*>EPA
EPA/635/R-16/184b
Public Comment Draft
www.epa.gov/iris
Toxicological Review of Ethyl Tertiary Butyl Ether
(CASRN 637-92-3]
Supplemental Information
August 2016
NOTICE
This document is a Public Comment Draft. This information is distributed solely for the purpose of
pre-dissemination 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
National Center for 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 pre-dissemination 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 of recommendation for use.
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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-2
B.l. TOXICOKINETICS B-2
B.l.l. Absorption B-2
B.l.2. Distribution B-7
B.1.3. Metabolism B-8
B.1.4. Elimination B-16
B.1.5. Physiologically Based Pharmacokinetic Models B-21
B.2. OTHER PERTINENT TOXICITY INFORMATION B-44
B.2.1. Other Toxicological Effects B-44
B.2.2. Genotoxicity Studies B-60
B.3. SUPPLEMENTAL ORGAN WEIGHT DATA B-65
B.3.1. Relative Kidney Weight Data B-65
B.3.2. Absolute Liver Weight Data B-68
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.l.l. Non-cancer Endpoints C-l
C.l.2. Cancer Endpoints C-70
APPENDIX D. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND EPA'S
DISPOSITION D-l
REFERENCES FOR APPENDICES R-l
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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]ETBE inhalation exposure B-5
Table B-2. Plasma radioactivity after a single oral or intravenous dose of [14C]ETBE to male
Crl:CD(SD) rats B-6
Table B-3. Blood:tissue partition coefficients for ETBE and ferf-butanol B-7
Table B-4. Unchanged 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 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-14
Table B-6. Elimination of [14C]ETBE-derived radioactivity from rats and mice within 96 hours
following a single 6-hour inhalation exposure B-18
Table B-7. PBPK model physiologic parameters and partition coefficients B-26
Table B-8. Rate constants determined by optimization of the model with experimental data.B-28
Table B-9. Evidence pertaining to kidney weight effects in animals exposed to ETBE B-44
Table B-10. Evidence pertaining to body weight effects in animals exposed to ETBE B-49
Table B-ll. Evidence pertaining to adrenal effects in animals exposed to ETBE B-52
Table B-12. Evidence pertaining to immune effects in animals exposed to ETBE B-53
Table B-13. Evidence pertaining to mortality in animals exposed to ETBE B-57
Table B-14. Summary of genotoxicity (both in vitro and in vivo) studies of ETBE B-63
Table B-15. Evidence pertaining to relative kidney weight effects in animals exposed to ETBEB-65
Table B-16. Evidence pertaining to absolute liver weight effects in animals exposed to ETBE .B-68
Table C-l. Non-cancer endpoints selected for dose-response modeling for ETBE C-3
Table C-2. Summary of BMD modeling results for urothelial hyperplasia of the renal pelvis in
male F344 rats exposed to ETBE in drinking water for 104 weeks (JPEC, 2010a)
modeled with doses as mg/kg-day (calculated by study authors); BMR = 10% extra
risk C-8
Table C-3. Summary of BMD modeling results for increased absolute kidney weight in male S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC, 2008d);
BMR = 10% relative deviation from the mean C-ll
Table C-4. Summary of BMD modeling results for increased relative kidney weight in male S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC, 2008d);
BMR = 10% relative deviation from the mean C-14
Table C-5. Summary of BMD modeling results for increased absolute kidney weight in female S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC, 2008d);
BMR = 10% relative deviation from the mean C-15
Table C-6. Summary of BMD modeling results for increased relative kidney weight in female S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC, 2008d);
BMR = 10% relative deviation from the mean C-18
Table C-7. Summary of BMD modeling results for increased absolute kidney weight in P0 male S-
D rats exposed to ETBE by daily gavage for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups (Gaoua, 2004a); BMR = 10% relative
deviation from the mean C-21
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Supplemental Information—ETBE
Table C-8. Summary of BMD modeling results for increased relative kidney weight in PO male S-D
rats exposed to ETBE by daily gavage for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups (Gaoua, 2004a); BMR = 10% relative
deviation from the mean C-24
Table C-9. Summary of BMD modeling results for increased absolute kidney weight in PO female
S-D rats exposed to ETBE by daily gavage for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups (Gaoua, 2004a); BMR = 10% relative deviation
from the mean C-27
Table C-10. Summary of BMD modeling results for increased relative kidney weight in P0 female
S-D rats exposed to ETBE by daily gavage for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups (Gaoua, 2004a); BMR = 10% relative
deviation from the mean C-29
Table C-ll. Summary of BMD modeling results for absolute kidney weight in F1 male Sprague-
Dawley rats exposed to ETBE by gavage in a two-generation study (Gaoua, 2004b);
BMR = 10% relative deviation from the mean C-30
Table C-12. Summary of BMD modeling results for relative kidney weight in F1 male Sprague-
Dawley rats exposed to ETBE by gavage in a two-generation study (Gaoua, 2004b);
BMR = 10% relative deviation C-32
Table C-13. Summary of BMD modeling results for absolute kidney weight in F1 female Sprague-
Dawley rats exposed to ETBE by gavage in a two-generation study (Gaoua, 2004b);
BMR = 10% relative deviation C-32
Table C-14. Summary of BMD modeling results for relative kidney weight in F1 female Sprague-
Dawley rats exposed to ETBE by gavage in a two-generation study (Gaoua, 2004b);
BMR = 10% relative deviation C-35
Table C-15. Summary of BMD modeling results for increased absolute kidney weight in P0 male
S-D rats exposed to ETBE by daily gavage for 16 weeks beginning 10 weeks prior to
mating (Fujii et al., 2010); BMR = 10% relative deviation from the mean C-36
Table C-16. BMD modeling results for increased relative kidney weight in P0 male S-D rats
exposed to ETBE by daily gavage for 16 weeks beginning 10 weeks prior to mating
(Fujii etal., 2010); BMR = 10% relative deviation from the mean C-39
Table C-17. Summary of BMD modeling results for increased absolute kidney weight in P0
female S-D rats exposed to ETBE by daily gavage for 17 weeks beginning 10 weeks
prior to mating until lactation day 21 (Fujii etal., 2010); BMR = 10% relative
deviation from the mean C-42
Table C-18. Summary of BMD modeling results for increased relative kidney weight in P0 female
S-D rats exposed to ETBE by daily gavage for 17 weeks beginning 10 weeks prior to
mating until lactation day 21 (Fujii etal., 2010); BMR = 10% relative deviation from
the mean C-45
Table C-19. Summary of BMD modeling results for urothelial hyperplasia of the renal pelvis in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for 104
wk (JPEC, 2010b); BMR = 10% extra risk C-47
Table C-20. Summary of BMD modeling results for increased absolute kidney weight in male S-D
rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk (JPEC,
2008b); BMR = 10% relative deviation from the mean C-49
Table C-21. Summary of BMD modeling results for increased relative kidney weight in male S-D
rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk (JPEC,
2008b); BMR = 10% relative deviation from the mean C-51
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Supplemental Information—ETBE
Table C-22. Summary of BMD modeling results for increased absolute kidney weight in female S-
D rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk (JPEC,
2008b); BMR = 10% relative deviation from the mean C-54
Table C-23. Summary of BMD modeling results for increased relative kidney weight in female S-
D rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk (JPEC,
2008b); BMR = 10% relative deviation from the mean C-57
Table C-24. Summary of BMD modeling results for increased absolute kidney weight in male
F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for 13 wk
(Medinsky et al., 1999; Bond et al., 1996b); BMR = 10% relative deviation from the
mean C-60
Table C-25. Summary of BMD modeling results for increased absolute kidney weight in female
F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for 13 wk
(Medinsky et al., 1999; Bond et al., 1996b); BMR = 10% relative deviation from the
mean C-63
Table C-26. Summary of derivation of oral PODs derived from route-to-route extrapolation from
inhalation exposures C-66
Table C-27. Summary of derivation of inhalation PODs derived from route-to-route extrapolation
from oral exposures C-68
Table C-28. Cancer endpoints selected for dose-response modeling for ETBE C-70
Table C-29. Summary of BMD modeling results for hepatocellular adenomas and carcinomas in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5d/wk, for 104
wk; modeled with doses as administered exposure concentration in ppm (JPEC,
2010b); BMR = 10% extra risk C-71
Table C-30. Summary of BMD modeling results for hepatocellular adenomas and carcinomas in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5d/wk, for 104
wk; modeled with doses as mg/m3 (JPEC, 2010b); BMR = 10% extra risk C-73
Table C-31. Summary of BMD modeling results for hepatocellular adenomas and carcinomas in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5d/wk, for 104
wk; modeled with PBPK doses as ETBE metabolized, mg/hr (JPEC, 2010b); BMR =
10% extra risk C-75
FIGURES
Figure B-l. Proposed metabolism of ETBE B-9
Figure B-2. Comparison of the ferf-butanol portions of existing MTBE models with ferf-butanol
blood concentrations from i.v. exposure by Poet and Borghoff (1997) B-23
Figure B-3. Schematic of the PBPK model for ETBE and its major metabolite ferf-butanol in
rats B-24
Figure B-4. Comparison of EPA model predictions with measured ferf-butanol blood
concentrations for i.v., inhalation and oral gavage exposure to ferf-butanol B-27
Figure B-5. Comparison of EPA model predictions with measured amounts of ferf-butanol after
oral gavage of ETBE B-29
Figure B-6. Comparison of EPA model predictions with measured amounts after a 4-hour
inhalation exposure to 4 and 40 ppm ETBE B-31
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Supplemental Information—ETBE
Figure B
Figure B
¦7. Comparison of EPA model predictions with measured amounts of A) ETBE and B)
ferf-butanol in exhaled breath after a 6-hour inhalation exposure to 5,000 ppm
ETBE B-32
8. Comparison of EPA model predictions with measured amounts of ferf-butanol in
blood after repeated inhalation exposure to ferf-butanol, 5 mg/kg-day ETBE oral
gavage for up to 14 days in male rats B-33
Figure B-9. Comparison of EPA model predictions with measured amounts of ferf-butanol in
blood after 5 mg/kg-day ETBE oral gavage for up to 14 days in male rats B-34
Figure B-10. Comparisons of relative kidney weights in male (A-C) and female rats (D-F) following
ETBE (black) or ferf-butanol (white) inhalation (square) or oral (circle) exposure with
internal dose metrics calculated from the PBPK model B-38
Figure B-ll. Comparisons of urothelial hyperplasia in male rats following ETBE (black) or ferf-
butanol (white) inhalation (square) or oral (circle) exposure with internal dose
metrics calculated from the PBPK model B-39
Figure B-12. Comparisons of marked or severe CPN in male and female rats following ETBE
(black) or ferf-butanol (white) inhalation (square) or oral (circle) exposure with
internal dose metrics calculated from the PBPK model B-40
Figure B-13. Comparisons of kidney tumors in male rats following 2 year oral or inhalation
exposure to ETBE or ferf-butanol with internal dose metrics calculated from the PBPK
model B-41
Figure B-14. Comparisons of liver tumors in male rats following 2 year oral or inhalation
exposure to ETBE or ferf-butanol with internal dose metrics calculated from the PBPK
model B-42
Figure B
Figure B
Figure C
Figure C
Figure C
Figure C
Figure C
Figure C
Figure C
Figure C
Figure C
Figure C
¦15. Exposure-response array of body weight effects following oral exposure to ETBE...B-
58
16. Exposure-response array of body weight effects following inhalation exposure to
ETBE B-59
1. Plot of incidence rate by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-9
2. Plot of mean response by dose, w
in mg/kg-day
3. Plot of mean response by dose, w
in mg/kg-day
4. Plot of mean response by dose, w
in mg/kg-day
5. Plot of mean response by dose, w
in mg/kg-day
6. Plot of mean response by dose, w
in mg/kg-day
7. Plot of mean response by dose, w
in mg/kg-day
¦8. Plot of mean response by dose, w
in mg/kg-day
¦9. Plot of mean response by dose, w
th fitted curve for selected mode
th fitted curve for selected mode
th fitted curve for selected mode
th fitted curve for selected mode
th fitted curve for selected mode
th fitted curve for selected mode
th fitted curve for selected mode
; dose shown
.C-12
; dose shown
.C-16
; dose shown
.C-19
; dose shown
.C-22
; dose shown
.C-25
dose shown
.C-27
; dose shown
.C-30
; dose shown
th fitted curve for selected model,
in mg/kg-day C-33
10. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-37
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Figure C-ll. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-40
Figure C-12. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-42
Figure C-13. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-45
Figure C-14. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-47
Figure C-15. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-49
Figure C-16. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-52
Figure C-17. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-55
Figure C-18. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-58
Figure C-19. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-61
Figure C-20. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-64
Figure C-21. Plot of incidence rate by dose, with fitted curve for selected model; dose shown
in ppm C-71
Figure C-22. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-73
Figure C-23. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/hr C-75
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Supplemental Information—ETBE
ABBREVIATIONS
AIC Akaike's information criterion
ARCO ARCO Chemical Company
AUC area under the curve
BMD benchmark dose
BMDL benchmark dose lower confidence
limit
BMDS Benchmark Dose Software
BMDU benchmark dose upper confidence
limit
BMR benchmark response
CASRN Chemical Abstracts Service Registry
Number
CUT Chemical Industry Institute of
Toxicology
CPN chronic progressive nephropathy
CYP450 cytochrome P450
DNA deoxyribonucleic acid
EPA U.S. Environmental Protection
Agency
GI gastrointestinal
HERO Health and Environmental Research
Online
HGPRT hypoxanthine-guanine
phosphoribosyl transferase
HIBA 2-hydroxyisobutyrate
HT heterogeneous
KO knockout
JPEC Japan Petroleum Energy Center
MN micronucleus, micronucleated
MNNCE mature normochromatic erythrocyte
population
MNPCE micronucleated polychromatic
erythrocyte
MNRETs micronucleated reticulocytes
MTBE methyl tertiary butyl ether
MPD 2-methyl-l, 2-propane diol
NADPH nicotinamide adenine dinucleotide
phosphate
PBPK physiologically-based
pharmacokinetic
PCE polychromatic erythrocytes
POD point of departure
RET reticulocyte
SD standard deviation
SRBC sheep red blood cell
TAME tertiary amyl methyl ether
TBA tert-butyl alcohol, tert-butanol
WT wild type
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1 APPENDIX A. OTHER AGENCY AND
2 INTERNATIONAL ASSESSMENTS
3 Table A-l. Health assessments and regulatory limits by other national and
4 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-day
Inhalation noncancer tolerable concentration in air: 1.9 mg/m3
American Conference of
Governmental Industrial
Hygienists
Threshold limit value: 20.9 mg/m3
5
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APPENDIX B. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-REPONSE
ANALYSIS
B.l. TOXICOKINETICS
B.l.l. Absorption
B.l.1.1. Human Studies
Most of the available human data on the uptake of ETBE were obtained from volunteers.
Nihlen etal. (19981 exposed eight healthy male volunteers (average age: 29 years) to 5, 25, and
50 ppm (20.9,104, and 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 by 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, 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 prior to 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 a graphical form by the authors).
Acetone levels were highly variable and appeared to reflect not only ETBE exposure, but the
physical activity of the volunteers. Nihlen etal. f 19981 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 [iM at 210,104, and 20.9 mg/m3, respectively. By 6 hours, the concentrations of ETBE had fallen
to very low levels (<1 [iM) even after the 210-mg/m3 exposure. Based on blood AUC values for
ETBE, the authors calculated two types of respiratory uptake: net respiratory uptake =
(concentration in inhaled air - concentration in exhaled air) multiplied by the pulmonary
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ventilation; and 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.
Amberg etal. (2000) 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 respectively. 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 until the end of the experiment 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. 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 et al. (1998).
These estimates of retained dose are lower than those reported during light exercise (Nihlen etal..
1998).
B.l.1.2. Animal Studies
Amberg etal. (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.
Immediately after the 4-hour exposure period, the authors reported that blood levels of ETBE were
lower in the rats than in humans, although exact values were not reported. The authors estimated
that the rats received doses of 20.5 and2.3 [imol atthe 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 (IPEC. 2008e. f). Seven-week-old
Crl:CD(SD) male rats (4/dose group) were administered either a single oral dose of 5, 50, or
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Supplemental Information—ETBE
400 mg/kg [14C]ETBE via gavage or 5 mg/kg-day [14C]ETBE daily for 14 days. In the single-dose
study by 1PEC f2008f). plasma levels were compared to 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 ofETBE/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 (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. 2008el. 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 Cmaxto 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 rats fSun and Beskitt. 1995b). 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 (1995a, b) 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 elimination in urine, feces, as volatile in expired air and as exhaled
CO2. Exhaled organic volatiles were trapped in charcoal filters. Exhaled CO2 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,
samples were collected at fewer time points; generally, at full-day intervals up to 96 hours. Animals
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were euthanized either immediately after exposure or after being removed from the metabolic
cages, and blood and kidneys were collected. 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 saturate absorption
mechanisms. Additional support for saturation of absorption is presented in Table B-l,
demonstrating the elimination of radiolabel from rats and mice in these studies (Sun and Beskitt.
1995a. hi
Table B-l. Radioactivity in blood and kidney of rats and blood and liver of
mice, following 6 hours of [14C]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.
bln mg [14C]ETBE equivalents per gram blood.
cln mg [14C]ETBE equivalents.
Sources: Sun and Beskitt (1995a) and Sun and Beskitt (1995b).
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 (1992) have proposed an equation to calculate the dermal permeability coefficient, Kp:
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Supplemental Information—ETBE
1 log Kp (cm/sec) = -6.3 + 0.71 x log Kow - 0.0061 x (molecular weight)
2 Using the log Kow [identified as Koct in Potts RO f 19921] values for ETBE (0.95-2.2) (Drogos
3 and Diaz. 2001) and converting cm/second values to cm/hour, the estimated Kp values are 0.0020-
4 0.016 cm/hour for ETBE.
5 Table B-2. Plasma radioactivity after a single oral or intravenous dose of
6 [14C]ETBE to male Crl:CD(SD) rats
Time (hours)
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.
aMean ± standard deviation; n = 4.
- = not measured, N.D. = not detected.
Source: JPEC(2008e).
7 ETBE is moderately absorbed following inhalation exposure in rats and humans, and blood
8 levels of ETBE approached—but did not reach—steady-state concentrations within 2 hours. Nihlen
9 etal. (1998) calculated the net respiratory uptake of ETBE in humans to be 26%. The AUC for the
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concentration-time curve was linearly related to the ETBE exposure level, suggesting linear kinetics
up to 209 mg/m3. The JPEC studies (IPEC. 2008e. 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.
B.1.2. Distribution
There are no in vivo data on the tissue distribution of ETBE in humans. Nihlen etal. (1995)
measured the partitioning of ETBE and tert-butanol in air into human blood from 10 donors (5
males, 5 females), saline, or oil inside of sealed vials. Also, human tissue-to-blood partitioning
coefficients were estimated in brain, fat, liver, kidney, lung, and muscle based upon their relative
water and fat contents. Kaneko etal. (2000) conducted a similar series of in vitro studies to
measure the partitioning of ETBE and tert-butanol in air to various rat tissues (5 male Wistar rats),
including blood, brain, fat, liver, kidney, lung, muscle, and testes. 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. Q9951 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 (2001) (namely, 0.35 for tert-butanol and 1.48-1.74 for ETBE). Nihlen etal. (1995)
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 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).
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The JPEC (2008e. f) 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 percent
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 dosing 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 through 168 hours after the last exposure
with the exception of 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
percent 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 (1995a) and Sun and Beskitt (1995b) studied the distribution of radiolabel
derived from [14C]ETBE in rats and mice, respectively. 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.
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 (CYP) enzymes via oxidative deethylation by the
addition of a hydroxyl group to the a-carbon of the ethyl ether group (Bernauer etal.. 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 etal.. 2001:
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Hong etal.. 1999a). Using data from rat hepatic microsome preparations, Turini etal. (1998)
suggested that CYP2B1 might be one of the primary enzymes responsible for this step in rats.
Acetaldehyde is oxidized to acetic acid by aldehyde dehydrogenase enzymes (some of which are
polymorphically expressed) and eventually to carbon dioxide (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 2-methyl-l,2-propane diol (MPD), and 2-
hydroxyisobutyrate (HIBA), acetone, and formaldehyde (Bernauer etal.. 1998). It should be noted
that these metabolites have been identified in studies using liver preparations from human or rat
studies using ETBE, MTBE, or tert-butanol (Bernauer et al.. 1998: Cederbaum and Cohen. 1980b):
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.I.4.).
glucuronide-0 —|—CH3
CH3
t-butyl glucuronide
CYP2A6
CYP3A4
O | CH3-
ch3
ETBE
CH,
0X0,
H*C—( CH3
OH
rats, humans
CH, OH
CYP450
HO—|—CH3 h c-
ETBE—hemi-acetal
ch3
t-butanol
rats,
humans
A
CH3 oh
2-methyl-1,2-propanediol
f
H3C—^OH
CH,
\ /0
CH,
oS \
acetaldehyde 0
ch3
t-butyl sulfate
HO O
[O]
ch3
2-hydroxyisobutyric acid
h2c=o
formaldehyde
h3c ch3
acetone
Figure B-l. Proposed metabolism of ETBE.
Source: Adapted from Dekant et al. (2001), NSF International (2003), ATS PR (1996), Bernauer et al.
(1998), Amberg et al. (1999), and Cederbaum and Cohen (1980a).
Zhang etal. f 199 71 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.
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B.l.3.1. Metabolism in Humans
Metabolism of ETBE in Humans in Vivo
Nihlen etal. f 19981 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
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 30 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 [iM 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 [iM 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 IV2 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.
Amberg etal. (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 [iM, although that
for tert-butanol was 13.9 ± 2.2 [iM. The corresponding values at 18.8 mg/m3 were 1.3 ± 0.7 and
1.8 ± 0.2 [iM, respectively. The time courses of metabolite appearance in urine after 170 mg/m3 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/m3and 1:17:45:435 after
18.8 mg/m3. Individual variations were large, but the authors did not report any gender differences
in the metabolism of ETBE based on data from only three subjects of each sex.
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In Vitro Metabolism of 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 1997bl 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
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 1999a) obtained hepatic microsomal protein preparations from 15 human
donors 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
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.
Following an evaluation of the activities of multiple different CYP forms in the 15 donor samples, it
was 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 co-
incubated with ETBE in the presence of chemical inhibitors or specific antibodies against either
CYP2A6 or CPY2E1. 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-[iM coumarin. Antibody against CYP2A6
inhibited metabolism by greater than 75%, but there was no inhibition by the antibody against
CYP2E1.
In the same paper, several specific human CYPs were expressed into human (3-
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
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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 not a major enzyme responsible for metabolism of ETBE. Le Gal etal. (2001)
used similar human cytochrome preparations as Hong etal. (1999a) (i.e., from human donors) or
genetically modified human p-lymphoblastoid cell lines transfected with CYP2A6, CYP2B6, CYP3A4,
or CYP2E1 and human CYP reductase to elucidate the metabolism of ETBE, MTBE, and TAME. They
identified acetaldehyde and tert-butanol as primary metabolites from ETBE.
B.l.3.2. Metabolism in Animals
Metabolism of ETBE in Animals In Vivo
Bernauer etal. f 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 [iM at 18.8 mg/m3, respectively. Peak levels of tert-butanol were higher in rats than in
humans. Similar to 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.
(2001) focused on aspects of ETBE metabolism which 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 mg/m3 or 167.1 mg/m3 ETBE), the relative concentration of tert-butanol to the received dose
was much smaller. This 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
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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.
The JPEC (2008e. f) 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. Metabolites were
measured in the plasma 8 hours after single or repeated dosing. Metabolites were 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 (1995a) did not identify the radiolabel eliminated, their
investigations do yield information pertinent to determining whether metabolic saturation might
occur under bioassay conditions. In their single-exposure protocol (see Section B.l.1.2), rats and
mice were exposed via inhalation to ETBE. These investigators reported the fraction of absorbed
dose that was eliminated 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 elimination was observed, as demonstrated by a marked decrease in the fraction of
radiolabel eliminated 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 envisioned that this shift in the elimination 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 authors, and the radiolabel has not been speciated as to chemical form. The unfortunate
limitation of the application of the PBPK model for human inhalation precludes its combination
with rat PBPK models to complete species extrapolation. The inhalation toxicity study by Saito et al.
f2013I 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
1 Table B-4. Unchanged ETBE and its metabolites in plasma 8 hours after a
2 single oral dose or repeated (7 or 14) daily oral dosing of [14C]ETBE to male
3 Crl:CD(SD) rats
Compound
Metabolite
% 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
te/t-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
te/t-butanol
12.9 ±3.1
55.0 ±2.9
18.2 ±3.8
22.2 ±6.0
4 aMean ± standard deviation; n = 4.
5 N.D. = not detected.
6
7 Source: JPEC (2QQ8e. f) unpublished reports.
8 Table B-5. Unchanged ETBE and its metabolites in the urine (measured 0-24
9 hours) after a single oral dose or repeated (7 or 14) daily oral dosing of
10 [14C]ETBE to male Crl:CD(SD) rats
Compound
Metabolite
% 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
11 aMean ± standard deviation; n = 4.
12 N.D. = not detected.
13
14 Source: JPEC (2Q08e, f) unpublished reports.
15
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Metabolism of ETBE in Animal Tissues in Vitro
Using microsomal protein isolated from the olfactory epithelium from male Sprague-
Dawley rats, Hongetal. f !997al measured ETBE metabolism as the formation of tert-butanol
(TBA). They found that metabolism occurred only in microsomal protein (not in cytosol) and only
in the presence of an NADPH- (nicotinamide adenine dinucleotide phosphate) 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 authors reported a Km value of 125 [J.M for ETBE.
Hong etal. f 1999bl 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 to
that observed in the Cyp2el wild-type parental strains (0.70 ± 0.12 for C57BL/6N mice, and
0.66 ± 0.14 for 129/Svmice). Therefore, microsomal protein from mice that did not express 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 P45 0 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
CYP3A or 1A forms. Studies of ETBE metabolism were based on high performance liquid
chromatography (HPLC) 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
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significant, approximate three-fold 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, 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 to the kinetic constants indicated by the
results of Hongetal. (1999b). it can be expected that the rate of ETBE metabolism at in vitro at
concentrations below 1 mM would 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
fDekant et al.. 20011.
B.1.4. Elimination
B.l.4.1. Elimination 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 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 that respiratory uptake was 32-34% in
humans, and net uptake (which excludes ETBE exhaled during exposure) was calculated 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 % of the
respiratory uptake of ETBE) accounted for even less: 0.12, 0.061, and 0.056% after the exposures to
20.9,104, and 209 mg/m3, respectively. The authors identified four phases of elimination of ETBE
from blood, with half-lives of about 2 and 20 minutes and 1.7 and 28 hours. Only one phase for
elimination of tert-butanol from blood was identified with a half-life of 12 hours compared to
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10 hours in another study with volunteers flohanson etal.. 19951. In urine, ETBE displayed two
phases of elimination, 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 flohanson etal.. 19951.
ETBE displayed a multi-phasic elimination from blood. The first phase likely indicates
uptake into highly-perfused tissues. The other phases could indicate uptake into less-perfused
tissues and fat, and 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 fNihlen et al.. 19981.
In the study by Amberg et al. (20001 described earlier (Section B.1.3.1), two elimination
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 thatfor tert-butanol was 8.2 ± 2.2 hours. The predominant urinary
metabolite identified was HIBA, 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 elimination after 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 gender differences in the
toxicokinetics of ETBE. Amberg etal. f 2 0 0 01 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.
B.l.4.2. Elimination in Animals
Amberg etal. (20001 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.
Similar to 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
eliminated ETBE considerably faster than humans. Urinary excretion accounted for 53 ± 15 and
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Supplemental Information—ETBE
1 50 ± 30% of the estimated dose at 170- and 18.8-mg/m3 exposures, respectively, with the
2 remainder of the dose being eliminated via exhalation, as suggested by the authors.
3 Bernauer etal. f 19981 studied the excretion of [13C]ETBE and MTBE in rats. F344 rats,
4 2/sex, were exposed via inhalation to 8,400 mg/m3 ETBE or 7,200 mg/m3 MTBE for 6 hours, or 3
5 male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours, and
6 ETBE metabolite prevalence in urine was MPD > HIBA > tert-butanol-sulfate > tert-butanol-
7 glucuronide. Oral administration of tert-butanol produced a similar metabolite profile, with relative
8 amounts of HIBA > tert-butanol-sulfate > MPD » tert-butanol-glucuronide ~ tert-butanol.
9 Although there are several unpublished reports relevant to the elimination of ETBE
10 following inhalation exposure, no additional peer-reviewed publications were identified.
11 Unpublished reports have not gone through the public peer-review process and are of unknown
12 quality. They are included here as additional information only.
13 Table B-6. Elimination of [14C] ETBE-derived radioactivity from rats and mice
14 within 96 hours following a single 6-hour inhalation exposure
Exposure level
Volatile
(mg/m3)
organics3
Exhaled C02a
Urine3
Feces3
Total"
F344 Raf
2,090
37
1
60
2
9.9
3,130
36
1
62
2
17.5
4,180
42
1
56
2
22.1
7,310
58
2
38
3
56.9
10,400
52
2
45
2
56.2
20,900d
63
2
34
1
97.5
(51)
(1)
(44)
(3)
(116)
CD-I Mousee
2,090
10
1
74
16
6.38
3,130
28
2
60
10
7.9
4,180
29
2
64
6
12.8
7,310
42
2
46
10
13.7
10,400
42
2
47
10
22.7
20,900d
44
5
39
12
18.9
(37)
(2)
(57)
(2)
(28)
15 aPercent of total eliminated radioactivity; mean of one male and one female.
16 bln mg [14C]ETBE equivalents.
17
18 Sources: cSun and Beskitt (1995b); dvalues in parentheses: Borghoff (1996); eSun and Beskitt (1995b).
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During 96 hours in metabolic cages, rats eliminated 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 elimination 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 eliminated
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 elimination 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 eliminated 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 eliminated to the
inhaled concentrations (Table B-6), a proportionate relationship is observed in rats at all
concentrations, but less than proportionate elimination of total radiolabel at the highest
concentration in mice. The complete data set led the authors of the report to conclude that
saturation of the inhalation absorptive processes might have occurred at concentrations of
approximately 7,310 mg/m3 (see Section B.1.1.2) The findings of Sun and Beskitt (1995a) in mice, at
20,900 mg/m3 were essentially confirmed by Borghoff (1996) (unpublished report) in a pilot study
that used the identical species, experimental protocol, materials, and methods but was conducted at
a different laboratory later (Table B-6).
Similarities between rats fSun and Beskitt. 1995bl and mice fSun and Beskitt. 1995al are
evident Both species demonstrate similar elimination pathways and present evidence of saturation
of metabolic pathways at concentrations lower than those which demonstrate saturation of
absorptive pathways. Metabolic saturation (evidenced as a shift from urine as the predominant
elimination 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 eliminated 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 (Table B-6) and excreted
about five times as much [14C]ETBE-derived radioactivity via feces than did rats. The total amounts
of eliminated 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 When
normalized to body weight, it is apparent that mice absorbed a higher dose than rats; however, the
total eliminated radioactivity at 20,900 mg/m3 showed no further increase over the values at
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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 1995a. b). The experimental protocol and materials were identical to the ones used by Sun
and Beskitt (1995a, b); however, in this pilot study, only three male F344 rats and three male CD-I
mice were used per experiment, with the 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 |iCi/mmol. It was found that rats, when assayed immediately after exposure, had 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 [iCi (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 [iCi immediately after exposure and 0.77 ± 0.16 [iCi for other mice placed in
metabolism cages. Elimination 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 elimination data were obtained, not on group means.
Mice had eliminated 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%
during the final 3 hours. Elimination of ETBE, tert-butanol, HIBA, and MPD in urine were assayed.
During 24 hours of collection, rats eliminated 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, eliminate ETBE faster than rats.
Unpublished reports by the IPEC (2008e) 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
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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
A physiologically based pharmacokinetic (PBPK) model of ETBE and its principal metabolite
tert-butanol has been developed for humans exposed while performing physical work fNihlen and
lohanson. 19991. The Nihlen and Johanson model is based on measurements of blood
concentrations of eight 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 were calculated from individual data on body weight and height. Additionally, to account for
physical activity, blood flows to tissues were 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 use of the model in
route-to-route extrapolation.
Many PBPK models have been developed for the structurally related substance, MTBE, in
rats and humans (Borghoff etal.. 2010: Leavens and Borghoff. 2009: Blancato etal.. 2007: Kim etal..
2007: Rao and Ginsberg. 1997: Borghoff etal.. 19961. These MTBE models can be modified for ETBE
by using the available toxicokinetic data described above. EPA's model evaluation and use for the
dose-response modeling in this assessment can be found below.
EPA evaluated a PBPK model of ETBE and its principle metabolite tert-butanol that was
developed for humans exposed while performing physical work fNihlen and lohanson. 19991. As
previously mentioned, the Nihlen and Johanson model is not appropriate for rodents or for oral
exposures, precluding cross-species or route-to-route extrapolations. Thus, EPA developed a PBPK
model for ETBE and its metabolite, tert-butanol, in the rat. This section present details on this
model and applicability to this assessment.
A PBPK model for ETBE and tert-butanol in rats was developed in acslX (Advanced
Continuous Simulation Language, Aegis, Inc., Huntsville, Alabama) by adapting information from
the many PBPK models that were developed in rats and humans for MTBE and the metabolite
tert- butanol that is common to both MTBE and ETBE (Borghoff etal.. 2010: Leavens and Borghoff.
2009: Blancato etal.. 2007: Kim etal.. 2007: Rao and Ginsberg. 1997: Borghoff etal.. 19961. A brief
description highlighting the similarities and differences in the Blancato etal. (20071 and Leavens
and Borghoff (20091 models is given, followed by an evaluation of the MTBE models and the
assumptions adopted from MTBE models or modified in the ETBE model.
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The Blancato etal. (2007) model is an update of the earlier Rao and Ginsberg (1997) model,
and the Leavens and Borghoff (2009) model is an update of the Borghoff et al. (1996) model. Both
the Blancato etal. f20071 and Leavens and Borghoff (2009) models are flow-limited models that
predict amounts and concentrations of MTBE and 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 (Andersen. 1991). The parent (MTBE) and metabolite
(tert-butanol) models are interlinked by the metabolism of MTBE to tert-butanol in the liver. Routes
of exposure included in the models are oral and inhalation for MTBE; Leavens and Borghoff (2009)
included 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 directly enter the
systemic blood supply, and the respiratory tract is assumed to be at a 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
2-methyl-l,2-propanediol and 2-hydroxyisobutyrate; both of these processes are described by a
single Michaelis-Menten equation in the models. All model assumptions are valid for tert-butanol
and were applied to the EPA-developed tert-butanol PBPK model, except for the separate brain
compartment The brain compartment was lumped with the compartment for other richly perfused
tissues in the EPA tert-butanol PBPK model.
In addition to differences in parameter values between the Blancato etal. (2007) and the
Leavens and Borghoff (2009) models, there were three differences in the model structure: (1) the
alveolar ventilation was reduced during exposure, (2) the rate of tert-butanol metabolism increased
over time due to induction of CYP enzymes, and (3) binding of MTBE and tert-butanol to
a2u-globulin was simulated in the kidney of male rats. The Blancato etal. f2007) model was
configured through EPA's PBPK modeling framework, ERDEM (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.
MTBE and tert-butanol binding to a2U-globulin in the kidneys of male rats was incorporated
in the PBPK model of MTBE by Leavens and Borghoff (2009). Binding to a2U-globulin is one
hypothesized mode of action for the observed kidney effects in MTBE-exposed animals. For a
detailed description of the role of a2U-globulin and other modes of action in kidney effects, see the
kidney Mode of Action section of the main volume (see Section 1.2.1). Binding of MTBE to ct2u-
globulin was applied to sex differences in kidney concentrations of MTBE and tert-butanol in the
Leavens and Borghoff (2009) model but acceptable estimates of MTBE and tert- butanol
pharmacokinetics in the blood are predicted in other models that did not consider a2U-globulin
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Supplemental Information—ETBE
binding. Moreover, as discussed below, the Leavens and Borghoff (2009) model did not adequately
fit the available tert-butanol i.v. dosing data, adding uncertainty to the binding parameters they
estimated. Given the lack of ETBE concentration data in kidney tissue following ETBE exposure,
binding to a2U-globulin could not be applied to the ETBE PPBK model; however, this binding does
not significantly affect blood concentrations, so this data gap is not considered critical to estimating
systemic concentration of ETBE.
B.l.5.1. Evaluation and Modification of Existing tert-Butanol Submodels
The Blancato etal. (2007) and Leavens and Borghoff (2009) models were evaluated by
comparing predictions from the tert-butanol portions of the models with the tert-butanol i.v. data of
Poet and Borghoff f 19971 (Figure B-2). 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.
A)
1000
100
10
0.1
B)
^
—300 mg/kg
~
mate
~
female
It
i 300 mg/kg
mate
o
female
[ 10000
-- 150 mg/kg
¦
mate
D
female
¦0000
| — 150 mg/kg
¦
male
o
female
75 mg/kg
•
male
O
female
1 ——7S mg/kg
•
male
0
female
37.5 mg/kg
A
male
A
female
1 37.5 mg/kg
A
male
A.
female
©
!
Q
¦
V «
N. •
A v o
~
X,
\ 1
0 2 4 6 S 10 12 14 16 18 20 22 24
time (hours)
1000 ~
0 2 4 6 8 10 12 14 16 18 20 22 24
time (hours)
Figure B-2. Comparison of the tert-butanol portions of existing MTBE models
with tert-butanol blood concentrations from i.v. exposure by Poet and
Borghoff f19971.
Neither the a) Blancato et al. (2007) nor the b) Leavens and Borghoff (2009) model adequately represents
the measured tert-butanol blood concentrations.
Attempts to reoptimize model parameters in the tert- butanol submodels of Blancato etal.
(2007) and Leavens and Borghoff (2009) to match blood concentrations from the i.v. dosing study
were unsuccessful. To account for the tert-butanol blood concentrations after i.v. tert-butanol
exposure, the model was modified by adding a pathway for reversible sequestration of tert-butanol
in the blood. This could represent binding of tert-butanol to proteins in blood (see Figure B-3). The
JPEC pharmacokinetic studies show that approximately 60% of the radiolabel in whole blood is in
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 the plasma, providing some limited evidence for association of tert-butanol with components in
2 blood. The PBPK model represented the rate of change of tert-butanol amount in the sequestered
3 blood compartment (Abi00d2) with the equation:
4 dAblood2/dt = Kon*CV* - KoFF*Cblood2
5 where Kon is the binding rate constant, CV is the free tert-butanol concentration in blood, Koff is the
6 unbinding rate constant, and Cbiood2 is the concentration of tert-butanol bound in blood (equal to
7 Ablood2/V blood)-
ETBE TBA
Inhalation Exhalation Inhalation Exhalation
IV Dose
'ON
'OFF
Urinary excretion
VMETBE, KMetbe,
VMETbe2' KMetbez
Oral Kas
Dose
Metabolism
Metabolism
Oral a$2
Liver
Kidney
Rapidly Perfused
Slowly Perfused
Fat
Liver
Kidney
Slowly Perfused
Sequestered
Rapidly Perfused
Fat
Alveolar Air
Blood
Alveolar Air
Blood
8 Dose
9 Figure B-3. Schematic of the PBPK model for ETBE and its major metabolite
10 tert-butanol in rats.
11 Exposure can be via multiple routes including inhalation, oral, or i.v. dosing. Metabolism of ETBE and tert-
12 butanol occur in the liver and are described by Michaelis-Menten equations with two pathways for ETBE
13 and one for tert-butanol. ETBE and tert-butanol are cleared via exhalation, and tert-butanol is additionally
14 cleared via urinary excretion. See Table B-7 for definitions of parameter abbreviations.
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The physiologic parameter values were obtained from the literature f Brown et al.. 19971
and are shown in Table B-7. tert-Butanol partition coefficients were obtained from literature that
determined the ratios of measured tissue:air and blood:air partition coefficients fBorghoff et al..
19961. The parameters describing rate constants of metabolism and elimination of tert-butanol
were obtained from the literature fBlancato etal.. 20071 and kept fixed because these have been
optimized to tert-butanol blood concentrations measured after MTBE exposure, which is also
metabolized to tert-butanol. The parameters describing tert-butanol absorption and tert-butanol
sequestration in blood were estimated by optimizing the model to the blood tert-butanol time-
course data simultaneously for rats exposed via i.v., inhalation, and oral routes (Leavens and
Borghoff. 2009: Poet and Borghoff. 1997: ARCO. 19831.
The model parameters were estimated with the acslX optimization routine to minimize the
log-likelihood function of estimated and measured tert-butanol concentrations. The Nedler-Mead
algorithm was used with heteroscedasticity allowed to vary between 0 and 2. The predictions of the
model with optimized parameters have a much improved fit to the tert-butanol blood
concentrations after tert-butanol i.v. as shown in panel A of Figure B-4. Additionally, the model
adequately estimates the tert-butanol blood concentrations after inhalation and oral gavage
exposures. The optimized parameter values are shown in Table B-8.
The ARCO (19831 study measured tert-butanol in plasma only, not whole blood like the Poet
and Borghoff f 19971 and Leavens and Borghoff f 20091 studies. Based on the measurements of
plasma and whole blood by 1PEC (2008f). the concentration of tert-butanol in plasma is
approximately 60% of the concentration in whole blood. The tert-butanol plasma concentrations
measured by ARCO were increased (divided by 60%) to the expected concentration in whole blood
for comparison with the PBPK model.
B.l.5.2. ETBE Model Parameterization and Fitting
The ETBE submodel used the same physiological parameters as tert-butanol obtained from
Brown etal. f 19971 and shown in Table B-7.
ETBE partition coefficients were obtained from Nihlen etal. (19951 which were calculated
for in tissues by relating measured blood:air, water:air, and oil:air partition coefficients to reported
compositions of water and lipids in rat tissues. The parameters describing ETBE absorption and
metabolism were optimized to fit the blood and urine time-course data for rats exposed to ETBE via
oral and inhalation routes flPEC. 2008f: Amberg et al.. 2000: Borghoff. 19961. During the
optimization, parameters describing tert-butanol were held constant. The model parameters were
estimated with the acslX optimization routine in the same way as the tert-butanol submodel. The
optimized parameter values are shown in Table B-8. The predictions of the model with optimized
parameters for ETBE oral gavage by 1PEC f2008f) are shown in Figure B-5.
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Supplemental Information—ETBE
1 Table B-7. PBPK model physiologic parameters and partition coefficients
Body weight and organ volumes as fraction of body weight
Body Weight (kg)
0.25
Brown et al. (1997)
Body fraction that is blood perfused (Fperf) 0.8995
Brown et al. (1997)
Liver
0.034
Brown et al. (1997)
Kidney
0.007
Brown et al. (1997)
Fat
0.07
Brown et al. (1997)
Rapidly perfused
0.04
Brown et al. (1997)
Slowly perfused
0.7485
a
Blood
0.074
Brown et al. (1997)
Cardiac output and organ blood flows as fraction of cardiac output
Cardiac output (L/hr)
5.38
Brown et al. (1997)b
Alveolar ventilation (L/hr)
5.38
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)
Rapidly perfused
0.279
e
Slowly perfused
0.336
Brown et al. (1997)
Partition coefficients for ETBE
Blood:air
11.7
Nihlen et al. (1995)
Liver:blood
1.68
Nihlen et al. (1995)
Fat:blood
12.3
Nihlen et al. (1995)
Rapidly perfused:blood
2.34
f
Slowly perfused:blood
1.71
g
Kidney:blood
1.42
Nihlen et al. (1995)
Partition coefficients for te/t-butanol
Blood:air
481
Borghoff et al. (1996)
Liver:blood
0.83
Borghoff et al. (1996)
Fat:blood
0.4
Borghoff et al. (1996)
Rapidly perfused:blood
0.83
Borghoff et al. (1996)
Slowly perfused:blood
1.0
Borghoff et al. (1996)
Kidney:blood
0.83
Borghoff et al. (2001)
aFperf - Z(other compartments).
b15.2*BW0-75.
cAlveolar ventilation is set equal to cardiac output.
dsum of liver and gastrointestinal (Gl) blood flows.
el - Z(all other compartments).
fSet equal to brain tissue.
gSet equal to muscle tissue.
2
3
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Supplemental Information—ETBE
A)
~
TBft inhalation exposure concentration!
TBA iv exposure
1000
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0.1
time (hours)
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Supplemental Information—ETBE
Table B-8. Rate constants determined by optimization of the model with
experimental data
Parameter
Value
Source or Reference
ferf-butanol rate constants
Metabolism (VMtba; mg/kg-hr)a
Metabolism (KMtba; mg/L)
Urinary elimination (Keum2; 1/hr)
te/t-butanol sequestration rate constant (Kon; L/hr)
te/t-butanol unsequestration rate constant (Koff; L/hr)
Absorption from gastrointestinal (Gl) (Kas2; l/hr)
ETBE rate constants
Metabolism high affinity (VMetbe; mg/L-hr)
Metabolism high affinity (KMetbe; mg/L)
Metabolism low affinity (VMetbe2; mg/L-hr)
Metabolism low affinity (KMetbe2; mg/L)
Absorption from Gl (Kas; l/hr)
8.0 Blancato et al. (2007)
28.8 Blancato et al. (2007)
0.5 Blancato et al. (2007)
0.148 Optimized
0.0134 Optimized
0.5 Optimized
1.89 Optimized
0.035 Optimized
15.2 Optimized
10.0 Optimized
0.5 Optimized
aScaled by BW07 (0.25°7 = 0.379).
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A)
• 400 mg/kg
~ 5 mg/kg
< 80
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20
0.2
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12
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Q
400 mg/kg ETBE gavage
< 0.3
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(2000) after ETBE inhalation in Figure B-6. Although the fit of the model to the data for the 4-ppm
exposure are sufficient, the prediction of the tert-butanol blood concentration after the 40-ppm
exposure is higher than was measured. The tert-butanol blood concentration would be reduced if
exposed animals were reducing their breathing rate or other breathing parameters but the
exposure concentration of 40-ppm ETBE exposure is unlikely to be high enough to cause a change
in breathing parameters because at the much higher ETBE concentration in the ARCO f 19831 study
(5,000 ppm), changes in breathing were not noted and the model predictions fit measured
concentrations well. The urinary elimination of tert-butanol is underestimated by the tert-butanol
submodel (Figure B-6). The rate constant for tert-butanol urinary elimination (Kelim2) 0.5/hour was
obtained from the literature [the same value was used by Blancato etal. f20071: Rao and Ginsberg
(1997). and Leavens and Borghoff (2009)]. which is supported by multiple studies of MTBE and
tert-butanol. To match the measured amount of tert-butanol in urine, the rate constant would need
to be increased to 1.5/hour as shown in Figure B-6. Urinary elimination of tert-butanol is the minor
elimination route; elimination is primarily by metabolism and exhalation, so increasing urinary
elimination does not noticeably change the fit to the tert-butanol blood concentrations.
Additionally, increasing the urinary elimination rate worsens the model predictions for urinary
elimination after oral gavage (Figure B-5); therefore, the rate constant obtained from the literature
(0.5/hour) was used for model predictions. The predictions of the model with optimized
parameters were compared with the amounts of ETBE and tert-butanol exhaled after exposure to
5,000-ppm ETBE as measured by ARCO (1983) in Figure B-7. The EPA model fits the measured
amounts well.
This document is a draft for review purposes only and does not constitute Agency policy.
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A)
B)
ETBE exposure concentration
ETBE exposure concentration
s»
E
I
=
s
o
4 6 8
time (hours)
10 12
40 ppm
4ppm
40 ppm
4 ppm
1
4 6 8
time (hours)
10 12
C)
D)
ETBE model A ETBE data
TBA mode
TBAdata
CUD 2.5
_ 0,04
00
1
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< 0.02
P
E
to
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data
¦ KEUM2=1.5/hr
¦ KELIM2=0.5/hr
0 5 10 15 20 25 30 35 40 45 50
ETBE exposure concentration (ppm)
0 4 8 12 16 20 24 28 32 36
time (hours)
Figure B-6. Comparison of EPA model predictions with measured amounts
after a 4-hour inhalation exposure to 4 and 40 ppm ETBE.
Concentrations in blood are shown in A) for ETBE, B) for te/t-butanol. In C) the measured ETBE and tert-
butanol blood concentrations for exposures to 4 and 40 ppm ETBE are compared with model predictions
of exposures from 0 to 50 ppm ETBE. The amount of te/t-butanol in urine is shown in D) for the 40 ppm
exposure for two values of Keum2, the rate constant for te/t-butanol urinary elimination. The value 0.5/hr
was obtained from Blancato et al. (2007) and is used in all other EPA model predictions (e.g. Figure B-5).
The increased rate constant 1.5/hr improves the fit of the model to urinary data. The 4 ppm exposure did
not significantly increase the amount of urine over background. The data are from Amberg et al. (2000).
The model predictions used the optimized parameter values as shown in Table B-8.
This document is a draft for review purposes only and does not constitute Agency policy.
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A)
B)
WJ
TS
.£
m
r
x
J5
3
3
u
0 1 2 3 4 5 6
time after exposure (hours)
0 1 2 3 4 5 6
time after exposure (hours)
Figure B-7. Comparison of EPA model predictions with measured amounts of
A) ETBE and B) tert-butanol in exhaled breath after a 6-hour inhalation
exposure to 5,000 ppm ETBE.
The data points show the individual measurements of the three rats in the ARCO (1983) study. The model
predictions used the optimized parameter values as shown in Table B-8.
Induction of tert-butanol metabolizing enzymes was included in the Leavens and Borghoff
(2009) model of MTBE based on their study of rats exposed for 8 days to tert-butanol via inhalation.
The enzyme induction equation and parameters developed in the Leavens and Borghoff f20091
model were applied to the tert-butanol submodel and are:
VmaxTBAIND = VmaxTBA*INDMAX(l-exp(-KIND*t))
where VmaxTBAIND is the maximum metabolic rate after accounting for enzyme induction,
VmaxTBA is the metabolism rate constant from Table B-8 for both tert-butanol pathways, INDMAX is
the maximum percent increase in VmaxTBA (124.9), and KIND is the rate constant for enzyme
induction (0.3977/day). The increased tert-butanol metabolism better estimates the measured
tert-butanol blood concentrations as shown in a comparison of the model predictions and
experimental measurements in Figure B-8. The model better predicted blood concentrations in
female rats than male rats. The male rats have lower tert-butanol blood concentrations after
repeated exposures than female rats and this difference could indicate greater induction of
tert-butanol metabolism in males or other physiologic changes such as ventilation, or urinary
excretion. The current data for tert-butanol metabolism do not provide sufficient information for
resolving this difference between male and female rats. The only repeat dose study with ETBE was
by oral gavage for 14 days at 5 mg/kg-day and tert-butanol blood concentrations did not decline
This document is a draft for review purposes only and does not constitute Agency policy.
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after repeated doses flPEC. 2008el The internal dose of tert-butanol after repeated ETBE dosing in
the IPEC f2008el study was much lower than in the tert-butanol repeated dosing study (Leavens
and Borghoff. 20091 and possibly the lower tert-butanol blood concentration was not sufficient to
cause significant induction of tert-butanol metabolizing enzymes. The comparison of the model
predictions and experimental measurements assuming no enzyme induction are shown in Figure
B-9. An alternative explanation of the repeat dose studies is that some tert-butanol metabolism
occurs in the respiratory tract and after inhalation exposure there is greater induction of enzymes
than after oral exposure.
600
_ 55C
"g son
nale rats without induction
6 so
1
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A
3S0
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72 96 120
time (hours)
144 168 192
male rats with induction
11726 pps"
\ \ >\ V* V\"\
time (hours)
female rats with induction
female rats without induction
400
300
100
f • /* ' * » \ ' V ' V \
- r*«W- -"<* • -"V
24 48 72: 96 :
lime (hours!
120 144 168 192
0
Figure B-8. Comparison of EPA model predictions with measured amounts of
tert-butanol in blood after repeated inhalation exposure to tert-butanol,
5 mg/kg-day ETBE oral gavage for up to 14 days in male rats.
The data show the individual measurements of the four rats in the JPEC (2010b) study. tert-Butanol blood
concentrations are not well predicted by the model at the highest tert-butanol exposure concentration
without enzyme induction.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
i th i kction without induction
^ time (hours)
2 Figure B-9. Comparison of EPA model predictions with measured amounts of
3 tert-butanol in blood after 5 mg/kg-day ETBE oral gavage for up to 14 days in
4 male rats.
5 The data show the individual measurements of the four rats in the JPEC (2010b) study. Adding enzyme
6 induction to the model has a small effect on the predicted tert-butanol blood concentrations and the
7 model predictions are closer to measured data when induction is not included.
8 B.l.5.3. Summary of the PBPK Model for ETBE
9 A PBPK model for ETBE and tert-butanol was developed by adapting previous models for
10 MTBE and tert-butanol (Leavens and Borghoff. 2009: Blancato etal.. 20071. Published tert-butanol
11 models (or sub-models) do not adequately represent the tert-butanol blood concentrations
12 measured in the i.v. study (Poet and Borghoff. 19971. The addition of a sequestered blood
13 compartment for tert-butanol substantially improved the model fit The alternative modification of
14 changing to diffusion-limited distribution between blood and tissues also improved the model fit,
15 but was considered less biologically plausible. Physiological parameters and partition coefficients
16 were obtained from published measurements. The rate constants for tert-butanol metabolism and
17 elimination were from a published PBPK model of MTBE with a tert-butanol subcompartment
18 (Blancato etal.. 20071. Additional model parameters were estimated by calibrating to data sets for
19 i.v., oral and inhalation exposures and repeated dosing studies for both ETBE and tert-butanol.
20 Although the model modestly overpredicted the tert-butanol blood concentration by approximately
21 1.5-fold in one case fAmbergetal.. 20001. overall, the model produced acceptable fits to multiple
22 rat time-course datasets of ETBE and tert-butanol blood levels following either inhalation or oral
23 gavage exposures.
This document is a draft for review purposes only and does not constitute Agency policy.
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B.l.5.4. ETBE Model Application
The PBPK model described above was applied to conduct route-to-route extrapolation
based on an equivalent internal dose. Cross chemical comparisons were made on an internal dose
basis using data from both the ETBE and tert-butanol (TBA) Toxicological Reviews. For simulating
studies where ETBE or tert-butanol was administered in drinking water, the consumption was
modeled as episodic, based on the pattern of drinking observed in rats by Spiteri (1982).
The PBPK model was used to calculate four internal dosimetrics: the daily average TBA
blood concentration (TBAblood), the daily amount of TBA metabolized in the liver (TBAmet), the
daily average of ETBE blood concentration (ETBEblood), and the daily amount of ETBE metabolized
in the liver (ETBEmet). The times to reach steady state for the dose metrics were much shorter than
the duration of the toxicity studies so the steady state values were considered representative of the
study and were used. To calculate steady state values for daily exposure to ETBE or TBA (i.e., the
oral exposure studies), the simulations were run until the daily average value had a < 1% change
between consecutive days. To calculate the steady state values in instances when study protocols
did not expose animals daily (inhalation exposure studies occurring five days per week or gavage
studies occurring 4 days per week), a full week was simulated with exposure days according to the
study protocol. The daily average after a full week exposure was used as the steady state value
because TBA blood concentration was negligible (< 0.1 ng/L) after two consecutive days without an
exposure. To better inform the contribution of the parent compound or metabolite of ETBE on
kidney and liver toxicity, kidney and liver responses were compared across studies based on these
internal doses. Each mechanistic question was evaluated for each of the following toxicity
endpoints:
• Relative change in kidney weights
• Extra risk of marked or severe CPN
• Extra risk of kidney urothelial hyperplasia
• Extra risk of kidney tumors
• Extra risk of liver tumors
Of the endpoints evaluated herein, absolute kidney weight, urothelial hyperplasia, and liver
tumors were considered for dose-response evaluation in Volume 1, Section 2 Dose-Response
Analysis.
For continuous endpoints, responses were expressed in terms of relative changes to
normalize for differences in control organ weights. For the quantal endpoints, comparisons are
made based on extra risk [(%incidence- control %incidence) 4- (1- control%incidence)] to
normalize for differences in control incidences. Because evaluating sex differences was not an
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
objective of this analysis and clear differences in sensitivity was observed between sexes, males and
females were evaluated separately.
Spearman's rank correlation coefficient ("rho") was calculated for all comparisons made
between kidney and liver endpoints and internal dose metrics. The strength of the dose-response
for the raw kidney and liver relative weight data was calculated using the Jonckheere-Terpstra
trend test
Relative kidney weights were compared based on internal doses calculated from the PBPK
model (Figure B-10). Oral and inhalation ETBE studies were found to be quantitatively consistent
across all dose metrics, with the strongest Spearman's rank correlations (rho > 0.93 males; > 0.79
females) for the tert-butanol dose metrics, tert-Butanol inhalation exposure yielded a consistently
weaker dose-response compared to oral tert-butanol exposure (panels A, B, D, and E); however, the
tert-butanol metabolized dose yielded the strongest correlations as the dose metric (rho = 0.92
males; 0.90 females). When ETBE and tert-butanol were combined, ETBE studies yielded a
consistent dose-response relationship with oral studies of tert-butanol (rho > 0.90), whereas
including the inhalation study of tert-butanol reduced the rho to 0.7~0.81.
Urothelial (transitional epithelial) hyperplasia is a renal lesion observed after 2 year
exposures in both ETBE and tert-butanol administration studies. Because females administered
ETBE did not have any reported hyperplasia, and female hyperplasia was observed in only one tert-
butanol study, only male data were analyzed (Figure B-ll). Oral and inhalation ETBE studies were
quantitatively consistent across all dose metrics, with the strongest rank correlations (>0.89) for
the tert-butanol dose metrics. When including the tert-butanol dataset, a consistent dose-response
relationship was observed using either tert-butanol blood concentration or tert-butanol
metabolized (rank correlations of 0.85-0.86).
CPN is a renal lesion observed after 2 years following both ETBE and TBA exposure in the
same studies as urothelial hyperplasia. A statistically significant dose-response for CPN was
observed in both males and females exposed to either compound, and all datasets were analyzed
based on internal dose (Figure B-12). In males, ETBE oral and inhalation studies were
quantitatively consistent across all analyzed dose metrics (rank correlations >0.8~1). In females,
the relationship was consistent across studies for the TBA dose metrics (rank correlations
>0.6~0.8), but not for the ETBE blood concentration dose metric (rank correlation of 0.37). When
including the TBA dataset in the analyses, a consistent dose-response relationship was observed
using either TBA blood concentration or TBA metabolized dose (rank correlations of 0.72~0.93),
with the strongest correlations occurring with the TBA blood concentration for males (rank
correlation = 0.93) and the metabolism rate of TBA (rank correlation = 0.87) for females.
Oral administration of TBA increased kidney tumors in male rats; however, no statistically
significant increase in kidney tumors was observed following oral or inhalation exposure to ETBE.
Conversely, inhalation ETBE exposure significantly increased liver tumors, but liver tumors were
not significantly increased following oral TBA exposure. No significant increase in liver or kidney
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
tumors were observed in females following ETBE or TBA administration, so the analyses were
confined to males.
The results indicate that studies administering ETBE either orally or inhalationally achieved
similar or higher levels of TBA blood concentrations or TBA metabolic rates as those induced by
direct TBA administration (Figure B-13). Neither dose metric yielded a consistent dose-response
for kidney tumors from TBA or ETBE studies, and as result, the correlation coefficients were low
and not significant (Figure B-13). Liver tumors following ETBE oral or inhalation exposure were not
consistent using either ETBE or tert-butanol metabolism rate dose metric (Figure B-14) and the
correlation coefficients were not significant These data indicate that internal dose is inadequate to
explain differences in tumor response across these studies.
Altogether, the PBPK model-based analysis indicates that kidney weight, urothelial
hyperplasia, and chronic progressive nephropathy (CPN) yielded a consistent dose-response
relationship using TBA blood concentration as the dose metric for both ETBE and TBA studies.
Kidney and liver tumors, however, were not consistent using 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
tert-butanol-oral
~
tert-butanol-in halation
1
2
JS
-------
Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
tert-butanol-oral
1.0
CO
'¦= 0.8
m
CO
Q- 0.4
CD
Q.
>s
— 0.2
"33
0.0
3
1
2
3 Figure B-ll. Comparisons of urothelial hyperplasia in male rats following
4 ETBE (black) or tert-butanol (white) inhalation (square) or oral (circle)
5 exposure with internal dose metrics calculated from the PBPK model.
6 Male urothelial hyperplasia is compared with tert-butanol blood concentration (A), the metabolism rate
7 of tert-butanol in the liver (B), and the blood concentration of ETBE (C).
A. rho=0.86 (all datasets)
rho=0.89 (ETBE only)
O
• O
o_
B. rho=0.85 (all datasets)
rho=0.94 (ETBE only)
O
O
C. rho= 0.71 (ETBE only)
0 20 40 60 0 1 2 3
tert-butanol blood concentration (mg/l) tert-butanol metabolized (mg/hr)
0 10 20 30
ETBE blood concentration (mg/l)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
•
ETBE oral
¦
ETBE inhalation
o
tert-butanol oral
0.8
A. rho= 0.93 (all datasets)
B. rho=0.80 (all datasets)
C. rho= 0.94 (all datasets)
° ¦
¦ °
¦
O »
•
• ¦
¦
¦
¦
••
• •
•
o
O
(/)
Q_
O
0.6 -
0.4 -
x 0.2
-------
Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
tert-butanol-oral
0.35 -
0.30 -
| 0.25 -
2
0.20 -
0
£
o 0.15 -
E
t °-10"
0
c
^ 0.05 -
0.00 -
1
2
3 Figure B-13. Comparisons of kidney tumors in male rats following 2 year oral
4 or inhalation exposure to ETBE or tert-butanol with internal dose metrics
5 calculated from the PBPK model.
6 Dose metrics represented are tert-butanol blood concentration (A) and the metabolism rate of tert-
7 butanol in the liver (B).
8
A. rho=0.39 (all datasets)
B. rho=0.10 (all datasets)
O
O
O
O
O
O
•
•
¦ •
¦
¦
•
¦ •
•
¦
•
0 20 40 60 0 1 2 3
tert-butanol blood concentration (mg/l) tert-butanol metabolized (mg/hr)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
tert-butanol-oral
C/J
X
0.25
0.20 -
0.15 -
0.10 -
A. rho= 0.099 (all datasets)
rho= 0.15 (ETBE only)
w 0.05
o
0.00
-0.05
-0.10 -
-0.15
O •
• •
B. rho= 0.33 (ETBE only)
• •
1 1 1 1
0 1 2 3 4 5
1 tert-butanol metabolized (mg/hr) ETBE metabolized (mg/hr)
2 Figure B-14. Comparisons of liver tumors in male rats following 2 year oral or
3 inhalation exposure to ETBE or tert-butanol with internal dose metrics
4 calculated from the PBPK model.
5 Dose metrics expressed are metabolism rate of tert-butanol (A) and metabolism rate of ETBE (B).
6
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 B.l.5.5. PBPK Model Code
2 The PBPK acslX model code is made available electronically through EPA's Health and
3 Environmental Research Online (HERO) database. All model files can be downloaded in a zipped
4 workspace from HERO (www, epa. gov/hero I
5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 B.2. OTHER PERTINENT TOXICITY INFORMATION
2 B.2.1. Other Toxicological Effects
3 B.2.1.1. Synthesis of Other Effects
4 The database for effects other than kidney, liver, reproductive, and cancer contain only 11
5 rodent studies. These included decreased body weight, increased adrenal weights, altered spleen
6 weights, and increased mortality. All selected studies used inhalation, oral gavage, or drinking
7 water exposures for >90 days. Shorter duration multiple exposure studies that examined
8 immunological endpoints were also included. No studies were removed for methodological
9 concerns.
10 Kidney effects
11 Numerical absolute kidney weight data are presented in Table B-9.
12 Table B-9. Evidence pertaining to kidney weight effects in animals exposed to
13 ETBE
Reference and study design
Results (percent change compared to control)
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
1,000 mg/kg-d
0
-
0
-
daily for 16 wk beginning 10 wk prior to
mating
100
5%
100
-2%
P0, female (24/group): 0,100, 300,
300
8%
300
0%
1,000 mg/kg-d
daily for 17 wk beginning 10 wk prior to
1,000
18%*
1,000
7%*
mating to lactation day 21
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
PO, male (25/group): 0, 250, 500,
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
1,000 mg/kg-d
0
-
0
-
daily for a total of 18 wks beginning 10 wk
before mating until after weaning of the
250
11%*
250
-1%
pups
500
15%*
500
2%
P0, female (25/group): 0, 250, 500,
1,000 mg/kg-d
1,000
21%*
1,000
5%
daily for a total of 18 wk beginning 10 wk
Fl, Male
Fl, Female
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
and lactation, then Fl doses beginning PND
0
-
0
-
22 until weaning of the F2 pups
250
10%
250
4%
500
22%*
500
3%
1,000
58%*
1,000
11%*
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
Dose
(mg/kg-d)
Absolute
weight
daily for 23 wk
0
1,000
19%*
Mivata et al. (2013); JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100,
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
400 mg/kg-d; female (15/group): 0, 5, 25,
0
-
0
-
100, 400 mg/kg-d
1%
1%
daily for 26 wk
O
O
25
6%
25
0%
100
5%
100
7%
400
25%*
400
10%*
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0,150, 500,1,500, 5,000 ppm
Dose (mg/m3)
Absolute
weight
Dose (mg/m3)
Absolute
weight
(0, 627, 2,090, 6,270, 20,900 mg/m3)b;
0
-
0
-
female (NR): 0,150, 500,1,500, 5,000 ppm
(0, 627, 2,090, 6,270, 20,900 mg/m3);
627
10%
627
1%
dynamic whole body chamber; 6 hr/d,
2,090
11%
2,090
-1%
5 d/wk for 13 wk; generation method,
analytical concentration and method were
6,270
18%*
6,270
4%
reported
20,900
16%*
20,900
7%
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (6/group): 0, 5,000 ppm (0,
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
20,900 mg/m3)a; female (6/group): 0,
0
-
0
-
5,000 ppm (0, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
20,900
19%
20,900
8%
5 d/wk for 13 wk followed by a 28 day
recovery period; generation method,
analytical concentration and method were
reported
Medinskv et al. (1999): Bond et al. (1996b)
Male
Female
rat, Fischer 344
inhalation - vapor
male (48/group): 0, 500,1,750, 5,000 ppm
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
(0, 2,090, 7,320, 20,900 mg/m3)a; female
0
-
0
-
(48/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
2,090
7%
2,090
4%
dynamic whole body chamber; 6 hr/d,
7,320
10%*
7,320
12%*
5 d/wk for 13 wk; generation method,
analytical concentration and method were
20,900
19%*
20,900
21%*
reported
Medinskv et al. (1999); Bond et al. (1996a)
Male
Female
mice, CD-I
inhalation - vapor
male (40/group): 0, 500,1,750, 5,000 ppm
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
(0, 2,090, 7,320, 20,900 mg/m3)a; female
0
-
0
-
(40/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
2,090
9%
2,090
0%
dynamic whole body chamber; 6 hr/d,
7,320
10%
7,320
6%
5 d/wk for 13 wk; generation method,
analytical concentration and method were
20,900
5%
20,900
4%
reported
1 a4.18 mg/m3 = 1 ppm.
2 *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
3 for controls, no response relevant; for other doses, no quantitative response reported.
4 (n): number evaluated from group.
5 Body weight
6 As presented in Table B-9, body weights were significantly reduced compared with vehicle
7 controls following 2-year oral and inhalation exposures to ETBE fSaito etal.. 2013: Suzuki etal..
8 2012:1PEC. 2010a. b). Reductions were also reported in studies of exposure durations shorter than
9 2 years f Ban ton etal.. 2011: Hagiwara etal.. 2011: Fuiii etal.. 2010:1PEC. 2008a. b; Gaoua. 2004b:
10 Medinskv etal.. 19991: however, these effects were frequently not statistically significant Food
11 consumption did not correlate well with body weight fSaito etal.. 2013: Suzuki etal.. 2012:1PEC.
12 2010a. b). Water consumption was reduced in the 2-year oral exposure study flPEC. 2010a).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Palatability and reduced water consumption due to ETBE exposure might contribute to the reduced
body weight, particularly for oral exposures. Ptyalism, which is frequently observed with
unpalatable chemicals following gavage, was observed in rats gavaged for 18 weeks fGaoua.
2004b). Body weight changes are poor indicators of systemic toxicity but are important when
evaluating relative organ weight changes. Because body weight was most severely affected in 2-
year studies, and 2-year kidney and liver weights are not appropriate for analysis as stated in
Sections 1.2.1 and 1.2.2, thus EPA concluded that body weight is not a hazard of ETBE exposure.
Adrenal weight
Adrenal weights were increased in 13-week and 23-week studies (see Table B-10). For
instance, a 13-week inhalation study found that absolute adrenal weights were increased in male
and female rats fMedinskv etal.. 19991. In another study, absolute and relative adrenal weights
were increased in male rats fHagiwara etal.. 20111. None of the observed organ weight changes
corresponded with functional or histopathological changes, thus EPA concluded that adrenal effects
were not 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 fWHO. 20121
(see Table B-12). 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 (Banton 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-12). 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.. 2013:1PEC. 2010bl and increased relative weights in female rats following 2 year oral
exposure fSuzuki etal.. 2012:1PEC. 2010a). Spleen weights are heavily influenced by the
proportion of red blood cells which do not impact immune function of the organ fElmore. 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
fLi etal.. 20111. 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 EPA
concluded that immune effects is not a hazard of ETBE exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Mortality
Mortality was significantly increased in male and female rats following a 2-year ETBE
inhalation exposure fSaito etal.. 2013:1PEC. 2010b) but not significantly affected following a 2-year
drinking water exposure fSuzuki etal.. 2012:1PEC. 2010al. Increased mortality in male rats
correlated with increased CPN severity in the kidney. Increased mortality in females was attributed
to pituitary tumors by the study authors; however, pituitary tumors were not dose responsively
increased by ETBE exposure. Survival was also reduced in a chronic 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 (Maltoni etal.. 1999). After 104 weeks, however, survival
in the controls was approximately 25% in males and 28% in females which is much lower than
anticipated 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. 2011). These data do not suggest that mortality was increased in these
studies due to excessively high exposure concentrations of ETBE, thus EPA concluded that mortality
was not a hazard of ETBE exposure.
B.2.1.2. Mechanistic Evidence
No relevant mechanistic data are available for these endpoints.
B.2.1.3. 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-10. Evidence pertaining to body weight effects in animals exposed to
ETBE
Reference and study design
Results (percent change compared to control)
Banton et al. (2011)
Female
rat, Sprague-Dawley
oral - gavage
female (10/group): 0, 250, 500,1,000 mg/kg-d
Dose
(mg/kg-d)
Bodv weight
daily for 28 consecutive days
0
250
500
1,000
3%
5%
-1%
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,1,000 mg/kg-d
Dose
(mg/kg-d)
Bodv weight
Dose
(mg/kg-d)
Bodv weight
daily for 16 wk beginning 10 wk prior to mating;
0
-
0
-
P0, female (24/group): 0,100, 300,
100
-4%
100
1%
1,000 mg/kg-d
daily for 17 wk beginning 10 wk prior to mating
300
-4%
300
1%
to lactation day 21
1,000
-7%
1,000
5%
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1,000 mg/kg-d
Dose
(mg/kg-d)
Final bodv
weight
Dose
(mg/kg-d)
Final bodv
weight
daily for a total of 18 wk beginning 10 wk before
0
-
0
-
mating until after weaning of the pups
P0, female (25/group): 0, 250, 500,
250
-1%
250
-7%
1,000 mg/kg-d
500
-3%
500
-2%
daily for a total of 18 wk beginning 10 wk before
1,000
-5%*
1,000
0%
mating until PND 21
Fl, male (25/group): 0, 250, 500,1,000 mg/kg-d
Fl, Male
Fl, Female
dams dosed daily through gestation and
lactation, then Fl doses beginning PND 22 until
weaning of the F2 pups
Dose
(mg/kg-d)
Final bodv
weight
Dose
(mg/kg-d)
Final bodv
weight
Fl, female (24-25/group): 0, 250, 500,
0
-
0
-
1,000 mg/kg-d
P0 dams dosed daily through gestation and
250
0%
250
-2%
lactation, then Fl dosed beginning PND 22 until
500
3%
500
-3%
weaning of the F2 pups
1,000
1%
1,000
2%
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
Dose
(mg/kg-d)
Final bodv
weight
daily for 23 wk
0
1,000
-5%*
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
Reference and study design
Results (percent change compared to control)
Mivata et al. (2013);JPEC (2008b)
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100, 400 mg/kg-d;
female (15/group): 0, 5, 25,100, 400 mg/kg-d
daily for 26 wk
Male Female
Dose Dose
(mg/kg-d) Body weight (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, 250,1,000 mg/kg-d; female
(60/group): 0, 250,1,000 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, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,10,000 ppm (0,
28,121, 542 mg/kg-d)a; female (50/group): 0,
625, 2,500, 10,000 ppm (0, 46, 171,
560 mg/kg-d)a
daily for 104 wk
Male Female
Dose Terminal bodv Dose Terminal bodv
(mg/kg-d) weight (mg/kg-d) weight
0 - 0 -
28 -4% 46 -10%*
121 -7%* 171 -11%*
542 -9%* 560 -17%*
JPEC (2008a)
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0,150, 500,1,500, 5,000 ppm (0,
627, 2,090, 6,270, 20,900 mg/m3)b; female (NR):
0, 150, 500, 1,500, 5,000 ppm (0, 627, 2,090,
6,270, 20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv 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, 5,000 ppm (0,
20,900 mg/m3)b; female (6/group): 0,
5,000 ppm (0, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk followed by a 28 day recovery period;
generation method, analytical concentration
and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
20,900 3% 20,900 4%
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
Reference and study design
Results (percent change compared to control)
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
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 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Body weight (mg/m3) Body weight
0 - 0 -
2,090 2% 2,090 -3%
7,320 4% 7,320 3%
20,900 2% 20,900 6%*
Medinskv et al. (1999); Bond et al. (1996b)
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, 50,00 ppm (0, 2,090,
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
2,090 0% 2,090 -2%
7,320 -1% 7,320 -1%
20,900 -3% 20,900 2%
Saito et al. (2013);JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b; female
(50/group): 0, 500, 1,500, 5,000 ppm (0, 2,090,
6,270, 20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d, 5 d/wk
for 104 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
2,090 -7%* 2,090 -6%*
6,270 -7%* 6,270 -10%*
20,900 -26%* 20,900 -23%*
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
6
This document is a draft for review purposes only and does not constitute Agency policy.
B-51 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table B-ll. Evidence pertaining to adrenal effects in animals exposed to ETBE
Reference and study design
Results (percent change compared to control)
Adrenal Weight
Hagiwara et al. (2011); JPEC (2008c)
rat, Fischer 344
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 16%* 19%*
Medinsky et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation - vapor
male (48/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(48/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male Female
Dose Dose
(mg/m3) Absolute weight (mg/m3) Absolute weight
0 - 0 -
2,090 11% 2,090 1%
7,320 9% 7,320 7%
20,900 34%* 20,900 18%*
Medinskv et al. (1999); Bond et al. (1996a)
mice, CD-I
inhalation - vapor
male (40/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(40/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6
hr/d, d/wk for 13 wk; generation
method, analytical concentration and
method were reported
Male Female
Dose Dose
(mg/m3) Absolute weight (mg/m3) Absolute weight
0 - 0 -
2,090 0% 2,090 -8%
7,320 50% 7,320 8%
20,900 0% 20,900 -8%
2 a4.18 mg/m3 = 1 ppm.
3 *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 (n): number evaluated from group.
6
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
Supplemental Information
Table B-12. Evidence pertaining to immune effects in animals exposed to ETBE
—ETBE
Reference and study design
Results (percent change compared to control)
Functional Immune Effects
Banton et al. (2011)
Female
rat, Sprague-Dawley
IgM antibody
IgM antibody
oral - gavage
Dose
forming cells/10A6 forming
female (10/group): 0, 250, 500,
(mg/kg-d)
spleen cells
cells/spleen
1,000 mg/kg-d
daily for 28 consecutive days
immunized i.v. 4 days prior to sacrifice
250
-21%
-20%
with sheep red blood cells
500
42%
36%
1,000
8%
8%
Immune Cell Populations
Li et al. (2011)
Male
mice, 129/SV
Dose
Number of
Number of
Number of
inhalation - vapor
(mg/m3)
CD3+T
CD4+T
CD8+T
male (6/group): 0, 500,1,750,
cells
cells
cells
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)a
u
whole body, 6 hr/d for 5 d/wk over
2,090
-18%*
-16%
-13%
6 wk; generation method not
7,320
-16%
-11%
-14%
reported; analytical concentration
20,900
-21%*
-17%*
-25%
and method were reported
Li et al. (2011)
Male
mice, C57BL/6
Dose
Number of
Number of
Number of
inhalation - vapor
(mg/m3)
CD3+T
CD4+T
CD8+T
male (6/group): 0, 500,1,750,
cells
cells
cells
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)a
u
whole body, 6 hr/d for 5 d/wk over
2,090
-14%
-15%
-12%
6 wk; generation method not
7,320
-13%
-11%
-13%*
reported; analytical concentration
20,900
-24%*
-23%*
-23%*
and method were reported
Li et al. (2011)
Male
mice, C57BL/6
Number of
Number of
Number of
inhalation - vapor
Dose
CD3+T
CD4+ T-
CD8+T
male (5/group): 0, 500,1,750,
(mg/m3)
cells
cells
cells
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)a
u
whole body, 6 hr/d for 5 d/wk over
2,090
-9%
-11%
-8%
13 wk; generation method not
7,320
-17%*
-28%*
-12%
reported; analytical concentration
20,900
-24%*
-37%*
-20%
and method were reported
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
Reference and study design
Results (percent change compared to control)
Spleen Weight
Banton et al. (2011)
rat, Sprague-Dawley
oral - gavage
female (10/group): 0, 250, 500,
1,000 mg/kg-d
daily for 28 consecutive days
Female
Dose
(mg/kg-d)
0
Absolute Relative
weight weight
250
-3%
0%
500
-15%
-18%
1,000
-9%
0%
P0, Male
P0, Female
Dose
Absolute
Relative
Dose
Absolute
Relative
(mg/kg-d)
weight
weight
(mg/kg-d)
weight
weight
0
-
-
0
-
-
100
-4%
-1%
100
0%
-2%
300
-2%
2%
300
-2%
-3%
1,000
0%
8%
1,000
-1%
-5%
Fuiiietal. (2010); JPEC (2008d)
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,
1,000 mg/kg-d
daily for 16 wk beginning 10 wk prior
to mating
P0, female (24/group): 0,100, 300,
1,000 mg/kg-d
daily for 17 weeks beginning 10 weeks
prior to mating to lactation day 21
Hagiwara et al. (2011); JPEC (2008c)
rat, Fischer 344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
daily for 23 wk
Male
Dose
(mg/kg-d)
0
Absolute Relative
weight weight
1,000
-5%
0%
Male
Female
Dose
Absolute
Relative
Dose
Absolute
Relative
(mg/kg-d)
weight
weight
(mg/kg-d)
weight
weight
0
-
-
0
-
-
628
-3%
-35%
46
-35%
2%
121
19%
3%*
171
-1%
28%
542
39%
-45%
560
-50%*
55%*
Male
Female
Dose
Absolute
Relative
Dose
Absolute
Relative
(mg/m3)
weight
weight
(mg/m3)
weight
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%
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121,
542 mg/kg-d)a; female (50/group): 0,
625, 2,500, 10,000 ppm (0, 46, 171,
560 mg/kg-d)a
daily for 104 wk
JPEC (2008a)
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0,150, 500,1,500,
5,000 ppm (0, 627, 2,090, 6,270,
20,900 mg/m3)b; female (NR): 0,150,
500, 1,500, 5,000 ppm (0, 627, 2,090,
6,270, 20,900 mg/m3)
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method
were reported
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
Reference and study design
Results (percent change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
Dose
Absolute
Relative
Dose
Absolute
Relative
inhalation - vapor
male (6/group): 0, 5,000 ppm (0,
(mg/m3)
0
weight
weight
(mg/m3)
0
weight
weight
20,900 mg/m3)b; female (6/group): 0,
5,000 ppm (0, 20,900 mg/m3)b
20,900
10%
6%
20,900
6%
0%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk followed by a 28 day
recovery period; generation method,
analytical concentration and method
were reported
Saito et al. (2013); JPEC (2010b)
Male
Female
rat, Fischer 344
Dose
Absolute
Relative
Dose
Absolute
Relative
inhalation - vapor
male (50/group): 0, 500,1,500,
(mg/m3)
0
weight
weight
(mg/m3)
0
weight
weight
5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b; female (50/group):
2,090
4%
15%
2,090
5%
30%
0, 500, 1,500, 5,000 ppm (0, 2,090,
6,270
32%
43%*
6,270
-39%
-31%
6,270, 20,900 mg/m3)b
dynamic whole body inhalation;
20,900
17%
66%*
20,900
-43%*
-25%
6 hr/d, 5 d/wk for 104 wk; generation
method, analytical concentration and
method were reported
Medinsky et al. (1999); Bond et al.
Male
Female
(1996b)
Dose
Absolute
Dose
Absolute
rat, Fischer 344
inhalation - vapor
(mg/m3)
0
weight
(mg/m3)
0
weight
male (48/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
2,090
6%
2,090
-3%
20,900 mg/m3)b; female (48/group):
7,320
3%
7,320
3%
0, 500, 1,750, 5,000 ppm (0, 2,090,
7,320, 20,900 mg/m3)b
20,900
5%
20,900
0%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method
were reported
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
Reference and study design
Results (percent change compared to control)
Medinskv et al. (1999); Bond et al.
Male
Female
(1996a)
Dose
Absolute
Dose
Absolute
mice, CD-I
(mg/m3)
weight
(mg/m3)
weight
inhalation - vapor
0
0
male (40/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
2,090
-5%
2,090
-11%
20,900 mg/m3)b; female (40/group):
7,320
0%
7,320
-2%
0, 500, 1,750, 5,000 ppm(0, 2,090,
20,900
-15%
20,900
-11%
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method
were reported
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 (n): number evaluated from group.
6
This document is a draft for review purposes only and does not constitute Agency policy.
B-56 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table B-13. Evidence pertaining to mortality in animals exposed to ETBE
Reference and study design
Results (percent change compared to control)
Maltoni et al. (1999)
rat, Sprague-Dawley
oral - gavage
male (60/group): 0, 250,1,000 mg/kg-d;
female (60/group): 0, 250,1,000 mg/kg-d
4 d/wk for 104 wk; observed until natural
death
Male Female
Dose (mg/m3) Survival at 104 Dose (mg/m3) Survival at 104
wk wk
0 - 0 -
250 -8% 250 -8%
1,000 -54% 1,000 18%
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121, 542 mg/kg-d)a;
female (50/group): 0, 625, 2,500,
10,000 ppm (0, 46,171, 560 mg/kg-d)a
daily for 104 wk
Male Female
Dose (mg/kg-d) % survival Dose (mg/kg-d) % survival
0 - 0 -
628 -3% 46 3%
121 -11% 171 6%
542 -11% 560 6%
Saito et al. (2013);JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm
(0, 2,090, 6,270, 20,900 mg/m3)b; female
(50/group): 0, 500, 1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d,
5 d/wk for 104 wk; generation method,
analytical concentration and method were
reported
Male Female
Dose (mg/m3) Survival at 104 Dose (mg/m3) Survival at 104
wk wk
0 - 0 -
2,090 -14% 2,090 3%
6,270 -9% 6,270 -21%*
20,900 -32%* 20,900 -21%*
2 Conversion performed by study authors.
3 b4.18 mg/m3 = 1 ppm.
4 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
5 for controls, no response relevant; for other doses, no quantitative response reported.
6 (n): number evaluated from group.
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
¦ = 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 (C)
F1 Male rat; GD 0-adult (C)
F1 Female rat; GD 0-adult (C)
Male rat; 23wks [D]
Female rat; 26wks (F)
Male rat; 26wks (F) ¦
B-
B-
0 B ~
B B El
~ DO
B—B-
B—B—B
~ ~ ~
B—B—B
a B O
a b a
chronic
Female rat; 104wks (G)
Male rat; 104wks (G)
Female rat; 104-wks [E)
Male rat; 104wks (E)
B 0
B-
10 100 1,000 10,000
Dose (mg/kg-day)
Sources: (A) Banton et al, 2011 (B) Fujiiet al, 2010; [PEC, 2008e (C) Gaoua, 2004b (O) 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-15. Exposure-response array of body weight effects following oral
exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
B-58 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
chronic
Female rats; 13 wks (B)
Male rats; 13 wks (B)
Female mice; 13 wks (B)
Male mice; 13 wks (B)
Female rats; !Q4wks (C)
Male rats; 104wks (C)
a—~ ¦ ~
B B
~
B B
B B ~
B Bi—~
1 10 100 1,000 10,000 100,000
Exposure Concentration (mg/m3)
""significantly increased body weight
Sources: (A) JPEC, 2008b (B)Medinsky etal, 1999; Bondet al, 1996 (C) Saito et al., 2013; JPEC,2010b
Figure B-16. Exposure-response array of body weight effects following
inhalation exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
B-59 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
33
34
35
Supplemental Information—ETBE
B.2.2. Genotoxicity Studies
B.2.2.1. Bacterial Systems
Mutagenic potential of ETBE has been tested by Zeiger etal. Q9921 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. One dose of 10,000 [ig/plate was tested
using different Salmonella strains including TA9 7, TA 9 8, TA10 0, TA15 3 5. 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 tert-butanol are discussed below, and the summary of the data is provided in
Table B-14.
B.2.2.2. In Vitro Mammalian Studies
Limited available studies (two) in in vitro mammalian systems were unpublished reports.
Vergnes and Kubena (1995b) evaluated the mutagenicity of ETBE using the hypoxanthine-guanine
phosphoribosyl transferase (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 author [(Vergnes and Kubena. 1995b) unpublished report] studied the
clastogenic potential of ETBE in vitro using chromosome aberration assay in Chinese hamster ovary
cells. The cells were exposed from 100 to 5,000 |J.g/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.
B.2.2.3. In Vivo Animal Studies
In vivo studies were conducted by same authors that tested ETBE for in vitro genotoxicity.
Vergnes and Kubena f!995al. unpublished report, performed an in vivo bone marrow
micronucleus (MN) test in mice in response to ETBE exposure. Male and female CD-I mice
(5 animals/sex/group) were exposed to ETBE by inhalation at target concentrations of 0, 400,
2,000, and 5,000 ppm (0,1,671, 8,357, and 20,894 mg/m3) for 6 hours/day, for 5 days. Following
treatment, polychromatic erythrocytes (PCE) from bone marrow were analyzed for micronucleus
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
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 co-authors, four animal studies were conducted by the JPEC in
rats using different routes of exposure (oral, inhalation, intraperitoneal or drinking water) to detect
micro nucleus as a result of exposure to ETBE [1PEC f2007c): 1PEC f2007a): 1PEC f2007d): 1PEC
(2007b) published as Noguchi etal. (2013)].
The first two studies (oral and intraperitoneal injection) were part of an acute (2 days)
exposure. In the first study, both male and female F344 rats (5 animals/sex/dose group) were
administered ETBE (99.3% pure) via gavage at doses of 0, 500,1,000, or 2,000 mg/kg-day
separated by 24 hours in olive oil [(IPEC. 2007a). unpublished report]. 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 polychromatic erythrocytes/total erythrocytes. There were
no treatment-related effects on the number of MNPCE or the ratio of PCE/total erythrocytes. 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
animals/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: IPEC.
2007b). 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/total erythrocytes. In addition, no dose-dependent tendencies for
increase in MNPCE/PCE or alterations in the ratios of PCE/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 where ETBE effects 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 animals/sex/dose group) were administered
drinking water containing 0,1,600, 4,000, or 10,000 ppm ETBE for 13 weeks fNoguchi etal.. 2013:
IPEC. 2007d). 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
the ratio of PCE/total erythrocytes. There were no treatment-related effects on the number of
MNPCEs or the ratio of PCE/total erythrocytes.
In the second 13-week study (inhalation), male and female F344 rats (10 animals/sex/dose
group) were exposed to ETBE (99.2-99.3% pure) through whole-body inhalation exposure at 0,
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Supplemental Information—ETBE
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 etal.. 2013: IPEC. 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/total erythrocytes. ETBE was determined to be negative for
micronuclei induction in rat bone marrow cells after a 13-week inhalation exposure.
Weng et al. conducted several studies evaluating the differential 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., to 0, 500,1,750 and 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 et al.. 20111.
Weng etal. (2012) studied the differential genotoxic effects of subchronic exposure to ETBE
in the liver of C57BL/6 wild-type and AIdh2 knockout mice. DNA strand breaks in the hepatocytes
of male and female with different AIdh2 genotypes were determined using 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 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.
In another study by the same authors (Weng etal.. 2013). the 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 (hOGGl)-modified oxidative base modification, and 8-
hydroxydeoxyguanosine. The mice (wild-type and knockout, males and females) were exposed to 0,
500,1,750 and 5,000 ppm ETBE for 6 hours/day, 5 days/week for 13 weeks. Peripheral blood
samples were obtained and processed to detect micronucleated reticulocytes (MN-RETs) and
micronuclei in the mature normochromatic erythrocyte population (MN-NCE). The results indicate
that ETBE significantly affected frequencies of MN-RETs in male and female mice. In knockout male
mice, the frequencies of MN-RETs of 1,750 ppm 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 MN-RETs than that of control group. In female mice,
there was no difference in the frequencies of MN-RETs between exposure groups and control group
in wild-type mice. In the same exposure group (5,000 ppm), the knock-out mice had a higher
frequency of MN-RETs compared to 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 (hOGGl)-modified oxidative base modification were measured in
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 sperm collected from the left caudia epididymis. In addition to the 13-week protocol used in the
2 other studies, Wengetal. (2014) also included an additional 9-week study where the male mice
3 (wild-type, knockout, and heterogeneous [HT]) were exposed to 0, 50, 200 and 500 ppm ETBE for
4 6 hours/day, 5 days/week for 9 weeks. In the 13-week study, there were significant increases in
5 damage in all three exposure groups in the knockout male mice, but only in the two highest dose
6 groups in the wild-type males. In the 9-week study, there was no change in the wild-type mice, but
7 both the heterogeneous and the knockout mice had significant increases in the two highest doses.
8 Table B-14. Summary of genotoxicity (both in vitro and in vivo) studies of
9 ETBE
Species
Test System
Dose/Cone.
Results3
Comments
Reference
Bacterial systems
-S9
+S9
Salmonella
typhimuriu
m (TA97,
TA98,
TA100,
TA1535)
Mutation
Assay
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 |Jg/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,
1670, 8,360,
20,900 mg/m3)b
Whole body Inhalation,
6 hr/d, 5 d, 5
animals/sex/group
Vergnes and Kubena
(1995a)
(unpublished report)
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 500, 1,000,
2,000 mg/kg-d
Oral gavage, 24 hr apart,
2 d, 5 animals/sex/group
JPEC (2007b)
(unpublished report)
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 250, 500, 1,000,
2,000 mg/kg-d
Intraperitoneal injection,
24 hr apart, 2 d, 5
animals/sex/group
Noguchi et al.
(2013); JPEC
(2007b), unpublished
report
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Supplemental Information—ETBE
Species
Test System
Dose/Cone.
Results3
Comments
Reference
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 1600, 4000,
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
animals/sex/group
Noguchi et al.
(2013); JPEC (2007c),
unpublished report
Fischer 344
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 hr/d, 5 d/wk, 13 wk. 10
animals/sex/group
Noguchi et al.
(2013); JPEC (2007c),
unpublished report
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay),
leukocytes
0, 500,1,750 and
5,000 ppm
Male -
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2011)
Female
WT/KO
-/+d
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay)
0, 500,1,750 and
5,000 ppm
Male -
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2012)
Female
WT/KO
-/+d
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
Micronucleus
assay,
erythrocytes
0, 500,1,750 and
5,000 ppm
Male*
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2013)
Female*
WT/KO
-/+
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 50, 200 and
500 ppm
WT/HT/
KO
-/+/+
Whole body inhalation,
6 hr/d, 5 d/wk, 9 wk
Weng et al. (2014)
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 500,1,750 and
5,000 ppm
WT/KO
+/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2014)
1 a+ = positive; - = negative; (+), equivocal.
2 b4.18 mg/m3 = 1 ppm.
3 Conversions performed by study authors.
4 dPositive in highest dose tested.
5 *When the data of ETBE-induced MN-RETs (micronucleated reticulocytes) were normalized with corresponding
6 control, the effect disappeared.
This document is a draft for review purposes only and does not constitute Agency policy.
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B.2.2.4. 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 co-authors seem to illustrate 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: (a) the coverage of the studies
across the genotoxicity tests needed for proper interpretation of the weight of evidence of the data;
(b) the quality of the available data. With respect to the array of types of genotoxicity tests
available, ETBE has only been tested in one bacterial assay. Limited (two) studies are available with
respect to in vitro studies. Existing in vivo studies have all been tested only for the micro nucleus
assay, DNA strand breaks, or both. Key studies in terms of chromosomal aberrations, DNA adducts
etc are missing. It should also be noted that the few existing studies are unpublished reports lacking
peer review. Given the above limitations; significant deficiencies; and sparse database both in terms
of quality and quantity; it is implicit that the database is inadequate or insufficient to draw any
conclusions on the effect of ETBE with respect to genotoxicity.
B.3. SUPPLEMENTAL ORGAN WEIGHT DATA
B.3.1. Relative Kidney Weight Data
Table B-15. Evidence pertaining to relative kidney weight effects in animals
exposed to ETBE
Reference and study design
Results (percent change compared to control)
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,
Dose
Dose
(mg/kg-d)
Relative weight
(mg/kg-d)
Relative weight
1,000 mg/kg-d
0
-
0
-
daily for 16 wk beginning 10 wk prior to
mating
100
8%*
100
-3%
P0, female (24/group): 0,100, 300,
300
12%*
300
-1%
1,000 mg/kg-d
daily for 17 weeks beginning 10 weeks prior
1,000
26%*
1,000
2%
to mating to lactation day 21
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
PO, male (25/group): 0, 250, 500,
Dose
(mg/kg-d)
Relative weight
Dose
(mg/kg-d)
Relative weight
1,000 mg/kg-d
0
-
0
-
daily for a total of 18 wk beginning 10 wk
before mating until after weaning of the
250
11%*
250
9%
pups
500
18%*
500
5%
P0, female (25/group): 0, 250, 500,
1,000 mg/kg-d
1,000
28%*
1,000
3%
daily for a total of 18 wk beginning 10 wk
Fl, Male
Fl, Female
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
Dose
(mg/kg-d)
Relative weight
Dose
(mg/kg-d)
Relative weight
and lactation, then Fl doses beginning PND
0
-
0
-
22 until weaning of the F2 pups
250
10%*
250
6%
500
19%*
500
6%
1,000
58%*
1,000
10%*
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
Dose
(mg/kg-d)
Relative weight
daily for 23 wk
0
1,000
25%*
Mivata et al. (2013);JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100,
Dose
(mg/kg-d)
Relative weight
Dose
(mg/kg-d)
Relative weight
400 mg/kg-d; female (15/group): 0, 5, 25,
0
-
0
-
100, 400 mg/kg-d
8%
7%
daily for 26 wk
O
O
25
6%
25
4%
100
12%*
100
11%*
400
21%*
400
15%*
Suzuki et al. (2012); JPEC (2010a)
Male
Female
rat, Fischer 344
oral - water
Dose (mg/kg-d)
Relative weight
Dose (mg/kg-d)
Relative weight
male (50/group): 0, 625, 2,500,10,000 ppm
0
-
0
-
(0, 28,121, 542 mg/kg-d)a; female
(50/group): 0, 625, 2,500, 10,000 ppm (0,
28
0%
46
13%*
46,171, 560 mg/kg-d )a
121
12%*
171
22%*
daily for 104 wk
542
31%*
560
37%*
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
Dose (mg/m3)
Relative weight
Dose (mg/m3)
Relative weight
male (NR): 0,150, 500,1,500, 5,000 ppm
0
-
0
-
(0, 627, 2,090, 6,270, 20,900 mg/m3)b;
female (NR): 0,150, 500,1,500, 5,000 ppm
627
10%
627
8%
(0, 627, 2,090, 6,270, 20,900 mg/m3)
2,090
9%
2,090
7%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
6,270
20%*
6,270
12%*
analytical concentration and method were
20,900
24%*
20,900
20%*
reported
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (6/group): 0, 5,000 ppm (0,
Dose
(mg/m3)
Relative weight
Dose
(mg/m3)
Relative weight
20,900 mg/m3)b; female (6/group): 0,
0
-
0
-
5,000 ppm (0, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d,
20,900
15%*
20,900
5%
5 d/wk for 13 wk followed by a 28 day
recovery period; generation method,
analytical concentration and method were
reported
Saito et al. (2013); JPEC (2010b)
Male
Female
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm
Dose
(mg/m3)
Relative weight
Dose
(mg/m3)
Relative weight
(0, 2,090, 6,270, 20,900 mg/m3)b; female
0
-
0
-
(50/group): 0, 500, 1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b
2,090
19%*
2,090
11%*
dynamic whole body inhalation; 6 hr/d,
6,270
26%*
6,270
16%*
5 d/wk for 104 wk; generation method,
analytical concentration and method were
20,900
66%*
20,900
51%*
reported
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
6
7
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
2 Table B-16. Evidence pertaining to absolute liver weight effects in animals
3 exposed to ETBE
Reference and study design
Results (percent change compared to control)
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,1,000 mg/kg-d
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
daily for 16 wk beginning 10 wk prior to mating
0
-
0
-
P0, female (24/group): 0,100, 300,1,000 mg/kg-d
daily for 17 wk beginning 10 wk prior to mating to
100
-3%
100
-1%
lactation day 21
300
-1%
300
3%
1,000
13%*
1,000
14%*
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1,000 mg/kg-d
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
daily for a total of 18 wk beginning 10 wk before
0
-
0
-
mating until after weaning of the pups
P0, female (25/group): 0, 250, 500,1,000 mg/kg-d
250
2%
250
-1%
daily for a total of 18 wk beginning 10 wk before
500
2%
500
4%
mating until PND 21
Fl, male (25/group): 0, 250, 500,1,000 mg/kg-d
1,000
17%*
1,000
6%
P0 dams dosed daily through gestation and
Fl, Male
Fl, Female
lactation, then Fl doses beginning PND 22 until
weaning of the F2 pups
Fl, female (24-25/group): 0, 250, 500,
Dose
Absolute
Dose
Absolute
(mg/kg-d)
weight
(mg/kg-d)
weight
1,000 mg/kg-d
0
-
0
-
P0 dams dosed daily through gestation and
lactation, then Fl dosed beginning PND 22 until
250
0%
250
1%
weaning of the F2 pups
500
14%*
500
3%
1,000
27%*
1,000
10%*
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
Dose
(mg/kg-d)
Absolute
weight
daily for 23 wk
0
1,000
21%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
Mivata et al. (2013); JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100, 400 mg/kg-d;
Dose
(mg/kg-d)
Absolute
weight
Dose
(mg/kg-d)
Absolute
weight
female (15/group): 0, 5, 25,100, 400 mg/kg-d
0
-
0
-
daily for 26 wk
5
-2%
5
-4%
25
7%
25
-1%
100
4%
100
2%
400
19%
400
9%
Suzuki et al. (2012); JPEC (2010a)
Male
Female
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,10,000 ppm (0, 28,
Dose
Absolute
Dose
Absolute
(mg/kg-d)
weight
(mg/kg-d)
weight
121, 542 mg/kg-day)a; female (50/group): 0, 625,
0
-
0
-
2,500, 10,000 ppm (0, 46,171, 560 mg/kg-day)a
daily for 104 wk
28
-11%*
46
-5%
121
-4%
171
-2%
542
2%
560
-10%
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0, 150, 500, 1,500, 5,000 ppm (0, 627,
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
2,090, 6,270, 20,900 mg/m3)b; female (NR): 0,150,
0
-
0
-
500, 1,500, 5,000 ppm (0, 627, 2,090, 6,270,
627
5%
627
-3%
20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk for
2,090
6%
2,090
-8%
13 wk; generation method, analytical
concentration and method were reported
6,270
4%
6,270
-2%
20,900
2%
20,900
5%
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b;
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
female (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b
0
-
0
-
dynamic whole body chamber; 6 hr/d, 5 d/wk for
20,900
13%
20,900
11%
13 wk followed by a 28 day recovery period;
generation method, analytical concentration and
method were reported
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and study design
Results (percent change compared to control)
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b; female (50/group):
0, 500, 1,500, 5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d, 5 d/wk for
104 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Absolute Dose Absolute
(mg/m3) weight (mg/m3) weight
0 - 0 -
2,090 1% 2,090 -3%
6,270 11%* 6,270 -8%
20,900 10% 20,900 1%
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
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 hr/d, 5 d/wk for
13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Absolute Dose Absolute
(mg/m3) weight (mg/m3) weight
0 - 0 -
2,090 6% 2,090 2%
7,320 14%* 7,320 9%
20,900 32%* 20,900 26%*
Medinskv et al. (1999); Bond et al. (1996a)
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 hr/d, 5 d/wk for
13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Absolute Dose Absolute
(mg/m3) weight (mg/m3) weight
0 - 0 -
2,090 4% 2,090 2%
7,320 13%* 7,320 19%*
20,900 18%* 20,900 33%*
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
6
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Supplemental Information—ETBE
1 APPENDIX C. DOSE-RESPONSE MODELING FOR
2 THE DERIVATION OF REFERENCE VALUES FOR
3 EFFECTS OTHER THAN CANCER AND THE
4 DERIVATION OF CANCER RISK ESTIMATES
5 C.l. Benchmark Dose Modeling Summary
6 This appendix provides technical detail on dose-response evaluation and determination of
7 points of departure (PODs) for relevant toxicological endpoints. The endpoints were modeled using
8 EPA's Benchmark Dose Software (BMDS, version 2.2). Sections C.l.1.1 and C.l.1.2 (non-cancer) and
9 Section C.l.2 (cancer) describe the common practices used in evaluating the model fit and selecting
10 the appropriate model for determining the POD, as outlined in the Benchmark Dose Technical
11 Guidance Document U.S. EPA (2012). In some cases, it might be appropriate to use alternative
12 methods based on statistical judgment; exceptions are noted as necessary in the summary of the
13 modeling results.
14 C.l.l. Non-cancer Endpoints
15 C.l.1.1. Evaluation of Model Fit
16 For each dichotomous endpoint, BMDS dichotomous models1 were fitted to the data using
17 the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-square
18 goodness-of-fit test (x2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess
19 model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the
20 vicinity of the benchmark response (BMR).
21 For each continuous endpoint, BMDS continuous models2 were fitted to the data using the
22 maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model,
23 first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test
24 2 was not rejected (x2 p-value > 0.10), the model was fitted to the data assuming constant variance.
25 If Test 2 was rejected (x2 p-value < 0.10), the variance was modeled as a power function of the
26 mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS
1 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.
2 Unless 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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2
3
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9
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12
Supplemental Information—ETBE
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.
C.1.1.2. Model Selection
For each endpoint, the BMDL estimate (95% lower confidence limit on the benchmark dose
(BMD), 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
estimates were "sufficiently close," that is, differed by at most three-fold, the model selected was
the one that yielded the lowest AIC value. If the BMDL estimates were not sufficiently close, the
lowest BMDL 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
1 Table C-l. Non-cancer endpoints selected for dose-response modeling for
2 ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
ORAL
Urothelial
hyperplasia of the
renal pelvis
Suzuki et al. (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
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 relative
kidney weight
Mivata 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
0.52 ± 0.04
0.56 ±0.05
0.55 ± 0.04
0.58 ±0.07
0.63 ±0.07
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 relative
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
0.54 ± 0.06
0.58 ±0.07
0.56 ± 0.04
0.6 ±0.06
0.62 ±0.06
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased relative
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
0.59628 ±
0.053
0.66246 ±
0.052
0.70569 ±
0.076
0.76341 ±
0.063
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
Increased relative
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
0.70673 ±0.11
0.77143 ±
0.198
0.74388 ±0.16
0.72691 ±0.06
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 relative
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
0.57406 ±
0.043
0.63368 ±
0.046
0.68399 ±
0.068
0.90836 ±
0.958
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased relative
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
0.69219 ±
0.061
0.73338 ±
0.075
0.7305 ±
0.048
0.76202 ±
0.097
Increased absolute
kidney weight
Fuiii et al. (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
Fuiii et al. (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
Fuiii et al. (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
Fuiii et al. (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
INHALATION
Urothelial
hyperplasia of the
renal pelvis
Saito et al. (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
This document is a draft for review purposes only and does not constitute Agency policy.
C-5 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
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 relative
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
0.584 ±
0.042
0.644 ±
0.064
0.638 ±
0.046
0.7 ±0.073
0.726 ±
0.047
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 relative
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
0.545 ±
0.04
0.587 ±
0.056
0.583 ±
0.035
0.613 ±0.06
0.656 ±
0.043
Increased absolute
kidney weight
Medinskv et al.
(1999); Bond et al.
(1996b)
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
This document is a draft for review purposes only and does not constitute Agency policy.
C-6 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased absolute
kidney weight
Medinskv et al.
(1999); Bond et al.
(1996b)
Female
F344 rats
Exposure
concentration
(ppm)
0
500
1,750
5,000
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
1
2
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 C.l.1.3. Modeling Results
2 Below are tables summarizing the modeling results for the noncancer endpoints modeled.
3 Oral Exposure Endpoints
4 Table C-2. Summary of BMD modeling results for urothelial hyperplasia of the
5 renal pelvis in male F344 rats exposed to ETBE in drinking water for
6 104 weeks flPEC. 201 Oal modeled with doses as mg/kg-day (calculated by
7 study authors); BMR = 10% extra risk
Model3
Goodness of fit
BMDioPct
(mg/kg-d)
BMDLioPct
(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
1.00E-03
139.54
217
177
LogLogistic
0.264
127.28
85.3
49.5
Probit
0.0015
138.30
197
162
LogProbit
0.374
126.14
85.8
51.3
Weibull
0.202
128.00
85.7
60.7
Multistage 3°b
Multistage 2°c
0.395
126.07
79.3
60.5
Quantal-Linearc
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-day were 0.000,
-1.377,1.024, and -0.187, respectively.
bFor the Multistage 3° model, the beta coefficient estimates were 0 (boundary of parameters space), and the
model reduced to the Multistage 2° model.
cThe Multistage 2° 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
Quantal Linear
BM D L BMD
13:10 09/10 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 + (l-background)*[l-EXP(-slope*dose)]
Benchmark Dose Computation.
BMR = 10% Extra risk
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)
# Param's
Deviance
Test d.f.
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
<.0001
AIC: =
126.074
This document is a draft for review purposes only and does not constitute Agency policy.
C-9 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
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 ChiA2= 2.98 d.f= 3 P-value = 0.3948
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-3. Summary of BMD modeling results for increased absolute kidney
2 weight in male S-D rats exposed to ETBE by daily gavage for 26 weeks (Mivata
3 etal.. 2013: IPEC. 2008d): 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
0.752
-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°e
Linear'
0.774
-48.055
176
115
Polynomial 3°g
0.774
-48.055
176
115
aModeled variance case presented (BMDS Test 2 p-value = <0.0001), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-day 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
the Linear and Polynomial 3° models appear equivalent, however differences exist in digits not displayed in the
table.
gThe Linear model, Polynomial 2° and 3° 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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3
4
5
6
7
8
9
10
11
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13
Supplemental Information—ETBE
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDI
4.6
4.4
4.2
CD
£ 4
o
Q_
(f>
CD
cz 3.8
CO
CD
S 3.6
3.4
3.2
3
0 50 100 150 200 250 300 350 400
dose
15:56 05/15 2014
Figure C-2. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Polynomial Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = beta_0 + beta_l*dose
A modeled variance is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 176.354
BMDL at the 95% confidence level = 114.829
Parameter Estimates
BMDL
Variable
Estimate
Default Initial
Parameter Values
la
-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-12 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
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 C-4. Summary of BMD modeling results for increased relative kidney
2 weight in male S-D rats exposed to ETBE by daily gavage for 26 weeks (Mivata
3 etal.. 2013: IPEC. 2008d): 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)b
0.0262
-339.53
242
174
No model adequately fit the data.
Exponential (M3)b
0.0262
-339.53
242
174
Exponential (M4)
Exponential (M5)c
0.0472
-340.67
113
45.6
Hill
0.0481
-340.71
112
47.2
Power
<0.0001
-315.18
40,000
4.00E-13
Polynomial 3°d
Polynomial 2°f
Linear
0.03
-339.83
231
161
aModeled variance case presented (BMDS Test 2 p-value = 0.0648, BMDS Test 3 p-value = 0.596), no model was
selected as a best-fitting model.
bThe Exponential (M2) model and the Exponential (M3) models appear equivalent, however differences exist in
digits not displayed in the table.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Polynomial 3° model, the b3 and or coefficient estimates was 0 (boundary of parameters space), and the
model reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-5. Summary of BMD modeling results for increased absolute kidney
2 weight in female S-D rats exposed to ETBE by daily gavage for 26 weeks
3 (Mivata et al.. 2013: IPEC. 2008d): BMR = 10% relative deviation from the
4 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°e
Polynomial 2°f
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-day 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
5
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
Supplemental Information—ETBE
Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
Exponential
2.2
2.1
2
9
8
BMD
BMD
0 50 100 150 200 250 300 350 400
dose
16:35 05/15 2014
Figure C-3. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Exponential Model. (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 223.57
BMDL at the 95% confidence level = 56.8917
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Ina
-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.
C-16 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-17 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-6. Summary of BMD modeling results for increased relative kidney
2 weight in female S-D rats exposed to ETBE by daily gavage for 26 weeks
3 (Mivata et al.. 2013: IPEC. 2008d): BMR = 10% relative deviation from the
4 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
0.111
-343.15
374
253
The Hill model is selected based
on lowest BMDL
Exponential (M4)
Exponential (M5)c
0.163
-343.53
170
41.1
Hill
0.158
-343.47
191
20.1
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.116
-343.25
369
244
aConstant variance case presented (BMDS Test 2 p-value = 0.335), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-day were -0.917,1.47, -0.738, 0.242, and -0.054,
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 row 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
5
This document is a draft for review purposes only and does not constitute Agency policy.
C-18 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hil
0.66
0.64
0.62
0.6
0.58
0.56
0.54
0.52
BMDL
0.5
BMD
0
50
100
150
200
250
300
350
400
16:44 05/15 2014
Figure C-4. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 190.577
BMDL at the 95% confidence level = 20.0557
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.00339206
0.00346
rho
n/a
0
intercept
0.553785
0.54
V
0.0828955
0.08
n
1
0.214814
k
94.6956
137.5
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
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
15
0.54
0.554
0.06
0.0582
-0.917
5
15
0.58
0.558
0.07
0.0582
1.47
25
15
0.56
0.571
0.04
0.0582
-0.738
100
15
0.6
0.596
0.06
0.0582
0.242
400
15
0.62
0.621
0.06
0.0582
-0.054
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
177.580484
6
-343.160967
A2
179.862753
10
-339.725506
A3
177.580484
6
-343.160967
fitted
175.736902
4
-343.473804
R
169.280788
2
-334.561576
3 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
21.1639
8
0.006724
Test 2
4.56454
4
0.335
Test 3
4.56454
4
0.335
Test 4
3.68716
2
0.1582
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-20 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-7. Summary of BMD modeling results for increased absolute kidney
2 weight in PO male S-D rats exposed to ETBE by daily gavage for a total of 18 wk
3 beginning 10 wk before mating until after weaning of the pups (Gaoua.
4 2004a); 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°e
Polynomial 2°f
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-day 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° model, the b3 coefficient estimates was 0 (boundary of parameters space, and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
5
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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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Hill
4.6
3.4
O 200 400 600 800 1'
dose
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.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 243.968
BMDL at the 95% confidence level = 93.9617
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
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
Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
BMDL
This document is a draft for review purposes only and does not constitute Agency policy.
C-22 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
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
1 Table C-8. Summary of BMD modeling results for increased relative kidney
2 weight in PO male S-D rats exposed to ETBE by daily gavage for a total of 18 wk
3 beginning 10 wk before mating until after weaning of the pups (Gaoua.
4 2004a); 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
0.0632
-449.45
415
355
The Hill model was selected
based on lowest AIC.
Exponential (M4)
Exponential (M5)c
0.871
-452.95
228
150
Hill
0.936
-452.97
224
137
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.127
-450.86
378
316
aConstant variance case presented (BMDS Test 2 p-value = 0.180), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-day were -0.0131, 0.0533, -0.0566, and 0.0164,
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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
5
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
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMD
0 200 400 600 800 1000
dose
1 15:07 05/15 2014
2 Figure C-6. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in mg/kg-day.
4 Hill Model. (Version: 2.17; Date: 01/28/2013)
5 The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
6 A constant variance model is fit
7 Benchmark Dose Computation.
8 BMR = 10% Relative deviation
9 BMD = 223.505
10 BMDL at the 95% confidence level = 137.393
11 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.00366216
0.0038145
rho
n/a
0
intercept
0.596439
0.59628
V
0.345283
0.16713
n
1
0.221145
k
1,070.38
649.462
12
13
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 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
25
0.596
0.596
0.053
0.0605
-0.0131
250
25
0.662
0.662
0.052
0.0605
0.0533
500
25
0.706
0.706
0.076
0.0605
-0.0566
1,000
25
0.763
0.763
0.063
0.0605
0.0164
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
230.488384
5
-450.976768
A2
232.931535
8
-449.86307
A3
230.488384
5
-450.976768
fitted
230.48514
4
-452.97028
R
195.370878
2
-386.741756
3 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
75.1213
6
<0.0001
Test 2
4.8863
3
0.1803
Test 3
4.8863
3
0.1803
Test 4
0.0064882
1
0.9358
4
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
1 Table C-9. Summary of BMD modeling results for increased absolute kidney
2 weight in PO female S-D rats exposed to ETBE by daily gavage for a total of 18
3 wk beginning 10 wk before mating until after weaning of the pups (Gaoua.
4 2004a); 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°
0.400
-212.81
1,409
1,035
Polynomial 2°
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-day 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.
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BT
Exponential
2.45
2.4
2.35
2.3
2.25
2.2
2.15
BMDL
BMiD
O
200
400
600
800
1 OOO
1200
1 400
1 600
1800
5 15:14 05/15 2014
6 Figure C-7. Plot of mean response by dose, with fitted curve for selected
7 model; dose shown in mg/kg-day.
8
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 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a * exp(sign * b * dose)
3 A constant variance model is fit
4
5 Benchmark Dose Computation.
6 BMR = 10% Relative deviation
7 BMD = 1,734.24
8 BMDL at the 95% confidence level = 1,030.08
9 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Ina
-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
10 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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
11 Likelihoods of Interest
Model
Log(likelihood)
# Param's
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
12 Tests of Interest
Test
-2* 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-28 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-10. Summary of BMD modeling results for increased relative kidney
2 weight in PO female S-D rats exposed to ETBE by daily gavage for a total of 18
3 wk beginning 10 wk before mating until after weaning of the pups (Gaoua.
4 2004a); 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 (M4)b
N/A
-283.41
1,258
829
No model adequately fit the data.
Exponential (M3)
N/A
-290.99
1,037
983
Exponential (M5)
N/Ac
-288.99
1,037
983
Hill
<0.0001
-276.90
errord
errord
Power
<0.0001
-296.86
1,648
1,056
Polynomial 3°
0.00528
-292.51
-9,999
976
Polynomial 2°
0.00236
-290.89
-9,999
945
Linear
1.92E-04
-285.88
40,622
errord
aModeled variance case presented (BMDS Test 2 p-value = <0.0001, BMDS Test 3 p-value = <0.0001), no model
was selected as a best-fitting model.
bFor the Exponential (M4) model, the estimate of c was 0 (boundary), and the model reduced to the Exponential
(M2) model.
cNo available degrees of freedom to calculate a goodness of fit value.
dBMD or BMDL computation failed for this model.
5
This document is a draft for review purposes only and does not constitute Agency policy.
C-29 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-ll. Summary of BMD modeling results for absolute kidney weight in
2 F1 male Sprague-Dawley rats exposed to ETBE by gavage in a two-generation
3 study (Gaoua. 2004b): 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)
6.30E-04
89.912
232
175
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Polynomial 3°
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°
0.374
77.965
318
235
Polynomial 2°
0.0943
79.973
330
251
Linear
<0.0001
96.039
263
179
aModeled variance case presented (BMDS Test 2 p-value = <0.0001), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-day were -0.584, 0.717, 0.225, and -0.837, respectively.
bNo available degrees of freedom to calculate a goodness of fit value.
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMD
8
Polynomial
BMDL
BMD
1 3:43 09/12 201 4
5 Figure C-8. Plot of mean response by dose, with fitted curve for selected
6 model; dose shown in mg/kg-day.
7 Polynomial Model. (Version: 2.19; Date: 06/25/2014)
8 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
This document is a draft for review purposes only and does not constitute Agency policy.
C-30
DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 A modeled variance is fit
2 Benchmark Dose Computation.
3 BMR = 10% Relative deviation
4 BMD = 318.084
5 BMDL at the 95% confidence level = 235.491
6 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
2.23248E-28
0
beta_3
0.00000000190507
0.000000000693333
7 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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
8 Likelihoods of Interest
Model
Log(likelihood)
# Param's
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
9
10
This document is a draft for review purposes only and does not constitute Agency policy.
C-31 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2* 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
2 Table C-12. Summary of BMD modeling results for relative kidney weight in F1
3 male Sprague-Dawley rats exposed to ETBE by gavage in a two-generation
4 study (Gaoua. 2004b): 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)
<0.0001
-298.20
249
194
No models provided an adequate
fit and a valid BMDL estimate,
therefore no model was selected.
Exponential (M3)
0.00994
-319.84
368
297
Exponential (M4)
<0.0001
-287.10
239
196
Exponential (M5)
N/Ab
-315.83
382
306
Hill
N/Ab
-315.82
382
317
Power
0.00326
-317.83
382
306
Polynomial 3°
0.0592
-322.92
352
281
Polynomial 2°
0.00360
-318.01
352
286
Linear
<0.0001
-291.10
239
196
aModeled variance case presented (BMDS Test 2 p-value = <0.0001, BMDS Test 3 p-value = 0.0558), no model was
selected as a best-fitting model.
bNo available degrees of freedom to calculate a goodness of fit value.
5 Table C-13. Summary of BMD modeling results for absolute kidney weight in
6 F1 female Sprague-Dawley rats exposed to ETBE by gavage in a two-
7 generation study (Gaoua. 2004b): 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)
Exponential (M3)
0.147
-178.46
1,016
679
Exponential (M4)
0.121
-178.16
980
654
This document is a draft for review purposes only and does not constitute Agency policy.
C-32 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Exponential (M5)
N/Ab
-176.44
1,019
613
model was selected based on
lowest AIC.
Hill
N/Ab
-176.44
1,019
611
Power
0.145
-178.44
1,019
666
Polynomial 3°
0.184
-178.80
1,001
684
Polynomial 2°
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-day were -0.05426, 0.8898, -1.173, and 0.3711,
respectively.
bNo available degrees of freedom to calculate a goodness of fit value.
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
Exponential
2.6
2.5
2.4
2.3
2.2
BM DL
BMD
O
200
400
600
800
1 000
1 13:47 09/12 2014
2 Figure C-9. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in mg/kg-day.
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-33 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a * exp(sign * b * 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
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
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
11
12
This document is a draft for review purposes only and does not constitute Agency policy.
C-34 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2* 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
2 Table C-14. Summary of BMD modeling results for relative kidney weight in F1
3 female Sprague-Dawley rats exposed to ETBE by gavage in a two-generation
4 study (Gaoua. 2004b): 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)
Exponential (M3)b
0.102
-412.25
1,064
702
No models provided an adequate
fit and a valid BMDL estimate,
therefore no model was selected.
Exponential (M4)
Exponential (M5)c
0.0333
-410.28
1,067
489
Hill
0.0335
-410.30
1,069
466
Power
1.02E-04
-398.44
6.5E+06
errord
Polynomial 3°
0.0333
-410.29
1,057
687
Polynomial 2°e
Linear
0.103
-412.26
1,063
686
aModeled variance case presented (BMDS Test 2 p-value = 0.00542, BMDS Test 3 p-value = 0.061), no model was
selected as a best-fitting model.
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.
dBMD or BMDL computation failed for this model.
eFor the Polynomial 2° 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.
C-35 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-15. Summary of BMD modeling results for increased absolute kidney
2 weight in PO male S-D rats exposed to ETBE by daily gavage for 16 weeks
3 beginning 10 weeks prior to mating fFuiii et al.. 20101: BMR = 10% relative
4 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 3-fold
difference.)
Exponential (M4)
Exponential (M5)c
0.600
-39.779
438
163
Hill
0.613
-39.799
435
139
Powerd
Polynomial 3°e
Polynomial 2°f
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-day 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° 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.
C-36 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
4.4 Hill' —
4.2
4
3.8
3.6
3.4
3.2
BMD
BMDI
0
200
400
600
800
1000
13:13 05/15 2014
Figure C-10. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Hill Model. (Version: 2.17; Date: 01/28/2013]
The form of the response function is: Y[dose] = intercept + v*doseAn/(kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 434.715
BMDL at the 95% confidence level = 139.178
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
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
This document is a draft for review purposes only and does not constitute Agency policy.
C-37 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-38 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-16. BMD modeling results for increased relative kidney weight in PO
2 male S-D rats exposed to ETBE by daily gavage for 16 weeks beginning 10
3 weeks prior to mating fFuiii et al.. 20101: BMR = 10% relative deviation from
4 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
0.0530
-460.12
471
401
The Hill model is selected as the
only adequately fitting model.
Exponential (M4)
Exponential (M5)c
0.0956
-461.22
256
150
Hill
0.108
-461.41
243
129
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.0720
-460.73
439
367
aConstant variance case presented (BMDS Test 2 p-value = 0.271), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-day were -0.602,1.25, -0.78, and 0.133, 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 row 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° 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.
C-39 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
B M DL
14:04 05/15 2014
400 600
dose
Figure C-ll. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 242.739
BMDL at the 95% confidence level = 128.617
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.0027678
0.0028115
rho
n/a
0
intercept
0.552461
0.546
V
0.251763
0.143
n
1
0.204461
k
863.449
1,625.63
This document is a draft for review purposes only and does not constitute Agency policy.
C-40 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
24
0.546
0.552
0.059
0.0526
-0.602
100
24
0.592
0.579
0.06
0.0526
1.25
300
24
0.609
0.617
0.042
0.0526
-0.78
1,000
24
0.689
0.688
0.049
0.0526
0.133
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
235.996644
5
-461.993287
A2
237.954442
8
-459.908884
A3
235.996644
5
-461.993287
fitted
234.705776
4
-461.411551
R
202.992245
2
-401.98449
3 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
69.9244
6
<0.0001
Test 2
3.9156
3
0.2707
Test 3
3.9156
3
0.2707
Test 4
2.58174
1
0.1081
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-41 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-17. Summary of BMD modeling results for increased absolute kidney
2 weight in PO female S-D rats exposed to ETBE by daily gavage for 17 weeks
3 beginning 10 weeks prior to mating until lactation day 21 (Fujii etal.. 2010):
4 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)
0.483
-199.73
1,135
781
Polynomial 2° is selected based
on most parsimonious model
with lowest AIC.
Exponential (M3)
0.441
-198.60
1,089
826
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 30d
Polynomial 2°
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-day 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model.
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the Bl
; Polynomial - ;
2.45 I- -
2.4
2.35
2.3
2.25
2.2
2.15
2.1
BMDL
2.05
BMD
O
200
400
600
800
1 000
5 14:09 05/15 2014
6 Figure C-12. Plot of mean response by dose, with fitted curve for selected
7 model; dose shown in mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
C-42 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
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
a
0.0323691
0.0337309
rho
n/a
0
beta_0
2.1504
2.15624
beta_l
7.16226E-28
0
beta_2
0.000000179719
0
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
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
11
12
This document is a draft for review purposes only and does not constitute Agency policy.
C-43 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2* 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
This document is a draft for review purposes only and does not constitute Agency policy.
C-44 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-18. Summary of BMD modeling results for increased relative kidney
2 weight in PO female S-D rats exposed to ETBE by daily gavage for 17 weeks
3 beginning 10 weeks prior to mating until lactation day 21 (Fujii et al.. 2010):
4 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)
0.367
-471.62
2,953
1,482
Polynomial 2° is selected based
Exponential (M3)
0.208
-470.04
1,573
1,026
on lowest AIC.
Exponential (M4)
0.156
-469.61
3,056
1,506
Exponential (M5)
N/Ab
-468.07
error0
0
Hill
N/Ab
-468.07
error0
error0
Power
0.208
-470.04
1,592
1,028
Polynomial 3°
0.207
-470.03
1,511
1,172
Polynomial 2°
0.450
-472.03
1,751
1,254
Linear
0.366
-471.61
3,055
1,506
aConstant variance case presented (BMDS Test 2 p-value = 0.665), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-day were 0.849, -0.925, 0.0742, and 0.00257, respectively.
bNo available degrees of freedom to calculate a goodness of fit value.
CBMD or BMDL computation failed for this model.
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the Bl
0 74 Polynomial
0.72
0.7
0.68
0.66
0.64
BMDL
BM;D
O
200
400
600
800
1 000
1200
1400
1600
1 800
5 14:31 05/15 2014
6 Figure C-13. Plot of mean response by dose, with fitted curve for selected
7 model; dose shown in mg/kg-day.
8 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
This document is a draft for review purposes only and does not constitute Agency policy.
C-45 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
2 A constant variance model is fit
3 Benchmark Dose Computation.
4 BMR = 10% Relative deviation
5 BMD = 1,751.45
6 BMDL at the 95% confidence level = 1,254.17
7 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.00253026
0.00259675
rho
n/a
0
beta_0
0.665286
0.668151
beta_l
2.84343E-27
0
beta_2
0.0000000216877
0
8 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
24
0.674
0.665
0.053
0.0503
0.849
100
24
0.656
0.666
0.048
0.0503
-0.925
300
24
0.668
0.667
0.057
0.0503
0.0742
1,000
24
0.687
0.687
0.045
0.0503
0.00257
9 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
239.810603
5
-469.621206
A2
240.598408
8
-465.196816
A3
239.810603
5
-469.621206
fitted
239.01285
3
-472.0257
R
237.463901
2
-470.927802
10 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
6.26901
6
0.3937
Test 2
1.57561
3
0.6649
Test 3
1.57561
3
0.6649
Test 4
1.59551
2
0.4503
This document is a draft for review purposes only and does not constitute Agency policy.
C-46 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Inhalation Exposure Endpoints
2 Table C-19. Summary of BMD modeling results for urothelial hyperplasia of
3 the renal pelvis in male F344 rats exposed to ETBE by whole-body inhalation
4 for 6 hr/d, 5 d/wk, for 104 wk (IPEC. 2010b): BMR = 10% extra risk
Model3
Goodness of fit
BMCiopct
(mg/m3)
BMCLiopct
(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
LogLogistic
0.814
164.40
3,010
1,831
Probit
0.202
165.59
4,059
3,365
LogProbit
0.633
164.57
3,050
1,896
Weibull
0.758
164.44
2,623
1,478
Multistage 3°
0.565
164.69
2,386
1,412
Multistage 2°
0.565
164.69
2,386
1,422
Quantal-Linear
0.269
165.16
1,541
1,227
aSelected model in bold; scaled residuals for selected model for doses 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 -f 24.45.
Gamma Multi-Hit Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for the
Gamma Multi-Hit
BMDL
O 5000 1OOOO
dose
1 3:40 09/1 O 201 4
6 Figure C-14. Plot of incidence rate by dose, with fitted curve for selected
7 model; dose shown in mg/m3.
This document is a draft for review purposes only and does not constitute Agency policy.
C-47
DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Gamma Model. (Version: 2.16; Date: 2/28/2013)
2 The form of the probability function is: P[response]= background+(l-
3 background]*CumGamma[slope*dose,power], where CumGammaQ is the cummulative Gamma distribution
4 function
5 Power parameter is restricted as power >=1
6 Benchmark Dose Computation.
7 BMR = 10% Extra risk
8 BMD = 2,734.41
9 BMDL at the 95% confidence level = 1,497.7
10 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0.0390054
0.0576923
Slope
0.000121504
0.000132454
Power
1.59019
1.84876
11 Analysis of Deviance Table
Model
Log( likelihood
)
# Param's
Deviance
Test d.f.
p-value
Full model
-79.1741
4
Fitted model
-79.1867
3
0.0253512
1
0.8735
Reduced
model
-124.987
1
91.626
3
<.0001
12 AIC: = 164.373
13 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
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
14 ChiA2 = 0.03 d.f= IP-value = 0.8737
This document is a draft for review purposes only and does not constitute Agency policy.
C-48 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-20. Summary of BMD modeling results for increased absolute kidney
2 weight in male S-D rats exposed to ETBE by whole-body inhalation for 6 hr/d,
3 5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord
(ppm)
BMDLiord
(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 BMDL
estimate, the Hill model was
selected based on lowest BMDL
(BMDLs 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°d
Polynomial 2°e
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 doses 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
4
3.8
3.6
3.4
3.2
3
BMDJ.
O
200
400
600
800
1000
1200
1400
5 Figure C-15. Plot of mean response by dose, with fitted curve for selected
6 model; dose shown in ppm.
This document is a draft for review purposes only and does not constitute Agency policy.
C-49 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
3 A constant variance model is fit
4 Benchmark Dose Computation.
5 BMR = 10% Relative deviation
6 BMD = 264.371
7 BMDL at the 95% confidence level = 15.4115
8 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
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
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
11
12
This document is a draft for review purposes only and does not constitute Agency policy.
C-50 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2* 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
2 Table C-21. Summary of BMD modeling results for increased relative kidney
3 weight in male S-D rats exposed to ETBE by whole-body inhalation for 6 hr/d,
4 5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from the mean
Model3
Goodness of fit
BMDiord (ppm)
BMDLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.00625
-225.68
2,954
2,226
The Hill model was selected
based on lowest AIC.
Exponential (M4)
Exponential (M5)c
0.152
-232.27
623
256
Hill
0.175
-232.55
470
133
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.00771
-226.13
2,792
2,051
aConstant variance case presented (BMDS Test 2 p-value = 0.321), selected model in bold; scaled residuals for
selected model for doses 0,150, 500,1,500, and 5,000 ppm were -0.599,1.37, -1.04, 0.241, and 0.0322,
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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° 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.
C-51 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hil
0.75
0.7
0.65
0.6
0.55
BMD
1 000
2000
3000
4000
5000
12:09 05/16 2014
Figure C-16. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 470.166
BMDL at the 95% confidence level = 132.528
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.00299441
0.0031028
rho
n/a
0
intercept
0.594365
0.584
V
0.149823
0.142
n
1
0.147616
k
714.991
2,225.81
This document is a draft for review purposes only and does not constitute Agency policy.
C-52 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
0.584
0.594
0.042
0.0547
-0.599
150
10
0.644
0.62
0.064
0.0547
1.37
500
10
0.638
0.656
0.046
0.0547
-1.04
1,500
10
0.7
0.696
0.073
0.0547
0.241
5,000
10
0.726
0.725
0.047
0.0547
0.0322
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
122.020272
6
-232.040543
A2
124.363765
10
-228.727531
A3
122.020272
6
-232.040543
fitted
120.275236
4
-232.550472
R
106.075094
2
-208.150188
3 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
36.5773
8
<0.0001
Test 2
4.68699
4
0.3209
Test 3
4.68699
4
0.3209
Test 4
3.49007
2
0.1746
4
5
This document is a draft for review purposes only and does not constitute Agency policy.
C-53 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table C-22. Summary of BMD modeling results for increased absolute kidney
2 weight in female S-D rats exposed to ETBE by whole-body inhalation for
3 6 hr/d, 5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from
4 the mean
Model3
Goodness of fit
BMDiord (ppm)
BMDLiord
(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 BMD is higher than the
maximum dose.
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°e
Polynomial 20f
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 doses 0,150, 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.
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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° 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.
C-54 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplemental Information—ETBE
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMC
Linear
2.1
2.05
1 .95
1 .85
1 .75
BMDL
0
1000
2000
3000
4000
5000
6000
7000
13:40 05/16 2014
Figure C-17. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
Polynomial Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = beta_0 + beta_l*dose
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 6,840.02
BMDL at the 95% confidence level = 3,978.09
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
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.
C-55 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-23. Summary of BMD modeling results for increased relative kidney
2 weight in female S-D rats exposed to ETBE by whole-body inhalation for 6
3 hr/d, 5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from the
4 mean
Goodness of fit
BMDLiord
(ppm)
Model3
p-value
AIC
BMDiord (ppm)
Basis for model selection
Exponential (M2)
Exponential (M3)b
0.147
-248.04
3,288
2,482
The Hill model was selected
based on lowest BMDL.
Exponential (M4)
Exponential (M5)c
0.240
-248.55
1,471
557
Hill
0.264
-248.74
1,330
316
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.162
-248.26
3,167
2,334
aConstant variance case presented (BMDS Test 2 p-value = 0.388), selected model in bold; scaled residuals for
selected model for doses 0,150, 500,1,500, and 5,000 ppm were -0.874,1.29, -0.235, -0.308, and 0.125,
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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
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Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.7
Hil
0.65
0.6
0.55
BMDL
BMD
0.5
0
1 000
2000
3000
4000
5000
13:54 05/16 2014
Figure C-18. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 1,329.5
BMDL at the 95% confidence level = 315.543
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.00216632
0.002282
rho
n/a
0
intercept
0.557859
0.545
V
0.130692
0.111
n
1
0.226907
k
1,785.17
1,916.67
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
0.545
0.558
0.04
0.0465
-0.874
150
10
0.587
0.568
0.056
0.0465
1.29
500
10
0.583
0.586
0.035
0.0465
-0.235
1,500
10
0.613
0.618
0.06
0.0465
-0.308
5,000
10
0.656
0.654
0.043
0.0465
0.125
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
129.701589
6
-247.403177
A2
131.770538
10
-243.541076
A3
129.701589
6
-247.403177
fitted
128.368125
4
-248.73625
R
117.090968
2
-230.181936
3 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
29.3591
8
0.0002742
Test 2
4.1379
4
0.3877
Test 3
4.1379
4
0.3877
Test 4
2.66693
2
0.2636
4
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Supplemental Information—ETBE
1 Table C-24. Summary of BMD modeling results for increased absolute kidney
2 weight in male F344 rats exposed to ETBE by whole-body inhalation for 6
3 hr/d, 5 d/wk, for 13 wk (Medinskv et al.. 1999: Bond et al.. 1996b): BMR =
4 10% relative deviation from the mean
Goodness of fit
Model3
p-value
AIC
BMCiord (ppm)
BMCLiord (ppm)
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 BMDL.
Exponential (M4)
Exponential (M5)c
0.199
-129.71
1,798
808
Hill
0.224
-129.89
1,667
603
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.208
-130.22
2,980
2,288
aConstant variance case presented (BMDS Test 2 p-value = 0.540), selected model in bold; scaled residuals for
selected model for doses 0, 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° 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° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
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Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
2.2
BMDL
14:00 05/16 2014
2000 3000
dose
Figure C-19. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 1,666.92
BMDL at the 95% confidence level = 603.113
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
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
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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
4
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Supplemental Information—ETBE
1 Table C-25. Summary of BMD modeling results for increased absolute kidney
2 weight in female F344 rats exposed to ETBE by whole-body inhalation for 6
3 hr/d, 5 d/wk, for 13 wk (Medinskv et al.. 1999: Bond et al.. 1996b): BMR =
4 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°f
Polynomial 2°g
Linear
0.0928
-188.45
2,552
2,111
aConstant variance case presented (BMDS Test 2 p-value = 0.428), selected model in bold; scaled residuals for
selected model for doses 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° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
gFor the Polynomial 2° 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
Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BT
1 .35
1 .3
1 .25
1 .1
1 .05
1 .2
1 .15
BMDL
14:13 05/16 201
Exponential
2 Figure C-20. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in ppm.
4 Exponential Model. (Version: 1.9; Date: 01/29/2013)
5 The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
6 A constant variance model is fit
7 Benchmark Dose Computation.
8 BMR = 10% Relative deviation
9 BMD = 1,341.66
10 BMDL at the 95% confidence level = 815.742
11 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Ina
-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
12
13
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
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)
# Param's
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* 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 PODs from Inhalation Studies - Use of PBPK Model for Route-to-route Extrapolation
5 A pharmacokinetic (PBPK) model for ETBE and its metabolite tert-butanol in rats has been
6 developed, as described in APPENDIX B of the Supplemental Information. Using this model, route-
7 to-route extrapolation of the inhalation benchmark concentration levels (BMCLs) to derive oral
8 PODs was performed as follows. First, the internal dose in the rat at each inhalation BMCLadj
9 (already adjusted to continuous exposure in mg/m3) was estimated using the PBPK model to derive
10 an "internal dose BMDL." Then, the oral dose concentration (assuming continuous exposure) that
11 led to the same internal dose in the rat was estimated using the PBPK model. The resulting BMDL
12 already reflects a continuous exposure so it is equivalent to a PODadj, described above. This value
13 was then converted to a human equivalent dose POD using the formula previously described in
14 "PODs from oral studies":
15 PODhed = PODadj (mg/kg-day) x DAF
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Supplemental Information—ETBE
A critical decision in the route-to-route extrapolation is the selection of the internal dose
metric to use that established "equivalent" oral and inhalation exposures. For ETBE-induced kidney
effects, the four options are the concentration of tert-butanol in blood, the rate of tert-butanol
metabolism, the rate of ETBE metabolism, and the concentration of ETBE in blood. Note that using a
kidney concentration for ETBE or tert-butanol will lead to the same route-to-route extrapolation
relationship as using blood concentration of ETBE or tert-butanol, respectively, because the
distribution from blood to kidney is independent of route. The major systemically available
metabolite of ETBE is tert-butanol, which has also been shown to cause kidney toxicity, so
tert-butanol is a plausible dose metric. There are no data to suggest that metabolites of tert-butanol
mediate its renal toxicity, so the rate of tert-butanol metabolism is not a supported dose metric. The
other metabolite of ETBE is acetaldehyde, but it is largely produced in the liver, and its systemic
availability is limited due to its rapid clearance. Therefore, the rate of metabolism of ETBE is not
supported as a dose metric for kidney toxicity. The final dose metric option is ETBE blood
concentration. Although it is possible that tert-butanol contributes to the kidney effects of ETBE, it
is clear that ETBE alone cannot fully account for the kidney effects, given the presence of
systemically available tert-butanol following ETBE exposure. As demonstrated in Appendix B,
comparing noncancer kidney effects following ETBE or tert-butanol administration based on
internal dose yielded consistent dose-response relationships using tert-butanol blood
concentration as the dose metric. Therefore, tert-butanol in blood was selected as the best available
dose metric for route-to-route extrapolation, although recognizing that some uncertainty remains
as to whether it can fully account for the kidney effects of ETBE.
Table C-26 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD for each inhalation data set discussed above.
Table C-26. Summary of derivation of oral PODs derived from route-to-route
extrapolation from inhalation exposures
Endpoint and reference
Species/sex
BMR
BMCLadj
(mg/m3)a
Internal
doseb
(mg/L)
Equivalent
PODadj
(mg/kg-d)
Equivalent
PODhed0
(mg/kg-d)
Kidney
Increased urothelial hyperplasia
Saito et al. (2013): JPEC (2010b)
Male F344 rats
10%
268
3.40
93.7
22.5
Increased absolute kidney
weight
JPEC (2008a)
Male Sprague-
Dawley rats
10%
112
1.35
39.5
9.18
Increased relative kidney
weight
JPEC (2008a)
Male Sprague-
Dawley rats
10%
99
1.19
34.9
8.38
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Supplemental Information—ETBE
Endpoint and reference
Species/sex
BMR
BMCLadj
(mg/m3)a
Internal
doseb
(mg/L)
Equivalent
PODadj
(mg/kg-d)
Equivalent
PODhed0
(mg/kg-d)
Increased absolute kidney
weight
JPEC (2008a)
Female
Sprague-
Dawley rats
10%
2,969
103
1,110
266
Increased relative kidney
weight
JPEC (2008a)
Female
Sprague-
Dawley rats
10%
236
2.96
82.8
19.9
Increased absolute kidney
weight
Medinskv et al. (1999)
Male F344 rats
10%
450
6.06
158
37.9
Increased absolute kidney
weight
Medinskv et al. (1999)
Female F344
rats
10%
609
8.60
213
51.1
1 Conversion factor used: 1 ppm = 4.17 mg/m3
2 bAverage blood concentration of te/t-butanol under continuous inhalation exposure to ETBE at the BMDL (from
3 Table 2-1).
4 Continuous ETBE oral human equivalent dose that leads to the same average blood concentration of te/t-butanol
5 as continuous inhalation exposure to ETBE at the BMCL (see text for details).
6 PODs from Oral Studies - Use of PBPK Model for Route-to-route Extrapolation
7 Because tert-butanol is the primary metabolite of ETBE and the evidence suggests it is
8 involved in kidney toxicity, a PBPK model for ETBE and its metabolite tert-butanol in rats was
9 developed, as described in Appendix B. Using this model, route-to-route extrapolation of the oral
10 BMDLs to derive inhalation PODs was performed as follows. First, the internal dose in the rat at
11 each oral BMDL (assuming continuous exposure) was estimated using the PBPK model to derive an
12 "internal dose BMDL." Then, the inhalation air concentration (again assuming continuous exposure)
13 that led to the same internal dose in the rat was estimated using the PBPK model. The resulting
14 BMCL already reflects a continuous exposure so it is equivalent to a BMCLadj, described above. This
15 value was then converted to a human equivalent dose POD using the formula previously described
16 in "PODs from inhalation studies":
17 BMCLhec = BMCLadj (mg/m3) x (LaLh) (interspecies conversion)
18 = BMCLadj (mg/m3) x (11.6 -h 11.7)
19 = BMCLadj (mg/m3) x (0.992)
20 A critical decision in the route-to-route extrapolation is the selection of the internal dose
21 metric to use that established "equivalent" oral and inhalation exposures. For ETBE-induced kidney
22 effects, the four options are the concentration of tert-butanol in blood, the rate of tert-butanol
23 metabolism, the rate of ETBE metabolism, and the concentration of ETBE in blood. Note that using a
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Supplemental Information—ETBE
kidney concentration for ETBE or tert-butanol will lead to the same route-to-route extrapolation
relationship as using blood concentration of ETBE or tert-butanol, respectively, because the
distribution from blood to kidney is independent of route. The major systemically available
metabolite of ETBE is tert-butanol, which has also been shown to cause kidney toxicity, so
tert-butanol is a plausible dose metric. There are no data to suggest that metabolites of tert-butanol
mediate its renal toxicity, so the rate of tert-butanol metabolism is not a supported dose metric. The
other metabolite of ETBE is acetaldehyde, but it is largely produced in the liver, and its systemic
availability is limited due to its rapid clearance. Therefore, the rate of metabolism of ETBE is not
supported as a dose metric. The final dose metric option is ETBE blood concentration. ETBE alone
cannot fully account for the kidney effects, given the presence of systemically available tert-butanol
following ETBE exposure and the relatively small concentrations of ETBE measured in the urine. As
demonstrated in Appendix B, comparing noncancer kidney effects following ETBE or tert-butanol
administration based on internal dose yielded consistent dose-response relationships using tert-
butanol blood concentration as the dose metric. Therefore, tert-butanol in blood was selected as the
best available dose metric for route-to-route extrapolation, although recognizing that some
uncertainty remains as to whether it can fully account for the kidney effects of ETBE.
Table C-27 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD for each inhalation data set discussed above.
Table C-27. Summary of derivation of inhalation PODs derived from route-to-
route extrapolation from oral exposures
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODhec15
(mg/m3)
Kidney
Increased urothelial hyperplasia
Suzuki et al. (2012); JPEC (2010a)
Male F344 rats
10%
60.5
2.11
171
Increased absolute kidney weight
JPEC (2008b); Mivata et al. (2013)
Male Sprague-
Dawley rats
10%
115
4.25
326
Increased relative kidney weight
JPEC (2008b); Mivata et al. (2013)
Male Sprague-
Dawley rats
NA
25°
1.99
70
Increased absolute kidney weight
JPEC (2008b); Mivata et al. (2013)
Female Sprague-
Dawley rats
10%
57
1.99
161
Increased relative kidney weight
JPEC (2008b); Mivata et al. (2013)
Female Sprague-
Dawley rats
10%
20
0.670
56
Increased absolute kidney weight
(PO generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
94
3.41
266
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Supplemental Information—ETBE
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODhec15
(mg/m3)
Increased relative kidney weight
(PO generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
137
5.17
388
Increased absolute kidney weight
(PO generation) Gaoua (2004b)
Female Sprague-
Dawley rats
10%
1,030
90.2
2,770
Increased relative kidney weight
(PO generation) Gaoua (2004b)
Female Sprague-
Dawley rats
NA
1,000°
85.5
2,700
Increased absolute kidney weight
(F1 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
235
9.7
667
Increased relative kidney weight
(F1 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
NA
250°
10.4
710
Increased absolute kidney weight
(F1 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
10%
670
42.4
1,900
Increased relative kidney weight
(F1 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
NA
500°
26.7
1,440
Increased absolute kidney weight
(P0 generation)
Fuiii et al. (2010)
Male Sprague-
Dawley rats
10%
139
5.25
394
Increased relative kidney weight
(P0 generation)
Fuiii et al. (2010)
Male Sprague-
Dawley rats
10%
129
4.83
365
Increased absolute kidney weight
(P0 generation)
Fuiii et al. (2010)
Female Sprague-
Dawley rats
10%
905
71.5
2,480
Increased relative kidney weight
(P0 generation)
Fuiii et al. (2010)
Female Sprague-
Dawley rats
10%
1,254
127
3,230
aAverage blood concentration of te/t-butanol under continuous oral exposure to ETBE at the BMDL (from
Table 2-1).
Continuous ETBE inhalation human equivalent concentration that leads to the same average blood
concentration of te/t-butanol as continuous oral exposure to ETBE at the BMDL (see text for details).
CBMD modeling failed to successfully calculate a BMD value (see Appendix C of the Supplemental Information).
NOAELor LOAELwas used for route-to-route extrapolation.
NA = not applicable
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Supplemental Information—ETBE
C.1.2. Cancer Endpoints
For the multistage cancer models, the coefficients were restricted to be non-negative (beta's
> 0). For each endpoint, multistage cancer models 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 < 0.053 indicates lack of fit). Other factors were 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 BMR.
For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as
estimated by the profile likelihood method) and AIC value were used to select a best-fit model from
among the models exhibiting adequate fit If the BMDL estimates were "sufficiently close," that is,
differed by more than three-fold, the model selected was the one that yielded the lowest AIC value.
If the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD.
The incidence of liver tumors in male F344 rats was found to be statistically significantly
increased following a 2-year inhalation exposure; hepatocellular adenomas and a single
hepatocellular carcinoma in the high-dose group were combined in modeling the dataset. The data
were modeled using three different exposure metrics: administered concentration as ppm,
administered concentration as mg/m3, and an internal PBPK exposure concentration of ETBE
metabolized.
Table C-28. Cancer endpoints selected for dose-response modeling for ETBE
Species / Sex
Endpoint
Doses and Effect Data
Hepatocellular
adenomas and
carcinomas
JPEC (2010b)
Exposure Concentration
(ppm)
0
500
1,500
5,000
Exposure Concentration
(mg/m3)
0
2,089
6,268
20,893
PBPK Concentration
(mg/hr)
0
1.145
2.7316
4.125
Incidence / Total
0/50
2/50
1/49
10/50
C.1.2.1. Modeling Results
Below are tables summarizing the modeling results for the cancer endpoints modeled.
'i
A significance level of 0.05 is used for selecting cancer models because the model family (multistage) is
selected a priori Benchmark Dose Technical Guidance Document, U.S. EPA (2012).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-29. Summary of BMD modeling results for hepatocellular adenomas
2 and carcinomas in male F344 rats exposed to ETBE by whole-body inhalation
3 for 6 hr/d, 5d/wk, for 104 wk; modeled with doses as administered exposure
4 concentration in ppm flPEC. 2010bl: BMR = 10% extra risk
Model3
Goodness of fit
BMCiopct (ppm)
BMCLiopct (ppm)
Basis for model
selection
P-
value
Scaled residuals
AIC
Three
0.0991
-0.030, 1.382, -0.898,
and 0.048
84.961
2,942
1,735
Multistage 1° was
selected based on
lowest AIC.
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
2,756
1,718
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.208
2,605
1,703
aSelected model in bold.
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
0.35
0.3
0.25
0.2
0.15
0.1
0.05
BMDL
14:57 05/16 2014
0 1000 2000 3000
dose
6 Figure C-21. Plot of incidence rate by dose, with fitted curve for selected
7 model; dose shown in ppm.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Multistage Model. (Version: 3.4; Date: 05/02/2014)
2 The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
3 beta2*doseA2...)]
4 The parameter betas are restricted to be positive
5 Benchmark Dose Computation.
6 BMR = 10% Extra risk
7 BMD = 2,604.82
8 BMDL at the 95% confidence level = 1,703.47
9 BMDU at the 95% confidence level = 4,634.52
10 Collectively, (1703.47,4634.52) is a 90% two-sided confidence interval for the BMD
11 Multistage Cancer Slope Factor = error
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
0.0000404483
0.0000438711
13 Analysis of Deviance Table
Model
Log( likelihood
)
# Param's
Deviance
Test d.f.
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
14 AIC:= 81.2084
15 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
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
16 ChiA2= 2.42 d.f= 3 P-value = 0.4898
17
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-30. Summary of BMD modeling results for hepatocellular adenomas
2 and carcinomas in male F344 rats exposed to ETBE by whole-body inhalation
3 for 6 hr/d, 5d/wk, for 104 wk; modeled with doses as mg/m3 (IPEC. 2010b):
4 BMR = 10% extra risk
Model3
Goodness of fit
BMDiopct (mg/m3)
BMDLiopct (mg/m3)
Basis for model
selection
P-
value
Scaled residuals
AIC
Three
0.0991
-0.040, 1.382, -0.897,
and 0.048
84.961
12,300
7,251
Multistage 1° was
selected based on
lowest AIC
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
11,514
7,179
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.209
10,884
7,118
aSelected model in bold.
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
0.35
0.3
0.25
0.2
0.15
0.1
0.05
BMDL
1 0000
dose
15:02 05/16 2014
6 Figure C-22. Plot of incidence rate by dose, with fitted curve for selected
7 model; dose shown in mg/m3.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Multistage Model. (Version: 3.4; Date: 05/02/2014)
2 The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
3 beta2*doseA2...)]
4 The parameter betas are restricted to be positive
5 Benchmark Dose Computation.
6 BMR = 10% Extra risk
7 BMD = 10,884.4
8 BMDL at the 95% confidence level = 7,118.08
9 BMDU at the 95% confidence level = 19,366.3
10 Collectively, (7,118.08,19,366.3) is a 90% two-sided confidence interval for the BMD
11 Multistage Cancer Slope Factor = error
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
9.6799E-06
0.0000104989
13 Analysis of Deviance Table
Model
Log( likelihood
)
# Param's
Deviance
Test d.f.
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
14 AIC:= 81.2087
15 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
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
16 ChiA2= 2.42 d.f= 3 P-value = 0.4897
17
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table C-31. Summary of BMD modeling results for hepatocellular adenomas
2 and carcinomas in male F344 rats exposed to ETBE by whole-body inhalation
3 for 6 hr/d, 5d/wk, for 104 wk; modeled with PBPK doses as ETBE metabolized,
4 mg/hr flPEC. 2010bl: BMR = 10% extra risk
Model3
Goodness of fit
BMCiopct (mg/hr)
BMCLiopct (mg/hr)
Basis for model
selection
P-
value
Scaled residuals
AIC
Three
0.177
0.000, 1.033, -1.433,
and 0.587
84.574
3.20
2.34
Multistage 1° was
selected based on
lowest AIC
Two
0.144
0.000, 0.871, -1.574,
and 0.798
85.271
3.09
2.19
One
0.184
0.000, 0.035, -1.713,
and 1.378
84.446
3.03
1.98
aSelected model in bold.
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
0.35
0.3
0.25
0.2
0.15
0.1
0.05
BMDL
15:14 05/16 2014
0 0.5 1 1.5 2 2.5
dose
6 Figure C-23. Plot of incidence rate by dose, with fitted curve for selected
7 model; dose shown in mg/hr.
8
This document is a draft for review purposes only and does not constitute Agency policy.
C-75 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Multistage Model. (Version: 3.4; Date: 05/02/2014)
2 The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
3 beta2*doseA2...)]
4 The parameter betas are restricted to be positive
5 Benchmark Dose Computation.
6 BMR = 10% Extra risk
7 BMD = 3.02863
8 BMDL at the 95% confidence level = 1.98128
9 BMDU at the 95% confidence level = 5.02417
10 Collectively, (1.98128, 5.02417) is a 90% two-sided confidence interval for the BMD
11 Multistage Cancer Slope Factor = error
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
0.0347882
0.0464377
13 Analysis of Deviance Table
Model
Log( likelihood
)
# Param's
Deviance
Test d.f.
p-value
Full model
-38.2989
4
Fitted model
-41.2229
1
5.84813
3
0.1192
Reduced
model
-48.0344
1
19.4711
3
0.0002184
14 AIC: = 84.4459
15 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0
0
0
50
0
1.145
0.039
1.952
2
50
0.035
2.7316
0.0907
4.442
1
49
-1.713
4.125
0.1337
6.684
10
50
1.378
16 ChiA2= 4.83 d.f= 3 P-value = 0.1844
17
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
2 APPENDIX D. SUMMARY OF EXTERNAL PEER
3 REVIEW AND PUBLIC COMMENTS AND EPA'S
4 DISPOSITION
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
REFERENCES FOR APPENDICES
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tert-butyl ether in rats and humans. Toxicol Sci 51: 1-8.
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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.
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CD-l(R) mice. Bond, JA; Medinsky, MA; Wolf, DC; Cattley, R; Farris, G; Wong, B; Janszen, D;
Turner, MJ; Sumner, SCJ.
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Institute of Toxicology under contract to ARCO Chemical Company.
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06B7EA0/$File/89970000047.pdf
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pharmacokinetic model for methyl tertiary-butyl ether and tertiary-butanol in male Fisher-
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Borghoff. ST: Parkinson. H: Leavens. TL. (2010). Physiologically based pharmacokinetic rat model
for methyl tertiary-butyl ether; comparison of selected dose metrics following various
This document is a draft for review purposes only and does not constitute Agency policy.
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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: lanszen. 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/61.1.176
Brown. RP: Delp. MP: Lindstedt. SL: Rhomberg. LR: Beliles. RP. (1997). Physiological parameter
values for physiologically based pharmacokinetic models [Review], Toxicol Ind Health 13:
407-484. http://dx.doi.Org/10.1177/074823379701300401
Cederbaum. AI: Cohen. G. (1980a). Oxidative demethylation of t-butyl alcohol by rat liver
microsomes. Biochem Biophys Res Commun 97: 730-736.
Cederbaum. AI: Cohen. G. (1980b). Oxidative demethylation of tert-butyl alcohol by rat-liver
microsomes. Biochem Biophys Res Commun 97: 730-736. http://dx.doi.org/10.1016/00Q6-
291XC80190325-3
Dekant. W: Bernauer. U: Rosner. E: Amberg. A. (2001). Toxicokinetics of ethers used as fuel
oxygenates [Review], Toxicol Lett 124: 37-45. http://dx.doi.org/10.1016/s0378-
4274C00100284-8
Drogos. PL: Diaz. AF. (2001). Oxygenates in Gasoline
Appendix A: Physical properties of fuel oxgenates and addititves. In ACS Symposium Series.
Washington, DC: American Chemical Society, http://dx.doi.org/10.1021/bk-2002-
0799.ch018
Elmore. SA. (2006). Enhanced histopathology of the spleen [Review], Toxicol Pathol 34: 648-655.
http://dx.doi.Org/10.1080/01926230600865523
Fuiii. S: Yabe. K: Furukawa. M: Matsulira. M: Aovama. H. (2010). A one-generation reproductive
toxicity study of ethyl tertiary butyl ether in rats. Reprod Toxicol 30: 414-421.
http://dx.doi.Org/10.1016/i.reprotox.2010.04.013
Gaoua. W. (2004a). Ethyl tertiary butyl ether (ETBE): Prenatal developmental toxicity study by the
oral route (gavage) in rats (pp. 1-280). (CIT Study No. 24860 RSR). unpublished study for
Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review was
conducted by EPA in November 2008 to evaluate the accuracy of experimental procedures,
results, and interpretation and discussion of the findings presented. A report of this peer
review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749
(fax), or hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
Gaoua. W. (2004b). Ethyl tertiary butyl ether (ETBE): Two-generation study (reproduction and
fertility effects) by the oral route (gavage) in rats. (CIT Study No. 24859 RSR). unpublished
study for Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review
was conducted by EPA in November 2008 to evaluate the accuracy of experimental
procedures, results, and interpretation and discussion of the findings presented. A report of
this peer review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202)
56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
Hagiwara. A: Doi. Y: Imai. N: Nakashima. H: Ono. T: Kawabe. M: Furukawa. F: Tamano. S: Nagano. K:
Fukushima. S. (2011). Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-
butyl ether in rats. Toxicology 289: 160-166. http://dx.doi.Org/10.1016/i.tox.20ri.08.007
Hong. IY: Wang. YY: Bondoc. FY: Lee. M: Yang. CS: Hu. WY: Pan. I. (1999a). Metabolism of methyl
tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously
expressed human cytochromes P450: Identification of CYP2A6 as a major catalyst. Toxicol
Appl Pharmacol 160: 43-48. http://dx.doi.org/10.1006/taap.1999.8750
Hong. IY: Wang. YY: Bondoc. FY: Yang. CS: Gonzalez. FI: Pan. Z: Cokonis. CD: Hu. WY: Bao. Z. (1999b).
Metabolism of methyl tert-butyl ether and other gasoline ethers in mouse liver microsomes
lacking cytochrome P450 2E1. Toxicol Lett 105: 83-88. http://dx.doi.org/ 10.1016/s0378-
4274f98100389-0
This document is a draft for review purposes only and does not constitute Agency policy.
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Hong. IY: Wang. YY: Bondoc. FY: Yang. CS: Lee. M: Huang. WO. (1997a). Rat olfactory mucosa
displays a high activity in metabolizing methyl tert-butyl ether and other gasoline ethers.
Toxicol Sci 40: 205-210. http://dx.doi.Org/10.1093/toxsci/40.2.205
Hong. 1Y: Yang. CS: Lee. M: Wang. YY: Huang. W0: Tan. Y: Patten. CI: Bondoc. FY. (1997b). Role of
cytochromes P450 in the metabolism of methyl tert-butyl ether in human livers. Arch
Toxicol 71: 266-269.
lohanson. G: Nihlen. A: Lof. A. (1995). Toxicokinetics and acute effects of MTBE and ETBE in male
volunteers. Toxicol Lett 82/83: 713-718. http://dx.doi.org/10.1016/0378-4274r95103589-
3
1PEC (Japan Petroleum Energy Center). (2007a). Micronucleus test of 2-ethoxy-2-methylpropane
(ETBE) using bone marrow in rats administered ETBE by gavage. (Study Number: 7049).
Japan: Japan Industrial Safety and Health Association.
1PEC (Japan Petroleum Energy Center). (2007b). Micronucleus test of 2-ethoxy-2-methylpropane
(ETBE) using bone marrow in rats administered ETBE intraperitoneally. (Study Number:
7048). Japan: Japan Bioassay Research Center, Japan Industrial Safety and Health
Association.
JPEC (Japan Petroleum Energy Center). (2007c). Micronucleus test of ETBE using bone marrow of
rats of the "13-week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (inhalation
study) [preliminary carcinogenicity study]". (Study Number: 7047). Japan Industrial Safety
and Health Association.
IPEC (Japan Petroleum Energy Center). (2007d). Micronucleus test of ETBE using bone marrow of
rats of the "13-week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (drinking
water study) [preliminary carcinogenicity study]". (Study Number: 7046). Japan Bioassay
Research Center, Japan Industrial Safety and Health Association.
IPEC (Japan Petroleum Energy Center). (2008a). A 90-day repeated dose toxicity study of ETBE by
whole-body inhalation exposure in rats. (Study Number: B061829). Mitsubishi Chemical
Safety Institute Ltd.
IPEC (Japan Petroleum Energy Center). (2008b). A 180-Day repeated dose oral toxicity study of
ETBE in rats. (Study Number: D19-0002). Japan: Hita Laboratory, Chemicals Evaluation and
Research Institute (CERI). An external peer review was conducted by EPA in November
2008 to evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and atwww.epa.gov/iris.
IPEC (Japan Petroleum Energy Center). (2008c). Medium-term mutli-organ carcinogenesis bioassay
of 2-ethoxy-2-methylpropane (ETBE) in rats. (Study Number: 0635). Ichinomiya, Japan:
DIMS Institute of Medical Science.
IPEC (Japan Petroleum Energy Center). (2008d). A one-generation reproduction toxicity study of
ETBE in rats. (Study Number: SR07060). Safety Research Institute for Chemical Compounds.
IPEC (Japan Petroleum Energy Center). (2008e). Pharmacokinetic study in rats treated with [14c]
ETBE repeatedly for 14 days. (P070497). Japan: Kumamoto Laboratory, Mitsubishi
Chemical Safety Institute Ltd. An external peer review was conducted by EPA in November
2008 to evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and atwww.epa.gov/iris.
IPEC (Japan Petroleum Energy Center). (2008f). Pharmacokinetic study in rats treated with single
dose of [14C] ETBE. (P070496). Japan: Kumamoto Laboratory, Mitsubishi Chemical Safety
Institute Ltd. An external peer review was conducted by EPA in November 2008 to evaluate
the accuracy of experimental procedures, results, and interpretation and discussion of the
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 (fax), orhotline.iris@epa.gov (e-mail address) and
at www.epa.gov/iris.
IPEC (Japan Petroleum Energy Center). (2010a). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Drinking water study). (Study No: 0691). Japan Industrial Safety and Health
Association, Japan Bioassay Research Center. An external peer review was conducted by
EPA in November 2008 to evaluate the accuracy of experimental procedures, results, and
interpretation and discussion of the findings presented. A report of this peer review is
available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or
hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
IPEC (Japan Petroleum Energy Center). (2010b). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Inhalation study). (Study No: 0686). Japan: Japan Industrial Safety and Health
Association. An external peer review was conducted by EPA in November 2008 to evaluate
the accuracy of experimental procedures, results, and interpretation and discussion of the
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and
at www.epa.gov/iris.
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tertiary-butyl ether in humans. Toxicol Sci 51: 184-194.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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Vergnes. IS: Kubena. MF. (1995b). Ethyl Tertiary Butyl Ether: Mutagenic Potential in the Cho/hgprt
Forward Mutation Assay [TSCA Submission], (Project ID 94N1424). Export, PA: Bushy Run
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metabolism and toxicological profiles of gasoline oxygenates. Inhal Toxicol 9: 237-254.
This document is a draft for review purposes only and does not constitute Agency policy.
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