of health effects studies reported in Table LS-1 and in the text.
Response: The number of studies identified has been corrected.
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Key Recommendation: The SAB recommended that the EPA clarify why ecological/non-mammalian
studies were apparently excluded from any consideration (despite the footnote in Table LS-3).
Response: Regarding the ETBE search strategy, ecological studies and non-mammalian studies
were not "excluded" but were instead considered supplemental data. The footnote in Table LS-3
was revised to be more transparent.
2. Chemical Properties and Toxicokinetics
Charge Question 2a. Chemical properties. Is the information on chemical properties accurate?
Key Recommendation: The SAB offered several specific recommendations for improvement of the
chemical properties table generally focused on increasing confidence and transparency in the
values presented. The SAB also recommended the use of a template focusing on the chemical
properties most relevant to the chemical and the assessment Several recommendations focused on
a preference for the citation of chemical properties from primary sources, for vetting the data in
cases in which more than one value is published, and for presenting rationales for the selected
values.
Response: In response to SAB comments, EPA has revised the ETBE chemical properties table
(Table 1-1) to present average experimental and predicted chemical properties from high quality
databases as curated by EPA's CompTox Chemicals Dashboard
(https://comptox.epa.gov/dashboard). The CompTox Chemicals Dashboard aggregates and
presents both experimental and predicted chemical property data, with links to the source and/or
model data [for details see Williams etal. f20171]. The experimental data are sourced from publicly
available databases as well PHYSPROP downloadable files fMansouri et al.. 20161. Predicted
chemical property data are obtained from EPISuite, OPERA models (Mansouri et al.. 2016)
(Mansouri et 2016), NICEATM models (Zang etal.. 2017). and the Toxicity Estimation Software Tool
(TEST) Models (https://www.epa.gov/chemical-research/toxicity-estimation-software-tool-test).
A key benefit of this aggregation of chemical properties over reporting an individual
measurement is a more robust estimate than is possible from an individual study, with each study
reporting measurements that are expected to have some degree of error.
Charge Question 2b. Toxicokinetic modeling. Section B.1.5 of Appendix B in the Supplemental
Information describes the application and modification of a physiologically-based
toxicokinetic model of ETBE in rats fBorghoffetal.. 20161. Is use of the model appropriate and
clearly described, including assumptions and uncertainties? Are there additional peer-
reviewed studies that should be considered for modeling?
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Key Recommendation: The SAB recommended that the EPA revise the model code to describe
metabolism as a function of the free liver concentration, CVL, and that metabolic parameters (e.g.,
Km or first order rate constants) be re-estimated. Metabolism based upon total liver concentration,
CL, is not scientifically correct.
Response: Model code has been revised to describe metabolism as a function of the free liver
concentration and the metabolic parameters have been re-estimated. The revised final code is
available on HERO fU.S. EPA. 20161.
Key Recommendation: The SAB recommended that the overall presentation of the PBPK modeling
should be cohesive, clear, and transparent, and should provide essential information, assumptions,
results and conclusions. The SAB recommended that the text of the PBPK model evaluation report
(U.S. EPA. 2017) be included in the Supplemental Information, in which case it would benefit from
adding a conclusions section.
Response: PBPK model evaluation for the IRIS assessments of ETBE and tert-butanol has been
added to the Supplemental Information to the Toxicological Review (See Appendix B.1.7).
Charge Question 2c. Choice of dose metric. Is the rate of ETBE metabolism an appropriate
choice for the dose metric?
Key Recommendation: The SAB recommended not implementing route extrapolation for the oral
cancer dose-response analysis for ETBE. Therefore, there was no need for the Agency to select a
dose metric.
Response: Route-to-route extrapolation is not implemented, in accordance with SAB
recommendation. Consequently, a dose metric wasn't warranted. See the response to Charge
Question 4d for further details.
3. Hazard Identification and Dose-Response Assessment
Charge Question 3a. Noncancer kidney toxicity (Sections 1.2.1,1.3.1). The draft assessment
identifies kidney effects as a potential human hazard of ETBE. EPA evaluated the evidence,
including the role of a2u-globulin and chronic progressive nephropathy, in accordance with
EPA guidance (U.S. EPA. 19911. Please comment on whether this conclusion is scientifically
supported and clearly described.
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Key Recommendation: The SAB was unable to reach consensus on whether noncancer kidney
effects should be considered a hazard relevant to humans based on the presented information in
the assessment The SAB recommended EPA strengthen the justification regarding the decision to
consider the observed kidney effects as a hazard relevant to humans.
Response: In response to concerns regarding the human relevance of the observed kidney
endpoints following ETBE exposure in rats, further expert consultation was requested from the
National Toxicology Program (see Appendix D). With this additional expert consultation, the
assessment has been revised to strengthen the justification regarding the human relevance of the
observed kidney effects. Briefly, dose-related increases in kidney effects (increased kidney weight,
increased severity of chronic progressive nephropathy, CPN) were observed in both male and
female rats treated with ETBE. While ETBE binds to alpha 2u-globulin and meets some but
not all the criteria in the EPA and International Agency for Research on Cancer (IARC)
alpha 2u-globulin framework [Capen et at f 19991: U.S. EPA (19911: see Section 1.2.1], U.S.
EPA (19911 noted that" [i]f a compound induces a^-globulin accumulation in hyaline droplets,
the associated nephropathy in male rats is not an appropriate endpointto determine noncancer
(systemic) effects potentially occuring in humans" (Section XVIII, p. 89). However, as a2u-globulin
nephropathy is strictly a male rat phenomenon, dose-related kidney effects in female rats are not
confounded by a2U-globulin nephropathy. With respect to CPN, this condition has no known
analogue in the aging human kidney (N ; ird et al. 20091 and its etiology is unknown
fNIEHS. 2019: Frazier et al. 2012: Hard and Khan. 2004: Peter et al. 19861. However, many of the
same lesions observed in CPN (e.g., thickening of tubule basement membranes, tubule atrophy,
tubule dilation, and glomerular sclerosis) are also observed in chronic kidney disease in humans
(Lusco et al. 2016: Zoia et al. 2015: Frazier et al. 2012: Abrass. 2000). As summarized in the NTP
pathology consultation, given that there is no definitive pathogenesis for CPN, it cannot be ruled out
that a chemical which exacerbates CPN in rats could also exacerbate existing disease processes in
the human kidney fNIEHS. 20191. Therefore, increased incidence of kidney effects with ETBE
exposure in the female rat (including increased kidney weight and increased severity of CPN) are
considered relevant to humans.
Key Recommendation: The SAB acknowledged the role of a2u-globulin in ETBE-induced
nephropathy in male rats was thoroughly considered according to the EPA 1991 criteria, however,
the SAB recommended also applying the criteria published by IARC in 1999 fCapen etal.. 19991.
Response: The U.S. EPA f!9911 and IARC criteria fCapen etal.. 19991 are very similar with the IARC
criteria having specific criteria pertaining to lack of genotoxicity of parent compound/metabolite
and male rat specificity for nephropathy and renal tumorigenicity whereas the EPA framework
considers these data as supplemental information (see Part 4, XVIIB. Additional Information Useful
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for the Analysis). The Assessment was revised in Section 1.2.1 (see Mode of Action Analysis for
Kidney Effects) to provide additional discussion of the IARC criteria not explicitly required in the
EPA 1991 a2u-globulin framework.
Charge Question 3b. Noncancer toxicity at other sites (Sections 1.2.2,1.2.3,1.2.4,1.2.6,1.3.1).
The draft assessment presents conclusions for noncancer toxicity at other sites that were not
used as the basis for deriving noncancer oral reference dose or inhalation reference
concentration purposes. Please comment on whether these conclusions are scientifically
supported and clearly described. If there are publicly available studies to associate other
health outcomes with ETBE exposure, please identify them and outline the rationale for
including them in the assessment
• Liver effects: Suggestive evidence
• Developmental toxicity: Inadequate evidence
• Male and female reproductive toxicity: Inadequate evidence
Key Recommendation: The SAB had suggestions for better describing the overall evidence for male
reproductive toxicity as "minimal effects at otherwise toxic dose levels," rather than "inadequate
evidence," since the SAB concludes there is an adequate amount of evidence that shows minimal
effects, at least in populations with normal ALDH2 function. The SAB also recommended female
reproductive toxicity be characterized as "no effects even at otherwise toxic dose levels," rather
than "inadequate evidence," since the SAB concludes there is an adequate amount of evidence,
which shows minimal effects.
Response: The description of male and female reproductive effects in section 1.2.3 has been
revised to be responsive to the SAB's suggested language. Regarding the overall evidence for male
reproductive toxicity, although minimal effects were observed at otherwise toxic dose levels, the
available evidence is considered insufficient to identify male reproductive effects as a potential
human hazard of ETBE exposure and male reproductive effects are not carried forward as a hazard.
While the ALDH2 knock out data suggest a potential sensitive subpopulation for male reproductive
effects, this evidence is considered preliminary (see discussion of these findings in Section 1.2.3,
and response to Key Recommendation below).
Regarding the overall evidence for female reproductive effects, although minimal effects
were observed at otherwise toxic dose levels, the available evidence is considered insufficient to
identify female reproductive effects as a potential human hazard of ETBE exposure and therefore,
female reproductive effects are not carried forward as a hazard.
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Key Recommendation: The SAB recommended that male reproductive effects in genetically
susceptible mice be clearly acknowledged in the assessment rather than treated as inconsistent
results. The SAB also suggested that an RfC be calculated for the male reproductive effects of ETBE
in ALDH2 KO mice, as this may be the most sensitive target for a sensitive subgroup.
Response: Based on the available mechanistic study indicating potentially increased susceptibility
for reproductive effects in male mice with impaired acetaldehyde metabolism, text in Sections 1.2.3
and 1.3.3 has been revised to clarify that effects observed in the studies in ALDH2 KO mice may be
indicative of increased susceptibility in a small percentage of the US population with inactive
ALDH2 variations. However, these findings are considered to be preliminary due to the small
sample size (n=5), single species, and the unconvincing magnitude of many of the statistically
significant effects, including the observation that the heterozygotes exhibited more robust changes
than the knockout mice. Thus, EPA did not calculate an RfC for these effects.
Charge Question 3 c. Oral reference dose for noncancer outcomes. Section 2.1 presents an oral
reference dose of 5x10~1 mg/kg-day, based on urothelial hyperplasia in male rats (Suzuki et
al.. 2012: IPEC. 2010a). Please comment on whether this value is scientifically supported and
its derivation clearly described. If an alternative data set or approach would be more
appropriate, please outline how such data might be used or how the approach might be
developed.
Key Recommendation: The SAB recommended that EPA carefully examine the question of the
validity and applicability of the endpoints chosen and analyzed for the oral RfD, including the
potential for CPN to serve as the mechanism of the kidney effects.
Response: CPN is not a defined mechanism or a more general MOA, rather it is a group of age-
related lesions observed in rats and of unknown etiology. However, many of the lesions observed
in CPN are also observed in chronic kidney disease in humans fLusco etal.. 2016: Zoiaetal.. 2015:
Frazier etal.. 2012: Abrass. 20001. In response to comments regarding the human relevance of the
observed kidney endpoints following ETBE exposure in rats (e.g., related to an alpha-2u-globulin
mechanism or exacerbation of CPN), further expert consultation was requested from the National
Toxicology Program [fNIEHS. 20191: see also Appendix D], With this additional expert
consultation, EPA evaluated the validity and applicability of the observed kidney effects and revised
the assessment to strengthen the discussion regarding the human relevance of the various kidney
effects. See Integration of Kidney Effects in Section 1.2.1 of the Toxicological Review. See also
response to Charge Question 3a.
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Key Recommendation: SAB recommended that the tables within this section include units for
completeness and interpretability and that EPA consider a more integrated presentation of the
current text, tables and graph so as to facilitate better tracking of information without much page-
flipping.
Response: Units have been added to the tables where missing, however, endpoints which display
changes as "% change relative to control" are unitless. A more integrated presentation of text, table
and figures is being implemented in future IRIS assessment templates.
Charge Question 3d. Inhalation reference concentration for noncancer outcomes. Section 2.2
presents an inhalation reference concentration of 9x10° mg/m3, based on urothelial
hyperplasia in male rats (Saito et al.. 20131. Please comment on whether this value is
scientifically supported and its derivation clearly described. If an alternative data set or
approach would be more appropriate, please outline how such data might be used or the
approach might be developed.
Key Recommendation: SAB recommended that EPA provide statistical analysis to make clear the
rationale for including or excluding studies. The SAB elaborated in the body of the final report that
"there does not seem to be any reporting of statistical analysis of individual studies (trend tests or
pair wise significance tests, and other statistical tests determined to be appropriate), and this
omission hampers consideration of the appropriateness of inclusion and use of studies."
The SAB also recommended the EPA provide statistical analysis to help elucidate differences in
response based on sex, further observing that sex differences in response appear more marked for
inhalation than for oral exposures. The SAB suggested that an evaluation of possible reasons for
this (including mere statistical fluctuation which, if responsible, would suggest averaging endpoint
values across sexes) would be informative.
Response: As discussed in the Preamble of this assessment, data analysis is part of the evaluation
of study quality of the available literature. Statistical analysis of data (including pairwise tests and
trend tests) is predominantly performed and reported by the study authors. Data informative for
EPA evaluation of organ/system findings are reported in the evidence tables and data arrays in
Section 1 (Hazard Identification) with positive statistical findings highlighted (for example, see
Table 1-2, Figure 1-4 and 1-5 for kidney histological effects). When additional data analysis is
deemed informative and/or is missing from the publication, further statistical tests will be
conducted by EPA and noted in the assessment. For example see "average severity of CPN" from
1PEC f2010a. denoted in Table 1-2. However, it is important to note that while endpoints
with statistically significant findings (especially those with significant dose response trends) inform
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the selection of endpoints for the derivation of candidate values, the biological significance and
coherence of an outcome is more important flJ.S. EPA. 20021. Decisions regarding the rationale for
endpoint and study inclusion for dose response assessment for ETBE are discussed in detail in
Section 1.3.1 and in Section 2.1.1 and 2.2.1 of the Toxicological Review.
Regarding the SAB recommendation for additional statistical analysis to inform averaging kidney
endpoints across sex, pooling kidney endpoints across sexes is not considered appropriate due to
biological considerations, specifically, the apparent increased susceptibility of male rats to ETBE-
induced kidney effects, potentially related to a2U-globulin binding with ETBE in male, but not female
rats. Additional consideration of the human relevance of the kidney effects observed in male and
female rats has been added to the assessment (see response to Charge Question 3a). Therefore, to
avoid the uncertainty and confounding by a2u-globulin-related processes in male rats, the
assessment has been revised to rely on data sets for kidney toxicity from female rats. Please see the
revised text in Section 1.3.1, 2.1.4, and Section 2.2.8.
Charge Question 4a. Cancer modes-of-action in the liver. As described in section 1.2.2, the draft
assessment evaluated the roles of the receptor pathways PPARa, PXR, and CAR in ETBE
tumorigenesis in male rats. The analysis, conducted in accordance with EPA's cancer
guidelines (U.S. EPA. 2005). considered the liver tumors in male rats to be relevant to human
hazard identification. Please comment on whether this conclusion is scientifically supported.
Key Recommendation: The SAB recommended that EPA should clarify the evidence needed to
conclude that a PPARa, CAR, and/or PXR MOA is operative and to indicate that liver tumors may
not be relevant to humans. The SAB suggested that examples, if provided, would be helpful to
illustrate the types of studies/information needed to satisfy each criterion, and that EPA should
revisit the evaluation of information available for ETBE using these criteria.
Response: Text has been added to clarify additional data gaps (see Section 1.2.2). Briefly, several
gaps in the receptor mediated effects data were explicitly noted such as evidence in only one
species, lack of any studies in receptor knock-out or humanized mice, lack of dose response
concordance between receptor mediated gene changes and tumors, and lack of any receptor
mediated data outside of the 1 and 2 week time points, which preclude establishing temporal
associations.
Key Recommendation: The SAB recommended that EPA may specifically want to reconsider
statements about transient hypertrophy.
This document is a draft for review purposes only and does not constitute Agency policy.
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Response: Statements regarding transient hypertrophy have been revised and additional
information on the observation of related endpoints, such as increased liver weight, has been added
for context.
Key Recommendation: The SAB recommended that EPA should revise Table 1-13 and
accompanying narrative to be more descriptive regarding availability of information for each MOA
and indicate whether studies relevant to the MOA exist, and where results are positive or negative,
instead of saying "no positive studies identified".
Response: The table and narrative text have been clarified to indicate the categories under which
no pertinent studies were identified.
Key Recommendation: The SAB commented that while acetaldehyde was proposed as a strong
candidate MOA for male rat liver tumors in the assessment, the plausibility of this MOA was not
well explored. The SAB recommended that evidence for this MOA should be developed and
presented more thoroughly; or, alternatively, encouraged the Agency to reduce emphasis on this
MOA in the final assessment.
Response: The data for an acetaldehyde based MOA for the observed liver tumors has been
evaluated in the Toxicological Review in Section 1.2.2. Although the available evidence suggests a
potential role for acetaldehyde in the increased liver tumor response observed in male rats exposed
to ETBE, the data are inadequate to conclude that ETBE induces liver tumors via acetaldehyde-
mediated mutagenicity. Therefore, emphasis on this MOA and its effect on the assessment
conclusions has been reduced throughout the document.
Charge Question 4b. Cancer characterization. As described in sections 1.2.1,1.2.2,1.2.5 and
1.3.2, and in accordance with EPA's cancer guidelines (U.S. EPA. 20051. the draft assessment
concludes that there is suggestive evidence of carcinogenic potential for ETBE by all routes of
exposure, based on liver tumors in male F344 rats via inhalation and on promotion of liver,
colon, thyroid, forestomach, and urinary bladder tumors in male rats via oral exposure. Please
comment on whether the decision to include 2-stage initiation-promotion studies in the human cancer
hazard characterization is sufficiently justified and if the amount of emphasis placed on the initiation
promotion data in the cancer hazard characterization is scientifically supported6. Please comment
on whether the "suggestive evidence" cancer descriptor is scientifically supported for all routes
of exposure. If another cancer descriptor should be selected, please outline how it might be
supported.
6 This unbolded segment of the charge question was added by the SAB-CAAC
This document is a draft for review purposes only and does not constitute Agency policy.
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Key Recommendation: The SAB recommended the use of the descriptor "Inadequate Information"
for oral ETBE, and "Suggestive Evidence" for inhaled ETBE.
Response: The EPA agrees with the SAB recommendation and has implemented these descriptors
in the revised assessment
Key Recommendation: The SAB recommended that the EPA not use the initiation-promotion assay
as key evidence to support a conclusion of carcinogenic potential.
Response: Section 1.2.5 of the Toxicological Review has been revised to clarify that initiation-
promotion assays are included only as supplemental studies informing carcinogenicity. Regarding
the animal database for carcinogenicity, EPA considers chronic bioassays as key evidence, and
other types of studies (including initiation promotion studies, co-carcinogenicity studies, studies in
genetically modified animals, etc) as supplemental lines of information which can aid in the
interpretation of more standard toxicological evidence, especially regarding potential modes of
action {U.S. EPA, 2005, 86237}.
Key Recommendation: The SAB recommended that the EPA explain within the assessment that the
assigned cancer classifications are an EPA Cancer Guidelines policy-based decision.
Response: "Cancer classifications" or cancer weight of evidence descriptors, are used as part of the
hazard narrative to express the conclusion regarding the weight of evidence for carcinogenic
hazard potential. Choosing a descriptor is a matter of scientific judgement, not policy, guided by
examples and considerations discussed in EPA's Cancer Guidelines fU.S. EPA. 20051.
Charge Question 4c. Cancer toxicity values. Section 3 of EPA's cancer guidelines (U.S. EPA.
20051 states: "When there is suggestive evidence, the Agency generally would not attempt a
dose-response assessment, as the data usually would not support one. However, when the
evidence includes a well-conducted study, quantitative analyses may be useful for some
purposes, for example, providing a sense of the magnitude and uncertainty of potential risks,
ranking potential hazards, or setting research priorities. In each case, the rationale for the
quantitative analysis is explained, considering the uncertainty in the data and the suggestive
nature of the weight of evidence."
Please comment on whether Sections 2.3 and 2.4 of the draft assessment adequately explain
the rationale for including a quantitative analysis given the "suggestive evidence" descriptor.
This document is a draft for review purposes only and does not constitute Agency policy.
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Also comment whether the Saito et al. (2013) study is a suitable basis for this quantitative
analysis.
Key Recommendation: The SAB recommended that the EPA refrain from conducting a quantitative
analysis for ETBE carcinogenicity or explain the limitations of the analysis and clearly state that the
intended purpose is to simply provide some sense of the magnitude of potential risks.
Response: No quantitative analysis of cancer risk is carried out for oral ETBE exposure. For
inhalation exposure, additional text has been added to the assessment to discuss the strengths and
limitations of a quantitative analysis of the tumor data and to clarify that the purpose is to provide a
sense of the magnitude of a potential cancer risk (this is useful because when no information on the
potential magnitude of risk is provided, it generally implies zero risk). See Section 1.3.2. and
Section 2.4.1. The assessment also notes the increased uncertainty in this risk estimate because of
the suggestive nature of the tumorigenic response (U.S. EPA, 2005a).
Charge Question 4d. Oral slope factor for cancer. Section 2.3 presents an oral slope factor of
lxlO-3 per mg/kg-day, based on liver tumors in male rats by inhalation fSaito et al.. 20131.
converted for oral exposure using a toxicokinetic model (Borahoff et al.. 20161. Please
comment on whether this value is scientifically supported and its derivation clearly described.
If an alternative approach would be more appropriate, please outline how it might be
developed.
Key Recommendation: The SAB recommended that since the only available ETBE inhalation cancer
bioassay (Saito etal.. 2013) (TPEC. 2010b) is not suitable for developing an oral cancer slope factor,
the EPA should not derive an oral slope factor by route extrapolation absent
pharmacokinetic/pharmacodynamic modeling that demonstrates consistency between the oral and
inhalation study results. The SAB indicated the following concerns about the use of Saito for route
to route extrapolation for developing an oral slope factor: (1) Oral studies did not demonstrate
cancer (2) EPA analysis indicated that a consistent dose response relationship could not be
observed when comparing across oral and inhalation exposures on the basis of any internal dose
measures.
Response: In response to the SAB recommendation, EPA is not carrying out a route to route
extrapolation for the derivation of the oral slope factor. Furthermore, there is uncertainty as to
whether the liver tumors observed in male rats following inhalation would be reasonably expected
following oral exposure as one high confidence oral cancer bioassay (Suzuki etal.. 2012: TPEC.
2010a). and a lower confidence chronic oral cancer bioassay (Maltoni et al.. 1999) did not observe
elevated liver tumors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Charge Question 4e. Inhalation unit risk for cancer. Section 2.4 presents an inhalation unit risk
of 8 xlO-5 per mg/m3, based on liver tumors in male rats by inhalation (Saito et al.. 20131.
Please comment on whether this value is scientifically supported and its derivation clearly
described. If an alternative approach would be more appropriate, please outline how it might
be developed.
Key Recommendation: The SAB had no specific recommendations; the SAB-CAAC did not reach
consensus on this charge question. Some members supported use of the fSaito etal.. 20131 study
for dose-response assessment recognizing it as a well-designed, well-conducted, and well-reported
study and also noting the liver metabolism of ETBE to acetaldehyde, a genotoxic carcinogen. Other
members believed the ETBE concentration which induced liver tumors to be excessively high, with
significantly elevated tumors only in one sex, at one dose, and questioned whether modeling a
single positive concentration would produce a meaningful inhalation unit risk.
Response: Text has been added to the assessment to more clearly denote the strengths and the
uncertainties in the data used to derive the inhalation unit risk (see Section 1.3.2, Biological
Considerations for Dose-Response). Briefly, while liver adenomas were primarily observed at the
highest dose in male rats, three liver adenomas were also observed at the lower two
concentrations, resulting in a significant positive exposure-response trend (p < 0.001 with Peto's
test). The Saito et al. (2013) study was considered appropriate for the basis of a quantitative
cancer estimate as it is a well-designed, conducted and reported study which included histological
examinations for tumors in many different tissues, contained three exposure levels and controls,
contained adequate numbers of animals per dose group (~50/sex/group), treated animals for up to
2 years, and included detailed reporting of methods and results. Decreased body weight gain and
survival was noted in the high dose males and females; however, the study authors did not detect
changes to the animals' general condition (e.g., abnormal behavior or clinical signs) associated with
ETBE. Similar decreases in body weight were observed in male (75% of control) and female
animals (78% of control), although significantly increased liver tumors were only observed in male
rats. Given the lack of overt toxicity and no alterations in toxicokinetics, the ETBE concentrations
were not considered to be excessively high. Additionally, text describing the suitability of the study,
it's utilization in deriving a cancer risk estimate, and the characterization of a cancer value
considering the suggestive nature of the cancer potential are further discussed in Section 2.4.
Charge Question 5. Question on Susceptible Populations and Lifestages
Section 1.3.3 identifies individuals with diminished ALDH2 activity as a susceptible population
due to an increased internal dose of acetaldehyde, a primary metabolite of ETBE. Please
comment on whether this conclusion is scientifically supported and clearly described. If there
This document is a draft for review purposes only and does not constitute Agency policy.
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are publicly available studies to identify other susceptible populations or lifestages, please
identify them and outline their impact on the conclusions.
Key Recommendation: The SAB recommends that the Agency clearly describe the uncertainty
between oral exposure and other routes of exposure in the ETBE assessment and provide relevant
positions with respect to differences in expected outcomes.
Response: Text has been added to this section to highlight the uncertainty pertaining to the fact that
the available database to inform early life susceptibility of ETBE is limited to the oral route of
exposure.
Key Recommendation: The SAB recommended the Agency identify susceptible populations and
incorporate information about them into the ETBE assessment to improve the scientific concepts of
the assessment Specifically, the SAB recommended discussing individuals with noncoding region
variants in adlh2, which could potentially affect gene expression, as well as discussing individuals
with other variants in alcohol metabolism who may be affected by ETBE exposure.
Response: Increased discussion of these additional potentially susceptible populations has been
added to the document.
Key Recommendation: The SAB recommends that information regarding life stages should be
included in the assessment
Response: Discussion of data informing potential early life susceptibility to ETBE has been added to
this section.
Charge Question 6. Question on the Executive Summary
The Executive Summary is intended to provide a concise synopsis of the key findings and
conclusions for a broad range of audiences. Please comment on whether the executive
summary clearly and appropriately presents the major conclusions of the draft assessment.
Key Recommendation: The SAB advises that it will be important for the final Executive Summary to
highlight the consequences of alternative choices for the final assessment, especially when these
hinge on decisions made about the interpretation and relevance of key toxicity endpoints that have
been contested.
This document is a draft for review purposes only and does not constitute Agency policy.
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Response: Text has been added to the Executive Summary to more clearly highlight the context
around the interpretation and relevance of key endpoints such as the human relevance of the
observed kidney effects (see Key Issues Addressed in Assessment).
Comments from two members of the Chartered SAB during the QA Review of the SAAB CAAC
Peer Review Report
The Chartered SAB is tasked with conducting quality reviews of draft SAB reports to
determine if they are ready for transmittal to the Administrator, reviewing whether the charge
questions were adequately addressed by the CAAC, whether the report has technical errors or
omissions, if the report is clear and logical, and if the CAAC recommendations in the report are
supported by the body of the draft report. During this quality review of the draft SAB-CAAC report
on the Draft IRIS assessments of ETBE and tert-butanol, two members of the chartered SAB (44
total members) disagreed with the CAAC regarding the recommendation for the cancer weight of
evidence descriptors for ETBE and tBA. These two members provided additional comments which
were included as Appendix C of the Final SAB report. A summary and response to their comments,
as they pertain to ETBE, are included below.
Comment: Two members of the chartered SAB disagreed with the SAB-CAAC's support of EPA's
cancer weight of evidence descriptor of "suggestive evidence" for ETBE. They stated ETBE should
be characterized as "insufficient evidence" (presumably analogous to EPA's cancer weight of
evidence descriptor for "inadequate evidence") because liver tumors were observed only in male
rats at the highest exposure concentration in the TPEC (2010b) inhalation bioassay, a concentration
they characterize as beyond the maximum tolerated dose (MTD) due to a 25% reduction in body
weight. In addition, two chronic oral bioassays were negative for liver tumors.
Response: The SAB-CAAC agreed with the cancer weight of evidence descriptor of "suggestive
evidence" (See Charge Question 4b) for the inhalation route of exposure, as the database was
consistent with this descriptor as illustrated in the Cancer Guidelines, based on the occurances of
tumors in one sex of one species. Briefly, a statistically significant increase in liver tumors was
observed in male rats exposed to ETBE by inhalation (primarily, but not exclusively at the high
dose) with the incidence of combined adenomas and carcinomas of 0/50, 2/50,1/50 and 10/50 at
0, 2,090, 6,270, 20,900 mg/m3, resulting in a statistically significant, positive exposure-response
trend (Peto's testp < 0.001).
Regarding the assertion that the highest inhalation dose in the Saito et al. (2013) study
exceeded the MTD, EPA's 2005 Cancer Guidelines discuss the determination of an "excessively high
dose" and describe the process as one of expert judgment which requires that "...adequate data
demonstrate that the effects are solely the result of excessive toxicity rather than carcinogenicity of
This document is a draft for review purposes only and does not constitute Agency policy.
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the tested agent" In the case of the Saito et al. (2013) inhalation study, the study authors did not
report any overt toxicity or altered toxicokinetics at the high dose. In addition, the high-dose
female rats had a similar reduction in body weight (22%) and no liver tumors were observed (see
discussion in Section 1.2.2). Discussion regarding the cancer descriptor for the inhalation route of
exposure, the rationale for deriving the inhalation unit risk (including consideration of potential
excessive high dose), and the characterization of the cancer risk estimate can be found in Sections
1.3.2 and 2.4.1, and in response to comments under Charge Questions 4b, 4c, and 4e.
With regard to the comments on the cancer descriptor and the oral cancer studies, the SAB-
CAAC recommended EPA's cancer weight of evidence descriptor of "inadequate evidence of
carcinogenic potential" for the oral route of exposure. EPA agreed and revised the assessment
accordingly. Thus, a cancer risk estimate for the oral route was not derived. See Sections 1.3.2 and
2.3, and responses under Charge Questions 4b and 4d.
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX F. QUALITY ASSURANCE (QA) FOR THE
IRIS TOXICOLOGICAL REVIEW OF ETHYL TERTIARY
BUTYL ETHER
This assessment was prepared under the auspices of the U.S. Environmental Protection
Agency's (EPA's) Integrated Risk Information System (IRIS) Program. The IRIS Program is housed
within the Office of Research and Development (ORD) in the Center for Public Health and
Environmental Assessment (CPHEA). EPA has an agency-wide quality assurance policy and that
policy is outlined in the EPA Quality Manual for Environmental Programs (see CIO 2105-P-01-0)
and follows the specifications outlined in EPA Order CIO 2105.0.
As required by CIO 2105.0, ORD maintains a Quality Management Program, which is
documented in an internal Quality Management Plan (QMP). The latest version was developed in
2013 and was developed using Guidance for Developing Quality Systems for Environmental
Programs fOA/G-11. An NCEA/CPHEA-specific QMP was also developed in 2013 as an appendix to
the ORD QMP. Quality Assurance for products developed within CPHEA is managed under the ORD
QMP and applicable appendices.
The IRIS Toxicological Review of Ethyl-Tertiary Butyl Ether has been designated as
Influential Scientific Information (ISI) and is classified as QA Category A. Category A designations
require reporting of all critical QA activities, including audits. The development of IRIS assessments
is done through a seven-step process. Documentation of this process is available on the IRIS
website: https://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#process.
Specific management of quality assurance within the IRIS Program is documented in a
Programmatic Quality Assurance Project Plan (PQAPP). APQAPP was developed using the EPA
Guidance for Quality Assurance Project Plans fOA/G-51. and the latest approved version is dated
March 2020. All IRIS assessments follow the IRIS PQAPP and all assessment leads and team
members are required to receive QA training on the IRIS PQAPP. During assessment development,
additional QAPPs may be applied for quality assurance management They include:
Title
Document Number
Date
Program Quality Assurance
Project Plan (PQAPP) for the
Integrated Risk Information
System (IRIS) Program
L-CPAD-0030729-QP-1-3
March 2020
This document is a draft for review purposes only and does not constitute Agency policy.
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An Umbrella Quality Assurance
Project Plan (QAPP] for PBPK
Models
B-003740-QP-1-0
Feb 2018
Quality Assurance Project Plan
[QAPP] for Enhancements to
Benchmark Dose Software
(BMDS]
B-003742-QP-1-0
Apr 2019
Contractor QAPP 1
B-IRISD-0030538
Contractor QAPP 2
B-IRISD-0030622
During assessment development, this project underwent one quality audit during
assessment development including:
Date
Type of audit
Major findings
Actions taken
June 2018
Technical System Audit
None
None
During Step 3 of the IRIS Process, IRIS toxicological review was subjected to external
reviews by other federal agency partners including the Executive Offices of the White House.
Comments during these IRIS Process steps are available in the Docket EPA-HO-QRD-2009-0229 on
regulations.gov.
During Step 4 of assessment development, the IRIS Toxicological Review of [Ethyl-Tertiary-
Butyl Ether] underwent public commentfrom May 16, 2016 to July 15, 2016. Following this
comment period, the toxicological review underwent external peer review by SAB on June 2017.
The peer review report is available on the
fhttps://yosemite.epa.gov/sab/sabproductnsf/0/8e4436d62dalfd2d85257e38006a3131!OpenDo
cument&TableRow=2.3#2.]. All public and peer review comments are available in the Docket EPA-
HO-ORD-2009-0229 on regulations.gov.
Prior to release (Step 7 of the IRIS Process), the final toxicological review is submitted to
management and QA clearance. During this step the CPHEA QA Director and QA Managers review
the project QA documentation and ensure that EPA QA requirements have been met.
This document is a draft for review purposes only and does not constitute Agency policy.
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REFERENCES FOR APPENDICES
Abrass. CK. (2000). The nature of chronic progressive nephropathy in aging rats [Review],
Advances in Renal Replacement Therapy 7: 4-10. http://dx.doi.org/10.lQ16/S1073-
4449C00170001-X
Amber g. A: Rosner. E: Dekant. W. (1999). Biotransformation and kinetics of excretion of methyl-
tert-butyl ether in rats and humans. Toxicol Sci 51: 1-8.
Amberg. A: Rosner. E: Dekant. W. (2000). Biotransformation and kinetics of excretion of ethyl tert-
butyl ether in rats and humans. Toxicol Sci 53: 194-201.
http ://dx. doi. or g/10.109 3 /toxsci/5 3.2.194
Andersen. ME. (1991). Physiological modelling of organic compounds. Ann Occup Hyg 35: 309-321.
http ://dx. doi.org/10.1093 /annhvg/3 5.3.309
ARCO (ARCO Chemical Company). (1983). Toxicologist's report on metabolism and
pharmacokinetics of radiolabeled TBA 534 tertiary butyl alcohol with cover letter dated
03/24/1994. (8EHQ86940000263). Newton Square, PA.
ATSDR (Agency for Toxic Substances and Disease Registry). (1996). Toxicological profile for
methyl-tert-butyl ether [ATSDR Tox Profile], Atlanta, GA: U.S. Department of Health and
Human Services, Public Health Service. http: //www.atsdr.cdc.gov/ToxProfiles/tp91.pdf
Banton. MI: Peachee. VL: White. KL: Padgett. EL. (2011). Oral subchronic immunotoxicity study of
ethyl tertiary butyl ether in the rat. J Immunotoxicol 8: 298-304.
http: / /dx.doi.org/10.3109/1547691 X.2011.598480
Bernauer. U: Amberg. A: Scheutzow. D: Dekant. W. (1998). Biotransformation of 12C- and 2-13C-
labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats:
Identification of metabolites in urine by 13C nuclear magnetic resonance and gas
chromatography/mass spectrometry. Chem Res Toxicol 11: 651-658.
http ://dx. doi. or g/10.10 21 /tx9 7 0 215v
Blancato. IN: Evans. MY: Power. FW: Caldwell. TC. (2007). Development and use of PBPK modeling
and the impact of metabolism on variability in dose metrics for the risk assessment of
methyl tertiary butyl ether (MTBE). J Environ Prot Sci 1: 29-51.
Bond. TA: Medinskv. MA: Wolf. DC: Cattlev. R: Farris. G: Wong. B: Tanszen. D: Turner. Ml: Sumner.
SCI. (1996). Ethyl tertiary butyl ether (ETBE): ninety-day vapor inhalation toxicity study in
CD-l(R) mice. Bond, JA; Medinsky, MA; Wolf, DC; Cattley, R; Farris, G; Wong B; Janszen, D;
Turner, MJ; Sumner, SCJ.
Borghoff. ST. (1996). Ethyl tertiary-butyl ether: Pilot/methods development pharmacokinetic study
in male F-344 rats & male cd-1 mice after single nose-only inhalation exposure, w/cvr ltr
dated 7/29/96. (EPA/OTS Doc #86960000579). Chemical Industry Institute of Toxicology
(CUT).
Borghoff. ST: Asgharian. B. (1996). Ethyl tertiary-butyl ether (ETBE): Pharmacokinetic study in male
and female CD-I mice after single inhalation exposure and male and female F-344 rats after
single and repeated inhalation exposure. (CUT Protocol 95026). La Palma, CA: ARCO
Chemical Company.
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Borghoff. ST: Murphy. IE: Medinskv. MA. (1996). Development of a physiologically based
pharmacokinetic model for methyl tertiary-butyl ether and tertiary-butanol in male Fischer-
344 rats. Toxicol Sci 30: 264-275. http://dx.doi.org/10.1006/faatl996.0Q64
Borghoff. ST: Parkinson. H: 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. http://dx.doi.org/10.1016/i.tox.2010.06.003
Borghoff. ST: Prescott. IS: Tanszen. DB: Wong. BA: Everitt. II. (2001). alpha2u-Globulin nephropathy,
renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-
344 rats. Toxicol Sci 61: 176-186. http://dx.doi.Org/10.1093/toxsci/61.l.176
Borghoff. ST: Ring. C: Banton. MI: Leavens. TL. (2016). Physiologically based pharmacokinetic model
for ethyl tertiary-butyl ether and tertiary-butyl alcohol in rats: Contribution of binding to
a2u-globulin in male rats and high-exposure nonlinear kinetics to toxicity and cancer
outcomes. J Appl Toxicol 37: 621-640. http://dx.doi.org/10.1002 /iat3412
Brown. RP: Delp. MP: Lindstedt. SL: Rhomberg. LR: Beliles. RP. (1997). Physiological parameter
values for physiologically based pharmacokinetic models. Toxicol Ind Health 13: 407-484.
http://dx.doi.Org/10.1177/074823379701300401
Capen. CC: Dvbing. E: Rice. TM: Wilbourn. ID. (1999). Species differences in thyroid, kidney and
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