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Toxicological Review of Ethyl Tertiary Butyl Ether
[CASRN 637-92-3]
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
Supplemental Information
September 2014
NOTICE
This document is an Interagency Science Consultation Review 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
CONTENTS
APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS A-l
APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-REPONSE
ANALYSIS B-2
B.l. CHEMICAL PROPERTIES B-2
B.2. TOXICOKINETICS B-3
B.2.1. Absorption B-3
B.2.2. Distribution B-7
B.2.3. Metabolism B-9
B.2.4. Elimination B-16
B.2.5. Physiologically based pharmacokinetic models B-22
B.3. GENOTOXICITY STUDIES B-36
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-63
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. Chemical identity and physicochemical properties of ETBE B-2
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-8
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. Radioactivity in blood and kidney of rats and blood and liver of mice, following 6
hours of [14C]-ETBE inhalation exposure B-19
Table B-8. PBPK model physiologic parameters and partition coefficients B-27
Table B-9. Rate constants determined by optimization of the model with experimental data. B-28
Table B-10. Summary of genotoxicity (both in vitro and in vivo) studies of ETBE B-39
Table C-l. Non-cancer endpoints selected for dose-response modeling for ETBE C-3
Table C-2. Summary of BMD modeling results for slight 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-d (calculated by study authors); BMR =
10% extra risk C-7
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 180 days (Miyata et al., 2013; JPEC,
2008c); BMR = 10% relative deviation from the mean C-10
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 180 days (Miyata et al., 2013; JPEC,
2008c); BMR = 10% relative deviation from the mean C-13
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 180 days (Miyata et al., 2013; JPEC,
2008c); BMR = 10% relative deviation from the mean C-14
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 180 days (Miyata et al., 2013; JPEC,
2008c); BMR = 10% relative deviation from the mean C-17
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 weeks beginning 10
weeks before mating until after weaning of the pups. Gaoua (2004a); BMR =
10% relative deviation from the mean C-20
Table C-8. Summary of BMD modeling results for increased relative kidney weight in P0 male S-D
rats exposed to ETBE by daily gavage for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups C-23
Table C-9. Summary of BMD modeling results for increased absolute kidney weight in P0 female
S-D rats exposed to ETBE by daily gavage for a total of 18 weeks beginning 10
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Supplemental Information—ETBE
weeks before mating until after weaning of the pups Gaoua (2004a); BMR = 10%
relative deviation from the mean C-26
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 weeks beginning 10
weeks 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 2-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 2-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 2-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 2-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 etal. (2010); BMR = 10% relative deviation from the mean..C-35
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-38
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-41
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 et al. (2010); BMR = 10% relative deviation
from the mean C-43
Table C-19. Summary of BMD modeling results for slight urothelial hyperplasia of the renal
pelvis in male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d,
5d/wk, for 104 wks (JPEC, 2010b)BMR = 10% extra risk C-45
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 wks
JPEC (2008b); BMR = 10% relative deviation from the mean C-47
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 wks
JPEC (2008b); BMR = 10% relative deviation from the mean C-49
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 wks
JPEC (2008b); BMR = 10% relative deviation from the mean C-51
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 wks
JPEC (2008b); BMR = 10% relative deviation from the mean C-54
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Supplemental Information—ETBE
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
wks (Medinsky et al., 1999; Bond et al., 1996); BMR = 10% relative deviation
from the mean C-57
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
wks (Medinsky et al., 1999; Bond et al., 1996); BMR = 10% relative deviation
from the mean C-60
Table C-26. Cancer endpoints selected for dose-response modeling for ETBE C-63
Table C-27. 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 wks; modeled with doses as administered exposure concentration in ppm
JPEC (2010b); BMR = 10% extra risk C-64
Table C-28. 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 wks; modeled with doses as mg/m3 JPEC (2010b); BMR = 10% extra risk. .C-
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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 wks; modeled with PBPK doses as ETBE metabolized, mg/hr {JPEC, 2010,
1517421}; BMR = 10% extra risk C-67
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-24
Figure B-3. Schematic of the PBPK model for ETBE and its major metabolite ferf-butanol in
rats B-25
Figure B-4. Comparison of the EPA model predictions with measured ferf-butanol blood
concentrations for i.v., inhalation and oral gavage exposure to ferf-butanol. B-28
Figure B-5. Comparison of the EPA model predictions with measured amounts of ferf-butanol
after oral gavage of ETBE B-29
Figure B-6. Comparison of the EPA model predictions with measured amounts after a 4-hour
inhalation exposure to 4 and 40 ppm ETBE B-31
Figure B-7 . Comparison of the EPA model predictions with measured amounts of A) ETBE and B)
ferf-butanol in exhaled breath after a 6-hour inhalation exposure to 5000 ppm
ETBE B-32
Figure B-8. Comparison of the 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 the 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 C-l. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-8
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Figure C-2. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-ll
Figure C-3. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-15
Figure C-4. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-18
Figure C-5. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-21
Figure C-6. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-24
Figure C-7. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-26
Figure C-8. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-30
Figure C-9. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-33
Figure C-10. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-36
Figure C-ll. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-39
Figure C-12. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-41
Figure C-13. Plot of mean response by dose, with fitted curve for selected model; dose shown in
mg/kg-d C-43
Figure C-14. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-46
Figure C-15. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-48
Figure C-16. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-50
Figure C-17. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-52
Figure C-18. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-55
Figure C-19. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-58
Figure C-20. Plot of mean response by dose, with fitted curve for selected model; dose shown in
ppm C-61
Figure C-21. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
ppm C-64
Figure C-22. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-66
Figure C-23. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/hr C-68
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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
CYP450 cytochrome P450
DNA deoxyribonucleic acid
EPA U.S. Environmental Protection
Agency
GI gastrointestinal
HBA 2-hydroxybutyrate
KO knockout
JPEC Japan Petroleum Energy Center
MN micronucleus, micronucleated
MNPCE micronucleated polychromatic
erythrocyte
MTBE methyl tertiary butyl ether
MPD 2-methyl-l, 2-propane diol
PCE polychromatic erythrocytes
POD point of departure
RET reticulocyte
SD standard deviation
TAME methyl tertiary butyl ether
WT wild type
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Supplemental Information—ETBE
1
2 APPENDIX A. OTHER AGENCY AND
3 INTERNATIONAL ASSESSMENTS
4 Table A-l. Health assessments and regulatory limits by other national and
5 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
6
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Supplemental Information—ETBE
1 APPENDIX B. INFORMATION IN SUPPORT OF
2 HAZARD IDENTIFICATION AND DOSE-REPONSE
3 ANALYSIS
4 B.l. CHEMICAL PROPERTIES
5 Table B-l. Chemical identity and physicochemical properties of ETBE.
Characteristic or property
Value
Reference
Chemical name
2-ethoxy-2-methylpropane
2-methyl-2-ethoxy propane
National Library of Medicine
Synonyms
ethyl tert-butyl ether
ethyl tert-butyl oxide
methyl-2-ethoxypropane
tert-butyl ethyl ether
ETBE
National Library of Medicine
Chemical formula
C6H14O
National Library of Medicine
CASRN (Chemical Abstracts Service
Registry Number)
637-92-3
National Library of Medicine
Molecular weight
102.17
National Library of Medicine
Melting point
-94°C
Drogos and Diaz (2001)
Boiling point
67-73°C
Drogos and Diaz (2001)
Density at 25°C
0.73-0.74 g/cm3 @ 25°C
Drogos and Diaz (2001)
Water solubility
7,650-26,000 mg/L
Drogos and Diaz (2001)
Partition coefficients:
Log oil/water
Log Kow
1.48
1.74
Montgomery (1994)
Drogos and Diaz (2001)
Vapor pressure
130-152 mm Hg @ 25°C
Drogos and Diaz (2001)
Henry's law constant
2.7 x 10"3 atm-m3/mol @ 25°C
Drogos and Diaz (2001)
Odor
Detection threshold
Recognition threshold
0.013 ppm (0.054 mg/m3)
0.024 ppm (0.1 mg/m3)
Vetrano (1993)
Taste detection threshold (in water)
0.047 ppm (47 Hg/L)
Vetrano (1993)
Odor detection threshold (in water)
0.049 ppm (49 Hg/L)
Vetrano (1993)
Odor detection threshold (in water)
0.005 ppm (5 Hg/L)
Vetrano (1993)
Conversion factors
1 ppm = 4.18 mg/m3
1 mg/m3 = 0.24 ppm
1 mg/m3 = 102,180 mmol/L
ppm = mg/m3 x 24.45 m3/mole -r-
molecular weight in g/mol
mmol/L = mg/m3 -r- molecular
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Characteristic or property
Value
Reference
weight in mg/mmol -r- 1,000
L/m3
B.2. TOXICOKINETICS
B.2.1. Absorption
B.2.1.1. Human Studies
Most of the available human data on the uptake of ETBE were obtained from volunteers.
Nihlen et al. f!998al 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 its primary
metabolite, 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 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. (1998a) 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
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
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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.
Amberg etal. f20001 exposed six volunteers (three males and three females, average age
28 ± 2 years) to 4.5 ppm (18.8 mg/m3) and 40.6 ppm (170 mg/m3) ETBE 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 received 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 Nihlenetal. (1998a).
B.2.1.2. Animal Studies
Amberg etal. f20001 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.
However, the Japan Petroleum Energy Center (JPEC) conducted an oral dosing study of the
absorption of ETBE in rats after single and repeated dosing for 14 days (TPEC. 2008d. e). Seven-
week-old Crl:CD(SD) male rats (4/dose group) were administered either a single oral dose of 5, 50,
or 400 mg/kg [14C]ETBE via gavage or 5 mg/kg-day [14C]ETBE daily for 14 days. In the single-dose
study TPEC (2008e). 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 TPEC f2008dl. 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 2nd 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 as well as after the single dose study.
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Plasma radioactivity levels increased following a single dose of [14C]ETBE; this increase was
not proportional as the dose increased, especially at the high dose (i.e., the peak plasma
radioactivity levels were 2,800, 22,100, and 89,900 ng equivalents of ETBE/mL [ng eq 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. 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 TPEC (2008d). the Cmax was achieved 6 hours after the first exposure and
increased until it reached a steady state around the 5th day of exposure. After the last exposure on
Day 14, the Cmax, of 6,660 ± 407 ng eq ETBE/mL was achieved 10 hours after administration of
[14C]ETBE, and plasma radioactivity steadily decreased after this point. The elimination plasma
half-life from Cmax to 24 hours was 17.9 hours after the first dose and 14.2 hours after the final dose.
The elimination half-life from Cmax to 168 hours after the final dose following repeated dosing was
24.7 hours. Based on radioactivity levels measured in urine and exhalation, over 90% of the
administered dose was absorbed.
Dekantetal. f20011 published a review article that presented an overview of their studies
of the toxicokinetics of ETBE, methyl tertiary butyl ether (MTBE), and methyl tertiary butyl ether
(TAME) in both humans and rats following inhalation exposure at 4 ppm (16.7 mg/m3 ETBE and
TAME; 14.4 mg/m3 MTBE) and 40 ppm (167.1 mg/m3 ETBE and TAME; 144.2 mg/m3 MTBE),
respectively [see also Ambergetal. (2000): Bernauer et al. (1998)]. In addition, MTBE and TAME
were administered to humans in aqueous solution at 5 and 15 mg, respectively. The authors
assumed 100% absorption of MTBE and TAME following ingestion. Table B-2 presents a synopsis of
their findings. A comparison of the MTBE, TAME, and ETBE data may provide some insight relative
to uptake of ETBE following ingestion.
A comparison of the percentage of oral dose excreted versus the percentage of inhalation
dose excreted suggests that the assumption of 100% absorption was correct for MTBE, but most
likely not for TAME. If air:blood partition coefficients were the only determinants of inhalation
uptake, one would expect the dose received for ETBE to be lower than those for both MTBE and
TAME because the air:blood partition coefficient for ETBE (11.7) is lower than that of MTBE (17.7)
and TAME (17.9) fNihlen et al.. 19951. and the uptake of ETBE is lower than that of MTBE based on
the data from this laboratory. If the log octanol:water partition coefficients (log Kow) were the only
determinants (approximately 1.1 for MTBE, 1.48-1.74 for ETBE, and 1.55 for TAME [Table B-3;
Drogos and Diaz (2001)]). then values for ETBE and TAME should be similar. Data in Table B-3
support the latter hypothesis, but there are limited data for the evaluation of either hypothesis. On
a body-weight basis, doses were about 500 times higher in rats than in humans, although exposures
were delivered under entirely identical conditions in the two species [e.g., Ambergetal. f20001].
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Supplemental Information—ETBE
1 No studies investigating dermal absorption of ETBE were identified. However, because
2 dermal absorption of homologous organic substances is thought to be a function of the
3 octanol:water partition coefficient, ETBE may be assumed to penetrate rat skin relatively well. For
4 humans, Potts and Guy (19921 have proposed an equation (3-1) to calculate the dermal
5 permeability coefficient, Kp:
6
7 log Kp (cm/sec) = -6.3 + 0.71 x log kow - 0.0061 x (molecular weight) (3-1)
8 Table B-2. Plasma radioactivity after a single oral or intravenous dose of
9 [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 ± 1007
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: IPEC f2008dl
10
11 Using the log kow [identified as Koct in Potts and Guy (1992)] values for ETBE (0.95-2.2) and
12 MTBE (0.55-1.91) from Drogos and Diaz (2001) and converting cm/second values to cm/hour, Kp
13 values yielded are 0.0020-0.016 cm/hour for ETBE and 0.0012-0.012 cm/hour for MTBE. These
14 calculations predict that the dermal absorption rate of ETBE in humans would be 1.3-1.7 times that
15 of MTBE. The Kp for MTBE (i.e., 0.028 cm/hour) calculated by Prah et al. f20041 was approximately
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twice as high as the Kp derived using equation 3-1. However, the data from Prah etal. (20041 were
derived from human subjects exposed to a single concentration, and the authors themselves
highlighted the importance of experimental variables such as temperature and exposure
concentration for dermal absorption.
ETBE is moderately absorbed following inhalation exposure in rats and humans, and blood
levels of ETBE approached—but did not reach—steady-state concentrations within 2 hours. Nihlen
etal. (1998a) calculated the net respiratory uptake of ETBE in humans to be 26% compared with
38% for MTBE, which, as the authors point out, parallels the lower blood:air partition coefficient for
ETBE (11.7) compared with MTBE (17.7). The AUC for the concentration-time curve was linearly
related to the ETBE exposure level, suggesting linear kinetics up to 209 mg/m3. The JPEC flPEC.
2008d. e) studies 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. Although comparison of log kow values suggests that dermal absorption
rates for ETBE would be higher than that of MTBE, no data are available on dermal absorption of
ETBE.
B.2.2. Distribution
In vivo data on the tissue distribution of ETBE in humans are not available. Nihlen etal.
f!9951 measured the partitioning of ETBE and tert-butanol in air into human blood, saline, or oil
inside of sealed vials, and the human tissue partitioning coefficients were estimated based upon the
relative water and fat contents in human tissues, including brain, fat, liver, kidney, lung, and muscle.
Blood samples were obtained from 10 human donors (5 males, 5 females). 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 for
humans and rats, respectively. Nihlen etal. (1995) also estimated oil:water partition (log kow)
coefficients and obtained values of-0.56 for tert-butanol and 1.36 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) also used these coefficients and
air:oil partition coefficients to calculate human blood:tissue partition coefficients. These values are
listed in Table B-3.
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Table B-3. Bloochtissue partition coefficients for ETBE and tert-butanol.
Partition coefficient
tert-butanol
ETBE
Blood:air
465
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
Nihlen etal. f!998al exposed eight healthy male volunteers (average age: 29 years) to 21,
104, and 209 mg/m3 ETBE by inhalation for 2 hours. The volunteers performed light physical
exercise during exposure. Profiles of ETBE, tert-butanol, and acetone were established for blood
throughout exposure and for up to 22 hours thereafter. The same laboratory conducted studies
with MTBE using the same experimental protocol. Net uptake of MTBE was 38% of the dose
(compared with 26% netuptake for ETBE), and net exhalation of MTBE was 28% ofthe net uptake
for MTBE (compared with 31% net exhalation for ETBE) fNihlen etal.. 1998bl. The results may
reflect the difference inblood:air partition coefficients between MTBE and ETBE (18 and 12,
respectively) (Nihlen etal.. 1995). suggesting that MTBE has a higher tendency to partition into
human blood and tissues and is less likely to be eliminated by exhalation compared with ETBE.
Therefore, the high volume of distribution for ETBE in humans, 6.4 L/kg, as compared to 3.9 L/kg
for MTBE (Nihlen et al.. 1998a) is indicative of the higher partition coefficients for blood:tissue for
ETBE relative to MTBE, particularly the over 2-fold greater blood:fat partition coefficient (11.6 and
4.98 for ETBE and MTBE, respectively).
The JPEC f2008d. e) 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
were at a maximum 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 7th administration when compared to 8 hours after the 14th administration. The levels of
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[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 bound to plasma proteins.
B.2.3. 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. On the
basis of structures of the metabolites elucidated, ETBE is initially metabolized by cytochrome P450
(CYP) enzymes via oxidative deethylation by the addition of a hydroxy group to the a-carbon of the
ethyl ether group fBernauer et al.. 19981. 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: Hong etal.. 1999al. Using data from rat hepatic
microsome preparations, Turini etal. (19981 suggested that CYP2B1 may be the lead enzyme for
this step in rats. Acetaldehyde is oxidized to acetic acid and eventually to carbon dioxide (CO2).
tert-Butanol can be sulfated, glucuronidated, and excreted into urine, or it can undergo further
oxidation to form 2-methyl-l,2-propane diol (MPD), 2-hydroxybutyrate (HBA), acetone, and
formaldehyde. It should be noted that these metabolites have been identified in human or rat liver
extracts for ETBE, MTBE, and tert-butanol fBernauer etal.. 1998: Cederbaum and Cohen. 1980bl:
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.
CYP2A6
CYP3A4
ON,
I C
P CH3-
CH,
ETBE
glucuronide-0 —|—CH3
CH3
t-butyl glucuronide
CH,
oXcH,
H*C—( CH3
OH
rats, humans
CH, OH
CYP450
HO—|—CH, h,C-
un,
-h
rats,
CH3 humans
t-butanol
CH, 0H
ETBE-hemi-acetal
2-methyl-1,2-propanediol
f
H3C—^OH
CH,
V* CH3
acetaldehyde
ch3
t-butyl sulfate
H°_°
[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), ATSDR (1996), Bernauer et
al. (1998), Amberg et al. (1999), and Cederbaum and Cohen (1980a).
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Zhang etal. (1997) used computer models to predict the metabolites of ETBE and their toxic
effects. The metabolism model correctly predicted cleavage into tert-butanol and acetaldehyde and
that tert-butanol would undergo glucuronidation and sulfation. However, for the further
metabolism of tert-butanol, 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 HBA, which have been found in vivo.
B.2.3.1. Metabolism in Humans
Metabolism of ETBE in Humans in Vivo
Nihlenetal. (1998a) 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.
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 13 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 HBA, were also assayed.
At an exposure level of 170 mg/m3, the peak concentration of tert-butanol in blood was
13.9 ± 2.2; the peak concentration was 1.8 ± 0.2 [iM at 18.8 mg/m3. The time courses of metabolite
appearance in urine after 170 and 18.8 mg/m3 were similar, but relative urinary levels of
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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:HBA) were
1:25:107:580 after 170 mg/m3 and 1:17:45:435 after 18.8 mg/m3. Individual variations were large,
but the authors did not report any gender differences in the metabolism of ETBE based on data
from only three subjects of each sex.
In Vitro Metabolism of ETBE Using Human Enzyme Preparations
The metabolism of ETBE has been studied in vitro using both human liver microsomes and
genetically engineered cells expressing individual human CYP isozymes. Hongetal. f1997b)
coexpressed human CYP2A6 or CYP2E1 with human CYP reductase in insect SF9 cells. In this
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. Corresponding activities with 1 mM MTBE as the
substrate were 6.1 and 0.7 nmol/min-nmol, respectively.
Hong etal. f!999al obtained 15 human liver microsome samples and used them to compare
metabolic activities with ETBE, MTBE, and TAME as the substrates. They found that the metabolism
of all three substrates was highly correlated with certain CYP isozymes. The highest degree of
correlation was found for CYP2A6, which also displayed the highest turnover numbers. The
15 samples displayed very large inter individual variations in metabolic activities, with turnover
numbers for ETBE ranging from 179-3,130 pmol/minute-mg protein. Michaelis constant (Km)
values, estimated in three human liver microsomal samples using MTBE, ranged from 28-89 [J.M,
with maximum substrate turnover velocity (Vmax) values ranging from 215-783 pmol/minute-mg
protein. The Vmax/Km ratios, however, varied only between 7.7 and 8.8.
As part of CYP inhibition studies in the same paper, human liver microsomes were co-
incubated with MTBE, ETBE, or TAME in the presence of chemicals or specific antibodies to inhibit
either CYP2A6 or CPY2E1. For chemical inhibition, coumarin was dissolved in 2 [J.L of methanol and
added to the liver microsomes prior to initiation of the reaction. For antibody inhibition,
monoclonal antibodies against human CPY2A6 and CYP2E1 were preincubated with liver
microsomes prior to incubation with the rest of the reaction mixture. Methanol alone caused
approximately 20% inhibition of MTBE, ETBE, and TAME. Coumarin, a CYP2A6 substrate, caused a
significant dose-dependent inhibition of all three oxidants with a maximal inhibition of ETBE of
99% at 100-[iM coumarin. Antibodies against CYP2A6 inhibited metabolism of MTBE, ETBE, and
TAME by 75-95%. In contrast, there was no inhibition by the antibody against CYP2E1. The same
anti-CYP2El antibody inhibited over 90% of CPY2E1 activity assayed as /V-nitrosodimethylamine in
the liver microsomes.
In the same paper, these authors introduced several specific human CYPs into human (3-
lymphoblastoid cells and measured metabolic activities with ETBE and MTBE as the substrates.
They established a correlation ranking for ETBE metabolism (relative to tert-butanol) by 10 human
CYP isozymes: 2A6 > 3A4 « 2B6 « 3A4/5 » 2C9 > 2E1 « 2C19 » 1A2 « 2D6 « 1A2. They characterized
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the correlation with CYP2A6 as high, 3A4; 3A5, and 2B6 as good; 2C9, 2E1, and 2C19 as poor; and
the remaining three CYP activities as not correlated with ETBE metabolism. They also reported
direct enzyme activities toward ETBE as the substrate (in pmol tert-butanol formed per minute per
pmol CYP enzyme): 2A6-1.61; 2E1-0.34; 2B6-0.18; and 1A2-0.13. CYPs 1B1, 2C8, 2C9, 2C19, and
2D6 were not investigated. CYP1A2, which showed activity toward ETBE, did not metabolize MTBE
to tert-butanol. CYP4A11 showed considerable activity toward MTBE but very low activity toward
ETBE and TAME. CYP3A4 and 1A1 did not metabolize ETBE or MTBE in this system but displayed
considerable activity toward TAME. The authors concluded that CYP2A6 is the major enzyme
responsible for the oxidative metabolism of MTBE, ETBE, and TAME 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 MTBE, ETBE, or TAME.
Le Gal etal. f20011 used similar human cytochrome preparations as Hong etal. f!999al
(i.e., from deceased human donors) or genetically modified human p-lymphoblastoid cells to
elucidate the metabolism of ETBE, MTBE, and TAME. They identified as primary metabolites
formaldehyde from MTBE and TAME, acetaldehyde from ETBE, tertiary amyl-alcohol from TAME,
and tert-butanol from ETBE and MTBE. The human microsomes showed higher catalytic activity
toward MTBE and TAME at 0.5 mM compared with ETBE, but very similar activities at substrate
concentrations of 10 mM. Le Gal etal. (2001) confirmed the wide interindividual variation of
activities previously reported by Hong etal. f!999al and Hong etal. fl997bl. Using MTBE as the
substrate, they found a highly significant correlation with CYP2A6 activities and a lesser, but still
significant, correlation with CYP3A4 activities. No correlations could be established for 1A1,1A2, or
2E1 activities. However, using substrate concentrations of 0.5 and 10 mM, they found that 2A6 and
3E4, but not 2E1 or 2B6, had high activity at 0.5 mM, while 2E1 and 2B6 displayed considerable
activity at 10 mM. Using the average levels and the turnover numbers of various CYPs in human
liver, they concluded that fuel oxygenate ethers were predominantly metabolized by CYP2A6, with
considerable contribution from CYP3A4. CYP2E1, they concluded, did not play a significant role in
human metabolism of these substances.
B.2.3.2. Metabolism in Animals
Metabolism of ETBE in Animals In Vivo
Bernauer etal. (1998) studied the metabolism and excretion of [13C]-ETBE, MTBE, and
tert-butanol in rats. F344 rats, 2/sex, were exposed via inhalation to 2,000 ppm (8,400 mg/m3)
ETBE or 2,000 ppm (7,200 mg/m3) MTBE for 6 hours; three male F344 rats received 250 mg/kg
tert-butanol by gavage. Urine was collected for 48 hours. The metabolic profiles for ETBE and MTBE
were essentially identical, with excretion of MPD > HBA > tert-butanol-sulfate > tert-butanol-
glucuronide. Oral administration of tert-butanol produced a similar metabolite profile, with HBA >
tert-butanol-sulfate > MPD » tert-butanol-glucuronide * tert-butanol. tert-Butanol could not be
detected in urine when ETBE or MTBE were administered by inhalation. Traces of acetone were
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also detected in urine. Amberg etal. (2000) exposed F344 NH rats, 5/sex/dose, to ETBE in the same
exposure chamber coincident with the 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 much lower than in humans: 5.3 ± 1.2 and 21.7 ± 4.9 [J.M at
170 mg/m3 and 1.0 ± 0.7 and 5.7 ± 0.8 [J.M at 18.8 mg/m3, respectively. Similar to humans, rats
excreted mostly HBA in urine, followed by MPD and tert-butanol. The molar ratios for total urinary
excretion of tert-butanol:MPD:HBA were 1:2.3:15 after exposure to 170 mg/m3 and 1:1.5:11 after
exposure to 18.8 mg/m3. Parent ETBE was not identified in rat urine in this study.
In a review covering mostly their own work on fuel oxygenate metabolism, Dekant et al.
f20011 focused on aspects of metabolism of MTBE and ETBE in humans and rats. They reported
that, at a high exposure level (8,400 mg/m3 ETBE; 7,200 mg/m3 MTBE), rats predominantly
excreted the glucuronide of tert-butanol in urine, which, at low levels (16.7 mg/m3 or 167.1 mg/m3
ETBE; 14.4 or 144.2 mg/m3 MTBE), had been barely detectable. They concluded that, at high
exposure levels, the normally rapid metabolism of tert-butanol to MPD and HBA became saturated,
forcing more of the initial metabolite of ETBE or MTBE through the glucuronidation pathway. The
apparent final metabolite of ETBE was HBA, although this substance can undergo further
metabolism to acetone. The latter process appeared to play a minor role in the overall metabolism
of ETBE or MTBE. The authors also pointed out that many metabolites of the fuel oxygenate ethers,
such as formaldehyde, acetaldehyde, tert-butanol, HBA, or acetone, occur naturally in normal
mammalian physiology, providing a highly variable background that needs to be accounted for in
metabolic experiments.
The JPEC (2008d. e) 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 both single and 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. These data indicate that ETBE
is quickly metabolized to tert-butanol, which is then metabolized to tert-butanol glucuronide, 2-
methyl-l,2-propanediol, and finally to 2-hydroxyisobutyrate.
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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-day
400 mg/kg-day
5 mg/kg-day
5 mg/kg-day
Unchanged ETBE
ETBE
N.D.
N.D.
N.D.
N.D.
P-l
2-
hydroxyisobutyrate
75.4 ±8.1a
35.7 ±2.5
71.4 ±4.7
69.8 ±7.3
P-2
ferf-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
ferf-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 f2008d. e) 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-day
400 mg/kg-day
5 mg/kg-day
5 mg/kg-day
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
ferf-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
ferf-butanol
1.5 ±0.5
3.7 ±0.6
1.9 ±0.2
1.8 ±0.
11 aMean ± standard deviation; n = 4
12 N.D. = not detected
13
14 Source: JPEC f2008d. e) unpublished reports
15
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Metabolism of ETBE in Animal Tissues in Vitro
Using isolated rat liver microsomes, Hong etal. f!997al found that metabolism occurred
only in the presence of an NADPH- (nicotinamide adenine dinucleotide phosphate) regenerating
system and that the metabolic activity was inhibited by 80% after treating the microsomal
preparation with carbon monoxide, indicating CYP involvement In another study investigating
potential target tissues for ETBE toxicity, Hong etal. (1997a) studied the metabolic activities of
olfactory mucosa, respiratory epithelium, liver, lung, and olfactory bulb from rats. They prepared
microsomes, added an NADPH-regenerating system, and evaluated enzyme kinetics at various
substrate concentrations. In olfactory mucosa, the authors derived Km values of 125 and 111 [J.M for
ETBE and MTBE, with corresponding Vmax values of 11.7 and 10.3 nmol/minute-mg protein,
respectively. Addition of TAME to the reaction mixture exerted a concentration-dependent
inhibition of ETBE or MTBE metabolism. Coumarin, a CYP2A6 substrate, also inhibited ETBE
metabolism. These results indicated that rat olfactory mucosa, on a per-weight basis, has 37 times
the capacity of liver to metabolize fuel oxygenate ethers, and hence, has the capacity for first-pass
metabolism.
Hong etal. f!999bl used CYP2E1 knockout mice to investigate whether this enzyme plays a
major role in fuel oxygenate ether metabolism. They compared the ether-metabolizing activity of
liver microsomes (30 minutes at37°C and 1 mM ether) between the CYP2E1 knockout mice and
their parental lineage strains using four or five female mice (7 weeks of age) per group. The ETBE-
metabolizing activities (nmol/minute-mg protein) were 0.51 ± 0.24 for CP2E1 knockout mice,
0.70 ± 0.12 for C57BL/6N mice, and 0.66 ± 0.14 for 129/Sv mice. The MTBE-metabolizing activities
(nmol/minute-mg protein) were 0.54 ± 0.17 for CP2E1 knockout mice, 0.67 ± 0.16 for C57BL/6N
mice, and 0.74 ± 0.14 for 129/Sv mice. The TAME-metabolizing activities (nmol/minute-mg
protein) were 1.14 ± 0.25 for CP2E1 knockout mice, 1.01 ± 0.26 for C57BL/6N mice, and 0.76 ± 0.25
for 129/Sv mice. Mice that did not express any CYP2E1 did not differ from wild-type animals in
their ability to metabolize ETBE, MTBE, or TAME, suggesting that CYP2E1 is unlikely to be
important in the metabolism of ETBE. Turini etal. T19981 investigated the influence of ETBE
exposure on hepatic microsomal enzyme activities (as measured using CYP isozyme-specific
substrates) and the effects of specific enzyme induction on ETBE metabolism in male Sprague-
Dawley rats. Moderate doses of ETBE (200 or 400 mg/kg) administered intraperitoneal^ for 4 days
did not induce any hepatic CYPs. However, ETBE (2 mL/kg) administered by gavage as a 50% corn
oil solution for 2 days almost doubled activities of 3A1 /2 and 2B1, doubled 2E1, and induced
CYP2B1/2 sixfold. CYP1A1 /2 activity was slightly reduced after 2 days of ETBE (2 mL/kg) by
gavage. The authors also estimated kinetic constants for various CYPs in rats and found the
following Km or Vmax values: controls (2C forms predominant), 6.3 mM/0.93 nmol/minute-mg
protein; 2A/2B induced, 4.1 mM/3.8 nmol/minute-mg protein; 2E1 induced, 4.7 mM/1.6
nmol/minute-mg protein; 3A induced, 4.4 mM/1.4 nmol/minute-mg protein; and 1A induced, not
determined/0.9 nmol/minute-mg protein. Using a system with reconstituted CYPs, the authors
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found that CYP2B1 displayed the lowest Km (2.3 mM), and the highest turnover number
(56 nmol/minute-nmol CYP) and concluded that this isoform was the principal CYP to metabolize
ETBE in the rat.
The enzymes that metabolize tert-butanol to MPD, HBA, and even acetone, have not been
fully characterized. However, it is clear that tert-butanol is not subject to metabolism by alcohol
dehydrogenases Dekantetal. f2001I
B.2.4. Elimination
B.2.4.1. Elimination in Humans
Nihlenetal. (1998a) exposed eight healthy male volunteers (average age, 29 years) to 20.9,
104, and 209 mg/m3 ETBE by inhalation for 2 hours. ETBE, 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 accounted for even less: 0.12, 0.061, and 0.056% of the
dose was retained after 20.9,104, and 209 mg/m3 exposures, 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 [10 hours in another study with volunteers: lohanson etal. fl9951]. 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 (lohanson etal.. 1995).
These data suggest complex toxicokinetics for ETBE in humans. The first phase of
elimination from blood likely indicates uptake into highly perfused tissues. The other phases may
indicate uptake into less perfused tissues and fat, as well as 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.
Amberg etal. (2000) exposed six volunteers (three males and three females, 28 ± 2 years
old) to 18.8 and 170 mg/m3 of ETBE, respectively. 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 and at 4 or 6 hours after exposure,
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and thereafter every 6 hours for 48 hours. Parent ETBE and tert-butanol were determined in blood
and urine. Two further metabolites of tert-butanol, HBA and MPD, were also determined in urine.
At 170 mg/m3, the peak concentration of ETBE in blood was 12.1 ±4.0 [J.M, while 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 [J.M, respectively. At the high exposure concentration, two elimination half-lives were
found for ETBE, 1.1 ± 0.1 and 6.2 ± 3.3 hours, tert-Butanol displayed only one half-life,
9.8 ± 1.4 hours. At the low exposure concentration, only the short half-life for ETBE could be
measured at 1.1 ± 0.2 hours, while that for tert-butanol was 8.2 ± 2.2 hours. The predominant
urinary metabolite identified was HBA, 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, respectively, were similar, but relative urinary levels
of HBA after 18.8 mg/m3 were higher, while those for MPD were lower, as compared to 170 mg/m3.
HBA 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, while 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. Individual variations were large, but the authors did not report gender differences in
the toxicokinetics of ETBE. Based on the dose estimates presented in Section B.2.3.1, Amberg et al.
(20001 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.2.4.2. Elimination in Animals
Amberg etal. f20001 exposed F344 NH rats, 5/sex/dose concurrent with the volunteers in
the same exposure chamber. Urine was collected for 72 hours following exposure. Similar to
humans, rats excreted mostly HBA 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 HBA. The authors concluded that rats
eliminated ETBE considerably faster than humans. Urinary excretion accounted for 53 ± 15 and
50 ± 30% of the estimated dose at 170- and 18.8-mg/m3 exposures, respectively, with the
remainder of the dose being eliminated via exhalation, as suggested by the authors.
Bernauer etal. (19981 studied the excretion of [13C]-ETBE and MTBE in rats. F344 rats,
2/sex, were exposed via inhalation to 8,400 mg/m3 ETBE or 7,200 mg/m3 MTBE for 6 hours, or
three male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours.
The metabolic profiles for ETBE and MTBE were essentially identical, with relative excreted
amounts of MPD > HBA > tert-butanol-sulfate > tert-butanol-glucuronide. Oral administration of
tert-butanol produced a similar metabolite profile, with relative amounts of HBA > tert-butanol-
sulfate > MPD » tert-butanol-glucuronide ~ tert-butanol.
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Although there are several unpublished reports relevant to the elimination of ETBE
following inhalation exposure, no additional peer-reviewed publications were identified.
Unpublished reports have not gone through the public peer-review process and are of unknown
quality. They are included here as additional information only.
Sun and Beskitt f!995bl investigated the pharmacokinetics of [14C]-ETBE in F344 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 (1995b) report for "Total" column in Table B-6 or for the specific
tissues in Table B-7. Of the three animals per sex exposed concurrently, two were used in the
further study, while the third was kept as a spare. One animal/sex was placed into a metabolic cage
and monitored for up to 118 hours. 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 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 14C02, and charcoal filter eluates were measured directly by liquid scintillation
spectrometry. Blood and kidney tissue were combusted in a sample oxidizer and analyzed by liquid
scintillation spectrometry.
Table B-6. Elimination of [14C]-ETBE-derived radioactivity from rats and mice
within 96 hours following a single 6-hour inhalation exposure.
Exposure level
(mg/m3)
Volatile organics3
Exhaled C02a
Urine3
Feces3
Total"
F344 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
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Supplemental Information—ETBE
Exposure level
(mg/m3)
Volatile organics3
Exhaled C02a
Urine3
Feces3
Total"
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)
aPercent of total eliminated radioactivity; mean of one male and one female.
bIn mg [14C]-ETBE equivalents.
Sources: cSun and Beskitt (1995b): dvalues in parentheses: Borghoff (19961: eSun and Beskitt
fl 995b")
1
2 Table B-7. Radioactivity in blood and kidney of rats and blood and liver of
3 mice, following 6 hours of [14C]-ETBE inhalation exposure.
Exposure level
(mg/m3)
F344 Rata,
CD-I Mousea,
Bloodb
Kidneyc
Bloodb
Liverc
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.
bIn mg [14C]-ETBE equivalents per gram blood.
cIn mg [14C]-ETBE equivalents.
Sources: Sun and Beskitt f!995bl.
4 During 96 hours in metabolic cages, approximately 60% of the eliminated radioactivity was
5 recovered from urine, and approximately 38% was recovered from exhaled organic volatiles. This
6 pattern was maintained at an exposure concentration of 4,180 mg/m3; above that, urinary
7 excretion of radioactivity decreased to 34% of the recovered radioactivity, while exhalation of
8 organic volatiles increased to 63%. Exhalation of 14C02 increased marginally, from 1% at
9 2,090 mg/m3 to 2% at 20,900 mg/m3, while fecal elimination remained fairly constant at about 2%
10 throughout the exposure concentrations. A compilation of these results, together with results from
11 mice from a parallel study (Sun and Beskitt. 1995b). is given in Table B-6. The authors concluded
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that the metabolic pathways leading to urinary excretion of ETBE degradation products became
saturated at an exposure concentration of approximately 7,310 mg/m3.
The time course of elimination indicated that exhalation of organic volatiles was essentially
complete by 24 hours, while 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. Levels of radioactivity in blood and kidneys after increasing
exposure concentrations of [14C]-ETBE are shown in Table B-7 (again combined with the mouse
data from the parallel study). The major finding was that radioactivity levels increased up to
10,450 mg/m3 but leveled off in kidney and fell considerably in blood at 20,900 mg/m3. To the
authors, these data were indicative of saturation of the absorption pathway at around
10,450 mg/m3. However, it is noteworthy that total elimination of ETBE-derived radioactivity
increased steadily from 2,090 to 20,900 mg/m3 (Table B-6). The authors reported no deaths
following 6 hours of ETBE exposure. The findings of Sun and Beskitt (1995a). unpublished report,
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.
In a parallel study with an identical experimental protocol, Sun and Beskitt (1995b). in an
unpublished report, exposed CD-I mice (3/sex/dose) to 2,090, 3,130, 4,180, 7,310,10,450, and
20,900 mg/m3 [14C]-ETBE. The only difference from the rat study in the Sun and Beskitt (1995a)
unpublished report was that, instead of kidneys, livers were harvested from mice. The
corresponding results from this study are shown in Tables B-6 and B-7, jointly with the results from
the rat study.
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 were
considerably higher, as reported, 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 and/or had a higher metabolic capacity.
However, the total eliminated radioactivity at 20,900 mg/m3 showed no further increase over the
values at 10,450 mg/m3, indicating that the absorptive and metabolic capacities of mice had
become saturated. Judging from the data in Table B-67, saturation of blood and liver had occurred
already at 7,310 mg/m3. The authors reported no deaths following 6 hours of ETBE exposure. It
may be noted here that Sun and Beskitt (1995a. b) did not state any estimates for absorbed dose.
The data in Table B-6, however, indicate that, given the rapid exhalation of [14C]-ETBE-derived
material, any attempt to estimate a level of inhalation absorption following a 6-hour exposure
without respiratory elimination control would be futile.
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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 (1995a, b). The experimental protocol and materials were identical to the ones used by Sun
and Beskitt (1995a. b) in their unpublished reports; however, in this pilot study, only three male
F344 rats and three male CD-I mice were used per experiment, with the only exposure level
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. 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, while the
balance of radioactivity after 96 hours in metabolic cages came to 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 animals 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, HBA, 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. HBA was detected in urine of both species but could not be quantified.
MPD was not detected. These results may be interpreted as suggesting that mice metabolize and,
hence, eliminate ETBE faster than rats.
Unpublished reports by the JPEC (2008d) determined that following oral exposure of
7-week-old Crl:CD(SD) male rats to [14C]ETBE, the largest amount of radioactivity was recovered in
expired air, followed by urinary excretion, with very little excretion occurring via the feces. With
increasing dose, increasing proportions of radioactivity were found in expired air. The total
radioactivity recovered by 168 hours after a single dose of 5 mg/kg [14C]ETBE was 39.16% in the
urine, 0.58% in the feces, and 58.32% in expired air, and, after a single dose of 400 mg/kg, 18.7% in
the urine, 0.15% in the feces, and 78.2% in expired air. With repeated dosing, the recovery of
radioactivity through excretion increased through day 6 when a steady state was achieved.
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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.2.5. Physiologically based pharmacokinetic models
A physiologically based pharmacokinetic (PBPK) model of ETBE and its principal metabolite
t-butanol (tert-butanol) has been developed for humans exposed while performing physical work
(Nihlen and Tohanson. 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.
The U.S. Environmental Protection Agency (EPA) evaluated a PBPK model of ETBE and its
principle metabolite tert-butanol that was developed for humans exposed while performing
physical work (Nihlen and Tohanson. 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 f20091 models is given, followed by an evaluation of the MTBE models and the
assumptions adopted from MTBE models or modified in the ETBE model.
The Blancato etal. (20071 model is an update of the earlier Rao and Ginsberg (19971 model,
and the Leavens and Borghoff (20091 model is an update of the Borghoff et al. (19961 model. Both
the Blancato etal. (20071 and Leavens and Borghoff (20091 models are flow-limited models that
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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. 19911. 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 f20091
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. f20071 and the
Leavens and Borghoff (20091 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. (20071 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 f20091. 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 MOA section of the main volume (see Section 1.1.1). Binding of MTBE to a2U-globulin was
applied to sex differences in kidney concentrations of MTBE and tert-butanol in the Leavens and
Borghoff (20091 model but acceptable estimates of MTBE and tert-butanol pharmacokinetics in the
blood are predicted in other models that did not consider a2U-globulin binding. Moreover, as
discussed below, the Leavens and Borghoff (20091 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
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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.2.5.1. Evaluation and Modification of Existing tert-butanol submodels
The Blancato etal. (20071 and Leavens and Borghoff (20091 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)
B)
10000
1000
300 mg/kg
~
male
female
- - 150 mg/kg
m
male
~
female
75 mg/kg
•
male
0
female
37.5 mg/kg
A
male
&
female
-300 mg/kg
150 mg/kg
-75 mg/kg
-37.5 mg/kg
~ male
¦ male
• male
* male
female
female
female
female
10000
1000
10 12 14 16 18 20 22 24
time (hours)
0.1
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 fl 9971.
Neither the a] Blancato etal. (20071 nor the b] Leavens and Borghoff (20091 model adequately
represents the measured tert-butanol blood concentrations.
Attempts to reoptimize model parameters in the tert- butanol submodels of Blancato etal.
(20071 and Leavens and Borghoff (20091 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-32).
The JPEC pharmacokinetic studies show that approximately 60% of the radiolabel in whole blood is
in the plasma, providing some limited evidence for association of tert-butanol with components in
blood. The PBPK model represented the rate of change of tert-butanol amount in the sequestered
blood compartment (Abiood2) with the equation:
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dAblood2/dt - KoN*CV* - KoFF*Cblood2
where Kon is the binding rate constant, CV is the free tert-butanol concentration in blood, Koff is the
unbinding rate constant, and Cbiood2 is the concentration of tert-butanol bound in blood (equal to
Ablood2/V blood} ¦
ETBE
Inhalation Exhalation
I t
Alveolar Air
B ood
Rapidly Perfused
Slowly Perfused
Fat
Kidney
Liver
Oral
Dose
Kas f
IV Dose
TBA
Inhalation Exhalation
A 1
Alveolar Air
Blood
^K°" f'<
OFF
Sequestered
Rapidly Perfused
Slowly Perfused
Fat
Kidney
VMetbe, KMetbe,
vmETBE2, kmETBE2
Liver
Metabolism
Oral Kas2
Dose
K
ELIM2
Urinary excretion
VMTBA/ KMtba
Metabolism
Figure B-3. Schematic of the PBPK model for ETBE and its major metabolite
tert-butanol in rats.
Exposure can be via multiple routes including inhalation, oral, or i.v. dosing. Metabolism of ETBE and
tert-butanol occur in the liver and are described by Michaelis-Menten equations with two pathways
for ETBE and one for tert-butanol. ETBE and tert-butanol are cleared via exhalation, and tert-butanol
is additionally cleared via urinary excretion. See Table B-8 for definitions of parameter abbreviations.
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The physiologic parameter values were obtained from the literature (Brown etal.. 19971
and are shown in Table B-8. tert-Butanol partition coefficients were obtained from literature where
they were determined by the ratios of measured tissue:air and blood:air partition coefficients
(Borghoff etal.. 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
fLeavens 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-9. The ARCO T19831 study
measured tert-butanol in plasma only, not whole blood like the Poet and Borghoff (19971 and
Leavens and Borghoff f20091 studies. Based on the measurements of plasma and whole blood by
TPEC (2008el. 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.2.5.2. ETBE Model Parameterization and Fitting
The ETBE submodel used the same physiological parameters as tert-butanol obtained from
the literature Brown etal. (1997) shown in Table B-8. ETBE partition coefficients were obtained
from literature Nihlen etal. T19951 where they were calculated for ETBE 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 (TPEC. 2008e: Ambergetal.. 2000: Borghoff. 1996). 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 for the tert-butanol submodel. The optimized
parameter values are shown in Table B-9. The predictions of the model with optimized parameters
for ETBE oral gavage by TPEC f2008el are shown in Figure B-5. This study measured tert-butanol in
plasma only, not whole blood like the Ambergetal. (2000) and other tert- butanol studies. Based on
the measurements of plasma and whole blood by TPEC (2008e). the concentration of tert-butanol in
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Supplemental Information—ETBE
1 Table B-8. 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 etal. (1997)b
Alveolar ventilation (L/hr)
5.38
Brown etal. (1997)c
Liver
0.174
Brown etal. (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
Liver:blood
Fat:blood
Rapidly perfused:blood
Slowly perfused:blood
Kidney:blood
Partition coefficients for ferf-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)
a Fperf- Z(other compartments)
b 15.2*BW0-75
c Alveolar ventilation is set equal to cardiac output
d sum of liver and gastrointestinal (Gl) blood flows
e 1 - Z(all other compartments)
f Set equal to brain tissue
g Set equal to muscle tissue
2
3
11.7 Nihlen et al. (1995)
1.68 Nihlen et al. (1995)
12.3 Nihlen et al. (1995)
2.34 f
1.71 g
1.42 Nihlen et al. (1995)
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Supplemental Information—ETBE
8 10 12 14 16 18 20 22 24
time (hours)
0.1
0 2 4 6 8 10 12 14 16 18 20 22 24
time (hours)
300 mg/kg ~male ~ female
— - 150 mg/kg ¦ male ~ female
---.75 mg/kg • male o female
— • —37.5 mg/kg k male A female
TBA iv exposure
— 1000
4
5
6
7
8
9
TBA gavage
¦500 mg/kg A
¦ 1 mg/kg •
tLO
F
r
100
o
10
r
aj
1
c
u
0.1
~a
o
o
0.01
-D
0 ppm male
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2S0ppm male
— — 2SO ppm female
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Supplemental Information—ETBE
Parameter
Value
Source or Reference
Metabolism high affinity (KMetbe; mg/L)
0.035
Optimized
Metabolism low affinity (VMEtbe2,' mg/L-hr)
15.2
Optimized
Metabolism low affinity (KMEtbe2,' mg/L)
10.0
Optimized
Absorption from Gl (KAs; 1/hr)
0. 5
Optimized
a scaled by BW07 (0.2507 = 0.379)
A) B)
• 400 mg/kg
A 5 mg/kg
~ 5 mg/kg
4 8
time (hours)
0.8
0.6
0.4
0.2
0
c
at
u
c
o
u
T3
O
_o
_n
<
03
< 80
cxo
JL 70
400 mg/kg ETBE gavage
12 16 20 24
time (hours)
4 8
time (hours)
4 8 12 16
time (hours)
5 mg/kg ETBE gavage
Figure B-5. Comparison of the EPA model predictions with measured amounts
of tert-butanol after oral gavage of ETBE.
The data points show the measurements from the four individual rats in the IPEC f2008e1 study. The
concentrations of tert-butanol in blood are shown in A) for the 5- and 400-mg/kg doses, and B] for
only the 5-mg/kg dose. The amount of tert-butanol in urine is shown in C] for the 400-mg/kg dose
and in D] for the 5-mg/kg dose. The model predictions used the optimized parameter values as
shown in Table B-9.
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plasma is approximately 60% of the concentration in whole blood. The tert-butanol plasma
concentrations measured by JPEC were increased (divided by 60%) to the expected concentration
in whole blood for comparison with the PBPK model. The predictions of the model with optimized
parameters are compared with amounts measured by Ambergetal. (20001 after ETBE inhalation in
Figure B-6. While 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 Chemical Industry Institute of Toxicology
(CUT) study (5000 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 (Relink)
0.5/hour was obtained from the literature (the same value was used by (Blancato etal.. 2007: Rao
and Ginsberg. 19971 and Leavens and Borghoff f20091. 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 Error! Reference source not found. 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 literature (0.5/hour) was used for model predictions. The predictions of the
model with optimized parameters are compared with the amounts of ETBE and tert-butanol
exhaled after exposure to 5000-ppm ETBE as measured by CUT in Figure B-7. The EPA model fits
the measured amounts well.
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A)
B)
Ofl
c
o
4->
ro
i_
4->
C
0)
u
c
o
u
T3
O
O
CO
ETBE exposure concentration
40 ppm
4 ppm
0.6
0.4
~
O
4 6 8
time (hours)
10 12
ETBE exposure concentration
— 40 ppm
¦-4 ppm
4 6 8
time (hours)
10 12
C)
D)
ETBE model A ETBE data
TBA model • TBAdata
oo 2.5
9 0.5
0 5 10 15 20 25 30 35 40 45 50
ETBE exposure concentration (ppm)
_ 0.04
(JJO
E_
c 0.03
c
< 0.02
CO
i o.oi
¦ data
KELIM2=1.5/hr
KELIM2=0.5/hr
0 4 8 12 16 20 24 28 32 36
time (hours)
Figure B-6. Comparison of the 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, EQ for tert-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 tert-butanol in urine is shown in D)
for the 40 ppm exposure for two values of Kelim2, the rate constant for tert-butanol urinary
elimination. The value 0.5/hr was obtained from Blancato et al. (20071 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 etal. (20001 The model predictions used the optimized
parameter values as shown in Table B-9.
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Supplemental Information—ETBE
time after exposure {hours)
time after exposure (hours)
Figure B-7 . Comparison of the EPA model predictions with measured amounts
of A) ETBE and B) tert-butanol in exhaled breath after a 6-hour inhalation
exposure to 5000 ppm ETBE.
The data points show the individual measurements of the three rats in the ARCO (19831 study. The
model predictions used the optimized parameter values as shown in Table B-9.
Induction of tert-butanol metabolizing enzymes was included in the Leavens and Borghoff
(20091 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-9 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 can be seen in the comparison of the model predictions and
experimental measurements shown 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
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Supplemen tal Information—ETBE
with ETBE was by oral gavage for 14 days at 5 mg/kg-day and tert-butanol blood concentrations
did not decline after repeated doses 1PEC f2010bl. The internal dose of tert-butanol after repeated
ETBE dosing in the 1PEC f2010bl study was much lower than in the tert-butanol repeated dosing
study (Leavens and Borghoff. 20091 and possibly the lower tert-butanol blood concentration wasn't
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 the induction of enzymes
occurs more than after oral exposure.
time (hours)
0 24 48 72 96 120 144 168 192 0 24 48 72 96 120 144 168 192
time (hours) time (hours)
Figure B-8. Comparison of the 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 1PEC ("2010b) study. tert-Butanol
blood concentrations are not well predicted by the model at the highest tert-butanol exposure
concentration without enzyme induction.
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with induction without induction
txo
E
c
o
c
0)
u
c
o
u
"O
o
_o
_Q
<
CD
0.1
0.01
48
96
144 192
time (hours)
240
288
336
Figure B-9. Comparison of the EPA model predictions with measured amounts
of tert-butanol in blood after 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 IPEC f2010bl study. Adding
enzyme induction to the model has a small effect on the predicted tert-butanol blood concentrations
and the model predictions are closer to measured data when induction is not included.
B.2.5.3. Summary of the PBPK Model for ETBE
A PBPK model for ETBE and tert-butanol was developed by adapting previous models for
MTBE and tert-butanol fBlancato etal. f20071: Leavens andBorghoff f20091I Published tert-
butanol models (or sub-models) do not adequately represent the tert-butanol blood concentrations
measured in the i.v. study (Poet et al. 1997). The addition of a sequestered blood compartment for
tert-butanol substantially improved the model fit The alternative modification of changing to
diffusion-limited distribution between blood and tissues also improved the model fit, but was
considered less biologically plausible. Physiological parameters and partition coefficients were
obtained from published measurements. The rate constants for tert-butanol metabolism and
elimination were from a published PBPK model of MTBE with a tert-butanol subcompartment
fBlancato etal. (2007)). Additional model parameters were estimated by calibrating to data sets
for i.v., oral and inhalation exposures as well as repeated dosing studies for both ETBE and TBA.
Although in one case (Ambert et al., 2000), the model modestly overpredicted the tert-butanol
blood concentration by approximately 1.5-fold, overall, the model produced acceptable fits to
multiple rat time-course datasets of ETBE and TBA blood levels following either inhalation or oral
gavage exposures.
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B.2.5.4. ETBE Model Application
The PBPK model described above was applied to conduct route-to-route extrapolation
based on an equivalent internal dose. The selection of the appropriate internal dose metric depends
on the endpoint and is discussed in the Volume 1, Section 2 Dose-Response Analysis. 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 fl9821.
B.2.5.5. PBPK Model Code08
The PBPK acslX model code is made available electronically through EPA's Health and
Environmental Research Online (HERO) database. All model files may be downloaded in a zipped
workspace from HERO (www.epa.gov/herol.
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B.3. GENOTOXICITY STUDIES
B.3.1.1. Bacterial Systems
Mutagenic potential of ETBE has been tested by Zeiger etal. (19921 using different
Salmonella typhimurium strains for 311 chemicals, including ETBE, both in the absence and
presence of metabolic activation (S9). Preincubation protocol was followed and precaution was
exercised to account for the volatility of the compound. One dose of 10,000 [ig/plate was tested
using different Salmonella strains including TA97, TA98, TA100, TA1535. The results showed that
the ETBE did not cause mutations in any of the Salmonella strains tested. It should be noted that
TA102, a sensitive strain for oxidative metabolite, was not used in this study. The available
genotoxicity data for tert-butanol are discussed below, and the summary of the data is provided in
Table B-10.
B.3.1.2. 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 5000[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 in vitro Chinese hamster ovary cells using chromosome aberration
assay. The cells were exposed from 100 to 5000[ig/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.3.1.3. In Vivo Animal Studies
In vivo studies were conducted by same authors that tested ETBE for in vitro genotoxicity.
Vergnes and Kubena (1995a). unpublished report, performed an in vivo bone marrow micronucleus
(MN) test in mice in response to ETBE exposure. Male and female CD-I mice (5animals/sex/group)
were exposed to ETBE by inhalation at target concentrations of 0, 400, 2000, and 5000 ppm (0,
1671, 8357, and 20894 mg/m3) for 6 hours/day, for 5 days. Following treatment, polychromatic
erythrocytes (PCE) from bone marrow were analyzed for micronucleus formation. The results
showed that no statistically significant increases in the mean percentages of micronucleated
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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
micronucleus as a result of exposure to ETBE [flPEC. 2007a. b, c, d) published as Noguchi etal.
("20131],
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 (5animals/sex/dose group) were
administered ETBE (99.3% pure) via gavage at doses of 0, 500,1000, or 2000 mg/kg-day separated
by 24 hours in olive oil TPEC f2007al. 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, 500,1000, or 2000 mg/kg/day in olive oil fNoguchi etal.. 2013: TPEC.
2007b). Animals were sacrificed, and bone marrow smears were collected and stained 24 hours
after the final injection. All animals in the 2000 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 0,1600, 4000, or 10000 ppm ETBE for 13 weeks fNoguchi etal.. 2013:
TPEC. 2007dl. 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,
500,1500, or 5000 ppm (0, 2089, 6268, or 20894 mg/m3) 6 hours/day, 5 days/week (Noguchi et
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al., 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 a number of 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 AIdh2 knockout mice after subchronic inhalation exposure. All studies used the same exposures
(i.e., to 0, 500,1750 and 5000 ppm ETBE for 6hrs/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 Wengetal. (2011).
Weng etal. f20121 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, while the increase was
only found in 5000ppm exposure group for the knockout female mice. In the wild-type, significant
DNA damage was seen only in males in the 5000 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 Wengetal. (2013). in addition to the DNA strand
breaks, 8-hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification, and 8-
hydroxydeoxyguanosine, the authors performed in vivo micronucleus tests on what appear to be
the same set of animals. The mice (wild-type and knockout, males and females) were exposed to 0,
500,1750 and 5000 ppm ETBE for 6h/day, 5days/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 1750ppm and 5000 ppm exposure groups were significantly increased
when compared with the control group. In the wild-type male mice, however, only the 5000 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 (5000 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 Wengetal. (2014). DNA strand breaks and 8-
hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification were measured in
sperm collected from the left caudia epididymis. In addition to the 13-week protocol used in the
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Supplemental Information—ETBE
1 other studies, Wengetal. (2014) also included an additional 9-week study where the male mice
2 (wild-type, knockout, and heterogeneous [HT]) were exposed to 0, 50, 200 and 500 ppm ETBE for
3 6h/day, 5days/week for 9 weeks. In the 13-week study, there were significant increases in damage
4 in all three exposure groups in the knockout male mice, but only in the two highest dose groups in
5 the wild-type males. In the 9-week study, there was no change in the wild-type mice, but both the
6 heterogeneous and the knockout mice had significant increases in the two highest doses.
7 Table B-10. Summary of genotoxicity (both in vitro and in vivo) studies of
8 ETBE.
Species
Test System
Dose/Cone.
Results3
Comments
Reference
Bacterial systems
-S9
+S9
Salmonella
Mutation
10,000 ng/plate
-
-
Preincubation procedure
Zeiger et al. (1992)
typhimuriu
Assay
was followed.
m (TA97,
Experiment was
TA98,
conducted in capped
TA100,
tubes to control for
TA1535)
volatility
In vitro systems
Chinese
Gene
100, 300, 1000,
-
-
Experiments conducted
Vergnes and
Hamster
Mutation
3000, 5,000
both with and without
Kubena (1995b)
Ovary cells
Assay
Hg/ml
metabolic activation
(unpublished
(hgprt
report)
locus)
Chinese
Chromosoma
100, 300, 1000,
Experiments conducted
Vergnes (1995)
Hamster
1 Aberration
3000, 5,000
-
-
both with and without
(unpublished
Ovary cells
Assay
Hg/ml
metabolic activation
report)
In vivo animal studies
CD-I mice
Bone
0, 400, 2000,
-
Whole body Inhalation,
Vergnes and
(male and
Marrow
5000 ppm (0,
6hrs/day, 5 days, 5
Kubena (1995a)
female)
Micronucleu
s test
1670, 8360,
20900 mg/m3)b
animals/ses/group
(unpublished
report)
Fisher 344
Bone
0, 500, 1000,
-
Oral gavage, 24h apart,
JPEC (2007b)
rats (male
Marrow
2000 mg/kg/day
2 days, 5
(unpublished
and
Micronucleu
animals/sex/group
report)
female)
s test
Fisher 344
Bone
0, 250, 500,
-
Intraperitoneal
Noguchi et al.
rats (male
Marrow
1000, 2000
injection, 24h apart, 2
(2013); JPEC
and
Micronucleu
mg/kg/day
days, 5
(2007b), unpublishe
female)
s test
animals/sex/group
d report
Fisher 344
Bone
0, 1600, 4000,
-
Drinking water, 13
Noguchi et al.
rats (male
Marrow
10000 ppm (0,
weeks, 10
(2013); JPEC
and
Micronucleu
101, 259, 626
animals/sex/group
(2007c),
female)
s test
mg/kg/day in
males; 0,120,
267, 629 mg/kg-d
in females)0
unpublished report
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Species
Test System
Dose/Cone.
Results3
Comments
Reference
Fisher 344
Bone
0, 500, 1500,
-
Whole body inhalation,
Noguchi et al.
rats (male
Marrow
5000 ppm (0,
6hrs/day, 5 days/week,
(2013); JPEC
and
Micronucleu
2090, 6270,
13 weeks. 10
(2007c),
female)
s test
20900 mg/m3)b
animals/sex/group
unpublished report
C57BL/6
DNA strand
0, 500,1750 and
Male -
+d/+
Whole body inhalation,
Weng et al. (2011)
wild-type
breaks
5000 ppm
WT/KO
6hrs/day, 5 days/week,
(WT) and
(alkaline
Female
-/+d
13 weeks
Aldh2
comet
WT/KO
knockout
assay),
(KO) mice
leukocytes
C57BL/6
DNA strand
0, 500,1750 and
Male -
+d/+
Whole body inhalation,
Weng et al. (2012)
wild-type
breaks
5000 ppm
WT/KO
6hrs/day, 5 days/week,
(WT) and
(alkaline
Female
-/+d
13 weeks
Aldh2
comet assay)
WT/KO
knockout
(KO) mice
C57BL/6
Micronucleu
0, 500,1750 and
Male*
+d/+
Whole body inhalation,
Weng et al. (2013)
wild-type
s assay,
5000 ppm
WT/KO
6hrs/day, 5 days/week,
(WT) and
erythrocytes
Female
-/+
13 weeks
Aldh2
*
knockout
WT/KO
(KO) mice
C57BL/6
DNA strand
0, 50, 200 and
WT/HT
-/+/+
Whole body inhalation,
Weng et al. (2014)
wild-type
breaks
500 ppm
/KO
6hrs/day, 5 days/week,
(WT) and
(alkaline
9 weeks
Aldh2
comet
knockout
assay);
(KO) mice
sperm
C57BL/6
DNA strand
0, 500,1750 and
WT/KO
+/+
Whole body inhalation,
Weng et al. (2014)
wild-type
breaks
5000 ppm
6hrs/day, 5 days/week,
(WT) and
(alkaline
13 weeks
Aldh2
comet
knockout
assay);
(KO) mice
sperm
fffa+ = positive; - = negative; (+), equivocal
b4.18 mg/m3 = lppm
Conversions performed by study authors
dpositive in highest dose tested
*when the data of ETBE-induced MN-RETs (micronucleated reticulocytes) were normalized with corresponding
control, the effect disappeared
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
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Supplemental Information—ETBE
1 across the genotoxicity tests needed for proper interpretation of the weight of evidence of the data;
2 (b) the quality of the available data. With respect to the array of types of genotoxicity tests
3 available, ETBE has only been tested in one bacterial assay. Limited (two) studies are available with
4 respect to in vitro studies. Existing in vivo studies have all been tested only for the micro nucleus
5 assay and/or DNA strand breaks. Key studies in terms of chromosomal aberrations, DNA adducts
6 etc are missing. It should also be noted that the few existing studies are unpublished reports lacking
7 peer review. Given the above limitations; significant deficiencies; and sparse database both in terms
8 of quality and quantity; it is implicit that the database is inadequate or insufficient to draw any
9 conclusions on the effect of ETBE with respect to genotoxicity.
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1
2 APPENDIX C. DOSE-RESPONSE MODELING FOR
3 THE DERIVATION OF REFERENCE VALUES FOR
4 EFFECTS OTHER THAN CANCER AND THE
s DERIVATION OF CANCER RISK ESTIMATES
6 C.l. Benchmark Dose Modeling Summary
7 This appendix provides technical detail on dose-response evaluation and determination of
8 points of departure (PODs) for relevant toxicological endpoints. The endpoints were modeled using
9 the U.S. EPA's Benchmark Dose Software (BMDS, version 2.2). Sections C.l.1.1 and C.l.1.2 (non-
10 cancer) and Section 0 (cancer) describe the common practices used in evaluating the model fit and
11 selecting the appropriate model for determining the POD, as outlined in the Benchmark Dose
12 Technical Guidance Document U.S. EPA (2012). In some cases, it may be appropriate to use
13 alternative methods based on statistical judgment; exceptions are noted as necessary in the
14 summary of the modeling results.
15 C.l.l. Non-cancer Endpoints
16 C.l.1.1. Evaluation of Model Fit
17 For each dichotomous endpoint, BMDS dichotomous models1 were fitted to the data using
18 the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-square
19 goodness-of-fit test (x2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess
20 model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the
21 vicinity of the benchmark response (BMR).
22 For each continuous endpoint, BMDS continuous models2 were fitted to the data using the
23 maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model,
24 first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test
25 2 was not rejected (x2 p-value > 0.10), the model was fitted to the data assuming constant variance.
26 If Test 2 was rejected (x2 p-value < 0.10), the variance was modeled as a power function of the
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.
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mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS
Test 3). For fitting models using either constant variance or modeled variance, models for the mean
response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with x2 p-value <
0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such as scaled
residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
C.l.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.
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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
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
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
1000
No. of
animals
25
25
25
25
Mean ± SD
3.58 ±0.413
3.96 ± 0.446
4.12 ±0.624
4.34 ± 0.434
Increased relative
kidney weight
Gaoua (2004b)
PO Male
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
250
500
1000
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
1000
No. of
animals
25
24
22
25
Mean ± SD
2.24 ±0.185
2.22 ±0.16
2.29 ±0.207
2.35 ±0.224
This document is a draft for review purposes only and does not constitute Agency policy.
B-3 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased relative
kidney weight
Gaoua (2004b)
PO Female
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
250
500
1000
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
1000
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
1000
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
1000
No. of
animals
25
24
25
23
Mean ± SD
2.24 ±0.178
2.34 ±0.242
2.3 ±0.226
2.49 ± 0.284
Increased relative
kidney weight
Gaoua (2004b)
F1 Female
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
250
500
1000
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 (2008c)
Male
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
100
300
1000
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 (2008c)
Male
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
100
300
1000
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 (2008c)
Female
Sprague-
Dawley
rats
Dose
(mg/kg-d)
0
100
300
1000
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
Female
Sprague-
Dose
(mg/kg-d)
0
100
300
1000
This document is a draft for review purposes only and does not constitute Agency policy.
B-4 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Fuiii et al. (2010);
JPEC (2008c)
Dawley
rats
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
2090
6270
20900
Incidence /
Total
2/50
5/50
16/49
41/50
Increased absolute
kidney weight
JPEC (2008a)
Male
Sprague-
Dawley
rats
Exposure
concentration
(ppm)
0
150
500
1500
5000
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
1500
5000
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
1500
5000
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
1500
5000
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.
(1996)
Male
F344 rats
Exposure
concentration
(ppm)
0
500
1750
5000
No. of
animals
11
11
11
11
Mean ± SD
1.73 ±0.155
1.85 ±0.137
1.903 ±0.1
2.067 ±0.124
Increased absolute
kidney weight
Medinskv et al.
(1999); Bond et al.
(1996)
Female
F344 rats
Exposure
concentration
(ppm)
0
500
1750
5000
No. of
animals
10
11
11
11
This document is a draft for review purposes only and does not constitute Agency policy.
B-5 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Mean ± SD
1.077 ± 0.069
1.125 ±0.048
1.208 ± 0.076
1.306 ± 0.055
1
2 C.l.1.3. Modeling Results
3 Below are tables summarizing the modeling results for the noncancer endpoints modeled.
4
5 Oral Exposure Endpoints
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 Table C-2. Summary of BMD modeling results for slight urothelial hyperplasia
2 of the renal pelvis in male F344 rats exposed to ETBE in drinking water for
3 104 weeks (IPEC. 2010a): modeled with doses as mg/kg-d (calculated by study
4 authors); BMR = 10% extra risk.
5
6
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-Lineard
0.395
126.07
79.3
60.5
a Selected model in bold; scaled residuals for selected model for doses 0, 28,121, and 542 mg/kg-d were 0.000, -
1.377, 1.024, and -0.187, respectively.
b For the Multistage 3° model, the beta coefficient estimates were 0 (boundary of parameters space). The models
in this row reduced to the Multistage 2° model.
c The Multistage 2° model may appear equivalent to the Quantal-Linear model, however differences exist in digits
not displayed in the table.
d The Quantal-Linear model may appear equivalent to the Multistage 3° model, however differences exist in digits
not displayed in the table. This also applies to the Multistage 2° model.
Data from JPEC (2010a)
7
This document is a draft for review purposes only and does not constitute Agency policy.
B-7 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
BMDL BMD
Quantal Linear Model,
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
o
13:10 09/10 2014
with BMR of 1 0% Extra Risk for the BMD arid 0.95 Lower Confidence Limit forthe
100 200 300 400 500
dose
Figure C-l. Plot of incidence rate by dose, with fitted curve for selected model;
dose shown in mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Supplemental Information—ETBE
Quanta I 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
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
ChiA2 = 2.98 d.f = 3 P-value = 0.3948
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1
2
3
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 180 days (Mivata
etal.. 2013: IPEC. 2008c): 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 on
the basis of 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
a Modeled 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-d were -0.421, -0.288,1.29, -0.669, and 0.15, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
f The Linear model may appear equivalent to the Polynomial 3° model, however differences exist in digits not
displayed in the table.
g The Polynomial 3° model may appear equivalent to the Power model, however differences exist in digits not
displayed in the table. This also applies to the Polynomial 2° model. This also applies to the Linear model.
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDI
4.6
4.4
4.2
: 4
t ,e
i „
3.4
3.2
3
15:56 05/15 2014
200
dose
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Supplemental Information—ETBE
Figure C-2. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-d.
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
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
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
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
Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
47.4275
8
<0.0001
This document is a draft for review purposes only and does not constitute Agency policy.
B-ll DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Test 2
24.6007
4
<0.0001
Test 3
2.34371
3
0.5042
Test 4
1.11251
3
0.7741
1
2
3
This document is a draft for review purposes only and does not constitute Agency policy.
B-12 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 180 days (Mivata
3 etal.. 2013: IPEC. 2008c): BMR = 10% relative deviation from the mean.
4
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)c
0.0262
-339.53
242
174
Exponential (M4)
Exponential (M5)d
0.0472
-340.67
113
45.6
Hill
0.0481
-340.71
112
47.2
Power
<0.0001
-315.18
40000
4.00E-13
Polynomial 3°e
Polynomial 2°f
Linear
0.03
-339.83
231
161
a Modeled 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.
b The Exponential (M2) model may appear equivalent to the Exponential (M3) model, however differences exist in
digits not displayed in the table.
c The Exponential (M3) model may appear equivalent to the Exponential (M2) model, however differences exist in
digits not displayed in the table.
d For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
5
6
This document is a draft for review purposes only and does not constitute Agency policy.
B-13 DRAFT—DO NOT CITE OR QUOTE
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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 180 days
3 (Mivata et al.. 2013: IPEC. 2008c): BMR = 10% relative deviation from the
4 mean.
5
6
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.369
-168.25
406
271
The Exponential (M4) model was
selected on the basis of 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
a Constant variance case presented (BMDS Test 2 p-value = 0.425), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25, 100, and 400 mg/kg-d were 0.2257, 0.2206, -0.737, 0.3806, and -0.08999,
respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c BMD or BMDL computation failed for this model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
7
This document is a draft for review purposes only and does not constitute Agency policy.
B-14 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
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
BMDL
BMD
0
50
1 00
150
200
250
300
350
400
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-d.
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
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
This document is a draft for review purposes only and does not constitute Agency policy.
B-15 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
100
15
2.02
2.002
0.21
0.1869
0.3806
400
15
2.07
2.074
0.23
0.1869
-0.08999
1
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
4 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
5
6
7
This document is a draft for review purposes only and does not constitute Agency policy.
B-16 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 180 days
3 (Mivata et al.. 2013: IPEC. 2008c): BMR = 10% relative deviation from the
4 mean.
5
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.111
-343.15
374
253
The Hill model is selected on the
basis of 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
a Constant 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-d were -0.917,1.47, -0.738, 0.242, and -0.054, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
Data from (Mivata et al., 2013; JPEC, 2008c)
6
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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-d.
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
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
100
15
0.6
0.596
0.06
0.0582
0.242
400
15
0.62
0.621
0.06
0.0582
-0.054
1
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
4 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
5
6
7
8
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-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
3 weeks beginning 10 weeks before mating until after weaning of the pups.
4 Gaoua f2004a1: BMR = 10% relative deviation from the mean.
5
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.155
-38.410
551
423
The Hill model is selected on the
basis of lowest BMDL.
Exponential (M4)c
0.727
-40.012
255
123
Exponential (M5)d
0.727
-40.012
255
123
Hill
0.811
-40.077
244
94.0
Power6
Polynomial 3°f
Polynomial 2°g
Linear
0.199
-38.902
517
386
a Constant 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 1000 mg/kg-d were -0.0247, 0.14, -0.181, and 0.0657, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c The Exponential (M4) model may appear equivalent to the Exponential (M5) model, however differences exist in
digits not displayed in the table.
d The Exponential (M5) model may appear equivalent to the Exponential (M4) model, however differences exist in
digits not displayed in the table.
e For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
f For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
g For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
Data from Gaoua (2004a)
6
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
4.6
Hil
4.4
4.2
3.8
3.6
3.4
BMDL
BMD
0
200
400
600
800
1000
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-d.
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
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1000
25
4.34
4.33
0.434
0.477
0.0657
1
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
4 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
5
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 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
3 weeks beginning 10 weeks before mating until after weaning of the pups.
4 Gaoua f2004al: BMR = 10% relative deviation from the mean.
5
6
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0632
-449.45
415
355
The Hill model was selected on the
basis of 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
a Constant 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 1000 mg/kg-d were -0.0131, 0.0533, -0.0566, and 0.0164, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
7
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hil
0.8
0.75
0.7
0.65
0.6
BMDL
BMD
O
200
400
600
800
1 000
8 15:07 05/15 2014
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Supplemental Information—ETBE
Figure C-6. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-d.
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 = 223.505
BMDL at the 95% confidence level = 137.393
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
1070.38
649.462
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
1000
25
0.763
0.763
0.063
0.0605
0.0164
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
Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
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
1
2
3
This document is a draft for review purposes only and does not constitute Agency policy.
B-25 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 weeks beginning 10 weeks before mating until after weaning of the pups
4 Gaoua f2004al: BMR = 10% relative deviation from the mean.
5
6
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
1734
1030
Exponential (M2) model is
selected on the basis of lowest
AIC; however, BMDL is higher than
the maximum dose.
Exponential (M3)
0.416
-212.86
1458
1040
Exponential (M4)
0.327
-212.56
1774
1032
Exponential (M5)
N/Ab
-211.39
error0
0
Hill
0.715
-213.39
error0
error0
Power
0.418
-212.87
1470
1041
Polynomial 3°
0.400
-212.81
1409
1035
Polynomial 2°
0.400
-212.81
1409
1037
Linear
0.619
-214.56
1774
1032
a Constant 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 1000 mg/kg-d were 0.5052, -0.7974, 0.1844, and 0.1033, respectively.
b No available degrees of freedom to calculate a goodness of fit value.
c BMD or BMDL computation failed for this model.
7
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bf
2.45
2.4
2.35
CD
(f)
a
o
Q_
(/) o o
CD 2.3
q:
a
CO
CD
S 2.25
2.2
2.15
O 200 400 600 800 1000 1200 1400 1600 1800
dose
8 15:14 05/15 2014
9
10 Figure C-7. Plot of mean response by dose, with fitted curve for selected
11 model; dose shown in mg/kg-d.
Exponential
BMDL BM:
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1
2 Exponential Model. (Version: 1.9; Date: 01/29/2013)
3 The form of the response function is: Y[dose] = a * exp(sign * b * dose)
4 A constant variance model is fit
5
6 Benchmark Dose Computation.
7 BMR = 10% Relative deviation
8 BMD = 1734.24
9 BMDL at the 95% confidence level = 1030.08
10
11 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
12
13 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
1000
25
2.35
2.346
0.224
0.1923
0.1033
14
15 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
16
17 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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Test 4
0.9403
2
0.6249
1
2
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-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 weeks beginning 10 weeks before mating until after weaning of the pups Gaoua
4 f2004a1: BMR = 10% relative deviation from the mean.
5
6
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M4)b
N/A
-283.41
1258
829
No model adequately fit the data.
Exponential (M3)
N/A
-290.99
1037
983
Exponential (M5)
N/Ac
-288.99
1037
983
Hill
<0.0001
-276.90
errord
errord
Power
<0.0001
-296.86
1648
1056
Polynomial 3°
0.00528
-292.51
-9999
976
Polynomial 2°
0.00236
-290.89
-9999
945
Linear
1.92E-04
-285.88
40622
errord
a Modeled 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.
b For the Exponential (M4) model, the estimate of c was 0 (boundary). The models in this row reduced to the
Exponential (M2) model.
c No available degrees of freedom to calculate a goodness of fit value.
d BMD or BMDL computation failed for this model.
7
8
9
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-ll. Summary of BMD modeling results for absolute kidney weight in
2 F1 male Sprague-Dawley rats exposed to ETBE by gavage in a 2-generation
3 study (Gaoua. 2004b): BMR = 10% relative deviation from the mean.
4
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
a Modeled 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 1000 mg/kg-d were -0.584, 0.717, 0.225, and -0.837, respectively.
b No available degrees of freedom to calculate a goodness of fit value.
Data from Gaoua (2004b)
5
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMD
8
O 200 400 600 800 10OO
dose
6 13:43 09/12 2014
7
8 Figure C-8. Plot of mean response by dose, with fitted curve for selected
9 model; dose shown in mg/kg-d.
10
Polynomial
BMDL
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Supplemental Information—ETBE
Polynomial Model. (Version: 2.19; Date: 06/25/2014)
The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
A modeled variance is fit
THE MODEL HAS PROBABLY NOT CONVERGED!!!
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 318.084
BMDL at the 95% confidence level = 235.491
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
lalpha
-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
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
1000
25
5.34
6.2
5.39
5.16
-0.837
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
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Test 3
2.13011
2
0.3447
Test 4
0.791648
1
0.3736
1 Table C-12. Summary of BMD modeling results for relative kidney weight in F1
2 male Sprague-Dawley rats exposed to ETBE by gavage in a 2-generation study
3 (Gaoua. 2004bl: BMR = 10% relative deviation.
4
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
a Modeled 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.
b No available degrees of freedom to calculate a goodness of fit value.
Data from Gaoua (2004b)
5
6
7 Table C-13. Summary of BMD modeling results for absolute kidney weight in
8 F1 female Sprague-Dawley rats exposed to ETBE by gavage in a 2-generation
9 study (Gaoua. 2004b): BMR = 10% relative deviation.
10
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
0.311
-180.23
978
670
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Exponential (M2)
model was selected based on
lowest AIC.
Exponential (M3)
0.147
-178.46
1016
679
Exponential (M4)
0.121
-178.16
980
654
Exponential (M5)
N/Ab
-176.44
1019
613
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Supplemental Information—ETBE
Hill
N/Ab
-176.44
1019
611
Power
0.145
-178.44
1019
666
Polynomial 3°
0.184
-178.80
1001
684
Polynomial 2°
0.159
-178.58
1002
673
Linear
0.301
-180.16
980
654
a Constant 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 1000 mg/kg-d were -0.05426, 0.8898, -1.173, and 0.3711, respectively.
b No available degrees of freedom to calculate a goodness of fit value.
Data from Gaoua (2004b)
1
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Level for BM
Exponential
2.6
2.5
2.4
2.3
2.2
BMDL
BMD
O
200
400
600
800
1 OOO
2 13:47 09/12 2014
3
4 Figure C-9. Plot of mean response by dose, with fitted curve for selected
5 model; dose shown in mg/kg-d.
6
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Supplemental Information—ETBE
Exponential Model. (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * exp(sign * b * dose)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 978.157
BMDL at the 95% confidence level = 669.643
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
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
1000
23
2.49
2.472
0.284
0.2322
0.3711
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
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
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Supplemental Information—ETBE
Test 3
5.186
3
0.1587
Test 4
2.336
2
0.311
1
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 2-generation
4 study (Gaoua. 2004b): BMR = 10% relative deviation.
5
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
1064
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
1067
489
Hill
0.0335
-410.30
1069
466
Power
1.02E-04
-398.44
6.5E+06
errord
Polynomial 3°
0.0333
-410.29
1057
687
Polynomial 2°e
Linear
0.103
-412.26
1063
686
a Modeled 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.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d BMD or BMDL computation failed for this model.
e For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
Data from Gaoua (2004b)
6
7
8 Table C-15. Summary of BMD modeling results for increased absolute kidney
9 weight in P0 male S-D rats exposed to ETBE by daily gavage for 16 weeks
10 beginning 10 weeks prior to mating Fuiii etal. f20101: BMR = 10% relative
11 deviation from the mean.
12
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Exponential (M2)
Exponential (M3)b
0.668
-41.247
648
479
The Hill model was selected on the
basis of 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
a Constant 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 1000 mg/kg-d were -0.202, 0.399, -0.232, and 0.0344, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
1
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
4.4
Hil
4.2
3.8
3.6
3.4
3.2
BMD
BMDI
O
200
400
600
800
1000
2 13:13 05/15 2014
3
4 Figure C-10. Plot of mean response by dose, with fitted curve for selected
5 model; dose shown in mg/kg-d.
6
7 Hill Model. (Version: 2.17; Date: 01/28/2013)
8 The form of the response function is: Y[dose] = intercept + v*doseAn/ (kAn + doseAn)
9 A constant variance model is fit
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1
2 Benchmark Dose Computation.
3 BMR = 10% Relative deviation
4 BMD = 434.715
5 BMDL at the 95% confidence level = 139.178
6
7 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
1122
1610
8
9 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
1000
24
4.07
4.07
0.53
0.473
0.0344
10
11 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
12
13 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
14
15
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-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 Fuiii etal. f20101: BMR = 10% relative deviation from the
4 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
0.0530
-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
a Constant 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 1000 mg/kg-d were -0.602,1.25, -0.78, and 0.133, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
5
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hil
0.7
0.65
0.6
0.55
BMDL
BMP
O 200 400 600 800 1000
dose
6 14:04 05/15 2014
7
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Supplemental Information—ETBE
Figure C-ll. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-d.
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
1625.63
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
1000
24
0.689
0.688
0.049
0.0526
0.133
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
Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
69.9244
6
<0.0001
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Test 2
3.9156
3
0.2707
Test 3
3.9156
3
0.2707
Test 4
2.58174
1
0.1081
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 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 Fuiii etal. f20101: BMR
4 = 10% relative deviation from the mean.
5
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
1135
781
Polynomial 2° is selected on the
basis of most parsimonious model
with lowest AIC.
Exponential (M3)
0.441
-198.60
1089
826
Exponential (M4)
0.217
-197.67
1144
771
Exponential (M5)
N/Ab
-196.66
error0
0
Hill
N/Ab
-196.66
error0
error0
Power
0.441
-198.60
1092
823
Polynomial 30d
Polynomial 2°
0.743
-200.60
1094
905
Linear
0.467
-199.67
1144
771
a Constant 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 1000 mg/kg-d were 0.499, -0.579, 0.0914, and -0.00282, respectively.
b No available degrees of freedom to calculate a goodness of fit value.
c BMD or BMDL computation failed for this model.
d For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model.
6
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the Bl
Polynomial
2.45
2.4
2.35
2.3
2.25
2.2
2.15
2.1
2.05
BM: D
BMDL
O
200
400
600
800
1 000
7 14:09 05/15 2014
8
9 Figure C-12. Plot of mean response by dose, with fitted curve for selected
10 model; dose shown in mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
B-41 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1
2 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
3 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
4 A constant variance model is fit
5
6 Benchmark Dose Computation.
7 BMR = 10% Relative deviation
8 BMD = 1093.86
9 BMDL at the 95% confidence level = 905.267
10
11 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
12
13 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
1000
19
2.33
2.33
0.24
0.18
-0.00282
14
15 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
16
17 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
18
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-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 Fuiii etal. f20101: BMR
4 = 10% relative deviation from the mean.
5
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
0.367
-471.62
2953
1482
Polynomial 2° is selected on the
basis of lowest AIC.
Exponential (M3)
0.208
-470.04
1573
1026
Exponential (M4)
0.156
-469.61
3056
1506
Exponential (M5)
N/Ab
-468.07
error0
0
Hill
N/Ab
-468.07
error0
error0
Power
0.208
-470.04
1592
1028
Polynomial 3°
0.207
-470.03
1511
1172
Polynomial 2°
0.450
-472.03
1751
1254
Linear
0.366
-471.61
3055
1506
a Constant 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 1000 mg/kg-d were 0.849, -0.925, 0.0742, and 0.00257, respectively.
b No available degrees of freedom to calculate a goodness of fit value.
c BMD or BMDL computation failed for this model.
6
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 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
7 14:31 05/15 2014
8
9 Figure C-13. Plot of mean response by dose, with fitted curve for selected
10 model; dose shown in mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
B-43 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1
2 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
3 The form of the response function is: Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
4 A constant variance model is fit
5
6 Benchmark Dose Computation.
7 BMR = 10% Relative deviation
8 BMD = 1751.45
9 BMDL at the 95% confidence level = 1254.17
10
11 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
12
13 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
1000
24
0.687
0.687
0.045
0.0503
0.00257
14
15 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
16
17 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
18
This document is a draft for review purposes only and does not constitute Agency policy.
B-44 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Inhalation Exposure Endpoints
2
3 Table C-19. Summary of BMD modeling results for slight urothelial
4 hyperplasia of the renal pelvis in male F344 rats exposed to ETBE by whole-
5 body inhalation for 6 hr/d, 5d/wk, for 104 wks flPEC. 2010blBMR = 10% extra
6 risk.
7
Model3
Goodness of fit
BMCiopct
(mg/m3)
BMCLiopct
(mg/m3)
Basis for model selection
p-value
AIC
Gamma
0.874
164.37
2734
1498
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
4329
3522
LogLogistic
0.814
164.40
3010
1831
Probit
0.202
165.59
4059
3365
LogProbit
0.633
164.57
3050
1896
Weibull
0.758
164.44
2623
1478
Multistage 3°
0.565
164.69
2386
1412
Multistage 2°
0.565
164.69
2386
1422
Quantal-Linear
0.269
165.16
1541
1227
a Selected model in bold; scaled residuals for selected model for doses 0, 2089, 6268, and 20893 mg/m3 were
0.036, -0.107, 0.104, and -0.040, respectively. Exposure concentrations were converted from 0, 500,1500, and
5000 ppm to mg/m3 using the calculation mg/m3 = (102.17, molecular weight of ETBE) x ppm -f 24.45.
Data from JPEC2010b
8
9
10
13:40 09/10 2014
10000
dose
Gamma Multi-Hit
BMD
Gamma Multi-Hit Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for the
1
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Figure C-14. Plot of incidence rate by dose, with fitted curve for selected
model; dose shown in mg/m3.
Gamma Model. (Version: 2.16; Date: 2/28/2013)
The form of the probability function is: P[response]= background+(l-
background)*CumGamma[slope*dose,power], where CumGamma(.) is the cummulative Gamma
distribution function
Power parameter is restricted as power >=1
Benchmark Dose Computation.
BMR = 10% Extra risk
BMD = 2734.41
BMDL at the 95% confidence level = 1497.7
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0.0390054
0.0576923
Slope
0.000121504
0.000132454
Power
1.59019
1.84876
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
AIC: = 164.373
Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0.039
1.95
2
50
0.036
2089
0.1046
5.231
5
50
-0.107
6268
0.3196
15.659
16
49
0.104
20893
0.8222
41.109
41
50
-0.04
ChiA2 = 0.03 d.f = 1 P-value = 0.8737
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
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 wks IPEC f2008b1: BMR = 10% relative deviation from the mean.
Model3
Goodness of fit
BMCiord (ppm)
BMCLiord (ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0115
-47.349
5505
3234
The Hill model was selected on the
basis of lowest AIC.
Exponential (M4)c
0.416
-54.646
327
39.2
Exponential (M5)d
0.416
-54.646
327
39.2
Hill
0.507
-55.041
218
16.2
Power6
Polynomial 3°f
Polynomial 2°g
Linear
0.0121
-47.465
5401
3086
a Constant variance case presented (BMDS Test 2 p-value = 0.662), selected model in bold; scaled residuals for
selected model for doses 0, 150, 500, 1500, and 5000 ppm were -0.0403, 0.29, -0.727, 0.792, and -0.315,
respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c The Exponential (M4) model may appear equivalent to the Exponential (M5) model, however differences exist in
digits not displayed in the table.
d The Exponential (M5) model may appear equivalent to the Exponential (M4) model, however differences exist in
digits not displayed in the table.
e For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
f For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
g For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hil
3.8
3.6
3.4
3.2
BMD
BMD
0
1 000
2000
3000
4000
5000
dose
Figure C-15. 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 = 217.735
BMDL at the 95% confidence level = 16.1532
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
a
0.104264
0.112741
rho
n/a
0
intercept
3.15411
3.15
V
0.533715
0.57
n
1
0.287502
k
150.7
157.5
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
3.15
0.243
0.323
-0.0403
150
10
3.45
3.42
0.385
0.323
0.29
500
10
3.49
3.56
0.314
0.323
-0.727
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1500
10
3.72
3.64
0.365
0.323
0.792
5000
10
3.64
3.67
0.353
0.323
-0.315
1
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
32.20061
6
-52.401221
A2
33.401145
10
-46.80229
A3
32.20061
6
-52.401221
fitted
31.520704
4
-55.041408
R
24.155193
2
-44.310386
3
4 Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
Test 1
18.4919
8
0.01783
Test 2
2.40107
4
0.6624
Test 3
2.40107
4
0.6624
Test 4
1.35981
2
0.5067
5
6
7 Table C-21. Summary of BMD modeling results for increased relative kidney
8 weight in male S-D rats exposed to ETBE by whole-body inhalation for 6 hr/d,
9 5 d/wk for 13 wks IPEC f2008bl: BMR = 10% relative deviation from the mean.
10
Model3
Goodness of fit
BMDiord (ppm)
BMDLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.00625
-225.68
2954
2226
The Hill model was selected on the
basis of 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
2792
2051
a Constant variance case presented (BMDS Test 2 p-value = 0.321), selected model in bold; scaled residuals for
selected model for doses 0,150,500,1500, and 5000 ppm were -0.599,1.37, -1.04,0.241, and 0.0322, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
n
1
0.147616
k
714.991
2225.81
1
2 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
1500
10
0.7
0.696
0.073
0.0547
0.241
5000
10
0.726
0.725
0.047
0.0547
0.0322
3
4 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
5
6 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
7
8 Table C-22. Summary of BMD modeling results for increased absolute kidney
9 weight in female S-D rats exposed to ETBE by whole-body inhalation for
10 6 hr/d, 5 d/wk for 13 wks IPEC f2008frl: BMR = 10% relative deviation from the
11 mean.
12
Model3
Goodness of fit
BMDiord (ppm)
BMDLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.8
-135.38
6790
4046
The Linear model is selected
based on lowest AIC; however, the
Exponential (M4)
0.731
-133.76
error0
0
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Exponential (M5)
0.760
-132.29
error0
0
BMD is higher than the maximum
dose.
Hill
0.760
-132.29
error0
error0
Powerd
Polynomial 30e
Polynomial 20f
Linear
0.806
-135.40
6840
3978
a Constant variance case presented (BMDS Test 2 p-value = 0.623), selected model in bold; scaled residuals for
selected model for doses 0, 150, 500, 1500, and 5000 ppm were -0.0742, 0.0535, -0.578, 0.774, and -0.176,
respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c BMD or BMDL computation failed for this model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
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
2
1 .95
.9
1.85
.8
1.75
.7
BMDL
O
1 000
2000
3000
4000
5000
6000
7000
dose
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 = 6840.02
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 BMDL at the 95% confidence level = 3978.09
2
3 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
4
5 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
1500
10
1.92
1.88
0.173
0.147
0.774
5000
10
1.97
1.98
0.16
0.147
-0.176
6
7 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
8
9 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
10
11
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 wks IPEC f2008b1: BMR = 10% relative deviation from the
4 mean.
5
Model3
Goodness of fit
BMDiord (ppm)
BMDLiord
(ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.147
-248.04
3288
2482
The Hill model was selected on the
basis of lowest BMDL.
Exponential (M4)
Exponential (M5)c
0.240
-248.55
1471
557
Hill
0.264
-248.74
1330
316
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.162
-248.26
3167
2334
a Constant variance case presented (BMDS Test 2 p-value = 0.388), selected model in bold; scaled residuals for
selected model for doses 0, 150, 500, 1500, and 5000 ppm were -0.874, 1.29, -0.235, -0.308, and 0.125,
respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
6
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
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 = 1329.5
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
1785.17
1916.67
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1500
10
0.613
0.618
0.06
0.0465
-0.308
5000
10
0.656
0.654
0.043
0.0465
0.125
1
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
4 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
5
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 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 wks (Medinskv et al.. 1999: Bond et al.. 1996): BMR = 10%
4 relative deviation from the mean.
5
Model3
Goodness of fit
BMCiord (ppm)
BMCLiord (ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.184
-129.97
3107
2439
The Hill model was selected on the
basis of lowest BMDL.
Exponential (M4)
Exponential (M5)c
0.199
-129.71
1798
808
Hill
0.224
-129.89
1667
603
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.208
-130.22
2980
2288
a Constant variance case presented (BMDS Test 2 p-value = 0.540), selected model in bold; scaled residuals for
selected model for doses 0, 500,1750, and 5000 ppm were -0.441, 0.91, -0.635, and 0.166, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
e For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row 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
2.2
BMDL
BMD
2000 3000
dose
14:00 05/16 2014
This document is a draft for review purposes only and does not constitute Agency policy.
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18
19
20
Supplemental Information—ETBE
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 = 1666.92
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
3309.8
1856.13
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
1750
11
1.9
1.93
0.1
0.127
-0.635
5000
11
2.07
2.06
0.124
0.127
0.166
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
Tests of Interest
Test
-2* log( Likelihood
Ratio)
Test df
p-value
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Supplemental Information—ETBE
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
1
2
3
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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 wks (Medinskv et al.. 1999: Bond et al.. 1996): BMR = 10%
4 relative deviation from the mean.
5
Model3
Goodness of fit
BMCiord (ppm)
BMCLiord (ppm)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0630
-187.67
2706
2275
The Exponential (M4) model was
selected as the most
parsimonious model of adequate
fit.
Exponential (M4)
Exponential (M5)c
0.956
-191.20
1342
816
Hill
N/Ad
-189.20
1325
741
Power6
Polynomial 3°f
Polynomial 2°g
Linear
0.0928
-188.45
2552
2111
a Constant variance case presented (BMDS Test 2 p-value = 0.428), selected model in bold; scaled residuals for
selected model for doses 0, 500,1750, and 5000 ppm were -0.0252, 0.043, -0.02385, and 0.004872, respectively.
b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
c For the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
d No available degrees of freedom to calculate a goodness of fit value.
e For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
f For the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space). The models in
this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates
were 0 (boundary of parameters space). The models in this row reduced to the Linear model.
g For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in
this row reduced to the Linear model.
Data from Medinskv et al. (1999)
6
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Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bl
Exponential
.35
.25
.05
BMDI
BMD
1
0
1 000
2000
3000
4000
5000
14:13 05/16 2014
Figure C-20. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
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 = 1341.66
BMDL at the 95% confidence level = 815.742
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
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
1750
11
1.208
1.208
0.076
0.05983
-0.02385
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Supplemental Information—ETBE
5000
11
1.306
1.306
0.055
0.05983
0.004872
1
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
4 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
5
6
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C.1.2. Cancer Endpoints
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-26. 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
1500
5000
Exposure Concentration
(mg/m3)
0
2089
6268
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. For
the multistage cancer models, the coefficients were restricted to be non-negative (beta's > 0).
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 f20121
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Supplemental Information—ETBE
1 Table C-27. 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 wks; modeled with doses as administered exposure
4 concentration in ppm IPEC f2010b"l: BMR = 10% extra risk.
5
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
2942
1735
Multistage 1° was
selected on the
basis of lowest AIC.
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
2756
1718
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.208
2605
1703
a Selected model in bold.
6
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for t
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
BMD
BMD
0
1 000
2000
3000
4000
5000
7 14:57 05/16 2014
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9 Figure C-21. Plot of incidence rate by dose, with fitted curve for selected
10 model; dose shown in ppm.
11
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Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
beta2*doseA2...)]
The parameter betas are restricted to be positive
Benchmark Dose Computation.
BMR = 10% Extra risk
BMD = 2604.82
BMDL at the 95% confidence level = 1703.47
BMDU at the 95% confidence level = 4634.52
Taken together, (1703.47, 4634.52) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = error
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
0.0000404483
0.0000438711
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
AIC: =81.2084
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
1500
0.0589
2.885
1
49
-1.144
5000
0.1831
9.155
10
50
0.309
ChiA2 = 2.42 d.f = 3 P-value = 0.4898
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Supplemental Information—ETBE
1 Table C-28. 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 wks; modeled with doses as mg/m3 IPEC f2010frl:
4 BMR = 10% extra risk.
5
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
12300
7251
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
11514
7179
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.209
10884
7118
a Selected model in bold.
Data from JPEC (2010b)
6
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for t
! ' Multistage Cancer ——- ' ' ' ' ' ' ' ""j
0.35 r Linear extrapolation -j
0.3
0.25
0.2
0.15
0.1
0.05
0
BMD
BMD
0
5000
1 0000
1 5000
20000
7 15:02 05/16 2014
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9 Figure C-22. Plot of incidence rate by dose, with fitted curve for selected
10 model; dose shown in mg/m3.
11
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Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
beta2*doseA2...)]
The parameter betas are restricted to be positive
Benchmark Dose Computation.
BMR = 10% Extra risk
BMD = 10884.4
BMDL at the 95% confidence level = 7118.08
BMDU at the 95% confidence level = 19366.3
Taken together, (7118.08,19366.3) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = error
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
9.6799E-06
0.0000104989
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
AIC: =81.2087
Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0
0
0
50
0
2089
0.02
1.001
2
50
1.009
6268
0.0589
2.885
1
49
-1.144
20893
0.1831
9.155
10
50
0.309
ChiA2 = 2.42 d.f = 3 P-value = 0.4897
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 wks; modeled with PBPK doses as ETBE
metabolized, mg/hr flPEC. 2010bl: BMR = 10% extra risk.
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Supplemental Information—ETBE
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 on the
basis of 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
a Selected model in bold.
1
Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for t
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
BMD
BMD
0
0.5
1
1 .5
2
2.5
3
3.5
4
2 15:14 05/16 2014
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4 Figure C-23. Plot of incidence rate by dose, with fitted curve for selected
5 model; dose shown in mg/hr.
6
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Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is: P[response] = background + (l-background)*[l-EXP(-betal*doseAl-
beta2*doseA2...)]
The parameter betas are restricted to be positive
Benchmark Dose Computation.
BMR = 10% Extra risk
BMD = 3.02863
BMDL at the 95% confidence level = 1.98128
BMDU at the 95% confidence level = 5.02417
Taken together, (1.98128, 5.02417) is a 90% two-sided confidence interval for the BMD
Multistage Cancer Slope Factor = error
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0
Beta(l)
0.0347882
0.0464377
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
AIC: = 84.4459
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
ChiA2 = 4.83 d.f = 3 P-value = 0.1844
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Supplemental Information—ETBE
1 APPENDIX D. SUMMARY OF EXTERNAL PEER
2 REVIEW AND PUBLIC COMMENTS AND EPA'S
3 DISPOSITION
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REFERENCES FOR APPENDICES
Amberg. A; Rosner, E; Dekant, W. (1999). Biotransformation and kinetics of excretion of methyl-tert-
butyl ether in rats and humans. Toxicol Sci 51: 1-8.
Amberg. A; Rosner. E; Dekant. W. (2000). Biotransformation and kinetics of excretion of ethyl tert-butyl
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Andersen. ME. (1991). Physiological modelling of organic compounds. Ann Occup Hyg 35: 309-321.
ARCO (ARCO Chemical Company). (1983). Toxicologist's report on metabolism and pharmacokinetics of
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ATSDR (Agency for Toxic Substances and Disease Registry). (1996). Toxicological profile for methyl-tert-
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Bernauer. U: Amberg. A: Scheutzow. D: Dekant. W. (1998). Biotransformation of 12C- and 2-13C-labeled
methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats: identification of
metabolites in urine by 13C nuclear magnetic resonance and gas chromatography/mass
spectrometry. Chem Res Toxicol 11: 651-658. http://dx.doi.org/10.1021/tx970215v
Blancato, JN; Evans. MV; Power. FW; Caldwell. JC. (2007). Development and use of PBPK modeling and
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Bond. JA; Medinsky, MA; Wolf. DC; Dorman, DC; Cattley, R; Farris, G; Wong. B; Morgan. K; Janszen, D;
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contract to ARCO Chemical Company.
http://vosemite.epa.gov/oppts/epatscat8.nsf/bv+Service/1332F4B209355DC785256F9E006B7E
A0/$File/89970000047.pdf
Borghoff. S; Murphy. J; Medinsky. M. (1996). Development of physiologically based pharmacokinetic
model for methyl tertiary-butyl ether and tertiary-butanol in male Fisher-344 rats. Fundam Appl
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Borghoff. S; Parkinson. H; Leavens. T. (2010). Physiologically based pharmacokinetic rat model for
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Borghoff. SJ. (1996). Ethyl tertiary-butyl ether: Pilot/methods development pharmacokinetic study in
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Borghoff. SJ; Prescott, JS; Janszen. DB; Wong. BA; Everitt, JI. (2001). alpha2u-Globulin nephropathy,
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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.
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Cederbaum. Al; Cohen. G. (1980a). Oxidative demethylation of t-butyl alcohol by rat liver microsomes.
Biochem Biophys Res Commun 97: 730-736.
Cederbaum. Al; Cohen. G. (1980b). Oxidative demethylation of tert-butyl alcohol by rat-liver
microsomes. Biochem Biophys Res Commun 97: 730-736.
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/s0378-4274(00)00284-8
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-Q799.ch018
Fujii, S; Yabe, K; Furukawa, M; Matsuura, M; Aoyama, 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. (CIT Study No. 24860 RSR). unpublished study for Totalfinaelf on behalf of
the ETBE Producers' Consortium.
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.
Hong. JY: Wang. YY: Bondoc. FY: Lee. M: Yang. CS: Hu. WY; Pan. J. (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. JY: Wang. YY: Bondoc. FY: Yang. CS: Gonzalez. FJ: 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/sQ378-
4274(98)00389-0
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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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This document is a draft for review purposes only and does not constitute Agency policy.
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