vycrM
EPA/635/R-17/016b
External Review Draft
www.epa.gov/iris
Toxicological Review of Ethyl Tertiary Butyl Ether
(CASRN 637-92-3]
Supplemental Information
June 2017
NOTICE
This document is an External 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
1 DISCLAIMER
2 This document is a preliminary draft for review purposes only. This information is
3 distributed solely for the purpose of pre-dissemination peer review under applicable information
4 quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
5 not be construed to represent any Agency determination or policy. Mention of trade names or
6 commercial products does not constitute endorsement of recommendation for use.
7
This document is a draft for review purposes only and does not constitute Agency policy.
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CONTENTS
APPENDIX A. OTHER AGENCY AND INTERNATIONAL ASSESSMENTS A-l
APPENDIX B INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-REPONSE ANALYSIS
B-l
B.l.TOXICOKINETICS B-l
B.l.l. Absorption B-l
B.1.2. Distribution B-7
B.1.3. Metabolism B-9
B.1.4. Elimination B-17
B.1.5. Physiologically Based Pharmacokinetic Models B-23
B.1.6. PBPK Model Code B-27
B.2.OTHER PERTINENT TOXICITY INFORMATION B-27
B.2.1. Other Toxicological Effects B-27
B.2.2. Genotoxicity Studies B-44
B.3.SUPPLEMENTAL ORGAN WEIGHT DATA B-50
B.3.1. Relative Kidney Weight Data B-50
B.3.2. Absolute Liver Weight Data B-52
APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR
EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK ESTIMATES C-l
C.l. BENCHMARK DOSE MODELING SUMMARY C-l
C.1.1. Noncancer Endpoints C-l
C.l.2. Cancer Endpoints C-42
APPENDIX D. SUMMARY OF PUBLIC COMMENTS AND EPA'S DISPOSITION D-l
REFERENCES FOR APPENDICES R-l
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TABLES
Table A-l. Health assessments and regulatory limits by other national and international health
agencies A-l
Table B-l. Radioactivity in blood and kidney of rats and blood and liver of mice, following 6
hours of [14C]ETBE inhalation exposure B-5
Table B-2. Plasma radioactivity after a single oral or intravenous dose of [14C]ETBE to male
Crl:CD(SD) rats B-6
Table B-3. Blood:tissue partition coefficients for ETBE and ferf-butanol B-7
Table B-4. Unchanged ETBE and its metabolites in plasma 8 hours after a single oral dose or
repeated (7 or 14) daily oral dosing of [14C]ETBE to male Crl:CD(SD) rats B-14
Table B-5. Unchanged ETBE and its metabolites in the urine (measured 0-24 hours) after a single
oral dose or repeated (7 or 14) daily oral dosing of [14C]ETBE to male Crl:CD(SD)
rats B-15
Table B-6. Elimination of [14C]ETBE-derived radioactivity from rats and mice within 96 hours
following a single 6-hour inhalation exposure B-20
Table B-7. Evidence pertaining to kidney weight effects in animals exposed to ETBE B-28
Table B-8. Evidence pertaining to body weight effects in animals exposed to ETBE B-33
Table B-9. Evidence pertaining to adrenal effects in animals exposed to ETBE B-36
Table B-10. Evidence pertaining to immune effects in animals exposed to ETBE B-37
Table B-ll. Evidence pertaining to mortality in animals exposed to ETBE B-41
Table B-12. Summary of genotoxicity (both in vitro and in vivo) studies of ETBE B-47
Table B-13. Evidence pertaining to relative kidney weight effects in animals exposed to ETBE B-50
Table B-14. Evidence pertaining to absolute liver weight effects in animals exposed to ETBE B-52
Table C-l. Noncancer endpoints selected for dose-response modeling for ETBE C-3
Table C-2. Summary of BMD modeling results for urothelial hyperplasia of the renal pelvis in
male F344 rats exposed to ETBE in drinking water for 104 weeks (JPEC, 2010a)
modeled with doses as mg/kg-day (calculated by study authors); BMR = 10%
extra risk C-6
Table C-3. Summary of BMD modeling results for increased absolute kidney weight in male S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC,
2008d); BMR = 10% relative deviation from the mean C-8
Table C-4. Summary of BMD modeling results for increased absolute kidney weight in female S-D
rats exposed to ETBE by daily gavage for 26 weeks (Miyata et al., 2013; JPEC,
2008d); BMR = 10% relative deviation from the mean C-ll
Table C-5. Summary of BMD modeling results for increased absolute kidney weight in P0 male S-
D rats exposed to ETBE by daily gavage for a total of 18 wk beginning 10 wk
before mating until after weaning of the pups (Gaoua, 2004a); BMR = 10%
relative deviation from the mean C-14
Table C-6. 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 wk beginning 10 wk
before mating until after weaning of the pups (Gaoua, 2004a); BMR = 10%
relative deviation from the mean C-17
Table C-7. Summary of BMD modeling results for absolute kidney weight in F1 male
Sprague-Dawley rats exposed to ETBE by gavage in a two-generation study
(Gaoua, 2004b); BMR = 10% relative deviation from the mean C-19
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Table C-8. Summary of BMD modeling results for absolute kidney weight in F1 female
Sprague-Dawley rats exposed to ETBE by gavage in a two-generation study
(Gaoua, 2004b); BMR = 10% relative deviation C-22
Table C-9. Summary of BMD modeling results for increased absolute kidney weight in P0 male
S-D rats exposed to ETBE by daily gavage for 16 weeks beginning 10 weeks prior
to mating (Fujii et al., 2010); BMR = 10% relative deviation from the mean C-24
Table C-10. 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 et al., 2010); BMR = 10%
relative deviation from the mean C-27
Table C-ll. Summary of BMD modeling results for urothelial hyperplasia of the renal pelvis in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for
104 wk (JPEC, 2010b); BMR = 10% extra risk C-29
Table C-12. Summary of BMD modeling results for increased absolute kidney weight in male S-D
rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk
(JPEC, 2008b); BMR = 10% relative deviation from the mean C-32
Table C-13. Summary of BMD modeling results for increased absolute kidney weight in female
S-D rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk for 13 wk
(JPEC, 2008b); BMR = 10% relative deviation from the mean C-34
Table C-14. Summary of BMD modeling results for increased absolute kidney weight in male
F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for 13
wk (Medinsky et al., 1999; Bond et al., 1996b); BMR = 10% relative deviation
from the mean C-37
Table C-15. Summary of BMD modeling results for increased absolute kidney weight in female
F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5 d/wk, for 13
wk (Medinsky et al., 1999; Bond et al., 1996b); BMR = 10% relative deviation
from the mean C-40
Table C-16. Cancer endpoints selected for dose-response modeling for ETBE C-43
Table C-17. Summary of BMD modeling results for hepatocellular adenomas and carcinomas in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5d/wk, for
104 wk; modeled with doses as administered exposure concentration in ppm
(JPEC, 2010b); BMR = 10% extra risk C-43
Table C-18. Summary of BMD modeling results for hepatocellular adenomas and carcinomas in
male F344 rats exposed to ETBE by whole-body inhalation for 6 hr/d, 5d/wk, for
104 wk; modeled with doses as mg/m3 (JPEC, 2010b); BMR = 10% extra risk C-45
FIGURES
Figure B-l. Proposed metabolism of ETBE B-9
Figure B-2. Example oral ingestion pattern for rats exposed via drinking water B-24
Figure B-3. Comparisons of liver tumors in male rats following 2-year oral or inhalation exposure
to ETBE or tert-butanol with internal dose metrics calculated from the PBPK
model. Results applying the model of Salazar et al. (2015) (top) and Borghoff et
al. (2016) (bottom) B-26
Figure B-4. Exposure-response array of body weight effects following oral exposure to ETBE B-42
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Figure B-5. Exposure-response array of body weight effects following inhalation exposure to
ETBE B-43
Figure C-l. Plot of incidence rate by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-6
Figure C-2. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-9
Figure C-3. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-12
Figure C-4. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-15
Figure C-5. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-17
Figure C-6. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-20
Figure C-7. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-22
Figure C-8. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-25
Figure C-9. Plot of mean response by dose, with fitted curve for selected model; dose shown
in mg/kg-day C-27
Figure C-10. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-30
Figure C-ll. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-32
Figure C-12. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-35
Figure C-13. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-38
Figure C-14. Plot of mean response by dose, with fitted curve for selected model; dose shown
in ppm C-41
Figure C-15. Plot of incidence rate by dose, with fitted curve for selected model; dose shown
in ppm C-44
Figure C-16. Plot of incidence rate by dose, with fitted curve for selected model; dose shown in
mg/m3 C-46
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ABBREVIATIONS
AIC
Akaike's information criterion
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HT
heterogeneous
ARCO
ARCO Chemical Company
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KO
knockout
AUC
area under the curve
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JPEC
Japan Petroleum Energy Center
BMD
benchmark dose
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MN
micronucleus, micronucleated
BMDL
benchmark dose lower confidence
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MNNCE
mature normochromatic erythrocyte
limit
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population
BMDS
Benchmark Dose Software
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MNPCE
micronucleated polychromatic
BMDU
benchmark dose upper confidence
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erythrocyte
limit
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MNRETs
micronucleated reticulocytes
BMR
benchmark response
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MTBE
methyl tertiary butyl ether
CASRN
Chemical Abstracts Service Registry
38
MPD
2-methyl-l,2-propane diol
Number
39
NADPH
nicotinamide adenine dinucleotide
CUT
Chemical Industry Institute of
40
phosphate
Toxicology
41
PBPK
physiologically based
CPN
chronic progressive nephropathy
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pharmacokinetic
CYP450
cytochrome P450
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PCE
polychromatic erythrocytes
DNA
deoxyribonucleic acid
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POD
point of departure
EPA
U.S. Environmental Protection
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RET
reticulocyte
Agency
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SD
standard deviation
GI
gastrointestinal
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SRBC
sheep red blood cell
HERO
Health and Environmental Research
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TAME
tertiary amyl methyl ether
Online
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TBA
tert-butyl alcohol, tert-butanol
HGPRT
hypoxanthine-guanine
50
WT
wild type
phosphoribosyl transferase
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HIBA
2-hydroxyisobutyrate
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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
6
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APPENDIX B. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND
DOSE-REPONSE ANALYSIS
B.l. TOXICOKINETICS
B.l.l. Absorption
Absorption in Humans
Most of the available human data on the uptake of ETBE were obtained from volunteers.
Nihlenetal. (19981 exposed eight healthy male volunteers (average age: 29 years) to 5, 25, and
50 ppm (20.9,104, and 210 mg/m3) ETBE by inhalation for 2 hours. Each volunteer was exposed at
each concentration in sequence with 2-week intervals between exposures. The study was
performed according to the Declaration of Helsinki after approval by the Regional Ethical
Committee of the institution where the study was performed, and written informed consent was
obtained from the volunteers. The volunteers performed light physical exercise (50 watts) on a
bicycle ergometer during exposure. Exhaled air was collected before exposure, every 30 minutes
during exposure, and 6 times after exposure. The concentrations of ETBE and one of its primary
metabolites, tert-butanol, were determined in exhaled air samples. Blood was drawn before
exposure, approximately every 10 minutes during exposure, approximately every 30 minutes from
1 to 4 hours after exposure, and an additional 4 times up to 48 hours after exposure. Urine was
collected prior to exposure, at 0 and 2 hours, and at approximately 4, 7,11, 20, 22, and 46 hours
after exposure. ETBE, tert-butanol, and acetone (an ETBE metabolite) concentrations were
determined in blood and urine. The blood profiles of the parent compound and metabolites were
similar at all three exposure levels and reflected exposure concentrations, as judged by linear
increases in blood area-under-the-curve (AUC) values for the concentration-time curve calculated
(but only reported in 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. (1998) calculated the ETBE doses to the volunteers
to be 0.58, 2.9, and 5.8 mmol for the 20.9-, 104-, and 210-mg/m3 exposure levels, respectively. The
concentrations of ETBE in blood rose sharply during the first 30 minutes of exposure and kept
rising at a lower rate until the end of exposure, reaching peak concentrations of about 10, 5.4, and
1.1 [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
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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
dose at all three exposure levels. Over 24 hours, the respiratory expiration was calculated as
45-50% of the respiratory uptake, and because the net respiratory uptake and expiration do not
consider the amount of ETBE cleared during exposure, the net respiratory excretion was lower, at
30-31% of the net respiratory uptake. These authors determined thatthe ETBE blood:air partition
coefficient in humans was 11.7.
Amberg etal. (2000) exposed six volunteers (three males and three females, average age
28 ± 2 years) to 4.5 ppm (18.8 mg/m3) and 40.6 ppm (170 mg/m3) ETBE respectively. The
exposures lasted 4 hours, and the two concentrations were administered to the same volunteers
4 weeks apart. These volunteers were healthy nonsmokers and were asked to refrain from alcohol
and medication intake from 2 days before until the end of the experiment The study was
performed according to the Declaration of Helsinki after approval by the Regional Ethical
Committee of the institution where the study was performed, and written informed consent was
obtained from the volunteers. Urine was collected at 6-hour intervals for 72 hours. Blood was
drawn immediately after exposure and thereafter every 6 hours for 48 hours. ETBE and its primary
metabolite, tert-butanol, were determined in blood; the same two substances, plus additional
metabolites of tert-butanol, were assessed in urine. The authors estimated the retained doses to be
1,090 [imol following 170-mg/m3 ETBE exposure and 121 [imol following 18.8-mg/m3 exposure.
These estimates were derived using a resting human respiratory rate of 9 L/minute (13 m3/day)
and a retention factor for ETBE of 0.3, which was based on data reported by Nihlenetal. fl9981.
These estimates of retained dose are lower than those reported during light exercise (Nihlen etal..
1998).
Absorption in Animals
Amberg etal. (2000) exposed F344 NH rats (5/sex/dose group) concurrent with the human
volunteers in the same exposure chamber. Blood was taken from the tail vein of each rat at the end
of the exposure period, and urine was collected for 72 hours at 6-hour intervals following exposure.
Immediately after the 4-hour exposure period, the authors reported that blood levels of ETBE were
lower in the rats than in humans, although exact values were not reported. The authors estimated
thatthe rats received doses of 20.5 and2.3 [imol atthe 170- and 18.8-mg/m3 exposures,
respectively, using an alveolar ventilation rate of 0.169 L/minute and a retention factor of 0.3 for
rats.
No published oral dosing studies of the absorption of ETBE in humans were identified. The
Japan Petroleum Energy Center (JPEC), however, conducted an oral dosing study of the absorption
of ETBE in rats after single and repeated dosing for 14 days flPEC. 2008e. f). Seven-week-old
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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 by TPEC f2008f). plasma levels were compared to those observed after a single intravenous
dose of 5 mg/kg-day [14C]ETBE. There is no indication that a similar comparison was conducted in
the repeated-dose study flPEC. 2008el Plasma radioactivity was measured in rats at 1, 2, 4, 6, 8,10,
and 24 hours after the first exposure in the repeated dose study; 8 and 24 hours after the second to
13th exposures; and at 1, 2, 4, 6, 8,10,12, 24, 32, 48, 72, 96,120,144, and 168 hours after the last
exposure in the repeated dose study and after the single dose study.
Plasma radioactivity levels increased following a single dose of [14C]ETBE; this increase was
not proportional as the dose increased, especially at the high dose [i.e., the peak plasma
radioactivity levels were 2,800, 22,100, and 89,900 ng equivalents of ETBE/mL (ng equivalent
ETBE/mL) in the 5-, 50-, and 400-mg/kg dose groups, respectively]. Maximum plasma [14C]ETBE
levels (Cmax) were estimated to be reached at 9.0,11.5, and 8.0 hours after administration in the 5-,
50-, and 400-mg/kg dose groups, respectively. The [14C]ETBE levels in the plasma were higher
following oral exposure than after intravenous exposure (see Table B-2). The estimated elimination
plasma half-lives were 17.5,19.8, and 9.9 hours for the 5-, 50-, and 400-mg/kg dose groups,
respectively. With repeated dosing of 5 mg/kg-day [14C]ETBE flPEC. 2008el. the Cmax was achieved
6 hours after the first exposure and increased until it reached a steady state around the fifth day of
exposure. After the last exposure on Day 14, the Cmax, of 6,660 ± 407 ng equivalent ETBE/mL was
achieved 10 hours after administration of [14C]ETBE, and plasma radioactivity steadily decreased
after this point The elimination plasma half-life from Cmax to 24 hours was 17.9 hours after the first
dose and 14.2 hours after the final dose. The elimination half-life from Cmax to 168 hours after the
final dose following repeated dosing was 24.7 hours. Based on radioactivity levels measured in
urine and exhalation, more than 90% of the administered dose was absorbed.
In two parallel studies, the pharmacokinetics of ETBE was studied in mice (Sun and Beskitt.
1995a) and male Fischer 344 rats fSun and Beskitt. 1995bl. Study authors investigated the
pharmacokinetics of [14C]ETBE in mice and rats (3/sex/dose) exposed by nose-only inhalation at
target concentrations of 500, 750,1,000,1,750, 2,500, and 5,000 ppm (2,090, 3,130, 4,180, 7,310,
10,450, and 20,900 mg/m3) for a single 6-hour period (the true doses differed by less than 10%
from the targets). Specific activity of the administered [14C]ETBE and localization of the label were
not reported. Note, that in the absence of the specific activity and localization of the label, it is not
clear how the "mg ETBE equivalents" were calculated in the Sun and Beskitt (1995a. b) report or for
the specific tissues. Of the three animals per sex exposed concurrently, two were used to determine
blood and tissue concentrations of radiolabel, and the third was kept in a metabolism cage for up to
118 hours to quantify radiolabel elimination in urine, feces, as volatile in expired air and as exhaled
CO2. Exhaled organic volatiles were trapped in charcoal filters. Exhaled CO2 was trapped in aqueous
1 M KOH. Samples from the 20,900-mg/m3 treated animals were collected at 3, 6,12,18, 24, 48, 72,
96, and 118 hours after termination of exposure. At the lower exposure concentrations listed above,
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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 14CC>2, and charcoal filter
eluates were measured directly by liquid scintillation spectrometry. Blood and kidney tissue from
rats and blood and liver tissue from mice were combusted in a sample oxidizer and analyzed by
liquid scintillation spectrometry.
Immediately upon cessation of exposure, radiolabel was quantified in the blood and kidneys
of two rats and in the blood and liver of two mice. Results in Table B-l demonstrate the absorption
of radiolabel expressed as mg equivalents of ETBE into blood. Because the ETBE carbon(s) bearing
the radiolabel was not identified, further speciation is not possible. The concentration of radiolabel
in rat blood is proportionate with exposure concentration to the highest concentration
(20,894 mg/m3), although in mice, such proportionality is absent at concentrations of
10,447 mg/m3 and above. These data indicate that ETBE is well absorbed following inhalation
exposure, but that higher concentrations (e.g., 10,447 mg/m3 and above) could result in reduced
respiration rates or otherwise affect mechanisms of inhalation uptake. Additional support for
reduction of absorption is presented in Table B-l, demonstrating the elimination of the radiolabel
from rats and mice in these studies (Sun and Beskitt. 1995a. b).
In contrast, Borghoff and Asgharian f 19961 evaluated the disposition of 14C radiolabel in
F344 rats and CD-I mice after whole-body and nose-only inhalation exposure to 500,1,750, or
5,000 ppm [14C]ETBE. Besides recovery of total radioactivity in urine, feces, and expired air, air and
urine samples were analyzed for ETBE and tert-butanol. Urine samples were also analyzed for
tert-butanol metabolites HBA and MPD, and 14CC>2 was measured in exhaled air. Results obtained
after both a single 6-hour exposure or after 13 days of pre-exposure to 0, 500, or 5,000 ppm ETBE
indicate that total inhalation uptake increases linearly with exposure concentration over this range,
although there are dose- and pre-exposure-related shifts in the form and route of elimination.
Because the later study used four rats per sex and exposure level, rather than just two, it should be
given higher weight
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Supplemental Information—ETBE
Table B-l. Radioactivity in blood and kidney of rats and blood and liver of
mice, following 6 hours of [14C]ETBE inhalation exposure
Exposure Level
(mg/m3)
F344 Rat3
CD-I Mouse3
Bloodb
Kidney0
Bloodb
Liver0
2,089
0.037
0.074
0.154
0.208
3,134
0.062
0.094
0.340
0.348
4,179
0.080
0.116
0.336
0.540
7,313
0.124
0.152
0.481
0.724
10,447
0.156
0.185
0.474
0.628
20,894
0.114
0.182
0.408
0.592
aMean values of one male and one female per rat/mouse.
bln mg [14C]ETBE equivalents per gram blood.
cln mg [14C]ETBE equivalents.
Sources: Sun and Beskitt (1995a) and Sun and Beskitt (1995b).
1
2 No studies investigating dermal absorption of ETBE were identified, but because dermal
3 absorption of homologous organic substances is thought to be a function of the octanohwater
4 partition coefficient, ETBE might be assumed to penetrate rat skin relatively well. For humans,
5 Potts RO T19921 have proposed an equation to calculate the dermal permeability coefficient, Kp:
6
7 log Kp (cm/sec) = -6.3 + 0.71 x log Kow - 0.0061 x (molecular weight) (B-l)
8
9 Using the logKow [identified as Knrt in Potts RO f 19921] values for ETBE (0.95-2.2) (Drogos
10 and Diaz. 2001) and converting cm/second values to cm/hour, the estimated Kp values are
11 0.0020-0.016 cm/hour for ETBE.
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Supplemental Information—ETBE
Table B-2. Plasma radioactivity after a single oral or intravenous dose of
[14C]ETBE to male Crl:CD(SD) rats
Time (hours)
Radioactive Concentration (ng eq of ETBE/mL)
Oral
Intravenous
Dose administered
5 mg/kg
50 mg/kg
400 mg/kg
5 mg/kg
0.083
-
-
-
918 ± 188a
0.25
-
-
-
822 ±165
0.5
-
-
-
914 ±156
1
2,150 ± 281
11,100 ± 1,007
47,000 ± 11,900
907 ±143
2
2,400 ±151
12,100 ± 883
58,200 ± 7,340
923 ±158
4
2,620 ± 109
14,800 ± 659
73,300 ± 6,800
929 ±193
6
2,750 ± 146
18,700 ± 1,550
82,900 ± 12,500
981 ±216
8
2,760 ± 265
19,900 ± 2,430
89,900 ± 16,300
973 ±196
10
2,710 ± 303
21,400± 2,830
87,300 ± 15,300
943 ± 203
12
2,660 ± 426
22,000± 3,060
78,500 ± 18,100
862 ± 205
24
1,330 ± 419
10,800 ± 2,820
17,200 ± 6,460
383 ±184
32
1,170 ± 424
9,310 ± 2,510
13,100 ± 6,580
334 ±190
48
443 ±271
3,900 ± 1,480
3,180 ± 1,480
144 ± 93.8
72
204 ±165
1,660 ± 845
2,000 ± 1,820
65.2 ± 34.0
96
81.3 ±70.3
792 ± 338
N.D.
31.3 ± 11.4
120
35.9 ±44.0
385 ±110
N.D.
16.1 ±3.8
144
19.6 ±26.0
179 ±129
N.D.
11.9 ± 13.8
168
N.D.
85.4 ± 103
N.D.
N.D.
aMean ± standard deviation; n = 4.
- = not measured, N.D. = not detected.
Source: JPEC(2008e).
1
2 ETBE is moderately absorbed following inhalation exposure in rats and humans, and blood
3 levels of ETBE approached—but did not reach—steady-state concentrations within 2 hours. Nihlen
4 etal. (19981 calculated the net respiratory uptake of ETBE in humans to be 26%. The AUC for the
5 concentration-time curve was linearly related to the ETBE exposure level, suggesting linear kinetics
6 up to 209 mg/m3. The JPEC studies (TPEC. 2008e. demonstrated that ETBE is readily absorbed
7 following oral exposure in rats with >90% of a single dose (5-400 mg/kg-day) or repeated doses
8 (5 mg/kg-day) estimated to be absorbed. In the repeated-dose study, peak plasma [14C]ETBE levels
9 were reached 6 hours after the first dose and 10 hours after the final (14th) dose, and the maximum
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plasma concentration reached a steady state on Day 5. No data are available on dermal absorption
of ETBE.
B.1.2. Distribution
There are no in vivo data on the tissue distribution of ETBE in humans. Nihlen etal. f 19951
measured the partitioning of ETBE and tert-butanol in air into human blood from 10 donors (5
males, 5 females), saline, or oil inside of sealed vials. Also, human tissue-to-blood partitioning
coefficients were estimated in brain, fat, liver, kidney, lung, and muscle based upon their relative
water and fat contents. Kaneko etal. f20001 conducted a similar series of in vitro studies to
measure the partitioning of ETBE and tert-butanol in air to various rat tissues (5 male Wistar rats),
including blood, brain, fat, liver, kidney, lung, muscle, and testes. The blood:air partition coefficients
for ETBE were much lower than for tert-butanol. Both studies reported efficient uptake of these
substances from air into blood, with blood:air partition coefficients of 11.7 and 11.6 for ETBE and
462 and 531 for tert- butanol in humans and rats, respectively. Nihlen etal. (1995) also estimated
oil:water partition (log Kow) coefficients and obtained values of 0.278 for tert-butanol and 22.7 for
ETBE. These values have a similar ranking, but are not identical, to those listed in a report by
Drogos and Diaz f20011 (namely, 0.35 for tert-butanol and 1.48-1.74 for ETBE). Nihlen etal. T19951
used the coefficients of tissue:air and blood:air partition coefficients to calculate human
tissue:blood partition coefficients. These values are listed in Table B-3.
Table B-3. Bloochtissue partition coefficients for ETBE and tert-butanol
Partition Coefficient
tert-Butanol
ETBE
Blood:air
462
11.7
Brain:blood
1.05
2.34
Muscle:blood
1.06
1.78
Fat:blood
0.646
11.6
Lung:blood
1.02
0.835
Kidney:blood
1.06
1.42
Liver:blood
1.05
1.44
Source: Nihlen et al. (1998).
The JPEC f2008e. f) examined the distribution of radioactivity in 7-week-old Crl:CD(SD)
male rats (4/dose group) following either a single oral dose of 5 or 400 mg/kg [14C]ETBE via gavage
or a repeated dose of 5 mg/kg-day for 7 or 14 days. Tissue samples were collected at 8, 24, 72, and
168 hours after a single dose; 8 and 24 hours after 7 days of repeated dosing; and 8, 24, 72, and
168 hours after 14 days of repeated dosing. Although the highest radioactivity levels were generally
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detected in plasma, [14C]ETBE was also detected in all tissues examined (brain, peripheral nerve,
eyes, submaxillary gland, thyroid gland, thymus, lungs, kidneys, heart, liver, adrenal glands, spleen,
pancreas, bone marrow, mesenteric lymph node, prostate, epididymis, testes, muscle, skin, adipose
tissue, stomach, large intestines, and small intestines). Tissue concentrations after a single
400 mg/kg dose of [14C]ETBE were higher than after a single 5 mg/kg dose; however, the
percentage distribution of radioactivity in tissues was lower with the higher dose. Tissue
radioactivity levels reached a maximum at 8 hours after a single dose of either 5 or 400 mg/kg
[14C]ETBE and rapidly decreased by 72 hours. In the repeated dosing study, the radioactivity was
the same 8 hours after the seventh administration when compared to 8 hours after the 14th
administration. The levels of [14C]ETBE in the tissues declined steadily from 8 hours through 168
hours after the last exposure with the exception of adipose tissue. In adipose tissue, there was a
rapid decline between 8 and 24 hours, but the levels remained consistent between the 24- and
168-hour time points. The percentage radioactivity found in red blood cells was estimated to be
20-27% within 72 hours of administration, and little was found to be bound to plasma proteins.
Sun and Beskitt (1995a) and Sun and Beskitt (1995b) studied the distribution of radiolabel
derived from [14C]ETBE in rats and mice, respectively. Animals were subjected to a single nose-only
inhalation exposure to [14C]ETBE for 6 hours. Immediately upon cessation of exposure, radiolabel
was quantified in the blood and kidneys of two rats and in the blood and liver of two mice. Results
in Table B-l (shown earlier) demonstrate the distribution of radiolabel expressed as mg
equivalents of ETBE from blood to kidney (rats) and liver (mice) during exposure. The
concentration of radiolabel in rat kidney and mouse liver parallels the concentration of radiolabel
in blood of the respective species, leading to an expectation of the proportionate distribution of 14C
from ETBE to rat kidney and mouse liver up to exposure concentrations of 7,313 mg/m3 in rats and
10,447 mg/m3 in mice. Because radiolabel levels do not distinguish between parent ETBE and its
metabolites, these results need to be interpreted with some caution, as the distribution of
individual chemical species may differ.
Leavens and Borghoff (2009) evaluated the distribution of the structurally similar
compound, MTBE, and the common metabolite, tert-butanol, after inhalation exposure to those two
compounds, specifically in the brain, kidney, and liver of male and female rats and testes of male
rats. Concentrations of MTBE and tert-butanol were similar in the female rat brain, kidney, and
liver, and concentrations in the male rat brain, liver, and testes, were similar for exposure level and
across time points, indicating an even distribution of MTBE and tert-butanol in those tissues/sexes.
While total concentrations of MTBE and tert-butanol were higher in male rat kidneys than other
tissues, consistent with the mechanism of binding to a2U-globulin for those two compounds
(Leavens and Borghoff. 2009). the overall observations are consistent with the conclusion that
unbound ETBE and tert-butanol distribute rapidly and evenly through the body, although
additional accumulation of material bound to a2U-globulin occurs for tert-butanol and may occur for
ETBE in the male rat kidney.
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B.1.3. Metabolism
The metabolism of ETBE has been studied in rats and humans using both in vivo and in
vitro methods. A schematic of the proposed metabolism of ETBE is presented in Figure B-l. Based
on elucidated structures of urinary metabolites from rats that were exposed to ETBE by inhalation,
ETBE is initially metabolized by cytochrome P450 (CYP) enzymes via oxidative deethylation by the
addition of a hydroxyl group to the a-carbon of the ethyl ether group fBernauer etal.. 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 (Le Gal etal.. 2001:
Hong etal.. 1999al. Using data from rat hepatic microsome preparations, Turini etal. T19981
suggested that CYP2B1 is the primary enzyme responsible for this step in rats but that CYP2A1 may
also have an important role. Acetaldehyde is oxidized to acetic acid by aldehyde dehydrogenase
enzymes (some of which are polymorphically expressed) and eventually to carbon dioxide (CO2).
tert-Butanol can be sulfated, glucuronidated, and excreted into urine, or it can undergo further
oxidation by the CYP enzymes (but not by alcohol dehydrogenases) to form 2-methyl-l,2-propane
diol (MPD), and 2-hydroxyisobutyrate (HIBA), acetone, and formaldehyde (Bernauer et al.. 1998). It
should be noted that these metabolites have been identified in studies using liver preparations from
human or rat studies using ETBE, MTBE, or tert- butanol fBernauer etal.. 1998: Cederbaum and
Cohen. 1980b): however, all the enzymes that perform these metabolic steps have not been fully
described. Excretion studies indicate that final metabolism to CO2 plays only a minor role (see
Section B.I.4.).
glucuronide-0 —|—CH3
CH3
t-butyl glucuronide
CYP2A6
CYP3A4
on,
I c
o ch3-
CH,
ETBE
ch3
O——CH,
h3c—( ch3
OH
ETBE-hemi-acetal
ch3 oh
CYP450
HO—|—CH, ^ h3C-
L/M-5
rats,
CH3 humans
A "
CH3 oh
t-butanol 2-methyl-1,2-propanediol
r
H3C—^OH
CH,
H3C ^ >C CH,
o \
acetaldehyde 0
ch3
t-butyl sulfate
H0^°
[O]
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. (19971 used computer models to predict the metabolites of ETBE. The
metabolism model correctly predicted cleavage into tert-butanol and acetaldehyde and that
tert-butanol would undergo glucuronidation and sulfation. For the further metabolism of
tert-butanol, however, the computer model predicted reductive steps leading to metabolites that
have not been identified in vivo or in vitro. The software did not predict the formation of MPD or
HIBA, which have been found in vivo.
Metabolism in Humans
Metabolism of ETBE in Humans In Vivo
Nihlenetal. T19981 exposed eight healthy male volunteers (average age: 29 years) to 0,
20.9,104, or 209 mg/m3 ETBE by inhalation for 2 hours. Profiles of ETBE, tert-butanol, and acetone
were established for blood throughout exposure and for up to 22 hours thereafter. The blood
profiles of parent compounds and metabolites were similar at all three exposure levels and
reflected exposure concentrations, as judged by linear increases in concentration-time AUC values
calculated by the authors (only reported graphically). Acetone levels were highly variable before,
during, and after the exposure period, and the variation could likely be due to variations in
endogenous acetone production due to diet or physical activity.
The concentration of ETBE in blood rose sharply during the first 30 minutes of exposure
and kept rising at a lower rate until the end of exposure to reach peak concentrations of about 10, 5,
and 1 [iM at 209,104, and 20.9 mg/m3, respectively. By 6 hours, ETBE concentrations had fallen to
low levels even after exposure to 209 mg/m3. The blood concentration of tert-butanol continued to
rise for the full 2-hour exposure period, with peak values of about 12 and 7 [iM at 209 and
104 mg/m3, respectively. Blood concentrations leveled off for 3-4 hours and then began a slow
decline to less than one-half maximum levels by 24 hours (tert-butanol levels could not be
determined following 20.9 mg/m3 exposure). Acetone blood levels began to increase after about
1 hour of exposure and continued to increase after the end of exposure (high dose) or leveled off for
about lVz hours after exposure (lower doses and controls). Blood acetone levels fell rapidly during
the next half hour but remained slightly above normal for the exposed volunteers until 4 hours
after exposure when measurements were terminated.
Amberg etal. (2000) exposed six volunteers (three males and three females; average age:
28 ± 2 years) to 18.8 and 170 mg/m3 of ETBE. The exposures lasted 4 hours, and the two
concentrations were administered to the same volunteers 4 weeks apart Urine was collected at
6-hour intervals for 72 hours. Blood was drawn immediately, at 4 or 6 hours after exposure, and
thereafter every 6 hours for 48 hours. Levels of parent ETBE and its primary metabolite,
tert-butanol, were determined in blood and urine. In urine, two further metabolites of tert-butanol,
MPD and HIBA, were also assayed.
At 170 mg/m3, the mean peak blood concentration of ETBE was 12.1 ± 4.0 [iM, although that
for tert-butanol was 13.9 ± 2.2 [iM. The corresponding values at 18.8 mg/m3 were 1.3 ± 0.7 and
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1.8 ± 0.2 [J.M, respectively. The time courses of metabolite appearance in urine after 170 mg/m3 and
18.8 mg/m3 were similar, but relative urinary levels of metabolites after 18.8 mg/m3 differed from
those after 170 mg/m3. Using parent ETBE as the reference, molar ratios for total urinary excretion
(ETBE:tert-butanol:MPD:HIBA) were 1:25:107:580 after 170 mg/m3and 1:17:45:435 after
18.8 mg/m3. Individual variations were large, but the authors did not report any gender differences
in the metabolism of ETBE based on data from only three subjects of each sex.
In Vitro Metabolism of ETBE Using Human Enzyme Preparations
The metabolism of ETBE has been studied in vitro using microsomal protein derived from
human liver and from genetically engineered cells expressing individual human CYP isozymes.
Hong etal. f!997bl coexpressed human CYP2A6 or CYP2E1 with human CYP reductase in insect SF9
cells. In this heterologous expression system, in the presence of 1 mM ETBE, tert-butanol was
formed at rates of 13.6 nmol/min-nmol CYP2A6 and 0.8 nmol/min-nmol CYP2E1, indicating a
greater capacity for ETBE metabolism by CYP2A6 than by CYP2E1 at high (e.g., 1 mM)
concentrations of ETBE.
Hong etal. f 1999a) obtained hepatic microsomal protein preparations from 15 human
donor liver microsomal samples and used them to evaluate the contributions of several CYP
enzymes to ETBE metabolism. The 15 samples displayed very large interindividual variations in
metabolic activities towards ETBE ranging from 179 to 3,130 pmol/minute-mg protein. Michaelis
constant (Km) values, estimated in three human liver microsomal samples using MTBE, ranged from
28 to 89 [iM, with maximum substrate turnover velocity (Vmax) values ranging from 215 to
783 pmol/minute-mg protein. The Vmax/Km ratios, however, varied only between 7.7 and 8.8.
Following an evaluation of the activities of multiple different CYP forms in the 15 donor samples, it
was demonstrated that the metabolism of ETBE was highly correlated with certain CYP forms. The
highest degree of correlation was found for CYP2A6, which also displayed the highest metabolic
capacity for ETBE.
As part of CYP inhibition studies in the same paper, human liver microsomes were
coincubated with ETBE in the presence of chemical inhibitors or specific antibodies against either
CYP2A6 or CPY2E1. For chemical inhibition, coumarin was added to the liver microsomes prior to
initiation of the reaction. For antibody inhibition, monoclonal antibodies against human CPY2A6 or
CYP2E1 were preincubated with liver microsomes prior to incubation with the rest of the reaction
mixture. Methanol alone caused approximately 20% inhibition of the metabolism of ETBE, and
coumarin, a CYP2A6 substrate, caused a significant dose-dependent inhibition of ETBE metabolism
which reached a maximal inhibition of 99% at 100-[iM coumarin. Antibody against CYP2A6
inhibited metabolism by greater than 75%, but there was no inhibition by the antibody against
CYP2E1.
In the same paper, several specific human CYPs were expressed into human
P-lymphoblastoid cells which were used to evaluate ETBE metabolism. Based on the ETBE
metabolizing activities in the 15 human liver microsomes and the enzyme activity profiles towards
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known CYP specific substrates, correlation coefficients (ranging from 0.94 for CYP2A6 to 0 for
CYP2D6) were calculated for each CYP enzyme. The correlation ranking for ETBE metabolism by
nine human CYP isozymes was as follows: 2A6 > 3A4 « 2B6 « 3A4/5 >> 2C9 > 2E1 « 2C19 » 1A2 «
2D6. The reported direct enzyme activities towards ETBE by the heterologous expression systems
(in pmol tert-butanol formed per minute per pmol CYP enzyme) were 1.61 for CYP2A6; 0.34 for
CYP2E1; 0.18 for CYP2B6; and 0.13 for CYP1A2. CYPs 1B1, 2C8, 2C9, 2C19, and 2D6 were not
investigated. CYP3A4 and 1A1 did not metabolize ETBE. The authors concluded that CYP2A6 is the
major enzyme responsible for the oxidative metabolism of ETBE in human livers. Furthermore,
they concluded that the results of the correlation analysis and antibody inhibition study strongly
suggest that CYP2E1 is not a major enzyme responsible for metabolism of ETBE. Le Gal etal. (20011
used similar human cytochrome preparations as Hong etal. (1999a) (i.e., from human donors) or
genetically modified human (3-lymphoblastoid cell lines transfected with CYP2A6, CYP2B6, CYP3A4,
or CYP2E1 and human CYP reductase to elucidate the metabolism of ETBE, MTBE, and TAME. They
identified acetaldehyde and tert-butanol as primary metabolites from ETBE.
Metabolism in Animals
Metabolism of ETBE in Animals In Vivo
Bernauer etal. f 19981 studied the metabolism and excretion of [13C]ETBE and tert- butanol
in rats. F344 rats, 2/sex, were exposed via inhalation to 2,000 ppm (8,400 mg/m3) ETBE; three
male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours. The
excretion profile for ETBE metabolites was MPD > HIBA > tert-butanol-sulfate > tert-butanol-
glucuronide. Oral administration of tert-butanol produced a similar metabolite profile, with HIBA >
tert-butanol-sulfate > MPD >> tert-butanol-glucuronide ~ tert-butanol. tert-Butanol could not be
detected in urine following inhalation exposure to ETBE. Traces of acetone were also detected in
urine. Ambergetal. f20001 exposed F344 NH rats, 5/sex/dose, to ETBE in the same exposure
chamber described earlier for the human volunteers. Urine was collected for 72 hours following
exposure. Blood samples were drawn from the tail vein every 6 hours up to 48 hours. Peak blood
levels of ETBE and tert-butanol were 5.3 ± 1.2 and 21.7 ± 4.9 [J.M at 170 mg/m3 and 1.0 ± 0.7 and
5.7 ± 0.8 [iM at 18.8 mg/m3, respectively. Peak levels of tert-butanol were higher in rats than in
humans. Similar to humans, rats excreted mostly HIBA in urine, followed by MPD and tert-butanol.
The molar ratios for total urinary excretion of tert-butanol:MPD:HIBA were 1:2.3:15 after exposure
to 170 mg/m3 and 1:1.5:11 after exposure to 18.8 mg/m3. Parent ETBE was not identified in rat
urine in this study.
In a review covering mostly their own work on fuel oxygenate metabolism, Dekant et al.
(2001) focused on aspects of ETBE metabolism which were considered quantitatively similar in
humans and rats, with no sex-dependent differences and no likely accumulation of metabolites or
parent compound. They reported that at a high exposure level (8,400 mg/m3 ETBE), rats
predominantly excreted the glucuronide of tert-butanol in urine; however at low exposure levels
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(16.7 mg/m3 or 167.1 mg/m3 ETBE), the relative concentration of tert-butanol to the received dose
was much smaller. This seems to indicate that at high exposure levels, the normally rapid
metabolism of tert-butanol to MPD and HIBA became saturated, forcing more of the tert-butanol
through the glucuronidation pathway. The apparent final metabolite of ETBE was HIBA which can
undergo further metabolism to acetone. The latter process appeared to play a minor role in the
overall metabolism of ETBE. Dekantetal. f20011 also noted that many metabolites of the fuel
oxygenate ethers, such as formaldehyde, acetaldehyde, tert-butanol, HIBA, or acetone, occur
naturally in normal mammalian physiology, providing a highly variable background that needs to
be accounted for in metabolic experiments.
The JPEC f2008e. f) measured metabolite distribution in the plasma and urine of 7-week old
Crl:CD(SD) male rats (4/dose group) following either a single oral dose of 5 or 400 mg/kg
[14C]ETBE via gavage or a repeated dose of 5 mg/kg-day for 7 or 14 days. Metabolites were
measured in the plasma 8 hours after single or repeated dosing. Metabolites were measured in
urine collected on Days 1, 7, and 14 after repeated dosing or during a 24-hour period after
administration of the single dose. The number of doses did not appear to affect the metabolic
pattern. The study authors determined the identities of five metabolites, and the results in plasma
and urine are summarized in Table B-4 and Table B-5, respectively. When combined with what is
known of the metabolic pathway for ETBE, these data indicate that ETBE is efficiently metabolized
to tert-butanol, which is then metabolized to tert-butanol glucuronide, 2-methyl-l,2-propanediol,
and finally to 2-hydroxyisobutyrate.
Although Sun and Beskitt (1995a) did not identify the radiolabel eliminated, their
investigations do yield information pertinent to determining whether metabolic saturation might
occur under bioassay conditions. In their single-exposure protocol (see Section 0), rats and mice
were exposed via inhalation to ETBE. These investigators reported the fraction of absorbed dose
that was eliminated in urine and feces, as expired volatiles, and as expired CO2 from one rat and one
mouse. At inhaled concentrations between 4,180 and 7,310 mg/m3 a shift in the primary route of
elimination was observed, as demonstrated by a marked decrease in the fraction of radiolabel
eliminated in urine and a marked increase in the fraction of radiolabel eliminated as volatiles in
expired air, and (in rats) a doubling of the fraction eliminated as exhaled CO2. Given the different
solubilities, molecular size and other characteristics of ETBE and its multiple metabolites, it is
envisioned that this shift in the elimination pattern of radiolabel is indicative of a shift in
metabolism at these exposure levels.
Considering the potential shift in metabolic pattern relative to the pattern of toxicity can be
informative, especially related to species and dose extrapolation. These data might still be
considered preliminary because they are from one animal of each species, have not been replicated
by other authors, and the radiolabel has not been speciated as to chemical form. The unfortunate
limitation of the application of the PBPK model for human inhalation precludes its combination
with rat PBPK models to complete species extrapolation. The inhalation toxicity study by Saito et al.
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Supplemental Information—ETBE
1 (20131. however, demonstrated an increased incidence of urothelial hyperplasia at an exposure
2 concentration of 6,270 mg/m3 and higher, and an increased incidence of hepatocellular adenoma or
3 carcinoma only at an exposure concentration of 20,900 mg/m3. Additional data are required to
4 determine whether increases in incidence could be related to pharmacokinetic effects (e.g.,
5 metabolic saturation).
6
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
Compound
Metabolite
Percentage 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. la
35.7 ±2.5
71.4 ±4.7
69.8 ±7.3
P-2
te/t-butanol glucuronide
N.D.
N.D.
N.D.
N.D.
P-3
Not enough to
determine
N.D.
N.D.
N.D.
N.D.
P-4
2-methyl-l,2-
propanediol
9.7 ±2.4
9.328 ±0.9
9.1 ±0.8
8.1 ± 1.4
P-5
te/t-butanol
12.9 ±3.1
55.0 ±2.9
18.2 ±3.8
22.2 ±6.0
aMean ± standard deviation; n = 4.
N.D. = not detected.
Source: JPEC (2008e, f) unpublished reports.
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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
Compound
Metabolite
Percentage 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
te/t-butanol glucuronide
29.2 ±3.0
25.9 ±4.6
22.8 ±3.2
25.2 ±5.8
P-3
Not enough to determine
2.5 ±0.2
1.7 ±0.4
2.2 ±0.3
1.7 ±0.4
P-4
2-methyl-l,2-
propanediol
13.1 ±0.6
13.3 ±2.5
13.4 ± 1.5
13.9 ±2.3
P-5
te/t-butanol
1.5 ±0.5
3.7 ±0.6
1.9 ±0.2
1.8 ±0.0
aMean ± standard deviation; n = 4.
N.D. = not detected.
Source: JPEC (2008e. f) unpublished reports.
Borghoff and Asgharian (19961 evaluated the disposition of a 14C radiolabel in F344 rats and
CD-I mice after whole-body and nose-only inhalation exposure to 500,1,750, or 5,000 ppm
[14C]ETBE. Besides recovery of total radioactivity in urine, feces, and expired air, air and urine
samples were analyzed for ETBE and tert-butanol. Urine samples were also analyzed for
tert-butanol metabolites, HBA and MPD. Results obtained after both a single 6-hour exposure or
after 13 days of pre-exposure to 0, 500, or 5,000 ppm ETBE indicated dose- and pre-exposure-
related shifts in the form and route, likely due to metabolic factors. Elimination shifted from being
primarily in the urine after 500 ppm exposure to primarily by exhalation at 5,000 ppm in naive rats,
indicating a saturation of metabolism of ETBE to TBA. This shift was greater in female rats than in
males. However, in rats pre-exposed to 5,000 ppm ETBE for 13 days, most of the excretion was in
the urine even at 5,000 ppm. Rats pre-exposed to 500 ppm ETBE also showed a shift from
exhalation to urinary excretion in comparison to naive rats, but to a smaller degree than elicited by
5,000 ppm pre-exposure. The changes in elimination after pre-exposure indicated an induction of
the metabolism of ETBE to tert-butanol. As with rats, the fraction of radiolabel in exhaled volatiles
in mice increased with exposure level, while the fraction excreted in urine decreased. The
exhalation pattern observed in rats showed levels of ETBE falling ~90% in the first 8 hours
postexposure, while levels of TBA exhaled actually rose between 0 and 3 hours postexposure and
then fell more slowly between 3 and 16 hours, particularly after 5,000 ppm ETBE exposure. The
increase in TBA between 0 and 3 hours postexposure can be explained by the continued
metabolism of ETBE during that period. The slower decline after 3 hours can be explained as a
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result of the generally slower clearance of TBA, which is saturated by the higher ETBE exposure
levels.
Metabolism of ETBE in Animal Tissues In Vitro
Using microsomal protein isolated from the olfactory epithelium from male
Sprague-Dawley rats, Hong etal. (1997a) measured ETBE metabolism as the formation of
tert-butanol (TBA). They found that metabolism occurred only in microsomal protein (not in
cytosol) and only in the presence of an NADPH- (nicotinamide adenine dinucleotide phosphate)
regenerating system. The metabolic activity was inhibited by 80% after treating the microsomal
preparation with carbon monoxide and by 87% in the presence of coumarin (a CYP2A6 inhibitor),
which indicates CYP involvement. Using an in vitro concentration of 1 mM ETBE, metabolic activity
could not be detected in microsomal protein from the olfactory bulb, lungs or kidneys. Activity
toward ETBE was 8.78, 0.95 and 0.24 nmol/minute/mg microsomal protein in olfactory mucosa,
respiratory mucosa and liver, respectively. In olfactory mucosa, the authors reported a Km value of
125 [J.M for ETBE.
Hong etal. (1999b) used hepatic microsomal protein derived from Cyp2el knockout mice to
investigate whether this enzyme plays a major role in ETBE metabolism. They compared the
metabolizing activity of liver microsomes (incubated for 30 minutes at 37°C and with 0.1 mM
ETBE) between the Cyp2el knockout mice and their parental lineage strains using four or five
female mice (7 weeks of age) per group. The ETBE-metabolizing activities were not significantly
different between the Cyp2el knockout strain (0.51 ± 0.24 nmol/minute-mg protein) compared to
that observed in the Cyp2el wild-type parental strains (0.70 ± 0.12 for C57BL/6N mice, and
0.66 ± 0.14 for 129/Svmice). Therefore, microsomal protein from mice that did not express any
CYP2E1 did not differ from microsomal protein derived from wild-type animals in their ability to
metabolize ETBE in vitro, suggesting that CYP2E1 might contribute only little to ETBE metabolism
in vivo. Furthermore, these authors evaluated potential sex- and age-dependent differences for the
metabolism of 1 mM concentrations of ETBE by hepatic microsomal protein. Although activities in
female knockout mice were approximately 60% of those in male knockout mice, the difference did
not reach the level of statistical significance. Finally, observed rates of ETBE metabolism
(approximately 0.5 to 0.9 nmol/min/mg microsomal protein) did not seem to differ when assayed
at 0.1 or 1 mM, indicating that for mouse hepatic microsomal ETBE metabolism, saturation can
occur at concentrations no higher than 0.1 mM in vitro, and that Km values would be expected to be
lower than 0.1 mM in vitro.
Turini etal. (1998) investigated the effects of ETBE exposure on P450 content and
activities, and characteristics of ETBE metabolism in hepatic microsomal protein from male
Sprague-Dawley rats in an attempt to elucidate the role of CYP2E1 in ETBE metabolism.
Administration of ETBE at 200 or 400 mg/kg for 4 days did not alter hepatic CYP profiles, but the
administration of 2 mL ETBE /kg resulted in significant increases of metabolic activities toward
substrates characteristic for CYP2B and CYP2E1 (p-NPH) forms, but not of activities catalyzed by
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CYP3A or 1A forms. Studies of ETBE metabolism were based on high performance liquid
chromatography (HPLC) detection of the acetaldehyde ETBE metabolite. Induction of CYP2B forms
in vivo via the administration of phenobarbital slightly reduced the Km value and produced a
significant, approximate threefold increase in Vmax; in these preparations, chemical inhibition of
CYP2B forms resulted in significant inhibition of ETBE metabolism. Studies with CYP enzymes
purified from rats confirmed metabolic competency of several CYP forms, with the activity of
purified rat CYP forms 2B1 > 2E1 > 1A1 > 2C11. Chemical inhibition ofCYP2El did not reduce ETBE
metabolic activity; CYP2A forms were not evaluated. In microsomal preparations from rats treated
with phenobarbital (a CYP2B inducer), incubation with chemical inhibitors of CYP2B forms
produced a significant decrease in ETBE metabolism. Pretreatment of rats with chemicals known as
inducers of CYP2E1, CYP3A and CYP1A forms did not result in significant changes in Km or Vmax
values for ETBE metabolism, measured in vitro. The results of these investigations indicate that, in
rats, CYP2E1 is apparently minimally involved in ETBE metabolism, and that under some
conditions, CYP2B forms can contribute to ETBE metabolism. The role of CYP2A forms was not
studied in this investigation. This study also investigated the kinetic constants for ETBE metabolism
in control rat hepatic microsomal protein, indicating a Km value of 6.3 mM and a Vmax value of
0.93 nmol/min/mg microsomal protein. When compared to the kinetic constants indicated by the
results of Hongetal. (1999b). it can be expected that the rate of ETBE metabolism at in vitro at
concentrations below 1 mM would be higher in mouse than in rat microsomal preparations.
The enzymes that metabolize tert-butanol to MPD, HIBA, and even acetone, have not been
fully characterized; however, tert-butanol is not subject to metabolism by alcohol dehydrogenases
(Dekantetal.. 20011.
B.1.4. Elimination
Elimination in Humans
Nihlenetal. (1998) exposed eight healthy male volunteers (average age, 29 years) to 20.9,
104, and 209 mg/m3 ETBE by inhalation for 2 hours. ETBE, and two metabolites [tert-butanol and
acetone) were measured in urine for up to 22 hours after exposure. The blood profiles of the parent
compound and metabolites were similar at all three exposure levels and reflected exposure
concentrations. The authors estimated the inhaled amount of ETBE in the volunteers to be 0.58, 2.9,
and 5.8 mmol for the 20.9-, 104-, and 209-mg/m3 exposure levels, respectively. Based on blood AUC
values for ETBE and metabolites, the authors calculated that respiratory uptake was 32-34% in
humans, and net uptake (which excludes ETBE exhaled during exposure) was calculated to be 26%
of the dose at all three exposure levels. During the 24 hours following the start of inhalation
exposure, respiratory expiration was calculated at 45-50% of the inhaled ETBE (respiratory
uptake), and net respiratory expiration was 31% (of the net respiratory uptake), of which
tert-butanol accounted for only 1.4-3.8%. Urinary excretion of parent ETBE (as percentage of the
respiratory uptake of ETBE) accounted for even less: 0.12, 0.061, and 0.056% after the exposures to
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20.9,104, and 209 mg/m3, respectively. The authors identified four phases of elimination of ETBE
from blood, with half-lives of about 2 and 20 minutes and 1.7 and 28 hours. Only one phase for
elimination of tert-butanol from blood was identified with a half-life of 12 hours compared to
10 hours in another study with volunteers flohanson etal.. 19951. In urine, ETBE displayed two
phases of elimination, with half-lives of about 8 minutes and 8.6 hours. The half-life of tert-butanol
in urine was determined to be 8 hours flohanson etal.. 19951.
ETBE displayed a multiphasic elimination from blood. The first phase likely indicates
uptake into highly perfused tissues. The other phases could indicate uptake into less-perfused
tissues and fat, and metabolism events. The apparent total body clearance of ETBE (based on the
net respiratory uptake) was 0.57 L/hour-kg (average of the three exposure levels). The metabolic
clearance was calculated as 0.39 L/hour-kg and the exhalation clearance as 0.35 L/hour-kg. These
authors reported that the kinetics of ETBE in humans was linear over the range of concentrations
studied (Nihlen etal.. 19981.
In the study by Amberg etal. (20001 described earlier (see Section 0), two elimination
half-lives were found for ETBE (1.1 ± 0.1 and 6.2 ± 3.3 hours) at the high exposure concentration
(170 mg/m3) although tert-butanol displayed only one half-life (9.8 ± 1.4 hours). Atthe low
exposure concentration (18.8 mg/m3), only the short half-life for ETBE could be measured at
1.1 ± 0.2 hours, although thatfor tert-butanol was 8.2 ± 2.2 hours. The predominant urinary
metabolite identified was HIBA, excreted in urine at 5-10 times the amount of MPD and
12-18 times the amount of tert-butanol (note: urine samples had been treated with acid before
analysis to cleave conjugates). Excretion of unchanged ETBE in urine was minimal. The time
courses of urinary elimination after 170 and 18.8 mg/m3 were similar, but relative urinary levels of
HIBA after 18.8 mg/m3 were higher, although those for MPD were lower, as compared to
170 mg/m3. HIBA in urine showed a broad maximum at 12-30 hours after exposure to both
concentrations, with a slow decline thereafter. MPD in urine peaked at 12 and 18 hours after 170
and 18.8 mg/m3, respectively, although tert-butanol peaked at 6 hours after both concentrations.
The time to peak of the three metabolites reflected the sequence of their formation and
interconversion as ETBE is metabolized. Interindividual variations were large, but the authors did
not report gender differences in the toxicokinetics of ETBE. Amberg etal. f20001 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.
Elimination in Animals
Amberg etal. (2000) exposed F344 NH rats, 5/sex/dose, concurrent with the human
volunteers in the same exposure chamber. Urine was collected for 72 hours following exposure.
Similar to humans, rats excreted mostly HIBA in urine, followed by MPD and tert-butanol. Parent
ETBE was not identified in rat urine. The half-life for tert-butanol in rat urine was 4.6 ± 1.4 hours at
170 mg/m3 but could not be calculated at 18.8 mg/m3. Corresponding half-lives were 2.6 ± 0.5 and
4.0 ± 0.9 hours for MPD, and 3.0 ± 1.0 and 4.7 ±2.6 hours for HIBA. The authors concluded that rats
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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 3
male F344 rats received 250 mg/kg tert-butanol by gavage. Urine was collected for 48 hours, and
ETBE metabolite prevalence in urine was MPD > HIBA > tert-butanol-sulfate > tert-butanol-
glucuronide. Oral administration of tert-butanol produced a similar metabolite profile, with relative
amounts of HIBA > tert-butanol-sulfate > MPD >> tert-butanol-glucuronide ~ tert-butanol.
Although there are several unpublished reports relevant to the elimination of ETBE
following inhalation exposure, no additional peer-reviewed publications were identified.
Unpublished reports have not gone through the public peer-review process and are of unknown
quality. They are included here as additional information only.
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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
Totalb
F344 Ratc
2,090d
37
[28, 32]
1
[1.2, 1.3]
60
[59, 59]
2
[2.8, 1.0]
9.9
[16.1, 13.6]
3,130
36
1
62
2
17.5
4,180
42
1
56
2
22.1
7,310d
58
[41, 52]
2
[1.5, 1.7]
38
[53, 41]
3
[0.7, 0.5]
56.9
[45, 31]
10,400
52
2
45
2
56.2
20,900de
63
(51)
[51, 64]
2
(1)
[1.6, 2.0]
34
(44)
[45, 30]
1
(3)
[0.2, 0.2]
97.5
(116)
[143, 94]
CD-I Mouse1
2,090d
10
[12.7, 26.8]
1
[1.2, 1.2]
74
[81.3, 67.2]
16
[3.2, 2.3]
6.38
[10.4, 6.8]
3,130
28
2
60
10
7.9
4,180
29
2
64
6
12.8
7,310d
42
[23, 36]
2
[2.2, 1.9]
46
[71, 61]
10
[1.1, 0.6]
13.7
[22.4, 17.3]
10,400
42
2
47
10
22.7
20,900de
44
(37)
[40, 47]
5
(2)
[2.9, 3.3]
39
(57)
[53, 47]
12
(2)
[0.6, 0.8]
18.9
(28)
[37.1, 25.2]
Percentage of total eliminated radioactivity; mean of one male and one female.
bln mg [14C]ETBE equivalents.
cSun and Beskitt (1995b);
dvalues in brackets: [males, females], nose-only exposures, elimination up to 48 hour Borghoff and Asgharian
(1996);
evalues in parentheses: Borghoff (1996); fSun and Beskitt (1995b).
1
2 During 96 hours in metabolic cages, rats eliminated approximately 60% of the radioactivity
3 in urine, approximately 38% was recovered as exhaled organic volatiles, and approximately 1% as
4 exhaled CO2. This pattern was maintained at an exposure concentration of 4,180 mg/m3; above that,
5 urinary excretion of radioactivity decreased to 34% of the recovered radioactivity, although
6 exhalation of organic volatiles increased to 63%. A shift in the elimination profile of radiolabel was
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seen at concentrations of 7,310 mg/m3 and above, which remained fairly constant to the highest
exposure of 20,900 mg/m3. In this range of concentrations, approximately 39% of the eliminated
radiolabel was found in urine, approximately 58% was exhaled as organic volatiles, and 2% was
eliminated as exhaled CO2.
A review of the data demonstrating the percentage of recovered radiolabel via various
routes of elimination demonstrate, in the rat and mouse, a pattern indicative of metabolic
saturation occurring at inhaled concentrations above 4,180 mg/m3.
In rats, the time course of elimination indicated that exhalation of organic volatiles was
essentially complete by 24 hours, although urinary excretion of ETBE-derived radioactivity
displayed a broad peak at 12-48 hours. The bulk of each dose was eliminated within 48 hours after
the end of exposure. At 20,900 mg/m3,14CC>2 exhalation and fecal excretion of radioactivity
remained rather constant from 12 to 118 hours. In comparing the total radiolabel eliminated to the
inhaled concentrations (see Table B-6), a proportionate relationship is observed in rats at all
concentrations, but less than proportionate elimination of total radiolabel at the highest
concentration in mice. The complete data set led the authors of the report to conclude that
saturation of the inhalation absorptive processes might have occurred at concentrations of
approximately 7,310 mg/m3 (see Section 0) The findings of Sun and Beskitt f!995al in mice at
20,900 mg/m3 were essentially confirmed by Borghoff (19961 (unpublished report, a pilot study)
and Borghoff and Asgharian f 19961 (unpublished report, final study) which used the identical
species, experimental protocol, materials, and methods but were conducted later at a different
laboratory (see Table B-6).
Similarities between rats (Sun and Beskitt. 1995b) and mice (Sun and Beskitt. 1995a) are
evident. Both species demonstrate similar elimination pathways and present evidence of saturation
of metabolic pathways at concentrations lower than those which demonstrate saturation of
absorptive pathways. Metabolic saturation (evidenced as a shift from urine as the predominant
elimination pathway and an increase in the fraction of dose eliminated via exhalation) occurred in
both species at concentrations approximating 7,310 mg/m3. Noteworthy differences between the
two species were that, in general, mice eliminated a smaller percentage of the dose in the form of
volatile organics and a higher amount in urine, at least up to 4,180 mg/m3 (see Table B-6) and
excreted about five times as much [14C]ETBE-derived radioactivity via feces than did rats. The total
amounts of eliminated radioactivity (mg equivalents) were considerably higher in rats than in mice;
however, the values in the respective columns of Table B-6 are not corrected for body weight. When
normalized to body weight, it is apparent that mice absorbed a higher dose than rats; however, the
total eliminated radioactivity at 20,900 mg/m3 showed no further increase over the values at
10,450 mg/m3, indicating that the absorptive capacities of mice had become saturated; however,
this analysis conducted in rats does not indicate a saturation of absorptive capacities over the range
of concentrations studied.
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Borghoff (19961. 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); however, in this pilot study, only three male F344 rats and three male CD-I
mice were used per experiment, with the only one exposure level at 20,900 mg/m3. Also, only blood
was collected from the animals, while the whole carcasses were liquefied and assayed for retained
radioactivity immediately after exposure and after the end of the animals' stay in metabolic cages.
The carbon at "the central position of the tert-butyl group" was radiolabeled. Radioactive ETBE was
obtained by mixing [14C]ETBE with unlabeled material in the gas phase for a specific activity of
2.74 |iCi/mmol. It was found that rats, when assayed immediately after exposure, had absorbed
2.57 ± 0.14 |iCi radioactivity, although the balance of radioactivity after 96 hours in metabolic cages
from other animals accounted for 3.17 ± 0.08 [iCi (mean ± standard deviation [SD], n = 3). The
authors could not make any suggestion as to the origin of this discrepancy. Absorbed doses in mice
were 0.85 ± 0.08 |iCi immediately after exposure and 0.77 ± 0.16 |iCi for other mice placed in
metabolism cages. Elimination values detected in these rats and mice are shown in parentheses in
Table B-6; the percentage values shown in this table were based on the total body burden of the
individual animals from which the elimination data were obtained, not on group means.
Mice had eliminated most of the dose within 12 hours after exposure, rats within 24 hours.
Organic volatiles collected on charcoal filters were analyzed for ETBE and tert-butanol contents.
Rats exhaled 22% of the absorbed ETBE within 1 hour after exposure, 12% during the following
2 hours, and only another 3% during the next 3 hours, tert-Butanol exhalation accounted for 1% of
the total during the first hour, 3% during the following 2 hours, and 4% during the last 3 hours of
the experimental period. Mice, on the other hand, exhaled 16% of the unmetabolized ETBE within
1 hour after exposure and 1% during the following 2 hours, with immeasurable amounts thereafter.
tert-Butanol exhalation made up 6% of total during the first hour, 8% in the next 2 hours, and 4%
during the final 3 hours. Elimination of ETBE, tert-butanol, HIBA, and MPD in urine were assayed.
During 24 hours of collection, rats eliminated about 7 times as much tert-butanol as ETBE in urine;
in mice, the ratio was >60. HIBA was detected in urine of both species but could not be quantified.
MPD was not detected. These results could be interpreted as suggesting that mice metabolize, and
hence, eliminate ETBE faster than rats.
A subsequent larger study by Borghoff and Asgharian f!9961 (see previous details)
essentially confirmed the results of the pilot study (Borghoff. 19961. F344 rats and CD-I mice were
exposed by inhalation to 500,1,750, or 5,000 ppm [14C]ETBE. Concentrations of ETBE and
tert-butanol were measured in exhaled breath up to 16 hours postexposure. The exhalation pattern
observed in rats showed levels of ETBE falling ~90% in the first 8 hours postexposure, while levels
of TBA exhaled actually rose between 0 and 3 hours postexposure and then fell more slowly
between 3 and 16 hours, particularly after 5,000 ppm ETBE exposure. The increase in TBA between
0 and 3 hours postexposure can be explained by the continued metabolism of ETBE during that
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
period. The slower decline after 3 hours can be explained as a result of the generally slower
clearance of TBA, which is saturated by the higher ETBE exposure levels. Exhaled breath levels
declined much more rapidly in mice than in rats.
Unpublished reports by the TPEC (2008el determined that following oral exposure of
7-week-old Crl:CD(SD) male rats to [14C]ETBE, the largest amount of radioactivity was recovered in
expired air, followed by urinary excretion, with very little excretion occurring via the feces. With
increasing dose, increasing proportions of radioactivity were found in expired air. The total
radioactivity recovered by 168 hours after a single dose of 5 mg/kg [14C]ETBE was 39.16% in the
urine, 0.58% in the feces, and 58.32% in expired air, and, after a single dose of 400 mg/kg, 18.7% in
the urine, 0.15% in the feces, and 78.2% in expired air. With repeated dosing, the recovery of
radioactivity through excretion increased through day 6 when a steady state was achieved;
however, the radioactivity level in the feces increased throughout the 14 days, but the level was too
low to affect the total recovery. After 14 days, 36.3% of the administered dose was recovered in the
urine, 2.33% was recovered in the feces, and 56.7% was recovered in expired air.
B.1.5. Physiologically Based Pharmacokinetic Models
Two physiologically based pharmacokinetic (PBPK) models have been developed
specifically for the administration of ETBE in rats (Borghoff etal.. 2016: Salazar etal.. 20151. A
detailed summary of these and other toxicokinetic models is provided in U.S. EPA f20171.The PBPK
model described in Borghoff et al. (20161 and in U.S. EPA (20171 was applied to conduct route-to-
route extrapolation based on an equivalent internal dose (the rate of ETBE metabolism in the liver).
While the model includes a possible adjustment for induction of tert-butanol metabolism, this
induction has only been observed in mice exposed directly to tert-butanol fMcComb and Goldstein.
19791. Further, implementing metabolic induction does not allow for dependence on exposure or
dose, nor for any de-induction that might occur during periods without exposure, such as weekends
during 5 days/week exposures. Finally, because induction is expected to have an equal impact on
oral and inhalation exposures—and only in the case that tert-butanol levels or metabolism were
used as a dose-metric—induction's potential impact on risk evaluation for ETBE is considered
minimal. Therefore, this adjustment was not turned off in the model; instead, the maximum
induction level was set to zero.
While model simulations accounted for variations during the day and week (e.g.,
6 hours/day, 5 days/week inhalation exposure), simulations reached a condition of "periodicity" by
the second week, such that the time-course of internal doses were identical in between the second
week and subsequent weeks of exposure with metabolic induction turned off. However, to ensure
applicability in the event that metabolic induction is considered (predicted to take 2-3 weeks),
simulations were generally run for 7 weeks, with results for the last 1-2 weeks used to estimate
average tissue or blood concentrations or metabolic rates.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
For simulating exposure to drinking water, the water consumption was modeled as
episodic, based on the pattern of drinking observed in rats fSpiteri. 19821. In particular, rats were
assumed to ingest water in pulses or "bouts," which were treated as periods of continuous
ingestion, interspersed with periods of no ingestion. During the active dark period (12 hours/day),
it is assumed that 80% of total daily ingestion occurs in 45-minute bouts alternating with 45
minutes of other activity. During the relatively inactive light period (12 hours/day), it is assumed
that the remaining 20% of daily ingestion occurs; the bouts are only assumed to last 30 minutes,
with 2.5 hours between. This pattern is thought to be more realistic than assuming continuous
24 hours/day ingestion. The resulting ingestion rate for one exposure is shown in Figure B-2.
15
12
9
6
3
o IIII IIII II—u—U—U—IIIII1111IIII111II—u—U—U—
0 6 12 18 24 30 36 42 48
Time (h)
Figure B-2. Example oral ingestion pattern for rats exposed via drinking
water.
PBPK modeling was also used to evaluate a variety of internal dose metrics (daily average
TBA blood concentration, daily amount of TBA metabolized in liver, daily average of ETBE blood
concentration, and daily amount of ETBE metabolized in liver) to assess the correlation with
different endpoints following exposure to ETBE or TBA (Salazar etal.. 20151. Administering ETBE
either orally or via inhalation achieved similar or higher levels of TBA blood concentrations or TBA
metabolic rates as those induced by direct TBA administration. Altogether, the PBPK model-based
analysis by Salazar etal. (20151 [which applied a model structurally similar to (Borghoffetal..
20161] indicates a consistent dose-response relationship between kidney weight, urothelial
hyperplasia, and chronic progressive nephropathy (CPN) and TBA blood concentration (as the dose
Ul
E
¦u
ro
c
0
'¦u
V)
0?
01
c
!_
o
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 metric for both ETBE and TBA). Kidney and liver tumors, however, were not consistently correlated
2 with any dose metric. These data are consistent with TBA mediating the noncancer kidney effects
3 following ETBE administration, but additional factors besides internal dose are necessary to explain
4 the induction of liver and kidney tumors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
•
ETBE-oral
¦
ETBE-inhalation
o
tert-butanol-oral
0.25
0.20 -
% 0.15 H
0.10 -
w 0.05 -
o
3 0.00 -
0
fj -0.05 -
-0.10 -
-0.15
A. rho= 0.099 (all datasets)
rho= 0.15 (ETBEonly)
B. rho= 0.33 (ETBE only)
• •
0 12 3
ferf-butanol metabolized (mg/hr)
12 3 4
ETBE metabolized (mg/hr)
Salazar etal. (20151
•
ETBE-oral
¦
ETBE-inhalation
O
ferf-butanol-oral
CO
0.25
0.20 -
0.15 -
0.10 -
0.05 -
0.00 -
-0.05 -
-0.10 -
-0.15
A rho= 0.14 (all datasets)
" rho= 0.33 (ETBE only)
¦
¦St
o
II
o
sz
cri
(ETBE only)
¦
o
o
¦
¦ :
o
¦
•
¦
•
• • •
• •
•
0.0
0.5 1.0 1.5 2.0 2.5 3.0 0
fert-butanol metabolized (mg/hr)
1 2 3
ETBE metabolized (mg/hr)
Borghoff et al. (20161
1
2
3
4
Figure B-3. Comparisons of liver tumors in male rats following 2-year oral or
inhalation exposure to ETBE or tert-butanol with internal dose metrics
calculated from the PBPK model. Results applying the model of Salazar et al.
f20151 (top) and Borghoff et al. f20161 (bottom)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Dose metrics expressed are metabolism rate of tert-butanol (A) and metabolism rate of
ETBE (B). Liver tumor incidences following ETBE oral or inhalation exposure did not present a
consistent dose-response relationship using either the ETBE or tert-butanol metabolism rate dose
metric, and the correlation coefficients were not statistically significant These data indicate that
internal dose is inadequate to explain differences in tumor response across these studies.
B.1.6. PBPK Model Code
The PBPK acslX model code is available electronically through EPA's Health and
Environmental Research Online (HERO) database. All model files may be downloaded in a zipped
workspace from HERO fU.S. EPA. 2016],
B.2. OTHER PERTINENT TOXICITY INFORMATION
B.2.1. Other Toxicological Effects
Synthesis of Other Effects
The database for effects other than kidney, liver, reproductive, and cancer contain only 11
rodent studies. These effects included decreased body weight, increased adrenal weights, altered
spleen weights, and increased mortality. All selected studies used inhalation, oral gavage, or
drinking water exposures for >90 days. Shorter duration, multiple-exposure studies that examined
immunological endpoints were also included. No studies were removed for methodological
concerns.
Kidney Effects
Numerical absolute kidney weight data are presented in Table B-7.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-7. Evidence pertaining to kidney weight effects in animals exposed to
ETBE
Reference and Study Design
Results (percent change compared to control)
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
PO, male (24/group): 0,100, 300,
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
1,000 mg/kg-day
0
-
0
-
daily for 16 wk beginning 10 wk prior to
mating
100
5%
100
-2%
P0, female (24/group): 0,100, 300,
300
8%
300
0%
1,000 mg/kg-day
daily for 17 wk beginning 10 wk prior to
1,000
18%*
1,000
7%*
mating to lactation day 21
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
P0, male (25/group): 0, 250, 500,
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
1,000 mg/kg-day
0
-
0
-
daily for a total of 18 wks beginning 10 wk
before mating until after weaning of the
250
11%*
250
-1%
pups
500
15%*
500
2%
P0, female (25/group): 0, 250, 500,
1,000 mg/kg-day
1,000
21%*
1,000
5%
daily for a total of 18 wk beginning 10 wk
Fl, Male
Fl, Female
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
and lactation, then Fl doses beginning PND
0
-
0
-
22 until weaning of the F2 pups
250
10%
250
4%
500
22%*
500
3%
1,000
58%*
1,000
11%*
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
Dose
(mg/kg-day)
Absolute
weight
daily for 23 wk
0
1,000
19%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Mivata et al. (2013); JPEC (2008b)
rat, CRL:CD(SD)
oral—gavage
male (15/group): 0, 5, 25,100,
400 mg/kg-day; female (15/group): 0, 5, 25,
100, 400 mg/kg-day
daily for 26 wk
Male
Dose
(mg/kg-day)
0
5
25
100
400
Absolute
weight
1%
6%
5%
25%*
Female
Dose
(mg/kg-day)
0
5
25
100
400
Absolute
weight
1%
0%
7%
10%*
JPEC (2008a)
rat, CRL:CD(SD)
inhalation—vapor
male (NR): 0,150, 500,1,500, 5,000 ppm
(0, 627, 2,090, 6,270, 20,900 mg/m3)b;
female (NR): 0,150, 500,1,500, 5,000 ppm
(0, 627, 2,090, 6,270, 20,900 mg/m3);
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Dose (mg/m3)
0
627
2,090
6,270
20,900
Absolute
weight
10%
11%
18%*
16%*
Female
Dose (mg/m3)
0
627
2,090
6,270
20,900
Absolute
weight
1%
-1%
4%
7%
JPEC (2008a)
rat, CRL:CD(SD)
inhalation—vapor
male (6/group): 0, 5,000 ppm (0,
20,900 mg/m3)a; female (6/group): 0,
5,000 ppm (0, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk followed by a 28 day
recovery period; generation method,
analytical concentration and method were
reported
Male
Dose
(mg/m3)
0
20,900
Absolute
weight
19%
Female
Dose
(mg/m3)
0
20,900
Absolute
weight
8%
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation—vapor
male (48/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(48/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male
Dose
(mg/m3)
0
2,090
7,320
20,900
Absolute
weight
7%
10%*
19%*
Female
Dose
(mg/m3)
0
2,090
7,320
20,900
Absolute
weight
4%
12%*
21%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Medinskv et al. (1999): Bond et al. (1996a)
Male
Female
mice, CD-I
inhalation—vapor
male (40/group): 0, 500,1,750, 5,000 ppm
Dose
Absolute
Dose
Absolute
(mg/m3)
weight
(mg/m3)
weight
(0, 2,090, 7,320, 20,900 mg/m3)a; female
0
-
0
-
(40/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
2,090
9%
2,090
0%
dynamic whole body chamber; 6 hr/d,
7,320
10%
7,320
6%
5 d/wk for 13 wk; generation method,
analytical concentration and method were
20,900
5%
20,900
4%
reported
1
2 a4.18 mg/m3 = 1 ppm.
3 * result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 - for controls, no response relevant; for other doses, no quantitative response reported.
5 (n) number evaluated from group.
6
7 Body Weight
8 As presented in Table B-7, body weights were significantly reduced compared with vehicle
9 controls following 2-year oral and inhalation exposures to ETBE fSaito etal.. 2013: Suzuki etal..
10 2012: TPEC. 2010a. b). Reductions were also reported in studies of exposure durations shorter than
11 2 years (Banton etal.. 2011: Hagiwara etal.. 2011: Fuiii etal.. 2010: TPEC. 2008a. b: Gaoua. 2004b:
12 Medinskv etal.. 19991: however, these effects were frequently not statistically significant. Food
13 consumption did not correlate well with body weight fSaito etal.. 2013: Suzuki etal.. 2012: TPEC.
14 2010a. b). Water consumption was reduced in the 2-year oral exposure study (TPEC. 2010al.
15 Palatability and reduced water consumption due to ETBE exposure might contribute to the reduced
16 body weight, particularly for oral exposures. Ptyalism, which is frequently observed with
17 unpalatable chemicals following gavage, was observed in rats gavaged for 18 weeks (Gaoua.
18 2004bl. Body weight changes are poor indicators of systemic toxicity but are important when
19 evaluating relative organ weight changes. Body weight was most severely affected in 2-year studies,
20 and 2-year kidney and liver weights are not appropriate for analysis as stated in Sections 1.2.1 and
21 1.2.2. Thus, the body weight effects data are inadequate to draw conclusions as a human hazard of
22 ETBE exposure.
23 Adrenal Weight
24 Adrenal weights were increased in 13-week and 23-week studies (see Table B-8). For
25 instance, a 13-week inhalation study found that absolute adrenal weights were increased in male
26 and female rats (Medinskv etal.. 19991. In another study, absolute and relative adrenal weights
27 were increased in male rats f Hagiwara etal.. 20111. None of the observed organ weight changes
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
corresponded with functional or histopathological changes; thus, adrenal effect data are inadequate
to draw conclusions as a human hazard of ETBE exposure.
Immune System
Functional immune assays represent clear evidence of immunotoxicity and generally
outweigh immune organ weight and cell population effects when establishing hazard (WHO. 20121
(see Table B-10). The single published functional assay available reported that the number of IgM+
sheep red blood cell (SRBC)-specific antibody forming cells was not significantly affected after a
28-day oral exposure to ETBE (Banton etal.. 20111. Relative spleen weights were inconsistently
affected in male and female rats following oral and inhalation >13-week exposures to ETBE
(see Table B-10). The only dose-responsive changes in spleen weights were increased relative
weights in male rats and decreased absolute weights in female rats following 2-year inhalation
exposure (Saito etal.. 2013: TPEC. 2010b) and increased relative weights in female rats following
2-year oral exposure (Suzuki etal.. 2012: TPEC. 2010a). Spleen weights are heavily influenced by
the proportion of red blood cells which do not impact immune function of the organ (Elmore.
20061. Thus, spleen weight changes must be correlated with histopathological and functional
changes for evidence of immunotoxicity (Elmore. 20061. none of which are observed for ETBE.
CD3+, CD4+, and CD8+ T cells were modestly reduced in male mice after 6 or 13 weeks of ETBE
exposure via inhalation but are not correlated with any change in T cell function as indicated by the
SRBC assay (Li etal.. 20111. No other indicators of histopathological or functional changes were
reported with a single chemical exposure. The ETBE database contains no evidence of altered
immune function that correlate with modest T cell population reductions and altered splenic organ
weights, thus the immune effect data are inadequate to draw conclusions as a human hazard of
ETBE exposure.
Mortality
Mortality was significantly increased in male and female rats following a 2-year ETBE
inhalation exposure (Saito etal.. 2013: TPEC. 2010b) but not significantly affected following a 2-year
drinking water exposure (Suzuki etal.. 2012: TPEC. 2010a). Increased mortality in male rats
correlated with increased CPN severity in the kidney. Increased mortality in females was attributed
to pituitary tumors by the study authors; however, pituitary tumors were not dose responsively
increased by ETBE exposure. Survival was also reduced in a lifetime gavage study at the highest
exposure in males and females after 72 weeks (data not shown), and after 104 weeks, survival was
reduced 54% in males at the highest dose (Maltoni etal.. 19991. After 104 weeks, however, survival
in the controls was approximately 25% in males and 28% in females which is much lower than
anticipated for a 2-year study (Maltoni etal.. 19991. The survival data in this study was likely
confounded by chronic respiratory infections which could have contributed to the reduced survival
fMalarkev and Bucher. 20111. These data do not suggest that mortality was increased in these
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 studies due to excessively high exposure concentrations of ETBE; thus, the mortality data are
2 inadequate to draw conclusions as a human hazard of ETBE exposure.
3 Mechanistic Evidence
4 No relevant mechanistic data are available for these endpoints.
5 Summary of Other Toxicity Data
6 EPA concluded that the evidence does not support body weight changes, adrenal and
7 immunological effects, and mortality as potential human hazards of ETBE exposure based on
8 confounding factors, lack of progression, and study quality concerns.
9
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-8. Evidence pertaining to body weight effects in animals exposed to
ETBE
Reference and Study Design
Results (percent change compared to control)
Banton et al. (2011)
Female
rat, Sprague-Dawley
oral—gavage
female (10/group): 0, 250, 500,
Dose
(mg/kg-dav)
Bodv weight
1,000 mg/kg-day
0
-
daily for 28 consecutive days
250
500
1,000
3%
5%
-1%
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
P0, male (24/group): 0,100, 300,
Dose
(mg/kg-day)
Bodv weight
Dose
(mg/kg-dav)
Bodv weight
1,000 mg/kg-day
0
-
0
-
daily for 16 wk beginning 10 wk prior to mating;
P0, female (24/group): 0,100, 300,
100
-4%
100
1%
1,000 mg/kg-day
300
-4%
300
1%
daily for 17 wk beginning 10 wk prior to mating
to lactation day 21
1,000
-7%
1,000
5%
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
P0, male (25/group): 0, 250, 500,
Dose
(mg/kg-day)
Final body
weight
Dose
(mg/kg-dav)
Final bodv
weight
1,000 mg/kg-day
0
-
0
-
daily for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups
250
-1%
250
-7%
P0, female (25/group): 0, 250, 500,
500
-3%
500
-2%
1,000 mg/kg-day
daily for a total of 18 wk beginning 10 wk before
1,000
-5%*
1,000
0%
mating until PND 21
Fl, Male
Fl, Female
Fl, male (25/group): 0, 250, 500,
1,000 mg/kg-day
dams dosed daily through gestation and
Dose
(mg/kg-day)
Final bodv
weight
Dose
(mg/kg-dav)
Final bodv
weight
lactation, then Fl doses beginning PND 22 until
0
-
0
-
weaning of the F2 pups
Fl, female (24-25/group): 0, 250, 500,
250
0%
250
-2%
1,000 mg/kg-day
500
3%
500
-3%
P0 dams dosed daily through gestation and
lactation, then Fl dosed beginning PND 22 until
1,000
1%
1,000
2%
weaning of the F2 pups
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Hagiwara et al. (2011); JPEC (2008c)
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
daily for 23 wk
Male
Dose Final bodv
(mg/kg-dav) weight
0
1,000 -5%*
Mivata et al. (2013);JPEC (2008b)
rat, CRL:CD(SD)
oral—gavage
male (15/group): 0, 5, 25,100, 400 mg/kg-day;
female (15/group): 0, 5, 25,100, 400 mg/kg-day
daily for 26 wk
Male Female
Dose Dose
(mg/kg-dav) Bodv weight (mg/kg-dav) Bodv weight
0 - 0 -
5 -6% 5 -5%
25 0% 25 -2%
100 -5% 100 -2%
400 2% 400 -3%
Maltoni et al. (1999)
rat, Sprague-Dawley
oral—gavage
male (60/group): 0, 250,1,000 mg/kg-day;
female (60/group): 0, 250,1,000 mg/kg-day;
4 d/wk for 104 wk; observed until natural death
Male
No significant difference at any dose
Female
No significant difference at any dose
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral—water
male (50/group): 0, 625, 2,500,10,000 ppm (0,
28,121, 542 mg/kg-day);a female (50/group): 0,
625, 2,500, 10,000 ppm (0, 46, 171,
560 mg/kg-day)a
daily for 104 wk
Male Female
Dose Terminal body Dose Terminal body
(mg/kg-dav) weight (mg/kg-dav) weight
0 - 0 -
28 -4% 46 -10%*
121 -7%* 171 -11%*
542 -9%* 560 -17%*
JPEC (2008a)
rat, CRL:CD(SD)
inhalation—vapor
male (NR): 0,150, 500,1,500, 5,000 ppm (0,
627, 2,090, 6,270, 20,900 mg/m3)b; female (NR):
0, 150, 500, 1,500, 5,000 ppm (0, 627, 2,090,
6,270, 20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
627 0% 627 -6%
2,090 1% 2,090 -7%
6,270 -1% 6,270 -7%
20,900 -7% 20,900 -11%
This document is a draft for review purposes only and does not constitute Agency policy.
B-34 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
JPEC (2008a)
rat, CRL:CD(SD)
inhalation—vapor
male (6/group): 0, 5,000 ppm (0,
20,900 mg/m3)b; female (6/group): 0,
5,000 ppm (0, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk followed by a 28 day recovery period;
generation method, analytical concentration
and method were reported
Male Female
Dose Dose
(mg/m3) Body weight (mg/m3) Body weight
0 - 0 -
20,900 3% 20,900 4%
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation—vapor
male (48/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female
(48/group): 0, 500, 1,750, 5,000 ppm (0, 2,090,
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
2,090 2% 2,090 -3%
7,320 4% 7,320 3%
20,900 2% 20,900 6%*
Medinskv et al. (1999); Bond et al. (1996b)
mice, CD-I
inhalation—vapor
male (40/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female
(40/group): 0, 500, 1,750, 50,00 ppm (0, 2,090,
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk
for 13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
2,090 0% 2,090 -2%
7,320 -1% 7,320 -1%
20,900 -3% 20,900 2%
Saito et al. (2013);JPEC (2010b)
rat, Fischer 344
inhalation—vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b; female
(50/group): 0, 500, 1,500, 5,000 ppm (0, 2,090,
6,270, 20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d, 5 d/wk
for 104 wk; generation method, analytical
concentration, and method were reported
Male Female
Dose Dose
(mg/m3) Bodv weight (mg/m3) Bodv weight
0 - 0 -
2,090 -7%* 2,090 -6%*
6,270 -7%* 6,270 -10%*
20,900 -26%* 20,900 -23%*
Conversion performed by study authors.
b4.18 mg/m3 = 1 ppm.
NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
for controls, no response relevant; for other doses, no quantitative response reported.
Percentage change compared to controls calculated as 100 x ((treated value—control value) -f control value).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-9. Evidence pertaining to adrenal effects in animals exposed to ETBE
Reference and Study Design
Results (percentage change compared to control)
Adrenal Weight
Hagiwara et al. (2011); JPEC (2008c)
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
daily for 23 wk
Male
Dose
(mg/kg-dav) Absolute weight Relative weight
0
1,000 16%* 19%*
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation—vapor
male (48/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(48/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male Female
Dose Dose
(mg/m3) Absolute weight (mg/m3) Absolute weight
0 - 0 -
2,090 11% 2,090 1%
7,320 9% 7,320 7%
20,900 34%* 20,900 18%*
Medinskv et al. (1999); Bond et al. (1996a)
mice, CD-I
inhalation—vapor
male (40/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(40/group): 0, 500, 1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration and method were
reported
Male Female
Dose Dose
(mg/m3) Absolute weight (mg/m3) Absolute weight
0 - 0 -
2,090 0% 2,090 -8%
7,320 50% 7,320 8%
20,900 0% 20,900 -8%
a4.18 mg/m3 = 1 ppm.
*: result is statistically significant (p < 0.05) based on analysis of data by study authors.
for controls, no response relevant; for other doses, no quantitative response reported,
(n): number evaluated from group.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-10. Evidence pertaining to immune effects in animals exposed to ETBE
Reference and Study Design
Results (percent change compared to control)
Functional Immune Effects
Banton et al. (2011)
Female
rat, Sprague-Dawley
oral—gavage
female (10/group): 0, 250, 500,
1,000 mg/kg-day
IgM antibodv
Dose forming cells/10
(mg/kg-dav) spleen cells
IgM antibodv
forming
cells/spleen
daily for 28 consecutive days
0
-
-
immunized i.v. 4 days prior to sacrifice
with sheep red blood cells
250
-21%
-20%
500
42%
36%
1,000
8%
8%
Immune Cell Populations
Li et al. (2011)
Male
mice, 129/SV
inhalation—vapor
male (6/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
Dose
(mg/m3)
Number of
CD3+T
cells
Number of
CD4+T
cells
Number of
CD8+T
cells
20,900 mg/m3)a
0
-
-
-
whole body, 6 hr/d for 5 d/wk over
2,090
-18%*
-16%
-13%
6 wk; generation method not
reported; analytical concentration
7,320
-16%
-11%
-14%
and method were reported
20,900
-21%*
-17%*
-25%
Li et al. (2011)
Male
mice, C57BL/6
inhalation—vapor
male (6/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
Dose
(mg/m3)
Number of
CD3+T
cells
Number of
CD4+T
cells
Number of
CD8+T
cells
20,900 mg/m3)a
0
-
-
-
whole body, 6 hr/d for 5 d/wk over
2,090
-14%
-15%
-12%
6 wk; generation method not
reported; analytical concentration
7,320
-13%
-11%
-13%*
and method were reported
20,900
-24%*
-23%*
-23%*
Li et al. (2011)
Male
mice, C57BL/6
inhalation—vapor
male (5/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
Number of
Number of
Number of
Dose
CD3+T
CD4+ T-
CD8+T
(mg/m3)
cells
cells
cells
20,900 mg/m3)a
0
-
-
-
whole body, 6 hr/d for 5 d/wk over
2,090
-9%
-11%
-8%
13 wk; generation method not
reported; analytical concentration,
7,320
-17%*
-28%*
-12%
and method were reported
20,900
-24%*
-37%*
-20%
This document is a draft for review purposes only and does not constitute Agency policy.
B-37 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Spleen Weight
Banton et al. (2011)
rat, Sprague-Dawley
oral—gavage
female (10/group): 0, 250, 500,
1,000 mg/kg-day
daily for 28 consecutive days
Female
Dose Absolute Relative
(mg/kg-dav) weight weight
0
250 -3% 0%
500 -15% -18%
1,000 -9% 0%
Fuiiietal. (2010); JPEC (2008d)
rat, Sprague-Dawley
oral—gavage
PO, male (24/group): 0,100, 300,
1,000 mg/kg-day
daily for 16 wk beginning 10 wk prior
to mating
P0, female (24/group): 0,100, 300,
1,000 mg/kg-day
daily for 17 weeks beginning 10 weeks
prior to mating to lactation day 21
P0, Male P0, Female
Dose Absolute Relative Dose Absolute Relative
(mg/kg-dav) weight weight (mg/kg-dav) weight weight
0 - - 0 - -
100 -4% -1% 100 0% -2%
300 -2% 2% 300 -2% -3%
1,000 0% 8% 1,000 -1% -5%
Hagiwara et al. (2011); JPEC (2008c)
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
daily for 23 wk
Male
Dose Absolute Relative
(mg/kg-dav) weight weight
0
1,000 -5% 0%
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral—water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121,
542 mg/kg-day)a; female (50/group):
0, 625, 2,500, 10,000 ppm (0, 46, 171,
560 mg/kg-day)a
daily for 104 wk
Male Female
Dose Absolute Relative Dose Absolute Relative
(mg/kg-dav) weight weight (mg/kg-dav) weight weight
0 - - 0 - -
628 -3% -35% 46 -35% 2%
121 19% 3%* 171 -1% 28%
542 39% -45% 560 -50%* 55%*
This document is a draft for review purposes only and does not constitute Agency policy.
B-38 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation—vapor
male (NR): 0,150, 500,1,500,
Dose
(mg/m3)
Absolute
weight
Relative
weight
Dose
(mg/m3)
Absolute
weight
Relative
weight
5,000 ppm (0, 627, 2,090, 6,270,
0
-
-
0
-
-
20,900 mg/m3)b; female (NR): 0,150,
627
0%
0%
627
-9%
-3%
500, 1,500, 5,000 ppm (0, 627, 2,090,
6,270, 20,900 mg/m3)
2,090
7%
5%
2,090
-2%
5%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
6,270
-1%
1%
6,270
-5%
1%
analytical concentration and method
20,900
-9%
-2%
20,900
1%
12%
were reported
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation—vapor
male (6/group): 0, 5,000 ppm (0,
Dose
(mg/m3)
Absolute
weight
Relative
weight
Dose
(mg/m3)
Absolute
weight
Relative
weight
20,900 mg/m3)b; female (6/group): 0,
0
-
-
0
-
-
5,000 ppm (0, 20,900 mg/m3)b
20,900
10%
6%
20,900
6%
0%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk followed by a 28-day
recovery period; generation method,
analytical concentration and method
were reported
Saito et al. (2013); JPEC (2010b)
Male
Female
rat, Fischer 344
inhalation—vapor
male (50/group): 0, 500,1,500,
Dose
(mg/m3)
Absolute
weight
Relative
weight
Dose
(mg/m3)
Absolute
weight
Relative
weight
5,000 ppm (0, 2,090, 6,270,
0
-
-
0
-
-
20,900 mg/m3)b; female (50/group): 0,
500, 1,500, 5,000 ppm (0, 2,090,
2,090
4%
15%
2,090
5%
30%
6,270, 20,900 mg/m3)b
6,270
32%
43%*
6,270
-39%
-31%
dynamic whole body inhalation;
6 hr/d, 5 d/wk for 104 wk; generation
20,900
17%
66%*
20,900
-43%*
-25%
method, analytical concentration and
method were reported
This document is a draft for review purposes only and does not constitute Agency policy.
B-39 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Medinskv et al. (1999); Bond et al.
Male
Female
(1996b)
rat, Fischer 344
inhalation—vapor
Dose
Absolute
Dose
Absolute
(mg/m3)
weight
(mg/m3)
weight
male (48/group): 0, 500,1,750,
0
-
0
-
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)b; female (48/group): 0,
2,090
6%
2,090
-3%
500, 1,750, 5,000 ppm (0, 2,090,
7,320
3%
7,320
3%
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d,
20,900
5%
20,900
0%
5 d/wk for 13 wk; generation method,
analytical concentration and method
were reported
Medinskv et al. (1999); Bond et al.
Male
Female
(1996a)
mice, CD-I
inhalation—vapor
Dose
Absolute
Dose
Absolute
(mg/m3)
weight
(mg/m3)
weight
male (40/group): 0, 500,1,750,
0
-
0
-
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)b; female (40/group): 0,
2,090
-5%
2,090
-11%
500, 1,750, 5,000 ppm(0, 2,090,
7,320
0%
7,320
-2%
7,320, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d,
20,900
-15%
20,900
-11%
5 d/wk for 13 wk; generation method,
analytical concentration and method
were reported
1
2 Conversion performed by study authors.
3 b4.18 mg/m3 = 1 ppm.
4 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
5 for controls, no response relevant; for other doses, no quantitative response reported.
6 (n): number evaluated from group.
7
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table B-ll. Evidence pertaining to mortality in animals exposed to ETBE
Reference and Study Design
Results (percentage change compared to control)
Maltoni et al. (1999)
Male
Female
rat, Sprague-Dawley
Dose (mg/m3)
Survival at
Dose (mg/m3)
Survival at
oral—gavage
104 wk
104 wk
male (60/group): 0, 250,1,000 mg/kg-day;
0
0
female (60/group): 0, 250,
1,000 mg/kg-day
250
-8%
250
-8%
4 d/wk for 104 wk; observed until natural
1,000
-54%
1,000
18%
death
Suzuki et al. (2012); JPEC (2010a)
Male
Female
rat, Fischer 344
oral—water
Dose
Percentage
Dose
Percentage
male (50/group): 0, 625, 2,500,
(mg/kg-day)
survival
(mg/kg-day)
survival
10,000 ppm (0, 28,121, 542 mg/kg-day)a;
0
-
0
-
female (50/group): 0, 625, 2,500,
628
-3%
46
3%
10,000 ppm (0, 46,171, 560 mg/kg-day)a
121
-11%
171
6%
daily for 104 wk
542
-11%
560
6%
Saito et al. (2013);JPEC (2010b)
Male
Female
rat, Fischer 344
Dose (mg/m3)
Survival at 104
Dose (mg/m3)
Survival at 104
inhalation—vapor
wk
wk
male (50/group): 0, 500,1,500, 5,000 ppm
0
0
(0, 2,090, 6,270, 20,900 mg/m3)b; female
(50/group): 0, 500, 1,500, 5,000 ppm (0,
2,090
-14%
2,090
3%
2,090, 6,270, 20,900 mg/m3)b
6,270
-9%
6,270
-21%*
dynamic whole body inhalation; 6 hr/d,
20,900
-32%*
20,900
-21%*
5 d/wk for 104 wk; generation method,
analytical concentration and method were
reported
1
2 Conversion performed by study authors.
3 b4.18 mg/m3 = 1 ppm.
4 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
5 for controls, no response relevant; for other doses, no quantitative response reported.
6 (n): number evaluated from group.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Decreased Body
Weight
subchronic/
reproductive
Female rat; 28d (A)
PO Female rat; 16wks (B)
PO Male rat; 16wks (B)
PO Male rat; 18wks (C)
PO Female rat; 18wks (Q
F1 Male rat; GD 0-adult (C)
F1 Female rat; CD 0-adult (CJ
Male rat; 23wks (D)
Female rat; 26wks (F)
Male rat; 26wks (F)
0-
~ B B
~ B B
B—B—B
B—B-
B—B—B
B—B—B
B—B—B
a b b
a b 0
chronic
Female rat; 104wks (G)
Male rat; 104wks (G)
Female rat; 104wks (E)
Male rat; 104wks (E)
~ 0
Q 0
1 10 100 1,000 10,000
Dose (mg/kg-day)
Sources: (A) Banton et al, 2011 (B) Fujii et al., 2010; JPEC, 2008e (C) Gaoua, 2004b CD] Hagiwara et al., 2011
(E) Maltoni et al., 1999 (F) Miyata et al., 2013; JPEC, 2008c (G) Suzuki et al., 2012; JPEC, 2010a
2
3
Figure B-4. Exposure-response array of body weight effects following oral
exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Decreased Body
Weight
Female rats; 13wks (A]
Male rats; 13wks (A)
Female rats; 13wks, 28d recovery (A]
Male rats; 13wks, 28d recovery (A)
subchronic
Female rats; 13 wks (B)
Male rats; 13 wks (B)
Female mice; 13 wks (B)
Male mice; 13 wks (B)
~ B B-
~ B B-
~ B
B B
B B
B B
~
-B
-B
chronic
Female rats; 104wks (C)
Male rats; 104wks (C)
1 10 100 1,000 10,000 100,000
Exposure Concentration (mg/mJ)
¦"significantly increased body weight
Sources: (A] JPEC, 2008b (B) Medinsky et al., 1999; Bond et al., 1996 [C] Saito et al, 2013; JPEC,2010b
2
3
Figure B-5. Exposure-response array of body weight effects following
inhalation exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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3
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5
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8
9
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13
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23
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26
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28
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Supplemental Information—ETBE
B.2.2. Genotoxicity Studies
Bacterial Systems
Mutagenic potential of ETBE has been tested by Zeiger etal. T19921 using different
Salmonella typhimurium strains for 311 chemicals, including ETBE, both in the absence and
presence of metabolic activation (S9). Preincubation protocol was followed and precaution was
exercised to account for the volatility of the compound. Five doses ranging from 100 to
10,000 [ig/plate were tested using different Salmonella strains including TA97, TA 98, TA100,
TA1535. The results showed thatthe 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-12.
In Vitro Mammalian Studies
Limited available studies (two) in in vitro mammalian systems were unpublished reports.
Vergnes and Kubena f!995bl evaluated the mutagenicity of ETBE using the hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) forward mutation assay in Chinese hamster ovary K1-BH4
cells. Duplicate cultures were treated with five concentrations of ETBE (>98% purity; containing
13 ppm A022, an antioxidant stabilizer) ranging from 100 to 5,000 [ig/mL, both in the presence and
absence of S9 activation. No statistically significant or concentration-related increase in the HGPRT
mutation frequencies were observed at any of the ETBE concentrations tested, either in the absence
or in the presence of metabolic (S9) activation.
The same authors [fVergnes and Kubena. 1995bl unpublished report] studied the
clastogenic potential of ETBE in vitro using chromosome aberration assay in Chinese hamster ovary
cells. The cells were exposed from 100 to 5,000 |J.g/mL of ETBE in culture medium, both in the
presence and absence of S9 metabolic activation system. No statistically significant or
concentration-related increase in the frequency of chromosomal aberrations, in the presence or
absence of the S9 metabolic activation system, was observed. Neither the effect of the antioxidant
stabilizer used in ETBE nor control for volatility of the compound was described for both studies
although capped glass bottles were used in the experiments.
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
(5 animals/sex/group) were exposed to ETBE by inhalation at target concentrations of 0, 400,
2,000, and 5,000 ppm (0,1,671, 8,357, and 20,894 mg/m3) for 6 hours/day, for 5 days. Following
treatment, polychromatic erythrocytes (PCE) from bone marrow were analyzed for micronucleus
formation. The results showed that no statistically significant increases in the mean percentages of
This document is a draft for review purposes only and does not constitute Agency policy.
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2
3
4
5
6
7
8
9
10
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12
13
14
15
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20
21
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25
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Supplemental Information—ETBE
micronucleated polychromatic erythrocytes (MNPCE) were observed in mice (male or female)
when exposed to ETBE.
In addition to Vergnes and Kubena f!995al. 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 [TPEC f2007cl: TPEC f2007al: TPEC f2007dl:
TPEC f2007bl published as Noguchi etal. f20131].
The first two studies (oral and intraperitoneal injection) were part of an acute (2-day)
exposure. In the first study, both male and female F344 rats (5 animals/sex/dose group) were
administered ETBE (99.3% pure) via gavage at doses of 0, 500,1,000, or 2,000 mg/kg-day
separated by 24 hours in olive oil [flPEC. 2007al. unpublished report]. Animals were sacrificed, and
bone marrow smears were collected and stained 24 hours after the final administration. Following
treatment, polychromatic erythrocytes from bone marrow were analyzed for MN formation. The
results were expressed as the ratio of polychromatic erythrocytes/total erythrocytes. There were
no treatment-related effects on the number of MNPCE or the ratio of PCE/total erythrocytes. ETBE
was determined to be negative for micronuclei induction in rat bone marrow cells after acute oral
exposure.
In the second study (intraperitoneal injection), male and female F344 rats (5
animals/sex/dose group) were administered two ETBE intraperitoneal injections separated by
24 hours at doses of 0, 250, 500,1,000, or 2,000 mg/kg-day in olive oil fNoguchi etal.. 2013: TPEC.
2007b). Animals were sacrificed, and bone marrow smears were collected and stained 24 hours
after the final injection. All animals in the 2,000 mg/kg-day group died on the first day of treatment
There were no treatment-related effects on either the number of MNPCEs or the ratio of
polychromatic erythrocytes/total erythrocytes. In addition, no dose-dependent tendencies for
increase in MNPCE/PCE or alterations in the ratios of PCE/total erythrocytes were noted in either
sex of the treated groups. ETBE was determined to be negative for micronuclei induction in rats
after acute intraperitoneal exposure.
The next two studies (drinking water and inhalation) were part of 13-week toxicity studies
in rats where ETBE effects on the micronuclei in PCE were examined at the end of the study. In the
first 13-week study, male and female F344 rats (10 animals/sex/dose group) were administered
drinking water containing 0,1,600, 4,000, or 10,000 ppm ETBE for 13 weeks (Noguchi 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,
500,1,500, or 5,000 ppm (0, 2,089, 6,268, or 20,894 mg/m3) for 6 hours/day, 5 days/week
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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23
24
25
26
27
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38
Supplemental Information—ETBE
(Noguchi etal.. 2013: TPEC. 2007b). Normochromatic and polychromatic erythrocytes and
micronuclei were counted as in the previous study. There were no treatment-related effects on the
number of MNPCE or the ratio of PCE/total erythrocytes. ETBE was determined to be negative for
micronuclei induction in rat bone marrow cells after a 13-week inhalation exposure.
Furthermore, NTP f!996a. 1996bl performed an in vivo bone marrow micronucleus test
both in B6C3F1 mice and Fischer rats. The animals were exposed through intraperitoneal injection
3 times in a period of 72 hours [n = 5). Doses for the mice were 0,1,300,1,700, 2,100 and
2,500 mg/kg, and the doses for rats were 0, 625,1,250, 2,500 mg/kg. No increase in micronucleated
PCEs were observed in either mice or rats. Two of five mice died in the 1,700 mg/kg dose group,
while 3 of 5 and 4 of 5 animals died in the 2,100 and 2,500 mg/kg dose groups, respectively, and the
surviving animals in the two highest dose groups were not scored. In the rat study, 2 of 5 animals
died in the highest dose group.
Weng etal. (20111 conducted several studies evaluating the differential genotoxicity of
ETBE in various tissues or systems (i.e., erythrocytes, leukocytes, liver, and sperm) in C57BL/6
wild-type and AIdh2 knockout mice after subchronic inhalation exposure. All studies used the same
exposures (i.e., to 0, 500,1,750 and 5,000 ppm ETBE for 6 hours/day, 5 days/week for 13 weeks).
Deoxyribonucleic acid (DNA) strand breaks were observed in leukocytes of male (all
concentrations) and female (high dose only) Aldh2 knockout mice and with the high dose in wild
type male mice fWengetal.. 20111.
Weng etal. (20121 studied the differential genotoxic effects of subchronic exposure to ETBE
in the liver of C57BL/6 wild-type and AIdh2 knockout mice. DNA strand breaks in the hepatocytes
of male and female with different AIdh2 genotypes were determined using alkaline comet assay. In
addition, 8-hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification, and
8-hydroxydeoxyguanosine were determined as endpoints for genetic damage. There was significant
increase in damage in all three exposure groups in the knockout male mice, although the increase
was only found in 5,000 ppm exposure group for the knockout female mice. In the wild-type,
significant DNA damage was seen only in males in the 5,000 ppm group, but not in females. This
indicates the sensitivity of sex differences both in knockout and wild-type mice.
In another study by the same authors (Weng etal.. 20131. the authors performed in vivo
micronucleus tests (on what appear to be the same set of animals), in addition to the DNA strand
breaks, 8-hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification, and
8-hydroxydeoxyguanosine. The mice (wild-type and knockout, males and females) were exposed to
0, 500,1,750 and 5,000 ppm ETBE for 6 hours/day, 5 days/week for 13 weeks. Peripheral blood
samples were obtained and processed to detect micronucleated reticulocytes (MN-RETs) and
micronuclei in the mature normochromatic erythrocyte population (MN-NCE). The results indicate
that ETBE significantly affected frequencies of MN-RETs in male and female mice. In knockout male
mice, the frequencies of MN-RETs of the 1,750- and 5,000-ppm exposure groups were significantly
increased when compared with the control group. In the wild-type male mice, however, only the
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3
4
5
6
7
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9
10
11
12
13
14
Supplemental Information—ETBE
5,000 ppm group had a higher frequency of MN-RETs than that of the control group. In female mice,
there was no difference in the frequencies of MN-RETs between exposure groups and the control
group in wild-type mice. In the same exposure group (5,000 ppm), the knock-out mice had a higher
frequency of MN-RETs compared to the wild-type. These results inform the influence of AIdh2 and
sex difference on genotoxicity as a result of exposure to ETBE.
In yet another study by the same authors fWeng etal.. 20141. DNA strand breaks and
8-hydroxyguanine DNA-glycosylase (hOGGl)-modified oxidative base modification were measured
in sperm collected from the left caudia epididymis. In addition to the 13-week protocol used in the
other studies, Weng etal. (20141 also included an additional 9-week study where the male mice
(wild-type, knockout, and heterogeneous [HT]) were exposed to 0, 50, 200 and 500 ppm ETBE for
6 hours/day, 5 days/week for 9 weeks. In the 13-week study, there were significant increases in
damage in all three exposure groups in the knockout male mice, but only in the two highest dose
groups in the wild-type males. In the 9-week study, there was no change in the wild-type mice, but
both the heterogeneous and the knockout mice had significant increases in the two highest doses.
Table B-12. Summary of genotoxicity (both in vitro and in vivo) studies of
ETBE
Species
Test System
Dose/Cone.
Results3
Comments
Reference
Bacterial systems
-S9
+S9
Salmonella
typhimuriu
m (TA97,
TA98,
TA100,
TA1535)
Mutation
Assay
100, 333, 1,000,
3,333, 10,000
Hg/plate
Preincubation procedure
was followed. Experiment
was conducted in capped
tubes to control for
volatility
Zeiger et al. (1992)
In vitro systems
Chinese
Hamster
Ovary cells
(hgprt locus)
Gene
Mutation
Assay
100, 300, 1,000,
3,000,
5,000 ng/m L
Experiments conducted
both with and without
metabolic activation
Vergnes and Kubena
(1995b)
(unpublished report)
Chinese
Hamster
Ovary cells
Chromosomal
Aberration
Assay
100, 300, 1,000,
3,000,
5,000 Hg/mL
-
-
Experiments conducted
both with and without
metabolic activation
Vergnes (1995)
(unpublished report)
In vivo animal studies
CD-I mice
(male and
female)
Bone Marrow
Micronucleus
test
0, 400, 2,000,
5,000 ppm (0,
1,670, 8,360,
20,900 mg/m3)b
Whole body Inhalation,
6 hr/d, 5 d, 5
animals/sex/group
Vergnes and Kubena
(1995a)
(unpublished report)
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Supplemental Information—ETBE
Species
Test System Dose/Cone.
Results3
Comments
Reference
B6C3F1 mice
(male)
Bone Marrow
Micronucleus
test
0,1,300, 1,700,
2,100,
2,500 mg/kg
Intraperitoneal injection
3x, 72 hr. Five
animals/group, 3 animals
in dose 1,700 mg/kg
dose. Surviving animals
were not scored at doses
of 2,100 and 2,500 mg/kg
NTP (1996a)
Fischer 344
rats (male)
Bone Marrow
Micronucleus
test
0, 625, 1,250,
2,500 mg/kg
Intraperitoneal injection
3x, 72 hr. Five
animals/group, 3 animals
in 2,500 mg/kg dose
group.
NTP(1996b)
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 500, 1,000,
2,000 mg/kg-day
Oral gavage, 24 hr apart,
2 d, 5 animals/sex/group
JPEC (2007b)
(unpublished report)
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 250, 500, 1,000,
2,000 mg/kg-day
Intraperitoneal injection,
24 hr apart, 2 d, 5
animals/sex/group
Noguchi et al.
(2013); JPEC
(2007b), unpublished
report
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0,1,600, 4,000,
10,000 ppm (0,
101, 259, 626
mg/kg-day in
males; 0,120, 267,
629 mg/kg-day in
females)0
Drinking water, 13 wk, 10
animals/sex/group
Noguchi et al.
(2013); JPEC (2007c),
unpublished report
Fischer 344
rats (male
and female)
Bone Marrow
Micronucleus
test
0, 500,1,500,
5,000 ppm (0,
2,090, 6,270,
20,900 mg/m3)b
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk. 10
animals/sex/group
Noguchi et al.
(2013); JPEC (2007c),
unpublished report
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay),
leukocytes
0, 500,1,750 and
5,000 ppm
Male—
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2011)
Female
WT/KO
-/+d
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Supplemental Information—ETBE
Species
Test System
Dose/Cone.
Results3
Comments
Reference
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay)
0, 500,1,750 and
5,000 ppm
Male—
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2012)
Female
WT/KO
-/+d
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
Micronucleus
assay,
erythrocytes
0, 500,1,750 and
5,000 ppm
Male*
WT/KO
+d/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2013)
Female*
WT/KO
-/+
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 50, 200 and
500 ppm
WT/HT/
KO
-/+/+
Whole body inhalation,
6 hr/d, 5 d/wk, 9 wk
Weng et al. (2014)
C57BL/6
wild-type
(WT) and
Aldh2
knockout
(KO) mice
DNA strand
breaks
(alkaline
comet assay);
sperm
0, 500,1,750 and
5,000 ppm
WT/KO
+/+
Whole body inhalation,
6 hr/d, 5 d/wk, 13 wk
Weng et al. (2014)
1
2 a+ = positive; - = negative; (+), equivocal.
3 b4.18 mg/m3 = 1 ppm.
4 Conversions performed by study authors.
5 dPositive in highest dose tested.
6 *When the data of ETBE-induced MN-RETs (micronucleated reticulocytes) were normalized with corresponding
7 control, the effect disappeared.
8
9 Summary
10 Limited studies have been conducted to understand the genotoxic potential of ETBE. Most
11 studies indicate that ETBE does not induce ge no toxicity in the systems tested. More recently, Weng
12 and coauthors seem to illustrate the influence of Aldh2 on the genotoxic effects of ETBE. With
13 respect to overall existing database, it should be noted that the array of genotoxic tests conducted
14 are limited. The inadequacy of the database is two dimensional: (a) the coverage of the studies
15 across the genotoxicity tests needed for proper interpretation of the weight of evidence of the data;
16 (b) the quality of the available data. With respect to the array of types of genotoxicity tests
17 available, ETBE has only been tested in one bacterial assay. Limited (two) studies are available with
18 respect to in vitro studies. Existing in vivo studies have all been tested only for the micro nucleus
19 assay, DNA strand breaks, or both. Key studies in terms of chromosomal aberrations and DNA
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Supplemental Information—ETBE
1 adducts are missing. It should also be noted that the few existing studies are unpublished reports
2 lacking peer review. Given the above limitations; significant deficiencies; and sparse database both
3 in terms of quality and quantity; it is implicit that the database is inadequate or insufficient to draw
4 any conclusions on the effect of ETBE with respect to genotoxicity.
5 B.3. SUPPLEMENTAL ORGAN WEIGHT DATA
6 B.3.1. Relative Kidney Weight Data
Table B-13. Evidence pertaining to relative kidney weight effects in animals
exposed to ETBE
Reference and Study Design
Results (percent change compared to control)
Fuiiietal. (2010); JPEC (2008d)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
P0, male (24/group): 0,100, 300,
Dose
(mg/kg-day)
Relative weight
Dose
(mg/kg-day)
Relative weight
1,000 mg/kg-day
0
-
0
-
daily for 16 wk beginning 10 wk prior to
mating
100
8%*
100
-3%
P0, female (24/group): 0,100, 300,
300
12%*
300
-1%
1,000 mg/kg-day
daily for 17 weeks beginning 10 weeks prior
1,000
26%*
1,000
2%
to mating to lactation day 21
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
P0, male (25/group): 0, 250, 500,
Dose
(mg/kg-day)
Relative weight
Dose
(mg/kg-day)
Relative weight
1,000 mg/kg-day
0
-
0
-
daily for a total of 18 wk beginning 10 wk
before mating until after weaning of the
250
11%*
250
9%
pups
500
18%*
500
5%
P0, female (25/group): 0, 250, 500,
1,000 mg/kg-day
1,000
28%*
1,000
3%
daily for a total of 18 wk beginning 10 wk
Fl, Male
Fl, Female
before mating until PND 21
Fl, males and females (25/group/sex): via
P0 dams in utero daily through gestation
Dose
(mg/kg-day)
Relative weight
Dose
(mg/kg-day)
Relative weight
and lactation, then Fl doses beginning PND
0
-
0
-
22 until weaning of the F2 pups
250
10%*
250
6%
500
19%*
500
6%
1,000
58%*
1,000
10%*
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
Dose
(mg/kg-day)
Relative weight
daily for 23 wk
0
1,000
25%*
Mivata et al. (2013);JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
oral—gavage
male (15/group): 0, 5, 25,100,
Dose
(mg/kg-day)
Relative weight
Dose
(mg/kg-day)
Relative weight
400 mg/kg-day; female (15/group): 0, 5, 25,
0
-
0
-
100, 400 mg/kg-day
daily for 26 wk
5
8%
5
7%
25
6%
25
4%
100
12%*
100
11%*
400
21%*
400
15%*
Suzuki et al. (2012); JPEC (2010a)
Male
Female
rat, Fischer 344
oral—water
male (50/group): 0, 625, 2,500,10,000 ppm
Dose
Dose
(mg/kg-day)
Relative weight
(mg/kg-day)
Relative weight
(0, 28,121, 542 mg/kg-day)a; female
0
-
0
-
(50/group): 0, 625, 2,500, 10,000 ppm (0,
46,171, 560 mg/kg-day)a
28
0%
46
13%*
daily for 104 wk
121
12%*
171
22%*
542
31%*
560
37%*
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation—vapor
Dose (mg/m3)
Relative weight
Dose (mg/m3)
Relative weight
male (NR): 0,150, 500,1,500, 5,000 ppm
0
-
0
-
(0, 627, 2,090, 6,270, 20,900 mg/m3)b;
627
10%
627
8%
female (NR): 0,150, 500,1,500, 5,000 ppm
(0, 627, 2,090, 6,270, 20,900 mg/m3)
2,090
9%
2,090
7%
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
6,270
20%*
6,270
12%*
analytical concentration and method were
20,900
24%*
20,900
20%*
reported
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Supplemental Information—ETBE
Reference and Study Design
Results (percent change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
Dose
(mg/m3)
Dose
(mg/m3)
inhalation—vapor
male (6/group): 0, 5,000 ppm (0,
Relative weight
Relative weight
20,900 mg/m3)b; female (6/group): 0,
0
-
0
-
5,000 ppm (0, 20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d,
20,900
15%*
20,900
5%
5 d/wk for 13 wk followed by a 28 day
recovery period; generation method,
analytical concentration and method were
reported
Saito et al. (2013); JPEC (2010b)
Male
Female
rat, Fischer 344
Dose
(mg/m3)
Dose
(mg/m3)
inhalation—vapor
male (50/group): 0, 500,1,500, 5,000 ppm
Relative weight
Relative weight
(0, 2,090, 6,270, 20,900 mg/m3)b; female
0
-
0
-
(50/group): 0, 500, 1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b
2,090
19%*
2,090
11%*
dynamic whole body inhalation; 6 hr/d,
6,270
26%*
6,270
16%*
5 d/wk for 104 wk; generation method,
analytical concentration and method were
20,900
66%*
20,900
51%*
reported
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 Percentage change compared to controls calculated as 100 x ((treated value—control value) -f control value).
6
7 B.3.2. Absolute Liver Weight Data
Table B-14. Evidence pertaining to absolute liver weight effects in animals
exposed to ETBE
Reference and Study Design
Fuiiietal. (2010); JPEC (2008d)
rat, Sprague-Dawley
oral—gavage
P0, male (24/group): 0,100, 300,1,000 mg/kg-day
daily for 16 wk beginning 10 wk prior to mating
P0, female (24/group): 0,100, 300,
1,000 mg/kg-day
daily for 17 wk beginning 10 wk prior to mating to
lactation day 21
Results (percentage change compared to control)
P0, Female
Absolute
Dose
Absolute
weight
(mg/kg-day)
weight
-
0
-
-3%
100
-1%
-1%
300
3%
13%*
1,000
14%*
P0, Male
Dose
(mg/kg-day)
0
100
300
1,000
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Supplemental Information—ETBE
Reference and Study Design
Results (percentage change compared to control)
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral—gavage
PO, male (25/group): 0, 250, 500,1,000 mg/kg-day
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
daily for a total of 18 wk beginning 10 wk before
0
-
0
-
mating until after weaning of the pups
P0, female (25/group): 0, 250, 500,
250
2%
250
-1%
1,000 mg/kg-day
500
2%
500
4%
daily for a total of 18 wk beginning 10 wk before
1,000
17%*
1,000
6%
mating until PND 21
Fl, male (25/group): 0, 250, 500,1,000 mg/kg-day
Fl, Male
Fl, Female
P0 dams dosed daily through gestation and
lactation, then Fl doses beginning PND 22 until
weaning of the F2 pups
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
Fl, female (24-25/group): 0, 250, 500,
0
-
0
-
1,000 mg/kg-day
P0 dams dosed daily through gestation and
250
0%
250
1%
lactation, then Fl dosed beginning PND 22 until
500
14%*
500
3%
weaning of the F2 pups
1,000
27%*
1,000
10%*
Hagiwara et al. (2011); JPEC (2008c)
Male
rat, Fischer 344
oral—gavage
male (12/group): 0,1,000 mg/kg-day
Dose
(mg/kg-day)
Absolute
weight
daily for 23 wk
0
1,000
21%*
Mivata et al. (2013); JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
oral—gavage
male (15/group): 0, 5, 25,100, 400 mg/kg-day;
Dose
(mg/kg-day)
Absolute
weight
Dose
(mg/kg-day)
Absolute
weight
female (15/group): 0, 5, 25,100, 400 mg/kg-day
0
-
0
-
daily for 26 wk
5
-2%
5
-4%
25
7%
25
-1%
100
4%
100
2%
400
19%
400
9%
Suzuki et al. (2012); JPEC (2010a)
Male
Female
rat, Fischer 344
oral—water
male (50/group): 0, 625, 2,500,10,000 ppm (0, 28,
Dose
Absolute
Dose
Absolute
(mg/kg-day)
weight
(mg/kg-day)
weight
121, 542 mg/kg-day)a; female (50/group): 0, 625,
0
-
0
-
2,500, 10,000 ppm (0, 46,171, 560 mg/kg-day)a
daily for 104 wk
28
-11%*
46
-5%
121
-4%
171
-2%
542
2%
560
-10%
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Supplemental Information—ETBE
Reference and Study Design
Results (percentage change compared to control)
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation—vapor
male (NR): 0, 150, 500, 1,500, 5,000 ppm (0, 627,
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
2,090, 6,270, 20,900 mg/m3)b; female (NR): 0,150,
0
-
0
-
500, 1,500, 5,000 ppm (0, 627, 2,090, 6,270,
627
5%
627
-3%
20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk for
2,090
6%
2,090
-8%
13 wk; generation method, analytical
concentration and method were reported
6,270
4%
6,270
-2%
20,900
2%
20,900
5%
JPEC (2008a)
Male
Female
rat, CRL:CD(SD)
inhalation—vapor
male (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b;
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
female (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b
0
-
0
-
dynamic whole body chamber; 6 hr/d, 5 d/wk for
13 wk followed by a 28 day recovery period;
20,900
13%
20,900
11%
generation method, analytical concentration and
method were reported
Saito et al. (2013); JPEC (2010b)
Male
Female
rat, Fischer 344
inhalation—vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
2,090, 6,270, 20,900 mg/m3)b; female (50/group):
0
-
0
-
0, 500, 1,500, 5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b
2,090
1%
2,090
-3%
dynamic whole body inhalation; 6 hr/d, 5 d/wk for
6,270
11%*
6,270
-8%
104 wk; generation method, analytical
concentration and method were reported
20,900
10%
20,900
1%
Medinsky et al. (1999); Bond et al. (1996b)
Male
Female
rat, Fischer 344
inhalation—vapor
male (48/group): 0, 500,1,750, 5,000 ppm (0,
Dose
(mg/m3)
Absolute
weight
Dose
(mg/m3)
Absolute
weight
2,090, 7,320, 20,900 mg/m3)b; female (48/group):
0
-
0
-
0, 500, 1,750, 5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)b
2,090
6%
2,090
2%
dynamic whole body chamber; 6 hr/d, 5 d/wk for
7,320
14%*
7,320
9%
13 wk; generation method, analytical
concentration and method were reported
20,900
32%*
20,900
26%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Reference and Study Design
Results (percentage change compared to control)
Medinskv et al. (1999); Bond et al. (1996a)
mice, CD-I
inhalation—vapor
male (40/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female (40/group):
0, 500, 1,750, 5,000 ppm(0, 2,090, 7,320,
20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk for
13 wk; generation method, analytical
concentration and method were reported
Male Female
Dose Absolute Dose Absolute
(mg/m3) weight (mg/m3) weight
0 - 0 -
2,090 4% 2,090 2%
7,320 13%* 7,320 19%*
20,900 18%* 20,900 33%*
1
2 Conversion performed by study authors.
3 b4.18 mg/m3 = 1 ppm.
4 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
5 for controls, no response relevant; for other doses, no quantitative response reported.
6 Percent change compared to controls calculated as 100 x ((treated value—control value) -f control value).
7
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1
2 APPENDIX C. DOSE-RESPONSE MODELING
3 FOR THE DERIVATION OF REFERENCE VALUES
4 FOR 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 EPA's Benchmark Dose Software (BMDS, version 2.2). Sections 0 and 0 (noncancer) and Section
10 C.l.2 (cancer) describe the common practices used in evaluating the model fit and selecting the
11 appropriate model for determining the POD, as outlined in the Benchmark Dose Technical Guidance
12 Document (U.S. EPA. 2012). In some cases, it might be appropriate to use alternative methods based
13 on statistical judgment; exceptions are noted as necessary in the summary of the modeling results.
14 C.l.l. Noncancer Endpoints
15 Evaluation of Model Fit
16 For each dichotomous endpoint, BMDS dichotomous models1 were fitted to the data using
17 the maximum likelihood method. Each model was tested for goodness-of-fit using a chi-square
18 goodness-of-fit test (x2 p-value < 0.10 indicates lack of fit). Other factors were also used to assess
19 model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in the
20 vicinity of the benchmark response (BMR).
21 For each continuous endpoint, BMDS continuous models2 were fitted to the data using the
22 maximum likelihood method. Model fit was assessed by a series of tests as follows. For each model,
23 first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). If Test
24 2 was not rejected (x2 p-value > 0.10), the model was fitted to the data assuming constant variance.
25 If Test 2 was rejected (x2 p-value < 0.10), the variance was modeled as a power function of the
26 mean, and the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS
'Unless otherwise specified, all available BMDS dichotomous models besides the alternative and nested
dichotomous models were fitted. The following parameter restrictions were applied: For the log-logistic model,
restrict slope > 1; for the gamma and Weibull models, restrict power > 1.
2Unless otherwise specified, all available BMDS continuous models were fitted. The following parameter
restrictions were applied: For the polynomial models, restrict the coefficients bl and higher to be nonnegative
or nonpositive if the direction of the adverse effect is upward or downward, respectively; for the Hill, power,
and exponential models, restrict power > 1.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Test 3). For fitting models using either constant variance or modeled variance, models for the mean
2 response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with x2 p-value <
3 0.10 indicating inadequate fit). Other factors were also used to assess the model fit, such as scaled
4 residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
5 Model Selection
6 For each endpoint, the BMDL estimate (95% lower confidence limit on the benchmark dose
7 (BMD), as estimated by the profile likelihood method and Akaike's information criterion (AIC) value
8 were used to select a best-fit model from among the models exhibiting adequate fit. If the BMDL
9 estimates were "sufficiently close," that is, differed by at most threefold, the model selected was the
10 one that yielded the lowest AIC value. If the BMDL estimates were not sufficiently close, the lowest
11 BMDL was selected as the POD.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose-response modeling for 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-day)
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-day)
0
5
25
100
400
No. of animals
15
15
14
15
13
Mean ± SD
3.27 ±0.34
3.29 ±0.3
3.47 ±0.32
3.42 ± 0.48
4.09 ±0.86
Increased absolute
kidney weight
Mivata et al.
(2013); JPEC
(2008b)
Female
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
5
25
100
400
No. of animals
15
15
15
15
15
Mean ± SD
1.88 ±0.2
1.89 ±0.16
1.88 ±0.15
2.02 ±0.21
2.07 ±0.23
Increased absolute
kidney weight
Gaoua (2004b)
PO Male
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
250
500
1,000
No. of animals
25
25
25
25
Mean ± SD
3.58 ±0.413
3.96 ±0.446
4.12 ±0.624
4.34 ± 0.434
Increased absolute
kidney weight
Gaoua (2004b)
PO
Female
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
250
500
1,000
No. of animals
25
24
22
25
Mean ± SD
2.24 ±0.185
2.22 ±0.16
2.29 ±0.207
2.35 ±0.224
Increased absolute
kidney weight
Gaoua (2004b)
F1 Male
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
250
500
1,000
No. of animals
24
25
24
25
Mean ± SD
3.38 ±0.341
3.73 ±0.449
4.13 ±0.64
5.34 ±5.39
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose response modeling for ETBE
(continued)
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased absolute
kidney weight
Gaoua (2004b)
F1 Female
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
250
500
1,000
No. of animals
25
24
25
23
Mean ± SD
2.24 ±0.178
2.34 ±0.242
2.3 ±0.226
2.49 ± 0.284
Increased absolute
kidney weight
Fuiii et al. (2010);
JPEC (2008d)
Male
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
3.46 ±0.57
3.62 ±0.45
3.72 ±0.35
4.07 ±0.53
Increased relative
kidney weight
Fuiii et al. (2010);
JPEC (2008d)
Male
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
0.546 ± 0.059
0.592 ±0.06
0.609 ± 0.042
0.689 ± 0.049
Increased absolute
kidney weight
Fuiii et al. (2010);
JPEC (2008d)
Female
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
100
300
1,000
No. of animals
21
22
23
19
Mean ± SD
2.17 ±0.18
2.13 ±0.14
2.17 ±0.17
2.33 ±0.24
Increased relative
kidney weight
Fuiii et al. (2010);
JPEC (2008d)
Female
Sprague-
Dawley
rats
Dose
(mg/kg-day)
0
100
300
1,000
No. of animals
24
24
24
24
Mean ± SD
0.674 ± 0.053
0.656 ± 0.048
0.668 ±0.057
0.687 ± 0.045
INHALATION
Urothelial
hyperplasia of the
renal pelvis
Saito et al. (2013);
JPEC (2010b)
Male
F344 rats
Exposure
concentration
(mg/m3)
0
2,090
6,270
20,900
Incidence/Tota
2/50
5/50
16/49
41/50
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-l. Noncancer endpoints selected for dose response modeling for ETBE
(continued)
Endpoint, Study
Sex,
Strain,
Species
Doses and Effect Data
Increased absolute
kidney weight
JPEC (2008a)
Male
Sprague-
Dawley
rats
Exposure
concentration
(ppm)
0
150
500
1,500
5,000
No. of animals
10
10
10
10
10
Mean ± SD
3.15 ±
0.243
3.45 ±
0.385
3.49 ±
0.314
3.72 ±
0.365
3.64 ±
0.353
Increased absolute
kidney weight
JPEC (2008a)
Female
Sprague-
Dawley
rats
Exposure
concentration
(ppm)
0
150
500
1,500
5,000
No. of animals
10
10
10
10
10
Mean ± SD
1.84 ±
0.129
1.85 ±0.18
1.83 ±
0.118
1.92 ±
0.173
1.97 ±0.16
Increased absolute
kidney weight
Medinskv et al.
(1999); Bond et al.
(1996b)
Male
F344 rats
Exposure
concentration
(ppm)
0
500
1,750
5,000
No. of animals
11
11
11
11
Mean ± SD
1.73 ±0.155
1.85 ±0.137
1.903 ±0.1
2.067 ±0.124
Increased absolute
kidney weight
Medinskv et al.
(1999); Bond et al.
(1996b)
Female
F344 rats
Exposure
concentration
(ppm)
0
500
1,750
5,000
No. of animals
10
11
11
11
Mean ± SD
1.077 ± 0.069
1.125 ±0.048
1.208 ± 0.076
1.306 ± 0.055
2 Modeling Results
3 Below are tables summarizing the modeling results for the noncancer endpoints modeled.
4 Oral Exposure Endpoints
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-2. Summary of BMD modeling results for urothelial hyperplasia of the
renal pelvis in male F344 rats exposed to ETBE in drinking water for
104 weeks (IPEC. 2010a) modeled with doses as mg/kg-day (calculated by
study authors); BMR = 10% extra risk
Model3
Goodness of Fit
BMDioPct
(mg/kg-day)
BMD LioPct
(mg/kg-day)
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
l.OOxlO"3
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-Linear0
0.395
126.07
79.3
60.5
aSelected model in bold; scaled residuals for selected model for doses 0, 28,121, and 542 mg/kg-day were 0.000,
-1.377,1.024, and -0.187, respectively.
bFor the Multistage 3° model, the beta coefficient estimates were 0 (boundary of parameters space), and the
model reduced to the Multistage 2° model.
cThe Multistage 2° model and Quantal-Linear models appear equivalent; however, differences exist in digits not
displayed in the table.
Quantal Linear Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit for the
0.7
Quantal Linear
0.6
0.5
0.4
0.3
0.2
0.1
BMDLJ BMP
O 100 200 300 400 500
dose
1 13:10 09/10 2014
2 Figure C-l. Plot of incidence rate by dose, with fitted curve for selected model;
3 dose shown in mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
C-6 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Quantal Linear Model using Weibull Model (Version: 2.16; Date: 2/28/2013)
2 The form of the probability function is: P[response] = background + (1-background) x [l-exp(- slope x dose)]
3 Benchmark Dose Computation.
4 BMR = 10% Extra risk
5 BMD = 79.3147
6 BMDL at the 95% confidence level = 60.5163
7 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0
0.0192308
Slope
0.00132839
0.00124304
Power
n/a
1
8 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
9 AIC = 126.074
10 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
11 X2 = 2.98; d.f= 3; p-value = 0.3948
12
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-3. Summary of BMD modeling results for increased absolute kidney
weight in male S-D rats exposed to ETBE by daily gavage for 26 weeks (Mivata
etal.. 2013: IPEC. 2008d): BMR = 10% relative deviation from the mean
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model Selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.752
-47.963
186
126
The linear model was selected
based on lowest AIC.
Exponential (M4)
Exponential (M5)c
0.603
-46.156
157
67.7
Hill
0.605
-46.161
156
63.6
Powerd
Polynomial 2°e
Linear'
0.774
-48.055
176
115
Polynomial 3°g
0.774
-48.055
176
115
aModeled variance case presented (BMDS Test 2 p-value = <0.0001), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-day were -0.421, -0.288,1.29, -0.669, and 0.15,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
the Linear and Polynomial 3° models appear equivalent; however, differences exist in digits not displayed in the
table.
gThe Linear model, Polynomial 2° and 3° models and the Power models appear equivalent; however, differences
exist in digits not displayed in the table.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
Supplemental Information—ETBE
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMD
Linear
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
BMD
BMD
0
50
1 00
150
200
250
300
350
400
15:56 05/15 2014
Figure C-2. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Polynomial Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = beta_0 + beta_l x 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
alpha
-13.8218
-1.41289
rho
9.65704
0
beta_0
3.30477
3.30246
beta_l
0.00187393
0.00193902
This document is a draft for review purposes only and does not constitute Agency policy.
C-9 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
15
3.27
3.3
0.34
0.32
-0.421
5
15
3.29
3.31
0.3
0.325
-0.288
25
14
3.47
3.35
0.32
0.343
1.29
100
15
3.42
3.49
0.48
0.418
-0.669
400
13
4.09
4.05
0.86
0.859
0.15
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
17.455074
6
-22.910149
A2
29.755425
10
-39.51085
A3
28.583571
7
-43.167142
fitted
28.027315
4
-48.05463
R
6.041664
2
-8.083328
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
47.4275
8
<0.0001
Test 2
24.6007
4
<0.0001
Test 3
2.34371
3
0.5042
Test 4
1.11251
3
0.7741
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-4. Summary of BMD modeling results for increased absolute kidney
weight in female S-D rats exposed to ETBE by daily gavage for 26 weeks
(Mivata et al.. 2013: IPEC. 2008d): BMR = 10% relative deviation from the
mean
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Model3
p-value
AIC
Basis for Model Selection
Exponential (M2)
Exponential (M3)b
0.369
-168.25
406
271
The Exponential (M4) model was
selected based on lowest BMDL.
Exponential (M4)
0.670
-168.60
224
56.9
Exponential (M5)
0.865
-167.37
error0
0
Hill
0.986
-169.37
error0
error0
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.382
-168.34
402
263
aConstant variance case presented (BMDS Test 2 p-value = 0.425), selected model in bold; scaled residuals for
selected model for doses 0, 5, 25,100, and 400 mg/kg-day were 0.2257, 0.2206, -0.737, 0.3806, and -0.08999,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
CBMD or BMDL computation failed for this model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
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
Supplemental Information—ETBE
Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Level for BM
Exponential
2.2
2.1
2
9
8
BMDL
BMD
0 50 100 150 200 250 300 350 400
dose
16:35 05/15 2014
Figure C-3. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Exponential Model. (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a* [c-(c-l) x exp(-b x 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
In alpha
-3.35462
-3.36529
rho(S)
n/a
0
a
1.86911
1.786
b
0.0100557
0.00368689
c
1.11181
1.21697
d
1
1
This document is a draft for review purposes only and does not constitute Agency policy.
C-12 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
15
1.88
1.869
0.2
0.1869
0.2257
5
15
1.89
1.879
0.16
0.1869
0.2206
25
15
1.88
1.916
0.15
0.1869
-0.737
100
15
2.02
2.002
0.21
0.1869
0.3806
400
15
2.07
2.074
0.23
0.1869
-0.08999
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
88.69837
6
-165.3967
A2
90.62918
10
-161.2584
A3
88.69837
6
-165.3967
R
82.20147
2
-160.4029
4
88.29837
4
-168.5967
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
16.86
8
0.03165
Test 2
3.862
4
0.4251
Test 3
3.862
4
0.4251
Test 6a
0.8
2
0.6703
4
5
This document is a draft for review purposes only and does not constitute Agency policy.
C-13 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-5. Summary of BMD modeling results for increased absolute kidney
weight in PO male S-D rats exposed to ETBE by daily gavage for a total of 18 wk
beginning 10 wk before mating until after weaning of the pups (Gaoua.
2004a); BMR = 10% relative deviation from the mean
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Model3
p-value
AIC
Basis for Model Selection
Exponential (M2)
Exponential (M3)b
0.155
-38.410
551
423
The Hill model is selected based
on lowest BMDL
Exponential (M4)c
0.727
-40.012
255
123
Exponential (M5)c
0.727
-40.012
255
123
Hill
0.811
-40.077
244
94.0
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.199
-38.902
517
386
aConstant variance case presented (BMDS Test 2 p-value = 0.119), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-day were -0.0247, 0.14, -0.181, and 0.0657, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
cThe Exponential (M4) model and the Exponential (M5) model appear equivalent; however, differences exist in
digits not displayed in the table.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space, and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
C-14 DRAFT—DO NOT CITE OR QUOTE
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1
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
4.6
Hill
4.4
4.2
4
3.8
3.6
3.4
BMDL
BMD
0
200
400
600
800
1 000
14:47 05/15 2014
Figure C-4. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Hill Model. (Version: 2.17; Date: 01/28/2013)
The form of the response function is: Y[dose] = intercept + v x dose"/ (k11 + dose11)
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
alpha
0.227462
0.236804
rho
n/a
0
intercept
3.58236
3.58
V
1.16337
0.76
n
1
0.647728
k
548.322
250
This document is a draft for review purposes only and does not constitute Agency policy.
C-15 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
25
3.58
3.58
0.413
0.477
-0.0247
250
25
3.96
3.95
0.446
0.477
0.14
500
25
4.12
4.14
0.624
0.477
-0.181
1,000
25
4.34
4.33
0.434
0.477
0.0657
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
24.067171
5
-38.134342
A2
26.992591
8
-37.985183
A3
24.067171
5
-38.134342
fitted
24.038627
4
-40.077253
R
9.48179
2
-14.963581
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
35.0216
6
<0.0001
Test 2
5.85084
3
0.1191
Test 3
5.85084
3
0.1191
Test 4
0.057089
1
0.8112
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-16 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-6. Summary of BMD modeling results for increased absolute kidney
weight in PO female S-D rats exposed to ETBE by daily gavage for a total of 18
wk beginning 10 wk before mating until after weaning of the pups (Gaoua.
2004a); BMR = 10% relative deviation from the mean
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model Selection
p-value
AIC
Exponential (M2)
0.625
-214.58
1,734
1,030
Exponential (M2) model is
selected based on lowest AIC;
however, BMDL is higher than the
maximum dose.
Exponential (M3)
0.416
-212.86
1,458
1,040
Exponential (M4)
0.327
-212.56
1,774
1,032
Exponential (M5)
N/Ab
-211.39
error0
0
Hill
0.715
-213.39
error0
error0
Power
0.418
-212.87
1,470
1,041
Polynomial 3°
0.400
-212.81
1,409
1,035
Polynomial 2°
0.400
-212.81
1,409
1,037
Linear
0.619
-214.56
1,774
1,032
aConstant variance case presented (BMDS Test 2 p-value = 0.391), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-day were 0.5052, -0.7974, 0.1844, and 0.1033,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
CBMD or BMDL computation failed for this model.
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bl
2.45
2.4
2.35
CD
-------
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12
Supplemental Information—ETBE
Exponential Model. (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a* exp(sign x b x dose)
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 1,734.24
BMDL at the 95% confidence level = 1,030.08
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
In alpha
-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
Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
25
2.24
2.221
0.185
0.1923
0.5052
250
24
2.22
2.251
0.16
0.1923
-0.7974
500
22
2.29
2.282
0.207
0.1923
0.1844
1,000
25
2.35
2.346
0.224
0.1923
0.1033
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
This document is a draft for review purposes only and does not constitute Agency policy.
C-18 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
9.572
6
0.1439
Test 2
3.005
3
0.3909
Test 3
3.005
3
0.3909
Test 4
0.9403
2
0.6249
Table C-7. Summary of BMD modeling results for absolute kidney weight in F1
male Sprague-Dawley rats exposed to ETBE by gavage in a two-generation
study (Gaoua. 2004b): BMR = 10% relative deviation from the mean
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model Selection
p-value
AIC
Exponential (M2)
6.30xl0"4
89.912
232
175
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Polynomial 3°
model was selected based on
lowest AIC.
Exponential (M3)
0.129
79.474
335
256
Exponential (M4)
<0.0001
98.039
263
179
Exponential (M5)
N/Ab
82.504
347
267
Hill
N/Ab
82.509
347
267
Power
0.0680
80.504
347
267
Polynomial 3°
0.374
77.965
318
235
Polynomial 2°
0.0943
79.973
330
251
Linear
<0.0001
96.039
263
179
aModeled variance case presented (BMDS Test 2 p-value = <0.0001), selected model in bold; scaled residuals for
selected model for doses 0, 250, 500, and 1,000 mg/kg-day were -0.584, 0.717, 0.225, and -0.837, respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
This document is a draft for review purposes only and does not constitute Agency policy.
C-19 DRAFT—DO NOT CITE OR QUOTE
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1
2
3
4
5
6
7
8
9
10
11
12
Supplemental Information—ETBE
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMD
8 p—¦—i—¦—¦—¦—¦—¦—¦ 11—¦—i—¦. ¦ ¦—¦—¦—¦—¦—¦—¦—i—¦—¦—¦—¦—¦—¦—¦—¦—¦—i—¦—¦—¦—¦—¦—¦—¦—¦—¦—i—¦—¦—¦—¦—¦—¦—¦—¦—¦—i—¦—"
Polynomial
EL
BM
13:43 09/12 2014
Figure C-6. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
Polynomial Model. (Version: 2.19; Date: 06/25/2014)
The form of the response function is: Y[dose] = beta_0 + beta_l x dose + beta_2 x dose2 + ...
A modeled variance is fit.
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
In alpha
-13.8779
2.02785
rho
9.40248
0
beta_0
3.41732
3.38
beta_l
0.000881597
0.00138667
beta_2
2.232xl0"28
0
beta_3
1.90507xl0"9
6.93333xl0"9
This document is a draft for review purposes only and does not constitute Agency policy.
C-20 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
24
3.38
3.42
0.341
0.313
-0.584
250
25
3.73
3.67
0.449
0.436
0.717
500
24
4.13
4.1
0.64
0.734
0.225
1,000
25
5.34
6.2
5.39
5.16
-0.837
2 Likelihoods of Interest
Model
Log(likelihood)
# Params
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
3
4 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
234.752
6
<0.0001
Test 2
227.602
3
<0.0001
Test 3
2.13011
2
0.3447
Test 4
0.791648
1
0.3736
5
This document is a draft for review purposes only and does not constitute Agency policy.
C-21 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-8. Summary of BMD modeling results for absolute kidney weight in F1
female Sprague-Dawley rats exposed to ETBE by gavage in a two-generation
study (Gaoua. 2004b): BMR = 10% relative deviation
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model selection
p-value
AIC
Exponential (M2)
0.311
-180.23
978
670
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Exponential (M2)
model was selected based on
lowest AIC.
Exponential (M3)
0.147
-178.46
1,016
679
Exponential (M4)
0.121
-178.16
980
654
Exponential (M5)
N/Ab
-176.44
1,019
613
Hill
N/Ab
-176.44
1,019
611
Power
0.145
-178.44
1,019
666
Polynomial 3°
0.184
-178.80
1,001
684
Polynomial 2°
0.159
-178.58
1,002
673
Linear
0.301
-180.16
980
654
'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 1,000 mg/kg-day were -0.05426, 0.8898, -1.173, and 0.3711,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
Exponential
2.6
2.5
2.4
2.3
2.2
BM DL
BMD
O
200
400
600
800
1000
dose
1 13:47 09/12 2014
2 Figure C-7. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
C-22 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Exponential Model. (Version: 1.9; Date: 01/29/2013)
2 The form of the response function is: Y[dose] = a* exp(sign x b x dose)
3 A constant variance model is fit.
4 Benchmark Dose Computation.
5 BMR = 10% Relative deviation
6 BMD = 978.157
7 BMDL at the 95% confidence level = 669.643
8 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
In alpha
-2.91989
-2.94397
rho(S)
n/a
0
a
2.24252
2.24321
b
0.0000974385
0.000096475
c
0
0
d
1
1
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
25
2.24
2.243
0.178
0.2322
-0.05426
250
24
2.34
2.298
0.242
0.2322
0.8898
500
25
2.3
2.354
0.226
0.2322
-1.173
1,000
23
2.49
2.472
0.284
0.2322
0.3711
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
94.28268
5
-178.5654
A2
96.87585
8
-177.7517
A3
94.28268
5
-178.5654
R
87.16418
2
-170.3284
2
93.11474
3
-180.2295
11
This document is a draft for review purposes only and does not constitute Agency policy.
C-23 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
19.42
6
0.003505
Test 2
5.186
3
0.1587
Test 3
5.186
3
0.1587
Test 4
2.336
2
0.311
Table C-9. Summary of BMD modeling results for increased absolute kidney
weight in PO male S-D rats exposed to ETBE by daily gavage for 16 weeks
beginning 10 weeks prior to mating (Fujii et al.. 2010): BMR = 10% relative
deviation from the mean
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model Selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.668
-41.247
648
479
The Hill model was selected
based on lowest BMDL (BMDLs
were greater than threefold
difference.)
Exponential (M4)
Exponential (M5)c
0.600
-39.779
438
163
Hill
0.613
-39.799
435
139
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.700
-41.342
625
448
aConstant variance case presented (BMDS Test 2 p-value = 0.102), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-day were -0.202, 0.399, -0.232, and 0.0344, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
C-24 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
4.4
Hill
4.2
3.8
3.6
3.4
3.2
BMD
BMDI
0
200
400
600
800
1 000
1 13:13 05/15 2014
2 Figure C-8. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in mg/kg-day.
4
5 Hill Model. (Version: 2.17; Date: 01/28/2013)
6 The form of the response function is: Y[dose] = intercept + v x dose"/ (k11 + dose11)
7 A constant variance model is fit.
8 Benchmark Dose Computation.
9 BMR = 10% Relative deviation
10 BMD = 434.715
11 BMDL at the 95% confidence level = 139.178
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.223598
0.2327
rho
n/a
0
intercept
3.47949
3.46
V
1.24601
0.61
n
1
0.27452
k
1,122
1,610
13
This document is a draft for review purposes only and does not constitute Agency policy.
C-25 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
24
3.46
3.48
0.57
0.473
-0.202
100
24
3.62
3.58
0.45
0.473
0.399
300
24
3.72
3.74
0.35
0.473
-0.232
1,000
24
4.07
4.07
0.53
0.473
0.0344
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
24.027112
5
-38.054223
A2
27.13071
8
-38.26142
A3
24.027112
5
-38.054223
fitted
23.899392
4
-39.798783
R
14.313578
2
-24.627156
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
25.6343
6
0.0002604
Test 2
6.2072
3
0.102
Test 3
6.2072
3
0.102
Test 4
0.25544
1
0.6133
4
This document is a draft for review purposes only and does not constitute Agency policy.
C-26 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—ETBE
Table C-10. Summary of BMD modeling results for increased absolute kidney
weight in PO 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
Model3
Goodness of Fit
BMDiord
(mg/kg-day)
BMDLiord
(mg/kg-day)
Basis for Model Selection
p-value
AIC
Exponential (M2)
0.483
-199.73
1,135
781
Polynomial 2° is selected based
on most parsimonious model
with lowest AIC.
Exponential (M3)
0.441
-198.60
1,089
826
Exponential (M4)
0.217
-197.67
1,144
771
Exponential (M5)
N/Ab
-196.66
error0
0
Hill
N/Ab
-196.66
error0
error0
Power
0.441
-198.60
1,092
823
Polynomial 30d
Polynomial 2°
0.743
-200.60
1,094
905
Linear
0.467
-199.67
1,144
771
aConstant variance case presented (BMDS Test 2 p-value = 0.103), selected model in bold; scaled residuals for
selected model for doses 0,100, 300, and 1,000 mg/kg-day were 0.499, -0.579, 0.0914, and -0.00282,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
CBMD or BMDL computation failed for this model.
dFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model.
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the B
Polynomial
2.45
2.35
2.25
2.15
2.05
BMDL
BIVHD
14:09 05/15 2014
2
3
Figure C-9. Plot of mean response by dose, with fitted curve for selected
model; dose shown in mg/kg-day.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = beta_0 + beta_l x dose + beta_2 x dose2 + ...
3 A constant variance model is fit.
4 Benchmark Dose Computation.
5 BMR = 10% Relative deviation
6 BMD = 1,093.86
7 BMDL at the 95% confidence level = 905.267
8 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.0323691
0.0337309
rho
n/a
0
beta_0
2.1504
2.15624
beta_l
7.16226xl0"28
0
beta_2
1.79719x10 s
0
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
21
2.17
2.15
0.18
0.18
0.499
100
22
2.13
2.15
0.14
0.18
-0.579
300
23
2.17
2.17
0.17
0.18
0.0914
1,000
19
2.33
2.33
0.24
0.18
-0.00282
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
103.595625
5
-197.191249
A2
106.684319
8
-197.368637
A3
103.595625
5
-197.191249
fitted
103.298361
3
-200.596722
R
96.89324
2
-189.78648
11
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2*log(Likelihoo
d 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
2
3 Inhalation Exposure Endpoints
4
Table C-ll. Summary of BMD modeling results for urothelial hyperplasia of
the renal pelvis in male F344 rats exposed to ETBE by whole-body inhalation
for 6 hr/d, 5 d/wk, for 104 wk (IPEC. 2010b): BMR = 10% extra risk
Model3
Goodness of Fit
BMCiopct
(mg/m3)
BMCLiopct
(mg/m3)
Basis for Model Selection
p-value
AIC
Gamma
0.874
164.37
2,734
1,498
Of the models that provided an
adequate fit and a valid BMCL
estimate, the Gamma model was
selected based on lowest AIC.
Logistic
0.146
166.30
4,329
3,522
LogLogistic
0.814
164.40
3,010
1,831
Probit
0.202
165.59
4,059
3,365
LogProbit
0.633
164.57
3,050
1,896
Weibull
0.758
164.44
2,623
1,478
Multistage 3°
0.565
164.69
2,386
1,412
Multistage 2°
0.565
164.69
2,386
1,422
Quantal-Linear
0.269
165.16
1,541
1,227
aSelected model in bold; scaled residuals for selected model for doses 0, 2,089, 6,268, and 20,893 mg/m3 were
0.036, -0.107, 0.104, and -0.040, respectively. Exposure concentrations were converted from 0, 500,1,500, and
5,000 ppm to mg/m3 using the calculation mg/m3 = (102.17, molecular weight of ETBE) x ppm -f 24.45.
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
Supplemental Information—ETBE
Gamma Multi-Hit Model, with BMR of 1 0% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the
1
1 3:40 09/1 O 201 4
Gamma Multi-Hit
BMDL BMD
10000
dose
Figure C-10. 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 + [1-background] x CumGamma[slopexdose,power], where CumGammaQ is the
cumulative Gamma distribution function.
Power parameter is restricted as power > = 1.
Benchmark Dose Computation.
BMR = 10% Extra risk
BMD = 2,734.41
BMDL at the 95% confidence level = 1,497.7
Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
Background
0.0390054
0.0576923
Slope
0.000121504
0.000132454
Power
1.59019
1.84876
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 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
2 AIC = 164.373
3 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0.039
1.95
2
50
0.036
2,089
0.1046
5.231
5
50
-0.107
6,268
0.3196
15.659
16
49
0.104
20,893
0.8222
41.109
41
50
-0.04
4 x2 = 0.03; d.f = 1; p-value = 0.8737
5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-12. Summary of BMD modeling results for increased absolute kidney
weight in male S-D rats exposed to ETBE by whole-body inhalation for 6 hr/d,
5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from the mean
Model3
Goodness of Fit
BMDiord
(ppm)
BMDLiord
(ppm)
Basis for Model Selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.168
-43.014
1,105
750
Of the models that provided an
adequate fit and a valid BMDL
estimate, the Hill model was
selected based on lowest BMDL
(BMDLs differed by more than 3).
Exponential (M4)
0.200
-42.943
380
1.73
Exponential (M5)
0.200
-42.943
380
2.61
Hill
0.294
-43.484
264
15.4
Power0
Polynomial 3°d
Polynomial 2°e
Linear
0.178
-43.133
1,071
703
aConstant variance case presented (BMDS Test 2 p-value = 0.506), selected model in bold; scaled residuals for
selected model for doses 0,150, 500, and 1,500 ppm were -0.13, 0.54, -0.8, 0.38, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
dFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
eFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
Hill Model, with BMR of 0.1 Rel. Dev. forthe BMD and 0.95 Lower Confidence Limit for the BMDL
4
3.8
3.6
3.4
3.2
3
200
400
600
800
1000
1200
1400
Figure C-ll. Plot of mean response by dose, with fitted curve for selected
model; dose shown in ppm.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Hill Model. (Version: 2.17; Date: 01/28/2013)
2 The form of the response function is: Y[dose] = intercept + v x dose11/ (kn + dose")
3 A constant variance model is fit.
4 Benchmark Dose Computation.
5 BMR = 10% Relative deviation
6 BMD = 264.371
7 BMDL at the 95% confidence level = 15.4115
8 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.101559
0.109774
rho
n/a
0
intercept
3.16295
3.15
V
0.600878
0.57
n
1
0.169179
k
237.864
157.5
9 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
3.15
3.16
0.24
0.32
-0.129
150
10
3.45
3.4
0.38
0.32
0.542
500
10
3.49
3.57
0.31
0.32
-0.795
1,500
10
3.72
3.68
0.36
0.32
0.381
10 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
26.293887
5
-42.587775
A2
27.46147
8
-38.922941
A3
26.293887
5
-42.587775
fitted
25.742228
4
-43.484456
R
19.334386
2
-34.668772
11
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
16.2542
6
0.01245
Test 2
2.33517
3
0.5058
Test 3
2.33517
3
0.5058
Test 4
1.10332
1
0.2935
Table C-13. Summary of BMD modeling results for increased absolute kidney
weight in female S-D rats exposed to ETBE by whole-body inhalation for
6 hr/d, 5 d/wk for 13 wk (IPEC. 2008b): BMR = 10% relative deviation from
the mean
Model3
Goodness of Fit
BMDiord (ppm)
BMDLiord
(ppm)
Basis for Model Selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.8
-135.38
6,790
4,046
The Linear model is selected
based on lowest AIC; however,
the BMD is higher than the
maximum dose.
Exponential (M4)
0.731
-133.76
error0
0
Exponential (M5)
0.760
-132.29
error0
0
Hill
0.760
-132.29
error0
error0
Powerd
Polynomial 3°e
Polynomial 20f
Linear
0.806
-135.40
6,840
3,978
aConstant variance case presented (BMDS Test 2 p-value = 0.623), selected model in bold; scaled residuals for
selected model for doses 0,150, 500,1,500, and 5,000 ppm were -0.0742, 0.0535, -0.578, 0.774, and -0.176,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
CBMD or BMDL computation failed for this model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMC
Linear
2.1
2.05
1 .95
1 .85
1 .75
BMDL
0
1000
2000
3000
4000
5000
6000
7000
1 13:40 05/16 2014
2 Figure C-12. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in ppm.
4
5 Polynomial Model. (Version: 2.17; Date: 01/28/2013)
6 The form of the response function is: Y[dose] = beta_0 + beta_l x dose
7 A constant variance model is fit.
8 Benchmark Dose Computation.
9 BMR = 10% Relative deviation
10 BMD = 6,840.02
11 BMDL at the 95% confidence level = 3,978.09
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.021752
0.0236988
rho
n/a
0
beta_0
1.84346
1.84346
beta_l
0.0000269511
0.0000269511
13
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
1.84
1.84
0.129
0.147
-0.0742
150
10
1.85
1.85
0.18
0.147
0.0535
500
10
1.83
1.86
0.118
0.147
-0.578
1,500
10
1.92
1.88
0.173
0.147
0.774
5,000
10
1.97
1.98
0.16
0.147
-0.176
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
71.192285
6
-130.384569
A2
72.502584
10
-125.005168
A3
71.192285
6
-130.384569
fitted
70.701239
3
-135.402478
R
67.96809
2
-131.93618
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
9.06899
8
0.3365
Test 2
2.6206
4
0.6232
Test 3
2.6206
4
0.6232
Test 4
0.982091
3
0.8056
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-14. Summary of BMD modeling results for increased absolute kidney
weight in male F344 rats exposed to ETBE by whole-body inhalation for 6
hr/d, 5 d/wk, for 13 wk (Medinskv et al.. 1999: Bond et al.. 1996b):
BMR = 10% relative deviation from the mean
Goodness of Fit
Model3
p-value
AIC
BMCiord (ppm)
BMCLiord (ppm)
Basis for Model Selection
Exponential (M2)
Exponential (M3)b
0.184
-129.97
3,107
2,439
The Hill model was selected
based on lowest BMDL.
Exponential (M4)
Exponential (M5)c
0.199
-129.71
1,798
808
Hill
0.224
-129.89
1,667
603
Powerd
Polynomial 3°e
Polynomial 2°f
Linear
0.208
-130.22
2,980
2,288
aConstant variance case presented (BMDS Test 2 p-value = 0.540), selected model in bold; scaled residuals for
selected model for doses 0, 500,1,750, and 5,000 ppm were -0.441, 0.91, —0.635, and 0.166, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
eFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Hill Model, with BMR of 0.1 Rel. Dev. for the BMD arid 0.95 Lower Confidence Limit for the BMDL
2.2 ¦ „m.
2.1
2
BMDL
BMD
0
1 000
2000
3000
4000
5000
1 14:00 05/16 2014
2 Figure C-13. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in ppm.
4
5 Hill Model. (Version: 2.17; Date: 01/28/2013)
6 The form of the response function is: Y[dose] = intercept + v x dose"/ (k11 + dose11)
7 A constant variance model is fit.
8 Benchmark Dose Computation.
9 BMR = 10% Relative deviation
10 BMD = 1,666.92
11 BMDL at the 95% confidence level = 603.113
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.0160221
0.0170425
rho
n/a
0
intercept
1.74684
1.73
V
0.521534
0.337
n
1
0.225826
k
3,309.8
1,856.13
13
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
11
1.73
1.75
0.155
0.127
-0.441
500
11
1.85
1.82
0.137
0.127
0.91
1,750
11
1.9
1.93
0.1
0.127
-0.635
5,000
11
2.07
2.06
0.124
0.127
0.166
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
69.681815
5
-129.36363
A2
70.76062
8
-125.521241
A3
69.681815
5
-129.36363
fitted
68.943332
4
-129.886663
R
55.026208
2
-106.052416
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
31.4688
6
<0.0001
Test 2
2.15761
3
0.5403
Test 3
2.15761
3
0.5403
Test 4
1.47697
1
0.2242
4
5
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Table C-15. Summary of BMD modeling results for increased absolute kidney
weight in female F344 rats exposed to ETBE by whole-body inhalation for 6
hr/d, 5 d/wk, for 13 wk (Medinskv et al.. 1999: Bond et al.. 1996b):
BMR = 10% relative deviation from the mean
Model3
Goodness of Fit
BMCiord (ppm)
BMCLiord (ppm)
Basis for Model Selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0630
-187.67
2,706
2,275
The Exponential (M4) model was
selected as the most
parsimonious model of adequate
fit.
Exponential (M4)
Exponential (M5)c
0.956
-191.20
1,342
816
Hill
N/Ad
-189.20
1,325
741
Power6
Polynomial 3°f
Polynomial 2°g
Linear
0.0928
-188.45
2,552
2,111
aConstant variance case presented (BMDS Test 2 p-value = 0.428), selected model in bold; scaled residuals for
selected model for doses 0, 500,1,750, and 5,000 ppm were -0.0252, 0.043, -0.02385, and 0.004872,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary), and the model reduced to the Exponential
(M4) model.
dNo available degrees of freedom to calculate a goodness-of-fit value.
eFor the Power model, the power parameter estimate was 1, and the model reduced to the Linear model.
fFor the Polynomial 3° model, the b3 coefficient estimates was 0 (boundary of parameters space), and the model
reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space), and the model reduced to the Linear model.
gFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space), and the model
reduced to the Linear model.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BT
Exponential
1 .35
1 .25
1
1 .05
BMD
BMD
0
1 000
2000
3000
4000
5000
1 14:13 05/16 2014
2 Figure C-14. Plot of mean response by dose, with fitted curve for selected
3 model; dose shown in ppm.
4
5 Exponential Model. (Version: 1.9; Date: 01/29/2013)
6 The form of the response function is: Y[dose] = a* [c-(c-l) x exp(-b x dose)]
7 A constant variance model is fit.
8 Benchmark Dose Computation.
9 BMR = 10% Relative deviation
10 BMD = 1,341.66
11 BMDL at the 95% confidence level = 815.742
12 Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
In alpha
-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
13
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—ETBE
1 Table of Data and Estimated Values of Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
10
1.077
1.077
0.069
0.05983
-0.0252
500
11
1.125
1.124
0.048
0.05983
0.043
1,750
11
1.208
1.208
0.076
0.05983
-0.02385
5,000
11
1.306
1.306
0.055
0.05983
0.004872
2 Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
99.60217
5
-189.2043
A2
100.9899
8
-185.9798
A3
99.60217
5
-189.2043
R
75.30605
2
-146.6121
4
99.60063
4
-191.2013
3 Tests of Interest
Test
-2*log(Likelihoo
d Ratio)
Test df
p-value
Test 1
51.37
6
<0.0001
Test 2
2.775
3
0.4276
Test 3
2.775
3
0.4276
Test 6a
0.003077
1
0.9558
4
5 C.1.2. Cancer Endpoints
6 For the multistage cancer models, the coefficients were restricted to be non-negative (beta's
7 > 0). For each endpoint, multistage cancer models were fitted to the data using the maximum
8 likelihood method. Each model was tested for goodness-of-fit using a chi-square goodness-of-fit test
9 (x2 p-value < 0.053 indicates lack of fit). Other factors were used to assess model fit, such as scaled
10 residuals, visual fit, and adequacy of fit in the low-dose region and in the vicinity of the BMR.
11 For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as
12 estimated by the profile likelihood method) and AIC value were used to select a best-fit model from
13 among the models exhibiting adequate fit If the BMDL estimates were "sufficiently close," that is,
14 differed by more than threefold, the model selected was the one that yielded the lowest AIC value. If
15 the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the POD.
3A significance level of 0.05 is used for selecting cancer models because the model family (multistage) is
selected a priori Benchmark Dose Technical Guidance Document, U.S. EPA (2012).
This document is a draft for review purposes only and does not constitute Agency policy.
C-42
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1 The incidence of liver tumors in male F344 rats was found to be statistically significantly
2 increased following a 2-year inhalation exposure; hepatocellular adenomas and a single
3 hepatocellular carcinoma in the high-dose group were combined in modeling the data set. The data
4 were modeled using two different exposure metrics: administered concentration as ppm, and
5 administered concentration as mg/m3.
6
Table C-16. Cancer endpoints selected for dose-response modeling for ETBE
Species/Sex
Endpoint
Doses and Effect Data
Hepatocellular
adenomas and
carcinomas in
male rats
JPEC (2010b)
Exposure Concentration
(ppm)
0
500
1,500
5,000
Exposure Concentration
(mg/m3)
0
2,089
6,268
20,893
Incidence/Total
0/50
2/50
1/49
10/50
7
8 Modeling Results
9 Below are tables summarizing the modeling results for the cancer endpoints modeled.
10
Table C-17. Summary of BMD modeling results for hepatocellular adenomas
and carcinomas in male F344 rats exposed to ETBE by whole-body inhalation
for 6 hr/d, 5d/wk, for 104 wk; modeled with doses as administered exposure
concentration in ppm flPEC. 2010bl: BMR = 10% extra risk
Model3
Goodness of Fit
BMCiopct (ppm)
BMCLiopct (ppm)
Basis for Model
Selection
P-
value
Scaled Residuals
AIC
Three
0.0991
-0.030, 1.382,
-0.898, and 0.048
84.961
2,942
1,735
Multistage 1° was
selected based on
lowest AIC.
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
2,756
1,718
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.208
2,605
1,703
aSelected model in bold.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
0.15
0.1
0.05
BMDL
BMD
0
1 000
2000
3000
4000
5000
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Figure C-15. Plot of incidence rate by dose, with fitted curve for selected
model; dose shown in ppm.
Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is:
P[response] = background + (1-background) x [l-exp(-betal x dose1-beta2 x dose2...)]
The parameter betas are restricted to be positive.
Benchmark Dose Computation.
BMR = 10% extra risk
BMD = 2,604.82
BMDL at the 95% confidence level = 1,703.47
BMDU at the 95% confidence level = 4,634.52
Collectively, (1,703.47,4,634.52) 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)
4.04483xl0"4
4.38711xl0"4
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1 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
2 AIC = 81.2084
3 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0
0
0
50
0
500
0.02
1.001
2
50
1.009
1,500
0.0589
2.885
1
49
-1.144
5,000
0.1831
9.155
10
50
0.309
4 x2 = 2.42; d.f = 3; p-value = 0.4898
5
Table C-18. Summary of BMD modeling results for hepatocellular adenomas
and carcinomas in male F344 rats exposed to ETBE by whole-body inhalation
for 6 hr/d, 5d/wk, for 104 wk; modeled with doses as mg/m3 flPEC. 2010bl:
BMR = 10% extra risk
Model3
Goodness of Fit
BMDiopct (mg/m3)
BMDLiopct (mg/m3)
Basis for Model
Selection
P-
value
Scaled Residuals
AIC
Three
0.0991
-0.040, 1.382,
-0.897, and 0.048
84.961
12,300
7,251
Multistage 1° was
selected based on
lowest AIC
Two
0.264
0.000, 1.284, -1.000,
and 0.137
83.093
11,514
7,179
One
0.490
0.000, 1.009, -1.144,
and 0.309
81.209
10,884
7,118
aSelected model in bold.
This document is a draft for review purposes only and does not constitute Agency policy.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD arid 0.95 Lower Confidence Limit fort
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
0.15
0.1
0.05
BMDL
BMD
0
5000
1 0000
15000
20000
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Figure C-16. Plot of incidence rate by dose, with fitted curve for selected
model; dose shown in mg/m3.
Multistage Model. (Version: 3.4; Date: 05/02/2014)
The form of the probability function is: P [response] = background +
(1-background) x [l-exp(-betal x dose1-beta2 x dose2...)]
The parameter betas are restricted to be positive.
Benchmark Dose Computation.
BMR = 10% extra risk
BMD = 10,884.4
BMDL at the 95% confidence level = 7,118.08
BMDU at the 95% confidence level = 19,366.3
Collectively, (7,118.08,19,366.3) 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)
9.6799xl0"6
1.04989xl0"4
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1 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
2 AIC = 81.2087
3 Goodness of Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled Resid
0
0
0
0
50
0
2,089
0.02
1.001
2
50
1.009
6,268
0.0589
2.885
1
49
-1.144
20,893
0.1831
9.155
10
50
0.309
4 x2 = 2.42; d.f = 3; p-value = 0.4897
5
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APPENDIX D. SUMMARY OF PUBLIC
COMMENTS AND EPA'S DISPOSITION
The T oxicological Review of ethyl tertiary butyl ether (ETBE) was released for a 60-day public
comment period on September 1, 2016. Public comments on the assessment were submitted to EPA
by:
• Japan Petroleum Energy Center (posted November 1 and November 3, 2016),
• Exponent, Inc. on behalf of LyondellBasell (posted October 24, 2016),
• LyondellBasell (posted October 20, 2016 and November 3, 2016),
• American Chemistry Council (posted October 28, 2016),
• Tox Strategies on behalf of LyondellBasell (posted October 24, 2016), and
• American Petroleum Institute (posted November 1, 2016).
A summary of major public comments provided in these submissions and EPA's response to
these comments are provided in the sections that follow. The comments have been synthesized and
paraphrased. Because several commenters often covered the same topic, the comment summaries
are organized by topic. Editorial changes and factual corrections offered by public commenters
were incorporated in the document as appropriate and are not discussed further. All public
comments provided were taken into consideration in revising the draft assessment prior to
releasing for external peer review.
Comments Related to Kidney Effects
Comment [LyondellBasell]: The selection of urothelial hyperplasia as the key endpoint reflecting a
potential human kidney hazard from ETBE exposure is inappropriate because urothelial
hyperplasia is associated with chronic progressive nephropathy (CPN). In addition, CPN should not
be considered relevant to humans because it is rat-specific with no known human counterpart
EPA Response: Section 1.21 shows that urothelial hyperplasia is weakly correlated with CPN. CPN is
a common and well-established constellation of age-related lesions in the kidney of rats, and there
is no known counterpart to CPN in aging humans. However, CPN is not a specific diagnosis but is a
spectrum of lesions. These individual lesions or processes (tubular degeneration/regeneration and
dilatation, glomerular sclerosis and atrophy, interstitial fibrosis and inflammation, etc.) could
certainly occur in a human kidney. Because they happen to occur as a group in the aged rat kidney
does not necessarily make them rat-specific individually if there is a treatment effect for one or
more of them. In addition, exacerbation of one or more of these processes likely reflects some type
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of cell injury/cytotoxicity, which is relevant to the human kidney. Different federal agencies have
considered CPN exacerbation not confounded by a2U-globulin to be a basis for reference values. For
instance, FDA also used CPN in their draft calculation of PDEs for MIBK
fhttp://www.fda.gov/ucm/groups/fdagov-public/@fdagov-drugs-
gen/documents/document/ucm467089.pdfl. EPA considers CPN exacerbation to be relevant for
human health.
Comment [LyondellBasell]: Dismissal of a2U-globulin nephropathy as an operative MOA for ETBE is
not scientifically justified. Multiple studies reported that ETBE induced hyaline droplets, and one
group observed that those droplets had angular profiles characteristic of accumulating a2U-globulin
fCohen etal.. 20111. Granular casts were observed in a 13-week study by two independent groups
(indicative of cell exfoliation), and linear papillary mineralization by several groups. Increased
tubule cell proliferation was reported to be sustained over a period of 1 to 13 weeks. Development
of renal tubule hyperplasia is not a necessary histopathological step for identifying a2U-globulin
nephropathy. When it does occur, it is an outcome of that histopathological sequence. Thus, the
absence of tubule hyperplasia (or renal tubule tumors) does not rule out an a2U-globulin MOA.
EPA Response: EPA does not discount the evidence for ETBE induction of hyaline droplets or
a2u-globulin. Granular casts were observed in one experiment observed by two independent sets of
pathologists, which does not offer an explanation of why other studies failed to observe them
despite similar durations and doses. Tubule cell proliferation was reported in both male and female
rats, which supports a non-a2U-globulin mechanism for this effect EPA agrees that absence of one
lesion does not rule out a2U-globulin; however, absence of several lesions may. The criteria for
establishing an a2U-globulin mechanism does not offer alternative criteria for weak inducers of
a2u-globulin to explain or allow for absence of evidence in the histopathological sequence.
Comments Related to Liver Effects
Regarding the Possible Mode of Action
Comment [American Petroleum Institute]: The draft review concludes that a mode of action (MOA)
for the high-dose male rat liver tumors could not be established, and in the absence of information
to indicate otherwise, the liver tumors induced by ETBE are considered to be relevant to humans.
We encourage the Agency to fully review the mode of action research of the Japan Petroleum
Energy Center (JPEC) provided in comments to EPA docket for ETBE. Interpretations from the JPEC
research program differ from those of EPA and conclude that the mode of action for ETBE high-dose
liver tumors in male rats is unlikely to be relevant to humans. The basis for this difference in data
interpretation is not clear in the draft IRIS review document. Although EPA states that data are
inadequate to conclude that ETBE induces liver tumors via a PPARa MOA, a CAR/PXR MOA, or an
acetaldehyde-mediated mutagenicity, the rationale underlying these Agency conclusions is not
This document is a draft for review purposes only and does not constitute Agency policy.
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clearly described in sufficient detail to understand EPA's views regarding the shortcomings of the
data set or how it could be improved.
EPA Response: The draft was modified in section 1.2.2 to clarify the rationale for why the data are
inadequate to establish a conclusion for these proposed MOAs. Specifically, all positive evidence
related to the 10 key characteristics of cancer were grouped and summarized in Table 1-13. This
summary of the evidence provides a more holistic approach for organizing and further discussing
cancer MOAs and is a more transparent presentation of potentially informative data gaps. In
addition, several gaps in the receptor-mediated effects data were explicitly noted such as evidence
in only one species, lack of any studies in PPAR KO mice, lack of dose-response concordance
between receptor-mediated gene changes and tumors, and lack of any receptor-mediated data
outside of the 1- and 2-week time-points, which preclude establishing temporal associations. These
data gaps led to the conclusion that the receptor-mediated MOA data are inadequate to establish
conclusions.
Comment [Japan Petroleum Energy Center]: Cellular hypertrophy was likely a result of microsome
proliferation and increased synthesis of microsomal cytochrome P450 enzymes. Significant
increase of hydroxyl radical levels by Week 2 of ETBE exposure accompanied the accumulation of
8-OHdG in the nucleus andP450 isoenzymes CYP2B1/2, CYP3A1/2 etc., and increase of
peroxisomes in the cytoplasm of hepatocytes. Examination of rat livers after 14 days of ETBE
treatment showed the high levels of concordance between induction of 8-OHdG and apoptosis
(ssDNA), which were inversely correlated with low cell proliferation. Increased 8-OHdG formation
is caused by developing oxidative stress and/or apoptotic degradation of DNA. Continuous P450
and hydroxyl radical elevation by high dose ETBE was coordinated with enhanced cell proliferation
at Day 3, followed by cell cycle arrest (low cell proliferation) and apoptosis at Day 14 (Week 2), and
regenerative cell proliferation at Day 28, as a continuing response to liver damage occurred at Day
14 (Week 2). Adaptive response to liver damage at Day 14 (Week 2) firstly include activation of
repair mechanisms, which contributes to protection of tissue against reactive oxygen
species-induced cell death (such as increase of DNA repair enzymes), and lastly, regenerative cell
proliferation. Elevation ofP450 has been proven to be associated with generation of reactive
oxygen species (ROS), which damage proteins and DNA. If the balance between the generation of
ROS and activity of repair system enzymes is disturbed, severe damage occurs on cellular and
molecular levels, what was reported to result in promotion of carcinogenesis if the damaged cell
was not eliminated by apoptosis. ETBE at high dose induced significant generation of hydroxyl
radicals, thus, the long-term exposure could result in promotion of hepatocarcinogenesis in
spontaneously initiated hepatocytes. Therefore, centrilobular hypertrophy is likely to be associated
with hepatocarcinogenesis.
This document is a draft for review purposes only and does not constitute Agency policy.
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EPA Response: The data do not provide evidence of a gradual increase in hydroxy! radicals because
radicals were only increased at one dose at both time points measured. Two time points are
insufficient data to establish a temporal trend in radical species formation, and a single 14-day
data-point that reports apoptosis and oxidative stress occurring at one dose is insufficient data to
establish either dose or temporal associations for a MOA. Thus, the conclusion that the available
evidence is inadequate appears to be the most appropriate for the database at this time.
Comments Related to Cancer Weight of Evidence
Comment [LyondellBasell]: The draft assessment inappropriately considers the ETBE two-stage
tumor promotion studies. The animal experimental data indicates that ETBE might be acting as a
promoter of mutagen induced liver tumors when administered at high doses of ETBE
(1,000 mg/kg-day), a dose level that also exceeds metabolic saturation. This promotional activity
has clear thresholds, with no evidence of promotion at a 300 mg/kg-day dose that is near or at an
oral dose reflecting onset on nonlinear toxicokinetics. Thus, the available data supports the
conclusion that the liver tumors observed following inhalation exposure of ETBE are most likely the
result of the promotion of spontaneously initiated cells and as such have clear threshold
dose-response relationships. Evidence of promotion of multi-mutagen-initiated tumorigenicity was
observed in thyroid at oral gavage doses of 300 and 1,000 mg/kg-day and in colon only at the high
dose. No evidence of tumor promotion was observed in kidney, forestomach, urinary bladder, or
urethra. In a later single mutagen-initiated study, ETBE-induced liver promotion was restricted to
the high dose of 1,000 mg/kg-day, while the incidence of kidney adenomas was increased at 500
and 1,000 mg/kg-day (however, combined adenoma/carcinoma incidence was not altered at any
dose). It is important to note, however, that these studies do not provide meaningful evidence of
ETBE carcinogenicity. Both assay designs were developed as screening assays of potential
carcinogenicity hazard (but not risk), and were validated for target organ predictability against
existing apical animal cancer bioassays. In the case of ETBE, however, which has two high quality
apical rat carcinogenicity studies conducted by two routes of administration, the possibility of
nonhepatic tumorigenicity (kidney, thyroid, colon) as suggested in the initiation-promotion assays
was not confirmed in two apical animal bioassays. This combined evidence indicates that
nonhepatic carcinogenicity identified in the two-stage carcinogenicity assays is not relevant to
ETBE carcinogen assessment, other than to provide possible supporting evidence that high doses of
ETBE exceeding metabolic saturation may have tumor-promoting activity. Such promotion
responses are generally regarded as threshold-based MOA events.
EPA Response: The two stage carcinogenicity bioassay data provide several instances of increased
tumors or preneoplastic lesions at doses below 1,000 mg/kg-day. These organ sites were in the
forestomach, thyroid, and kidney (see Tables 1-4,1-17,1-18). These data indicate that ETBE has the
potential to induce tumorigenic responses below 1,000 mg/kg-day. Furthermore, no MOA was
This document is a draft for review purposes only and does not constitute Agency policy.
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identified for liver tumors so it is not possible to conclude that the induction of liver tumors by
ETBE at one dose is also not operative at lower doses.
Comment [LyondellBasell]: The conclusion that the 5,000 ppm inhalation exposure concentration
was an excessively high test concentration in rats is further evidenced by the Saito etal. f20131
study that reported that male and female body weights were significantly decreased to 75 and 78%,
respectively, of controls at the terminal 104-week sacrifice. This severe weight loss exceeds the
10% body weight loss recommended for achievement of a maximum tolerated dose. The potential
that excessive toxicity was uniquely associated with the high dose (5,000 ppm) exposure condition
under which nonlinear toxicokinetics were apparent is further evidenced by the observation that
male and female terminal body weights were a nonstatistically significant 94 and 91% of controls,
respectively, in next lower (1,500 ppm) ETBE exposure. In addition, the significantly increased
incidence of preneoplastic eosinophilic and basophilic liver foci was limited to the 5,000 ppm
treatment group, indicating that tumorigenic responses would be unlikely at the 1,500 ppm
mid-dose. These findings further indicate that the high-dose-specific ETBE male rat liver tumors
were secondary to use of an excessively high top bioassay dose.
EPA Response: Although loss of body weight occurred in the 2-year study, this was not the case for
the two-stage initiation-promotion cancer bioassays, which observed increased tumors at multiple
organ sites and multiple doses. This suggests that the increase in tumors were not related to a
maximum tolerated dose as indicated by loss of body weight
Comments Related to Reproductive and Developmental Effects
Comment [LyondellBasell]: Numerous detailed questions and specific concerns related to the
organization, presentation, and interpretation of rodent evidence relevant to the determination of
reproductive and developmental effects following ETBE exposure were received.
EPA Response: EPA appreciates the detailed comments regarding evidence summarized in evidence
tables and exposure-response arrays and discussed in the associated synthesis text. The
reproductive effects discussion and associated tables and figures (see Section 1.2.3) has now been
reorganized and revised to separately present and evaluate evidence relevant to male and female
reproductive effects, including an expanded and more detailed presentation and discussion of all
the pertinent endpoints reported in the identified literature. Likewise, the discussion of evidence
for developmental effects (see Section 1.2.4) has been revised to more clearly present and discuss
all pertinent endpoints reported in the assembled database. Revisions emphasized a transparent
consideration of all available data, integrated into a conclusion statement for each possible effect:
male reproductive effects, female reproductive effects, and developmental effects.
This document is a draft for review purposes only and does not constitute Agency policy.
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Comments Related to the Physiologically Based Pharmacokinetic Model and Toxicokinetics
Comment [LyondellBasell]: Numerous questions and concerns related to specific aspects of PBPK
modeling described by Salazar etal. f20151 and as implemented in public comment draft were
received.
EPA Response: EPA has adopted the newly available Borghoff et al. f20161 model, as summarized in
Appendix B.1.5-B.1.6 and U.S. EPA ("20171.
Comment [LyondellBasell]: Several comments noted that the results of Sun and Beskitt (1995a, b)
and a preliminary study by Borghoff f!9961 were presented and discussed, while the more
comprehensive study of Borghoff and Asgharian (19961 was not
EPA Response: A summary of Borghoff and Asgharian (19961 has been added to the ADME
discussion in Appendix B; furthermore, results from Borghoff and Asgharian (19961 are now
incorporated into the ADME/TK review, and model simulations are compared to those in U.S. EPA
C20171.
Comment [LyondellBasell]: The section of the draft assessment does not describe the interpretative
implications of the finding that liver tumors were only observed at an inhalation dose level
exhibiting nonlinear toxicokinetic behavior. As implied by the title of this section of the draft
assessment ("Toxicokinetic Considerations Relevant to Liver Toxicity and Tumors"), such data
should be a key consideration in the overall MOA evaluation. Importantly, in the Supplementary
Information provided for the draft assessment it is stated that: "A review of the data demonstrating
the percentage of recovered radiolabel via various routes of elimination demonstrate, in the rat and
mouse, a pattern indicative of metabolic saturation occurring at inhaled concentrations above 4,180
mg/m3 [1,000 ppm]" (p. B-19). This key conclusion is not carried over to the main draft assessment
document.
EPA Response: Metabolic saturation would only lead to a disproportionate increase in toxicity if it is
the parent chemical, ETBE in this case, that is the proximate agent. Figure 11, panels A and C, in
Borghoff et al. (1996) shows that extent of nonlinearity in the blood AUC of ETBE in the dose range
evaluated is modest; there is not a sudden sharp inflection upward of AUC versus exposure at
1,000 ppm. Further, the MOA analysis indicates a probability that it is not parent ETBE that is the
proximate agent, leading to the choice of ETBE metabolic rate as a measure of internal dose for
route-to-route extrapolation. And while Figure 13-A of Borghoff etal. (19961 shows that the
metabolic rate is predicted to be approaching saturating (becoming flatter) in the range of
5,000 ppm, it is close to linear at 1 /5th that level. This result is not consistent with an argument
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that toxicity appears due to a significant shift in toxicokinetics at 1,000 ppm, let alone increase
disproportionately with exposure at higher levels.
This document is a draft for review purposes only and does not constitute Agency policy.
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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 ether in rats and humans. Toxicol Sci. 53: 194-201.
http://dx.doi.Org/10.1093/toxsci/53.2.194.
ATSDR (Agency for Toxic Substances and Disease Registry). (1996). Toxicological profile for
methyl-tert-butyl ether [ATSDR Tox Profile], Atlanta, GA: U.S. Department of Health and
Human Services, Public Health Service. http: //www.atsdr.cdc.gov/ToxProfiles/tp91.pdf.
Banton. MI: Peachee. VL: White. KL: Padgett. EL. (2011). Oral subchronic immunotoxicity study of
ethyl tertiary butyl ether in the rat. J Immunotoxicol. 8: 298-304.
http://dx.doi.org/10.3109/1547691X.2011.598480.
Bernauer. U: Amberg. A: Scheutzow. D: Dekant. W. (1998). Biotransformation of 12C- and 2-13C-
labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats:
Identification of metabolites in urine by 13C nuclear magnetic resonance and gas
chromatography/mass spectrometry. Chem Res Toxicol. 11: 651-658.
http: / /dx. do i. o r g /10.10 21 /tx9 7 0 215v.
Bond. TA: Medinskv. MA: Wolf. DC: Cattlev. R: Farris. G: Wong. B: Tanszen. D: Turner. Ml: Sumner.
SCI. (1996a). Ethyl tertiary butyl ether (ETBE): ninety-day vapor inhalation toxicity study in
CD-l(R) mice. Bond, JA; Medinsky, MA; Wolf, DC; Cattley, R; Farris, G; Wong, B; Janszen, D;
Turner, MJ; Sumner, SCJ.
Bond. TA: Medinsky. MA: Wolf. DC: Dorman. DC: Cattley. R: Farris. G: Wong. B: Morgan. K: Tanszen. D:
Turner. Ml: Sumner. SCI. (1996b). Ethyl tertiary butyl ether (ETBE): ninety-day vapor
inhalation toxicity study with neurotoxicity evaluations in Fischer 344 rats [TSCA
Submission] (pp. 1-90). (89970000047). Research Triangle Park, NC: Chemical Industry
Institute of Toxicology under contract to ARCO Chemical Company.
http://yosemite.epa.gov/oppts/epatscat8.nsf/by+Service/1332F4B209355DC785256F9E0
06B7EA0/$File/89970000047.pdf.
Borghoff. ST. (1996). Ethyl tertiary-butyl ether: Pilot/methods development pharmacokinetic study
in male F-344 rats & male cd-1 mice after single nose-only inhalation exposure, w/cvr ltr
dated 7/29/96. (TSCATS/444664). Chemical Industry Institute of Toxicology (CUT).
Borghoff. ST: Asgharian. B. (1996). Ethyl tertiary-butyl ether (ETBE): Pharmacokinetic study in male
and female CD-I mice after single inhalation exposure and male and female F-344 rats after
single and repeated inhalation exposure. (CUT Protocol 95026). La Palma, CA: ARCO
Chemical Company.
Borghoff. ST: Murphy. IE: Medinsky. MA. (1996). Development of physiologically based
pharmacokinetic model for methyl tertiary-butyl ether and tertiary-butanol in male Fisher-
344 rats. Fundam Appl Toxicol. 30: 264-275. http://dx.doi.org/10.1006/faatl996.0064.
Borghoff. ST: Ring. C: Banton. MI: Leavens. TL. (2016). Physiologically based pharmacokinetic model
for ethyl tertiary-butyl ether and tertiary-butyl alcohol in rats: Contribution of binding to
This document is a draft for review purposes only and does not constitute Agency policy.
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a2u-globulin in male rats and high-exposure nonlinear kinetics to toxicity and cancer
outcomes. J Appl Toxicol, http://dx.doi.org/10.1002/iat.3412.
Cederbaum. AI: Cohen. G. (1980a). Oxidative demethylation of t-butyl alcohol by rat liver
microsomes. Biochem Biophys Res Commun. 97: 730-736.
Cederbaum. AI: Cohen. G. (1980b). Oxidative demethylation of tert-butyl alcohol by rat-liver
microsomes. Biochem Biophys Res Commun. 97: 730-736.
http://dx.doi. org/10.1016/0006-291XC8019032S-3.
Cohen. SM: Hard. GC: Regan. KS: Seelv. TC: Bruner. RH. (2011). Pathology working group review of
selected histopathologic changes in the kidneys of rats assigned to toxicology and
carcinogenicity studies of ethyl tertiary butyl ether (ETBE): Japan Bioassay Research Center
studies no.: 0065 and 0691 [Unpublished report] (pp. 1-30). Research Triangle Park, NC:
Research Pathology Associates under contract to Lyondell Chemical Company.
Dekant. W: Bernauer. U: Rosner. E: Amberg. A. (2001). Toxicokinetics of ethers used as fuel
oxygenates [Review], Toxicol Lett. 124: 37-45. http://dx.doi.org/10.1016/s0378-
4274C00100284-8.
Drogos. PL: Diaz. AF. (2001). Oxygenates in Gasoline Appendix A: Physical properties of fuel
oxgenates and addititves. In ACS Symposium Series. Washington, DC: American Chemical
Society, http://dx.doi.org/10.1021/bk-2002-0799.ch018.
Elmore. SA. (2006). Enhanced histopathology of the spleen [Review], Toxicol Pathol. 34: 648-655.
http://dx.doi.Org/10.1080/01926230600865523.
Fuiii. S: Yabe. K: Furukawa. M: Matsuura. M: Aovama. H. (2010). A one-generation reproductive
toxicity study of ethyl tertiary butyl ether in rats. Reprod Toxicol. 30: 414-421.
http://dx.doi.Org/10.1016/i.reprotox.2010.04.013.
Gaoua. W. (2004a). Ethyl tertiary butyl ether (ETBE): Prenatal developmental toxicity study by the
oral route (gavage) in rats (pp. 1-280). (CIT Study No. 24860 RSR). unpublished study for
Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review was
conducted by EPA in November 2008 to evaluate the accuracy of experimental procedures,
results, and interpretation and discussion of the findings presented. A report of this peer
review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749
(fax), or hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
Gaoua. W. (2004b). Ethyl tertiary butyl ether (ETBE): Two-generation study (reproduction and
fertility effects) by the oral route (gavage) in rats. (CIT Study No. 24859 RSR). unpublished
study for Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review
was conducted by EPA in November 2008 to evaluate the accuracy of experimental
procedures, results, and interpretation and discussion of the findings presented. A report of
this peer review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202)
56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
Hagiwara. A: Doi. Y: Imai. N: Nakashima. H: Ono. T: Kawabe. M: Furukawa. F: Tamano. S: Nagano. K:
Fukushima. S. (2011). Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-
butyl ether in rats. Toxicology. 289: 160-166. http://dx.doi.Org/10.1016/i.tox.2011.08.007.
Hong. TY: Wang. YY: Bondoc. FY: Lee. M: Yang. CS: Hu. WY: Pan. I. (1999a). Metabolism of methyl
tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously
expressed human cytochromes P450: Identification of CYP2A6 as a major catalyst. Toxicol
Appl Pharmacol. 160: 43-48. http://dx.doi.org/10.1006/taap.1999.8750.
This document is a draft for review purposes only and does not constitute Agency policy.
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Hong. TY: Wang. YY: Bondoc. FY: Yang. CS: Gonzalez. FT: 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-
4274C98100389-0.
Hong. TY: Wang. YY: Bondoc. FY: Yang. CS: Lee. M: Huang. W0. (1997a). Rat olfactory mucosa
displays a high activity in metabolizing methyl tert-butyl ether and other gasoline ethers.
Toxicol Sci. 40: 205-210. http://dx.doi.org/10.1093/toxsci/40.2.205.
Hong. TY: Yang. CS: Lee. M: Wang. YY: Huang. WO: Tan. Y: Patten. CI: Bondoc. FY. (1997b). Role of
cytochromes P450 in the metabolism of methyl tert-butyl ether in human livers. Arch
Toxicol. 71: 266-269.
Tohanson. G: Nihlen. A: Lof. A. (1995). Toxicokinetics and acute effects of MTBE and ETBE in male
volunteers. Toxicol Lett. 82/83: 713-718. http://dx.doi.org/10.1016/0378-4274r95103589-
3.
TPEC (Japan Petroleum Energy Center). (2007a). Micronucleus test of 2-ethoxy-2-methylpropane
(ETBE) using bone marrow in rats administered ETBE by gavage. (Study Number: 7049).
Japan: Japan Industrial Safety and Health Association.
TPEC (Japan Petroleum Energy Center). (2007b). Micronucleus test of 2-ethoxy-2-methylpropane
(ETBE) using bone marrow in rats administered ETBE intraperitoneally. (Study Number:
7048). Japan: Japan Bioassay Research Center, Japan Industrial Safety and Health
Association.
JPEC (Japan Petroleum Energy Center). (2007c). Micronucleus test of ETBE using bone marrow of
rats of the "13-week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (inhalation
study) [preliminary carcinogenicity study]". (Study Number: 7047). Japan Industrial Safety
and Health Association.
TPEC (Japan Petroleum Energy Center). (2007d). Micronucleus test of ETBE using bone marrow of
rats of the "13-week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (drinking
water study) [preliminary carcinogenicity study]". (Study Number: 7046). Japan Bioassay
Research Center, Japan Industrial Safety and Health Association.
TPEC (Japan Petroleum Energy Center). (2008a). A 90-day repeated dose toxicity study of ETBE by
whole-body inhalation exposure in rats. (Study Number: B061829). Mitsubishi Chemical
Safety Institute Ltd. An external peer review was conducted by EPA in November 2008 to
evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and atwww.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008b). A 180-Day repeated dose oral toxicity study of
ETBE in rats. (Study Number: D19-0002). Japan: Hita Laboratory, Chemicals Evaluation and
Research Institute (CERI). An external peer review was conducted by EPA in November
2008 to evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and atwww.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008c). Medium-term mutli-organ carcinogenesis bioassay
of 2-ethoxy-2-methylpropane (ETBE) in rats. (Study Number: 0635). Ichinomiya, Japan:
DIMS Institute of Medical Science.
This document is a draft for review purposes only and does not constitute Agency policy.
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TPEC (Japan Petroleum Energy Center). (2008d). A one-generation reproduction toxicity study of
ETBE in rats. (Study Number: SR07060). Safety Research Institute for Chemical Compounds.
TPEC (Japan Petroleum Energy Center). (2008e). Pharmacokinetic study in rats treated with [14c]
ETBE repeatedly for 14 days. (P070497). Japan: Kumamoto Laboratory, Mitsubishi
Chemical Safety Institute Ltd. An external peer review was conducted by EPA in November
2008 to evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and atwww.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008f). Pharmacokinetic study in rats treated with single
dose of [14C] ETBE. (P070496). Japan: Kumamoto Laboratory, Mitsubishi Chemical Safety
Institute Ltd. An external peer review was conducted by EPA in November 2008 to evaluate
the accuracy of experimental procedures, results, and interpretation and discussion of the
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and
at www.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2010a). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Drinking water study). (Study No: 0691). Japan Industrial Safety and Health
Association, Japan Bioassay Research Center. An external peer review was conducted by
EPA in November 2008 to evaluate the accuracy of experimental procedures, results, and
interpretation and discussion of the findings presented. A report of this peer review is
available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or
hotline.iris@epa.gov (e-mail address) and atwww.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2010b). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Inhalation study). (Study No: 0686). Japan: Japan Industrial Safety and Health
Association. An external peer review was conducted by EPA in November 2008 to evaluate
the accuracy of experimental procedures, results, and interpretation and discussion of the
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and
at www.epa.gov/iris.
Kaneko. T: Wang. P. -Y: Sato. A. (2000). Partition coefficients for gasoline additives and their
metabolites. J Occup Health. 42: 86-87. http://dx.doi.org/10.1539/ioh.42.86.
Le Gal. A: Dreano. Y: Gervaso. PG: Berthou. F. (2001). Human cytochrome P450 2A6 is the major
enzyme involved in the metabolism of three alkoxyethers used as oxyfuels [Review], Toxicol
Lett. 124: 47-58. http://dx.doi.org/10.1016/s0378-4274r00100286-l.
Leavens. TL: Borghoff. ST. (2009). Physiologically based pharmacokinetic model of methyl tertiary
butyl ether and tertiary butyl alcohol dosimetry in male rats based on binding to alpha2u-
globulin. Toxicol Sci. 109: 321-335. http://dx.doi.org/10.1093/toxsci/kfpQ49.
Li. 0: Kobavashi. M: Inagaki. H: Hirata. Y: Hirata. K: Shimizu. T: Wang. R. -S: Suda. M: Kawamoto. T:
Nakajima, T; Kawada, T. (2011). Effects of subchronic inhalation exposure to ethyl tertiary
butyl ether on splenocytes in mice. Int J Immunopathol Pharmacol. 24: 837-847.
https://doi.org/10.1177/0394632011024004Q3.
Malarkev. DE: Bucher. TR. (2011). Summary report of the National Toxicology Program and
Environmental Protection Agency-sponsored review of pathology materials from selected
Ramazzini Institute rodent cancer bioassays [NTP], Research Triangle Park: National
Toxicology Program.
This document is a draft for review purposes only and does not constitute Agency policy.
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http://ntp.niehs.nih.gov/ntp/about ntp/partnerships/international/summarvpwg report
ri bioassavs.pdf.
Maltoni. C: Belpoggi. F: Soffritti. M: Minardi. F. (1999). Comprehensive long-term experimental
project of carcinogenicity bioassays on gasoline oxygenated additives: plan and first report
of results from the study on ethyl-tertiary-butyl ether (ETBE). Eur J Oncol. 4: 493-508.
McComb. TA: Goldstein. DB. (1979). Quantitative comparison of physical dependence on tertiary
butanol and ethanol in mice: Correlation with lipid solubility. Journal of Pharmacology and
Experimental Therapeutics. 208: 113-117.
Medinskv. MA: Wolf. DC: Cattlev. RC: Wong. B: Tanszen. DB: Farris. GM: Wright. GA: Bond. TA. (1999).
Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on Fischer-344
rats and CD-I mice. Toxicol Sci. 51: 108-118. http://dx.doi.Org/10.1093 /toxsci/51.l.108.
Mivata. K: Koga. T: Aso. S: Hoshuvama. S: Aiimi. S: Furukawa. K. (2013). A subchronic (180-day) oral
toxicity study of ethyl tertiary-butyl ether, a bioethanol, in rats. Drug Chem Toxicol.
http://dx.doi. org/10.3109 /01480545.2013.851690.
Nihlen. A: Lof. A: Tohanson. G. (1995). Liquid/air partition coefficients of methyl and ethyl t-butyl
ethers, t-amyl methyl ether, and t-butyl alcohol. J Expo Anal Environ Epidemiol. 5: 573-582.
Nihlen. A: Lof. A: Tohanson. G. (1998). Controlled ethyl tert-butyl ether (ETBE) exposure of male
volunteers: I Toxicokenetics. Toxicol Sci. 46: 1-10.
http://dx.doi.org/10.1006/toxs.1998.2516.
Noguchi. T: Kamigaito. T: Katagiri. T: Kondou. H: Yamazaki. K: Aiso. S: Nishizawa. T: Nagano. K:
Fukushima. S. (2013). Lack of micronucleus induction activity of ethyl tertiary-butyl ether in
the bone marrow of F344 rats by sub-chronic drinking-water treatment, inhalation
exposure, or acute intraperitoneal injection. J Toxicol Sci. 38: 913-924.
http://dx.doi.org/10.2131/its.38.913.
NSF International. (2003). t-Butanol: Oral Risk Assessment Document (CAS 75-65-0) (pp. 81). Ann
Arbor, MI. http://www.documents.dgs.ca.gov/bsc/pex/exibit nsf t butanol.pdf.
NTP (National Toxicology Program). (1996a). Genetic toxicity evaluation of 2-methyl-2-
ethoxypropane (ETBE) (637-92-3) in micronucleus study A70000 in B6C3F1 mice.
Research Triangle Park, NC: National Institute of Environmental Health Sciences.
NTP (National Toxicology Program). (1996b). Genetic toxicity evaluation of 2-methyl-2-
ethoxypropane (ETBE) (637-92-3) in micronucleus study A87074 in F344 rats. Research
Triangle Park, NC: National Institute of Environmental Health Sciences.
Potts RO. G. uv RH. (1992). Predicting skin permeability. Pharm Res. 9: 663-669.
Saito. A: Sasaki. T: Kasai. T: Katagiri. T: Nishizawa. T: Noguchi. T: Aiso. S: Nagano. K: Fukushima. S.
(2013). Hepatotumorigenicity of ethyl tertiary-butyl ether with 2-year inhalation exposure
in F344 rats. Arch Toxicol. 87: 905-914. http://dx.doi.org/10.1007/s00204-012-Q997-x.
Salazar. KD: Brinkerhoff. CI: Lee. IS: Chiu. WA. (2015). Development and application of a rat PBPK
model to elucidate kidney and liver effects induced by ETBE and tert-butanol. Toxicol Appl
Pharmacol. 288: 439-452. http://dx.doi.Org/10.1016/i.taap.2015.08.015.
Spiteri. NT. (1982). Circadian patterning of feeding, drinking and activity during diurnal food access
in rats. Physiol Behav. 28:139-147. http: / /dx.doi.org/10.1016/0031-9384C82190115-9.
Sun. ID: Beskitt. TL. (1995a). Ethyl tertiary-butyl ether (ETBE): Pharmacokinetics after single and
repeated inhalation exposures of mice, with cover letter dated 06/21/95 [TSCA
This document is a draft for review purposes only and does not constitute Agency policy.
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Submission], (Project ID 94N1455). Export, PA: Bushy Run Research Center, Union Carbide
Corporation under contract to ARCO Chemical Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuery=OTS0557696.
Sun. TP: Beskitt. TL. (1995b). Ethyl tertiary-butyl ether (ETBE): Pharmacokinetics after single and
repeated inhalation exposures of rats [TSCA Submission], (Project ID 94N1454). Export, PA:
Bushy Run Research Center, Union Carbide Corporation under contract to ARCO Chemical
Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuery=OTSQ557695.
Suzuki. M: Yamazaki. K: Kano. H: Aiso. S: Nagano. K: Fukushima. S. (2012). No carcinogenicity of
ethyl tertiary-butyl ether by 2-year oral administration in rats. J Toxicol Sci. 37: 1239-1246.
Turini. A: Amato. G: Longo. V: Gervasi. PG. (1998). Oxidation of methyl- and ethyl-tertiary-butyl
ethers in rat liver microsomes: role ofthe cytochrome P450 isoforms. Arch Toxicol. 72: 207-
214. http://dx.doi.Org/10.1007/s002040050490.
U.S. EPA (U.S. Environmental Protection Agency). (2012). Benchmark dose technical guidance (pp.
1-99). (EPA/100/R-12/001). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum.
U.S. EPA (U.S. Environmental Protection Agency). (2016). Model files for tert-butanol and ETBE.
U.S. EPA (U.S. Environmental Protection Agency). (2017). PK/PBPK model evaluation for the IRIS
assessments of ethyl tertiary butyl ether (CASRN 637-92-3) and tert-butyl alcohol (CAS No.
75-65-0) (Draft) [EPA Report], Washington, DC: U.S. Environmental Protection Agency,
Pharmacokinetics Working Group.
Vergnes. IS. (1995). Ethyl tertiary butyl ether: In vitro chromosome aberrations assay in Chinese
hamster ovary cells. (Project ID 94N1425). Export, PA: Bushy Run Research Center, Union
Carbide Corporation under contract to ARCO Chemical Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuery=OTSQ557635.
Vergnes. IS: Kubena. MF. (1995a). Ethyl tertiary butyl ether: Bone marrow micronucleus test in
mice. (Project ID 94N1426). Export, PA: Bushy Run Research Center, Union Carbide
Corporation under contract to ARCO Chemical Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuery=OTSQ557636.
Vergnes. IS: Kubena. MF. (1995b). Ethyl Tertiary Butyl Ether: Mutagenic Potential in the Cho/hgprt
Forward Mutation Assay [TSCA Submission], (Project ID 94N1424). Export, PA: Bushy Run
Research Center, Union Carbide Corporation under contract to ARCO Chemical Company.
Weng. Z: Ohtani. K: Suda. M: Yanagiba. Y: Kawamoto. T: Nakaiima. T: Wang. RS. (2014). Assessment
of the reproductive toxicity of inhalation exposure to ethyl tertiary butyl ether in male mice
with normal, low active and inactive ALDH2. Arch Toxicol. 88: 1007-1021.
http://dx.doi. org/10.1007 /s00204-014-1192-z.
Weng. Z: Suda. M: Ohtani. K: Mei. N. an: Kawamoto. T: Nakaiima. T: Wang. R. (2013). Subchronic
exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice.
Toxicology. 311: 107-114. http://dx.doi.Org/10.1016/i.tox.2013.06.005.
Weng. Z: Suda. M: Ohtani. K: Mei. N: Kawamoto. T: Nakaiima. T: Wang. RS. (2012). Differential
genotoxic effects of subchronic exposure to ethyl tertiary butyl ether in the livers of Aldh2
knockout and wild-type mice. Arch Toxicol. 86: 675-682.
http://dx.doi.Org/10.1007/s00204-011-0779-x.
This document is a draft for review purposes only and does not constitute Agency policy.
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Weng. ZO: Suda. M: Ohtani. K: Mei. N: Kawamoto. T: Nakajima. T: Wang. RS. (2011). Aldh2 Knockout
Mice Were More Sensitive to DNA Damage in Leukocytes due to Ethyl Tertiary Butyl Ether
Exposure. Ind Health. 49: 396-399.
WHO (World Health Organization). (2012). Guidance for immunotoxicity risk assessment for
chemicals. (Harmonization Project Document No. 10). Geneva, Switzerland.
http://www.inchem.org/documents/harmproi/harmproi/harmproilO.pdf.
Zeiger. E: Anderson. B: Haworth. S: Lawlor. T: Mortelmans. K. (1992). Salmonella mutagenicity
tests: V Results from the testing of 311 chemicals. Environ Mol Mutagen. 19: 2-141.
http://dx.doi.Org/10.1002/em.2850190603.
Zhang. YP: Macina. OT: Rosenkranz. HS: Karol. MH: Mattison. DR. (1997). Prediction of the
metabolism and toxicological profiles of gasoline oxygenates. Inhal Toxicol. 9: 237-254.
This document is a draft for review purposes only and does not constitute Agency policy.
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