EPA/635/R-16/079a
Public Comment Draft
www.epa.gov/iris
Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
(CAS No. 75-65-0)
April 2 016
NOTICE
This document is a Public Comment Draft. This information is distributed solely for the purpose of
pre-dissemination peer review under applicable information quality guidelines. It has not been
formally disseminated by EPA. It does not represent and should not be construed to represent any
Agency determination or policy. It is being circulated for review of its technical accuracy and
science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of tert-Butyl Alcohol
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-ButyI Alcohol
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE x
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xiv
EXECUTIVE SUMMARY ES-1
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION LS-1
1. HAZARD IDENTIFICATION 1-1
1.1. Overview of Chemical Properties and Toxicokinetics 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-2
1.1.3. Description of Toxicokinetic Models 1-3
1.1.4. Chemicals Extensively Metabolized to tert-Butanol 1-4
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-4
1.2.1. Kidney Effects 1-4
1.2.2. Thyroid Effects 1-38
1.2.3. Developmental Effects 1-46
1.2.4. Neurodevelopmental Effects 1-53
1.2.5. Reproductive Effects 1-56
1.2.6. Other Toxicological Effects 1-61
1.3. INTEGRATION AND EVALUATION 1-61
1.3.1. Effects Other Than Cancer 1-61
1.3.2. Carcinogenicity 1-63
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-65
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-2
2.1.3. Derivation of Candidate Values 2-4
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-8
2.1.5. Selection of the Overall Reference Dose 2-8
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2.1.6. Confidence Statement 2-9
2.1.7. Previous IRIS Assessment 2-9
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-9
2.2.1. Identification of Studies and Effects for Dose-Response Analysis 2-9
2.2.2. Methods of Analysis 2-10
2.2.3. Derivation of Candidate Values 2-13
2.2.4. Derivation of Organ/System-Specific Reference Concentrations 2-16
2.2.5. Selection of the Overall Reference Concentration 2-16
2.2.6. Confidence Statement 2-17
2.2.7. Previous IRIS Assessment 2-17
2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference
Concentration 2-17
2.3. ORAL SLOPE FACTOR FOR CANCER 2-18
2.3.1. Analysis of Carcinogenicity Data 2-18
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods 2-19
2.3.3. Derivation of the Oral Slope Factor 2-20
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-21
2.3.5. Previous IRIS Assessment: Oral Slope Factor 2-23
2.4. INHALATION UNIT RISK FOR CANCER 2-23
2.4.1. Previous IRIS Assessment: Inhalation Unit Risk 2-24
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-24
REFERENCES R-l
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Toxicological Review of tert-Butyl Alcohol
TABLES
Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol ES-2
Table ES-2. Organ/system-specific RfCs and overall RfCfor tert-butanol ES-3
Table LS-1. Details of the search strategy employed for tert-butanol LS-4
Table LS-2. Summary of additional search strategies for tert-butanol LS-4
Table LS-3. Inclusion-exclusion criteria LS-5
Table LS-4. Considerations for evaluation of experimental animal studies LS-8
Table LS-5. Summary of experimental animal database LS-8
Table 1-1. Physicochemical properties and chemical identity of tert-butanol 1-1
Table 1-2. Changes in kidney histopathology in animals following exposure to tert-butanol 1-12
Table 1-3. Changes in kidney tumors in animals following exposure to tert-butanol 1-15
Table 1-4. Summary of data on the a2u-globulin process in male rats exposed to tert-butanol 1-22
Table 1-5. Proposed empirical criteria for attributing renal tumors to CPN 1-33
Table 1-6. Evidence pertaining to thyroid effects in animals following oral exposure to tert-
butanol 1-39
Table 1-7. Evidence pertaining to developmental effects in animals following exposure to tert-
butanol 1-48
Table 1-8. Evidence pertaining to neurodevelopmental effects in animals following exposure to
tert-butanol 1-54
Table 1-9. Evidence pertaining to reproductive effects in animals following exposure to tert-
butanol 1-56
Table 2-1. Summary of derivations of points of departure following oral exposure for up to 2
years 2-4
Table 2-2. Effects and corresponding derivation of candidate values 2-6
Table 2-3. Organ/system-specific RfDs and overall RfD for tert-butanol 2-8
Table 2-4. Summary of derivation of PODs following inhalation exposure 2-12
Table 2-5. Summary of derivation of inhalation points of departure derived from route-to-route
extrapolation from oral exposures 2-13
Table 2-6. Effects and corresponding derivation of candidate values 2-14
Table 2-7. Organ/system-specific RfCs and overall RfCfor tert-butanol 2-16
Table 2-8. Summary of the oral slope factor derivation 2-21
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor for tert-butanol 2-22
FIGURES
Figure LS-1. Summary of literature search and screening process for tert-butanol LS-3
Figure 1-1. Biotransformation of tert-butanol in rats and humans 1-3
Figure 1-2. Comparison of absolute kidney weight change in male and female rats across oral
and inhalation exposure based on internal blood concentration. Spearman rank
correlation coefficient (rho) was calculated to evaluate the direction of a
monotonic association (e.g., positive value = positive association) and the
strength of association 1-10
Figure 1-3. Comparison of absolute kidney weight change in male and female mice following oral
exposure based on administered concentration. Spearman rank correlation
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Toxicological Review of tert-Butyl Alcohol
coefficient (rho) was calculated to evaluate the direction of a monotonic
association (e.g., positive value = positive association) and the strength of
association 1-11
Figure 1-4. Comparison of absolute kidney weight change in male and female mice following
inhalation exposure based on administered concentration. Spearman rank
correlation coefficient (rho) was calculated to evaluate the direction of a
monotonic association (e.g., positive value = positive association) and the
strength of association 1-11
Figure 1-5. Exposure response array for kidney effects following oral exposure to te/t-butanol 1-17
Figure 1-6. Exposure-response array of kidney effects following inhalation exposure to tert-
butanol (13-week studies, no chronic studies available) 1-18
Figure 1-7. Temporal pathogenesis of a2u-globulin-associated nephropathy in male rats. a2u-
Globulin synthesized in the livers of male rats is delivered to the kidney, where
it can accumulate in hyaline droplets and be retained by epithelial cells lining
the S2 (P2) segment of the proximal tubules. Renal pathogenesis following
continued te/t-butanol (TBA) exposure and increasing droplet accumulation can
progress step-wise from increasing epithelial cell damage, death and
dysfunction leading to the formation of granular casts in the corticomedullary
junction, linear mineralization of the renal papillae, and carcinogenesis of the
renal tubular epithelium (Adapted from Swenberg and Lehman-McKeeman
(1999) and U.S. EPA (1991a) 1-21
Figure 1-8. Exposure-response array for effects potentially associated with a2u-globulin renal
tubule nephropathy and tumors in male rats after oral exposure to te/t-butanol 1-24
Figure 1-9. Exposure-response array for effects potentially associated with a2u-globulin renal
tubule nephropathy and tumors in male rats after inhalation exposure to
te/t-butanol 1-25
Figure 1-10. Exposure-response array of thyroid follicular cell effects following chronic oral
exposure to te/t-butanol. (Note: Only one carcinoma was observed in male mice
at the high-dose group.) 1-41
Figure 1-11. Exposure-response array of developmental effects following oral exposure to te/t-
butanol 1-51
Figure 1-12. Exposure-response array of developmental effects following inhalation exposure to
te/t-butanol 1-52
Figure 1-13. Exposure-response array of reproductive effects following oral exposure to te/t-
butanol 1-59
Figure 1-14. Exposure-response array of reproductive effects following inhalation exposure to
te/t-butanol 1-60
Figure 2-1. Candidate values with corresponding POD and composite UF. Each bar corresponds
to one data set described in Table 2-1 and Table 2-2 2-7
Figure 2-2. Candidate RfC values with corresponding POD and composite UF 2-15
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ABBREVIATIONS
Toxicological Review of tert-ButyI Alcohol
AIC Akaike's information criterion MNPCE
ALD approximate lethal dosage
ALT alanine aminotransferase MTD
AST aspartate aminotransferase NAG
atm atmosphere NCEA
ATSDR Agency for Toxic Substances and
Disease Registry NCI
BMD benchmark dose NOAEL
BMDL benchmark dose lower confidence limit NTP
BMDS Benchmark Dose Software NZW
BMR benchmark response OCT
BW body weight ORD
CA chromosomal aberration PBPK
CASRN Chemical Abstracts Service Registry POD
Number POD[ADj]
CBI covalent binding index QSAR
CHO Chinese hamster ovary (cell line)
CL confidence limit RDS
CNS central nervous system RfC
CPN chronic progressive nephropathy RfD
CYP450 cytochrome P450 RGDR
DAF dosimetric adjustment factor RNA
DEN diethylnitrosamine SAR
DMSO dimethylsulfoxide SCE
DNA deoxyribonucleic acid SD
EPA Environmental Protection Agency SDH
FDA Food and Drug Administration SE
FEVi forced expiratory volume of 1 second SCOT
GD gestation day
GDH glutamate dehydrogenase SGPT
GGT yglutarnyl transferase
GSH glutathione SSD
GST glutathione-S-transferase TCA
Hb/g-A animal blood:gas partition coefficient TCE
Hb/g-H human blood:gas partition coefficient TWA
HEC human equivalent concentration UF
HED human equivalent dose UFA
i.p. intraperitoneal UFn
IRIS Integrated Risk Information System UFi
IVF in vitro fertilization UFs
LCso median lethal concentration
LDso median lethal dose UFD
LOAEL lowest-observed-adverse-effect level U.S.
MN micronuclei
micronucleated polychromatic
erythrocyte
maximum tolerated dose
N-acetyl-p-D-glucosaminidase
National Center for Environmental
Assessment
National Cancer Institute
no-observed-adverse-effect level
National Toxicology Program
New Zealand White (rabbit breed)
ornithine carbamoyl transferase
Office of Research and Development
physiologically based pharmacokinetic
point of departure
duration-adjusted POD
quantitative structure-activity
relationship
replicative DNA synthesis
inhalation reference concentration
oral reference dose
regional gas dose ratio
ribonucleic acid
structure activity relationship
sister chromatid exchange
standard deviation
sorbitol dehydrogenase
standard error
glutamic oxaloacetic transaminase, also
known as AST
glutamic pyruvic transaminase, also
known as ALT
systemic scleroderma
trichloroacetic acid
trichloroethylene
time-weighted average
uncertainty factor
animal-to-human uncertainty factor
human variation uncertainty factor
LOAEL-to-NOAEL uncertain factor
subchronic-to-chronic uncertainty
factor
database deficiencies uncertainty factor
United States
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-ButyI Alcohol
AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Janice S. Lee, Ph.D. (Chemical Manager)
Keith Salazar, Ph.D.* (Co-Chemical
Manager)
Chris Brinkerhoff, Ph.D.
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
*Washington, DC
ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Currently with U.S. EPA, Office of Chemical Safety
and Pollution Prevention, Office of Pollution
Prevention and Toxics
Washington, DC
Contributors
Andrew Hotchkiss, Ph.D.
Channa Keshava, Ph.D.
Amanda Persad, Ph.D.
Vincent Cogliano, Ph.D.*
Jason Fritz, Ph.D.*
Catherine Gibbons, Ph.D. *
Samantha Jones, Ph.D. *
Kathleen Newhouse *
Karen Hogan *
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
*Washington, DC
Production Team
Maureen Johnson
Vicki Soto
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Contractor Support
Robyn Blain, Ph.D.
Michelle Cawley*
William Mendez, Jr., Ph.D.
Pam Ross
ICF International
Fairfax, VA
*Research Triangle Park, NC
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director)
John Vandenberg, Ph.D,# (National Program Director, Human
Health Risk Assessment)
Lynn Flowers, Ph.D., DABT (Associate Director for Health)
Vincent Cogliano, Ph.D. (IRIS Program Director)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Weihsueh A. Chiu, Ph.D. (Branch Chief, Toxicity Pathways
Branch) formerly with the U.S. EPA
U.S. EPA/ORD/NCEA
Washington, DC
*Cincinnati, OH
# Research Triangle Park, NC
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Toxicological Review of tert-ButyI Alcohol
Andrew Hotchkiss, Ph.D.# (Acting Branch Chief, Toxicity
Pathways Branch)
Jason Lambert, Ph.D., DABT* (Acting Branch Chief, Biological
Risk Assessment Branch)
Ted Berner (Assistant Center Director)
Karen Hogan (former Acting Branch Chief, Toxicity Effects
Branch)
Internal Review Team
General Toxicology Workgroup U.S. EPA
Inhalation Workgroup Office of Research and Development
Neurotoxicity Workgroup National Center for Environmental Assessment
Pharmacokinetics Workgroup Washington, DC
Reproductive and Developmental Research Triangle Park, NC
Toxicology Workgroup Cincinnati, OH
Statistical Workgroup
Toxicity Pathways Workgroup
Executive Review Committee
2
Reviewers
3 This assessment was provided for review to scientists in EPA's Program and Region Offices.
4 Comments were submitted by:
5 Office of the Administrator/Office of Children's Health Protection
6 Office of Land and Emergency Management
7 Region 2, New York, NY
8 Region 8, Denver, CO
9 This assessment was provided for review to other federal agencies and the Executive Office of the
10 President Comments were submitted by:
11 Department of Health and Human Services/Agency for Toxic Substances and Disease Registry,
12 Department of Health and Human Services/National Institute of Environmental Health
13 Sciences/National Toxicology Program,
14 Executive Office of the President/Office of Management and Budget,
15 Executive Office of the President/Office of Science and Technology Policy
This document is a draft for review purposes only and does not constitute Agency policy.
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PREFACE
3 This Toxicological Review critically reviews the publicly available studies on tert-butyl
4 alcohol (tert-butanol) to identify its adverse health effects and to characterize exposure-response
5 relationships. The assessment examined all effects by oral and inhalation routes of exposure and
6 includes an oral noncancer reference dose (RfD), an inhalation noncancer reference concentration
7 (RfC), a cancer weight of evidence descriptor, and a cancer dose-response assessment. It was
8 prepared under the auspices of the U.S. Environmental Protection Agency's (EPA's) Integrated Risk
9 Information System (IRIS) program. This is the first IRIS assessment for this chemical.
10 Toxicological Reviews for tert-butanol and ethyl tert-butyl ether (ETBE) were developed
11 simultaneously because they have several overlapping scientific aspects.
12 • tert-Butanol is one of the primary metabolites of ETBE, and some of the toxicological effects
13 of ETBE are attributed to tert-butanol. Therefore, data on ETBE are considered informative
14 for the hazard identification and dose-response assessment of tert-butanol, and vice versa.
15 • The scientific literature for the two chemicals includes data on (X2u-globulin-related
16 nephropathy; therefore, a common approach was employed to evaluate these data as they
17 relate to the mode of action for kidney effects.
18 • A combined physiologically based pharmacokinetic (PBPK) model for tert-butanol and
19 ETBE in rats was modified to support the dose-response assessments for these chemicals
20 [Salazaretal.. 2015].
21 A public meeting was held in December 2013 to obtain input on preliminary materials for
22 tert-butanol, including draft literature searches and associated search strategies, evidence tables,
23 and exposure-response arrays prior to the development of the IRIS assessment All public
24 comments provided were taken into consideration in developing the draft assessment. The
25 complete set of public comments is available on the docket at http: / /www.regulations.gov (Docket
26 ID No. EPA-HQ-ORD-2013-0111).
27 Organ/system-specific reference values are calculated based on kidney and thyroid toxicity
28 data. These reference values could be useful for cumulative risk assessments that consider the
29 combined effect of multiple agents acting on the same biological system.
30 This assessment was conducted in accordance with EPA guidance, which is cited and
31 summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and
32 related documents produced during its development are available on the IRIS website
33 (http: //www. epa. gov/iris). Appendices for toxicokinetic information, PBPK modeling, genotoxicity
34 study summaries, dose-response modeling, and other information are provided as Supplemental
35 Information to this Toxicological Review. For additional information about this assessment or for
This document is a draft for review purposes only and does not constitute Agency policy.
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1 general questions regarding IRIS, please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-
2 566-1749 (fax), or hotline.iris@epa.gov.
3 Uses
4 tert-Butanol primarily is an anthropogenic substance that is produced in large quantities
5 (HSDB, 2007) from several precursors, including 1-butene, isobutylene, acetyl chloride and
6 dimethylzinc, and tert-butyl hydroperoxide. The domestic production volume of tert-butanol,
7 including imports, was approximately 4 billion pounds in 2012 (U.S. EPA. 2014).
8 tert-Butanol has been used as a fuel oxygenate, an octane booster in unleaded gasoline, and
9 a denaturant for ethanol. From 1997 to 2005, the annual tert-butanol volume found in gasoline
10 ranged from approximately 4 million to 6 million gallons. During that time, larger quantities were
11 used to make methyl tert-butyl ether (MTBE) and ETBE. MTBE and ETBE are fuel oxygenates that
12 were used in the United States prior to 2007 at levels of more than 2 billion gallons annually.
13 Current use levels of MTBE and ETBE in the United States are much lower, but use in Europe and
14 Asia remains strong.1
15 tert-Butanol has been used for a variety of other purposes, including as a dehydrating agent
16 and solvent. As such, it is added to lacquers, paint removers, and nail enamels and polishes.
17 tert-Butanol also is used to manufacture methyl methacrylate plastics and flotation devices.
18 Cosmetic and food-related uses include the manufacture of flavors, and, because of its camphor-like
19 aroma, it also is used to create artificial musk, fruit essences, and perfume (HSDB, 2007). It is used
20 in coatings on metal and paperboard food containers (Cal/EPA. 1999) and industrial cleaning
21 compounds, and can be used for chemical extraction in pharmaceutical applications (HSDB. 2007).
22 Fate and Transport
23 5oi7
24 tert-Butanol is expected to be highly mobile in soil due to its low affinity for soil organic
25 matter. Rainwater or other water percolating through soil is expected to dissolve and transport
26 most tert-butanol present in soil, potentially leading to groundwater contamination. Based on its
27 vapor pressure, tert-butanol's volatilization from soil surfaces is expected to be an important
28 dissipation process (HSDB. 2007). As a tertiary alcohol, tert-butanol is expected to degrade more
29 slowly in the environment compared to primary (e.g., ethanol) or secondary (e.g., isopropanol)
30 alcohols. In anoxic soil conditions, the half-life of tert-butanol is estimated to be months
31 (approximately 200 days). Microbial degradation rates are increased in soils supplemented with
32 nitrate and sulfate nutrients (HSDB. 2007).
1 http://www.ihs.com/products/chemical/planning/ceh/gasoline-octane-improvers.aspx.
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1 Water
2 tert-Butanol is expected to volatilize from water surfaces within 2 to 29 days and does not
3 readily adsorb to suspended solids and sediments in water [HSDB. 2007). Biodegradation in
4 aerobic water occurs over weeks to months and in anaerobic aquatic conditions, the biodegradation
5 rate decreases. Bioconcentration of tert-butanol in aquatic organisms is low [HSDB. 2007).
6 Air
7 tert-Butanol primarily exists as a vapor in the ambient atmosphere. Vapor-phase tert-
8 butanol is degraded in the atmosphere by reacting with photochemically produced hydroxyl
9 radicals with a half-life of 14 days [HSDB. 20071.
10 Occurrence in the Environment
11 The Toxics Release Inventory (TRI) Program National Analysis Report estimated that more
12 than 1 million pounds of tert-butanol has been released into the soil from landfills, land treatment,
13 underground injection, surface impoundments, and other land disposal sources. The TRI program
14 also estimated that 476,266 pounds of tert-butanol was released into the atmosphere from fugitive
15 emissions and point sources [U.S. EPA. 2012c]. In California, air emissions of tert-butanol from
16 stationary sources are estimated to be at least 27,000 pounds per year, based on data reported by
17 the state's Air Toxics Program [Scorecard, 2014]. The TRI National Analysis Report estimated 7,469
18 pounds of tert-butanol was released into surface waters from point and nonpoint sources in 2011
19 [U.S. EPA. 2012c).
20 tert-Butanol has been identified in drinking water wells throughout the United States
21 [HSDB. 20071. California's Geotracker Database2 lists 3,496 detections of tert-butanol in
22 groundwater associated with contaminated sites in that state since 2011. tert-Butanol also has been
23 detected in drinking water wells in the vicinity of landfills [U.S. EPA. 2012c]. Additionally, tert-
24 Butanol leaking from underground storage tanks could be a product of MTBE and ETBE, which can
25 degrade to form tert-butanol in soils [HSDB. 2007]. The industrial chemical tert-butyl acetate also
26 can degrade to form tert-butanol in animals post exposure and in the environment
27 Ambient outdoor air concentrations of tert-butanol vary according to proximity to urban
28 areas [HSDB. 20071.
29 General Population Exposure
30 tert-Butanol exposure can occur in many different settings. Releases from underground
31 storage tanks could potentially result in exposure for people who get their drinking water from
32 wells. Due to its high environmental mobility and resistance to biodegradation, tert-butanol has the
33 potential to contaminate and persist in groundwater and soil [HSDB. 2007].
2 http://geotracker.waterboards.ca.gov/.
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1 Ingestion of contaminated food can be a source of tert-butanol exposure through its use as a
2 coating in metallic and paperboard food containers [Gal/EPA, 1999], and tert-butanol has been
3 detected in food [HSDB. 2007). Internal exposure to tert-butanol also can occur as a result of
4 ingestion of MTBE or ETBE, as tert-butanol is a metabolite of these compounds [NSF International,
5 20031
6 Other human exposure pathways include inhalation, lactation and, to a lesser extent, dermal
7 contact. Inhalation exposure can occur due to the chemical's volatility and release from industrial
8 processes, consumer products, and contaminated sites [HSDB. 2007]. tert-Butanol has been
9 identified in mother's milk [HSDB. 2007]. Dermal contact is a viable route of exposure through
10 handling consumer products containing tert-butanol [NSF International. 2003].
11 Assessments by Other National and International Health Agencies
12 Toxicity information on tert-butanol has been evaluated by the National Institute for
13 Occupational Safety and Health [NIOSH, 2007], the Occupational Safety and Health Administration
14 [OSHA. 2006], and the Food and Drug Administration [FDA. 201 la. b]. The results of these
15 assessments are presented in Appendix A of the Supplemental Information to this Toxicological
16 Review. Of importance to recognize is that these earlier assessments could have been prepared for
17 different purposes and might use different methods. In addition, newer studies have been included
18 in the IRIS assessment.
4 ^
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
3 Note: The Preamble to IRIS assessments is
4 being revised based on comments received
5 from external peer reviewers and the
6 public, and based on IRIS Program
1 experience with the implementation of
8 systematic review methods. Subsequent
9 drafts of th e tert-b utan ol assessm en t will
10 include the revised Preamble.
11 1. Scope of the IRIS Program
12 Soon after the EPA was established in
13 1970, itwas atthe forefront of developing risk
14 assessment as a science and applying it in
15 decisions to protect human health and the
16 environment. The Clean Air Act, for example,
17 mandates that the EPA provide "an ample
18 margin of safety to protect public health"; the
19 Safe Drinking Water Act, that "no adverse
20 effects on the health of persons may
21 reasonably be anticipated to occur, allowing
22 an adequate margin of safety." Accordingly,
23 the EPA uses information on the adverse
24 effects of chemicals and on exposure levels
25 below which these effects are not anticipated
26 to occur.
27 IRIS assessments critically review the
28 publicly available studies to identify adverse
29 health effects from exposure to chemicals and
30 to characterize exposure-response
31 relationships. In terms set forth by the
32 National Research Council fNRC. 19831 IRIS
33 assessments cover the hazard identification
34 and dose-response assessment steps of risk
35 assessment, not the exposure assessment or
36 risk characterization steps that are conducted
37 by the EPA's program and regional offices and
38 by other federal, state, and local health
39 agencies that evaluate risk in specific
40 populations and exposure scenarios. IRIS
41 assessments are distinct from and do not
42 address political, economic, and technical
43 considerations that influence the design and
44 selection of risk management alternatives.
45 An IRIS assessment may cover a single
46 chemical, a group of structurally or
47 toxicologically related chemicals, or a complex
48 mixture. These agents may be found in air,
49 water, soil, or sediment Exceptions are
50 chemicals currently used exclusively as
51 pesticides, ionizing and non-ionizing
52 radiation, and criteria air pollutants listed
53 under Section 108 of the Clean Air Act (carbon
54 monoxide, lead, nitrogen oxides, ozone,
55 particulate matter, and sulfur oxides).
56 Periodically, the IRIS Program asks other
57 EPA programs and regions, other federal
58 agencies, state health agencies, and the
59 general public to nominate chemicals and
60 mixtures for future assessment or
61 reassessment Agents may be considered for
62 reassessment as significant new studies are
63 published. Selection is based on program and
64 regional office priorities and on availability of
65 adequate information to evaluate the potential
66 for adverse effects. Other agents may also be
67 assessed in response to an urgent public
68 health need.
69 2. Process for developing and peer-
70 reviewing IRIS assessments
71 The process for developing IRIS
72 assessments (revised in May 2009 and
73 enhanced in July 2013) involves critical
74 analysis of the pertinent studies, opportunities
75 for public input, and multiple levels of
76 scientific review. The EPA revises draft
77 assessments after each review, and external
78 drafts and comments become part of the
79 public record (U.S. EPA. 2009).
80 Before beginning an assessment, the IRIS
81 program discusses the scope with other EPA
82 programs and regions to ensure that the
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1 assessment will meet their needs. Then a
2 public meeting on problem formulation
3 invites discussion of the key issues and the
4 studies and analytical approaches that might
5 contribute to their resolution.
6 Step 1. Development of a draft
7 Toxicological Review. The draft
8 assessment considers all pertinent
9 publicly available studies and applies
10 consistent criteria to evaluate study
11 quality, identify health effects, identify
12 mechanistic events and pathways,
13 integrate the evidence of causation for
14 each effect, and derive toxicity values. A
15 public meeting prior to the integration of
16 evidence and derivation of toxicity values
17 promotes public discussion of the
18 literature search, evidence, and key issues.
19 Step 2. Internal review by scientists in EPA
20 programs and regions. The draft
21 assessment is revised to address the
22 comments from within the EPA.
23 Step 3. Interagency science consultation
24 with other federal agencies and the
25 Executive Offices of the President. The
26 draft assessment is revised to address the
27 interagency comments. The science
28 consultation draft, interagency comments,
29 and the EPA's response to major
30 comments become part of the public
31 record.
32 Step 4. Public review and comment,
33 followed by external peer review. The
34 EPA releases the draft assessment for
35 public review and comment A public
36 meeting provides an opportunity to
37 discuss the assessment prior to peer
38 review. Then the EPA releases a draft for
39 external peer review. The peer review
40 meeting is open to the public and includes
41 time for oral public comments. The peer
42 reviewers assess whether the evidence
43 has been assembled and evaluated
44 according to guidelines and whether the
45 conclusions are justified by the evidence.
46 The peer review draft, written public
47 comments, and peer review report
48 become part of the public record.
49 Step 5. Revision of draft Toxicological
50 Review and development of draft IRIS
51 summary. The draft assessment is revised
52 to reflect the peer review comments,
53 public comments, and newly published
54 studies that are critical to the conclusions
55 of the assessment. The disposition of peer
56 review comments and public comments
57 becomes part of the public record.
58 Step 6. Final EPA review and interagency
59 science discussion with other federal
60 agencies and the Executive Offices of
61 the President The draft assessment and
62 summary are revised to address the EPA
63 and interagency comments. The science
64 discussion draft, written interagency
65 comments, and EPA's response to major
66 comments become part of the public
67 record.
68 Step 7. Completion and posting. The
69 Toxicological Review and IRIS summary
70 are posted on the IRIS website
71 [http://www.epa.gov/iris/].
72 The remainder of this Preamble addresses step 1,
73 the development of a draft Toxicological
74 Review. IRIS assessments follow standard
75 practices of evidence evaluation and peer
76 review, many of which are discussed in
77 EPA guidelines [U.S. EPA.
78 2005a. b, 2000b. 1998b. 1996. 1991b. 198
79 6a, b) and other methods [U.S. EPA.
80 2012a. b, 2011. 2006a. b, 2002. 19941
81 Transparent application of scientific
82 judgment is of paramount importance. To
83 provide a harmonized approach across
84 IRIS assessments, this Preamble
85 summarizes concepts from these
86 guidelines and emphasizes principles of
87 general applicability.
88 3. Identifying and selecting
89 pertinent studies
90 3.1. Identifying studies
91 Before beginning an assessment, the EPA
92 conducts a comprehensive search of the
93 primary scientific literature. The literature
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1 search follows standard practices and includes
2 the PubMed and ToxNet databases of the
3 National Library of Medicine, Web of Science,
4 and other databases listed in the EPA's HERO
5 system (Health and Environmental Research
6 Online, http://hero.epa.gov/]. Searches for
7 information on mechanisms of toxicity are
8 inherently specialized and may include
9 studies on other agents that act through
10 related mechanisms.
11 Each assessment specifies the search
12 strategies, keywords, and cut-off dates of its
13 literature searches. The EPA posts the results
14 of the literature search on the IRIS web site
15 and requests information from the public on
16 additional studies and ongoing research.
17 The EPA also considers studies received
18 through the IRIS Submission Desk and studies
19 (typically unpublished) submitted under the
20 Toxic Substances Control Act or the Federal
21 Insecticide, Fungicide, and Rodenticide Act
22 Material submitted as Confidential Business
23 Information is considered only if it includes
24 health and safety data that can be publicly
25 released. If a study that may be critical to the
26 conclusions of the assessment has not been
27 peer-reviewed, the EPA will have it peer-
28 reviewed.
29 The EPA also examines the toxicokinetics
30 of the agent to identify other chemicals (for
31 example, major metabolites of the agent) to
32 include in the assessment if adequate
33 information is available, in order to more fully
34 explain the toxicity of the agent and to suggest
35 dose metrics for subsequent modeling.
36 In assessments of chemical mixtures,
37 mixture studies are preferred for their ability
38 to reflect interactions among components.
39 The literature search seeks, in decreasing
40 order of preference (U.S. EPA.
41 2000b. 32.2: 1986b. 32.11:
42 - Studies of the mixture being assessed.
43 - Studies of a sufficiently similar
44 mixture. In evaluating similarity, the
45 assessment considers the alteration of
46 mixtures in the environment through
47 partitioning and transformation.
48 - Studies of individual chemical
49 components of the mixture, if there are
50 not adequate studies of sufficiently
51 similar mixtures.
52 3.2. Selecting pertinent epidemiologic
53 studies
54 Study design is the key consideration for
55 selecting pertinent epidemiologic studies from
56 the results of the literature search.
57 - Cohort studies, case-control studies,
58 and some population-based surveys
59 (for example, NHANES) provide the
60 strongest epidemiologic evidence,
61 especially if they collect information
62 about individual exposures and
63 effects.
64 - Ecological studies (geographic
65 correlation studies) relate exposures
66 and effects by geographic area. They
67 can provide strong evidence if there
68 are large exposure contrasts between
69 geographic areas, relatively little
70 exposure variation within study areas,
71 and population migration is limited.
72 - Case reports of high or accidental
73 exposure lack definition of the
74 population at risk and the expected
75 number of cases. They can provide
76 information about a rare effect or
77 about the relevance of analogous
78 results in animals.
79 The assessment briefly reviews ecological
80 studies and case reports but reports details
81 only if they suggest effects not identified by
82 other studies.
83 3.3. Selecting pertinent experimental
84 studies
85 Exposure route is a key design
86 consideration for selecting pertinent
87 experimental animal studies or human clinical
88 studies.
89 - Studies of oral, inhalation, or dermal
90 exposure involve passage through an
91 absorption barrier and are considered
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1
2
3
4
5
6
7
8
9
10
most pertinent to
environmental exposure.
human
45 quality characteristics
46 similar design.
across studies of
Injection or implantation studies are
often considered less pertinent but
may provide valuable toxicokinetic or
mechanistic information. They also
may be useful for identifying effects in
animals if deposition or absorption is
problematic (for example, for particles
and fibers).
11 Exposure duration is also a key design
12 consideration for selecting pertinent
13 experimental animal studies.
14 - Studies of effects from chronic
15 exposure are most pertinent to
16 lifetime human exposure.
17 - Studies of effects from less-than-
18 chronic exposure are pertinent but
19 less preferred for identifying effects
20 from lifetime human exposure. Such
21 studies may be indicative of effects
22 from less-than-lifetime human
23 exposure.
24 Short-duration studies involving animals
25 or humans may provide toxicokinetic or
26 mechanistic information.
27 For developmental toxicity and
28 reproductive toxicity, irreversible effects may
29 result from a brief exposure during a critical
30 period of development. Accordingly,
31 specialized study designs are used for these
32 effects [U.S. EPA. 2006b. 1998b. 1996.1991b).
33 4. Evaluating the quality of
34 individual studies
35 After the subsets of pertinent
36 epidemiologic and experimental studies have
37 been selected from the literature searches, the
38 assessment evaluates the quality of each
39 individual study. This evaluation considers the
40 design, methods, conduct, and documentation
41 of each study, but not whether the results are
42 positive, negative, or null. The objective is to
43 identify the stronger, more informative
44 studies based on a uniform evaluation of
47 4.1. Evaluating the quality of
48 epidemiologic studies
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
The assessment evaluates design and
methodological aspects that can increase or
decrease the weight given to each
epidemiologic study in the overall evaluation
fU.S. EPA. 2005a. 1998b. 1996.1994.1991bl:
- Documentation of study design,
methods, population characteristics,
and results.
- Definition and selection of the study
group and comparison group.
- Ascertainment of exposure to the
chemical or mixture.
Ascertainment of disease or health
effect
- Duration of exposure and follow-up
and adequacy for assessing the
occurrence of effects.
Characterization of exposure during
critical periods.
- Sample size and statistical power to
detect anticipated effects.
70 - Participation rates and potential for
71 selection bias as a result of the
72 achieved participation rates.
73 - Measurement error (can lead to
74 misclassification of exposure, health
75 outcomes, and other factors) and other
76 types of information bias.
77 - Potential confounding and other
78 sources of bias addressed in the study
79 design or in the analysis of results. The
80 basis for consideration of confounding
81 is a reasonable expectation that the
82 confounder is related to both exposure
83 and outcome and is sufficiently
84 prevalent to result in bias.
85 For developmental toxicity, reproductive
86 toxicity, neurotoxicity, and cancer there is
87 further guidance on the nuances of evaluating
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1 epidemiologic studies of these effects [U.S.
2 EPA. 2005a. 1998b. 1996.1991bl
3 4.2. Evaluating the quality of
4 experimental studies
5 The assessment evaluates design and
6 methodological aspects that can increase or
7 decrease the weight given to each
8 experimental animal study, in-vitro study, or
9 human clinical study [U.S. EPA.
10 2005a. 1998b. 1996. 1991bl Research
11 involving human subjects is considered only if
12 conducted according to ethical principles.
13 - Documentation of study design,
14 animals or study population, methods,
15 basic data, and results.
16 - Nature of the assay and validity for its
17 intended purpose.
18 - Characterization of the nature and
19 extent of impurities and contaminants
20 of the administered chemical or
21 mixture.
22 - Characterization of dose and dosing
23 regimen (including age at exposure)
24 and their adequacy to elicit adverse
25 effects, including latent effects.
26 - Sample sizes and statistical power to
27 detect dose-related differences or
28 trends.
29 - Ascertainment of survival, vital signs,
30 disease or effects, and cause of death.
31 - Control of other variables that could
32 influence the occurrence of effects.
33 The assessment uses statistical tests to
34 evaluate whether the observations may be due
35 to chance. The standard for determining
36 statistical significance of a response is a trend
37 test or comparison of outcomes in the exposed
38 groups against those of concurrent controls. In
39 some situations, examination of historical
40 control data from the same laboratory within
41 a few years of the study may improve the
42 analysis. For an uncommon effect that is not
43 statistically significant compared with
44 concurrent controls, historical controls may
45 show that the effect is unlikely to be due to
46 chance. For a response that appears significant
47 against a concurrent control response that is
48 unusual, historical controls may offer a
49 different interpretation [U.S. EPA.
50 2005a. §2.2.2.1.31
51 For developmental toxicity, reproductive
52 toxicity, neurotoxicity, and cancer there is
53 further guidance on the nuances of evaluating
54 experimental studies of these effects [U.S. EPA.
55 2005a. 1998b. 1996. 1991bl In multi-
56 generation studies, agents that produce
57 developmental effects at doses that are not
58 toxic to the maternal animal are of special
59 concern. Effects that occur at doses associated
60 with mild maternal toxicity are not assumed to
61 result only from maternal toxicity. Moreover,
62 maternal effects may be reversible, while
63 effects on the offspring may be permanent
64 [U.S. EPA. 1998b. §3.1.2.4.5.4: 1991b.
65 §3.1.1.41.
66 4.3. Reporting study results
67 The assessment uses evidence tables to
68 present the design and key results of pertinent
69 studies. There may be separate tables for each
70 site of toxicity or type of study.
71 If a large number of studies observe the
72 same effect, the assessment considers the
73 study quality characteristics in this section to
74 identify the strongest studies or types of study.
75 The tables present details from these studies,
76 and the assessment explains the reasons for
77 not reporting details of other studies or
78 groups of studies that do not add new
79 information. Supplemental information
80 provides references to all studies considered,
81 including those not summarized in the tables.
82 The assessment discusses strengths and
83 limitations that affect the interpretation of
84 each study. If the interpretation of a study in
85 the assessment differs from that of the study
86 authors, the assessment discusses the basis for
87 the difference.
88 As a check on the selection and evaluation
89 of pertinent studies, the EPA asks peer
90 reviewers to identify studies that were not
91 adequately considered.
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i 5. Evaluating the overall evidence of
2 each effect
3 5.1. Concepts of causal inference
4 For each health effect, the assessment
5 evaluates the evidence as a whole to
6 determine whether it is reasonable to infer a
7 causal association between exposure to the
8 agent and the occurrence of the effect This
9 inference is based on information from
10 pertinent human studies, animal studies, and
11 mechanistic studies of adequate quality.
12 Positive, negative, and null results are given
13 weight according to study quality.
14 Causal inference involves scientific
15 judgment, and the considerations are nuanced
16 and complex. Several health agencies have
17 developed frameworks for causal inference,
18 among them the U.S. Surgeon General [CDC.
19 2004: HEW. 1964). the International Agency
20 for Research on Cancer [IARC. 2006]. the
21 Institute of Medicine (IOM. 2008). and the EPA
22 (2010. S1.6: 2005a. 52.51 Although developed
23 for different purposes, the frameworks are
24 similar in nature and provide an established
25 structure and language for causal inference.
26 Each considers aspects of an association that
27 suggest causation, discussed by Hill (Hill.
28 1965] and elaborated on by Rothman and
29 Greenland (Rothman and Greenland. 1998].
30 and U.S. EPA f2005a. §2.2.1.7: 1994. Appendix
31 C].
32 Strength of association: The finding of a large
33 relative risk with narrow confidence
34 intervals strongly suggests that an
35 association is not due to chance, bias, or
36 other factors. Modest relative risks,
37 however, may reflect a small range of
38 exposures, an agent of low potency, an
39 increase in an effect that is common,
40 exposure misclassification, or other
41 sources of bias.
42 Consistency of association: An inference of
43 causation is strengthened if elevated risks
44 are observed in independent studies of
45 different populations and exposure
46 scenarios. Reproducibility of findings
47 constitutes one of the strongest arguments
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xix DRAFT—DO NOT CITE OR QUOTE
48 for causation. Discordant results
49 sometimes reflect differences in study
50 design, exposure, or confounding factors.
51 Specificity of association: As originally
52 intended, this refers to one cause
53 associated with one effect Current
54 understanding that many agents cause
55 multiple effects and many effects have
56 multiple causes make this a less
57 informative aspect of causation, unless the
58 effect is rare or unlikely to have multiple
59 causes.
60 Temporal relationship: A causal
61 interpretation requires that exposure
62 precede development of the effect.
63 Biologic gradient (exposure-response
64 relationship): Exposure-response
65 relationships strongly suggest causation. A
66 monotonic increase is not the only pattern
67 consistent with causation. The presence of
68 an exposure-response gradient also
69 weighs against bias and confounding as
70 the source of an association.
71 Biologic plausibility: An inference of
72 causation is strengthened by data
73 demonstrating plausible biologic
74 mechanisms, if available. Plausibility may
75 reflect subjective prior beliefs if there is
76 insufficient understanding of the biologic
77 process involved.
78 Coherence: An inference of causation is
79 strengthened by supportive results from
80 animal experiments, toxicokinetic studies,
81 and short-term tests. Coherence may also
82 be found in other lines of evidence, such as
83 changing disease patterns in the
84 population.
85 "Natural experiments": A change in exposure
86 that brings about a change in disease
87 frequency provides strong evidence, as it
88 tests the hypothesis of causation. An
89 example would be an intervention to
90 reduce exposure in the workplace or
91 environment that is followed by a
92 reduction of an adverse effect
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Toxicological Review of tert-Butyl Alcohol
1 Analogy: Information on structural analogues
2 or on chemicals that induce similar
3 mechanistic events can provide insight
4 into causation.
5 These considerations are consistent with
6 guidelines for systematic reviews that
7 evaluate the quality and weight of evidence.
8 Confidence is increased if the magnitude of
9 effect is large, if there is evidence of an
10 exposure-response relationship, or if an
11 association was observed and the plausible
12 biases would tend to decrease the magnitude
13 of the reported effect Confidence is decreased
14 for study limitations, inconsistency of results,
15 indirectness of evidence, imprecision, or
16 reporting bias [Guyatt et al., 2008b: Guyatt et
17 al.. 2008a].
is 5.2. Evaluating evidence in humans
19 For each effect, the assessment evaluates
20 the evidence from the epidemiologic studies as
21 a whole. The objective is to determine whether
22 a credible association has been observed and,
23 if so, whether that association is consistent
24 with causation. In doing this, the assessment
25 explores alternative explanations (such as
26 chance, bias, and confounding) and draws a
27 conclusion about whether these alternatives
28 can satisfactorily explain any observed
29 association.
30 To make clear how much the
31 epidemiologic evidence contributes to the
32 overall weight of the evidence, the assessment
33 may select a standard descriptor to
34 characterize the epidemiologic evidence of
35 association between exposure to the agent and
36 occurrence of a health effect
37 Sufficient epidemiologic evidence of an
38 association consistent with causation:
39 The evidence establishes a causal
40 association for which alternative
41 explanations such as chance, bias, and
42 confounding can be ruled out with
43 reasonable confidence.
44 Suggestive epidemiologic evidence of an
45 association consistent with causation:
46 The evidence suggests a causal association
47 but chance, bias, or confounding cannot be
48 ruled out as explaining the association.
49 Inadequate epidemiologic evidence to infer
50 a causal association: The available
51 studies do not permit a conclusion
52 regarding the presence or absence of an
53 association.
54 Epidemiologic evidence consistent with no
55 causal association: Several adequate
56 studies covering the full range of human
57 exposures and considering susceptible
58 populations, and for which alternative
59 explanations such as bias and confounding
60 can be ruled out, are mutually consistent
61 in not finding an association.
62 5.3. Evaluating evidence in animals
63 For each effect, the assessment evaluates
64 the evidence from the animal experiments as a
65 whole to determine the extent to which they
66 indicate a potential for effects in humans.
67 Consistent results across various species and
68 strains increase confidence that similar results
69 would occur in humans. Several concepts
70 discussed by Hill [Hill. 1965] are pertinent to
71 the weight of experimental results:
72 consistency of response, dose-response
73 relationships, strength of response, biologic
74 plausibility, and coherence [U.S. EPA,
75 2005a. 52.2.1.7: 1994. Appendix C1.
76 In weighing evidence from multiple
77 experiments, U.S. EPA [2005a. §2.5]
78 distinguishes:
79 Conflicting evidence (that is, mixed positive
80 and negative results in the same sex and
81 strain using a similar study protocol] from
82 Differing results (that is, positive results and
83 negative results are in different sexes or
84 strains or use different study protocols].
85 Negative or null results do not invalidate
86 positive results in a different experimental
87 system. The EPA regards all as valid
88 observations and looks to explain differing
89 results using mechanistic information (for
90 example, physiologic or metabolic differences
91 across test systems] or methodological
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
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18
19
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39
40
41
42
43
differences (for example, relative sensitivity of
the tests, differences in dose levels,
insufficient sample size, or timing of dosing or
data collection).
It is well established that there are critical
periods for some developmental and
reproductive effects (U.S. EPA.
2006b. 2005a. b, 1998b. 1996. 1991b).
Accordingly, the assessment determines
whether critical periods have been adequately
investigated. Similarly, the assessment
determines whether the database is adequate
to evaluate other critical sites and effects.
In evaluating evidence of genetic toxicity:
- Demonstration of gene mutations,
chromosome aberrations, or
aneuploidy in humans or experimental
mammals [in vivo] provides the
strongest evidence.
This is followed by positive results in
lower organisms or in cultured cells
[in vitro] or for other genetic events.
- Negative results carry less weight,
partly because they cannot exclude the
possibility of effects in other tissues
(IARC. 2006).
For germ-cell mutagenicity, The EPA has
defined categories of evidence, ranging from
positive results of human germ-cell
mutagenicity to negative results for all effects
of concern (U.S. EPA. 1986a. §2.3).
5.4. Evaluating mechanistic data
Mechanistic data can be useful in
answering several questions.
- The biologic plausibility of a causal
interpretation of human studies.
The generalizability of animal studies
to humans.
The susceptibility of particular
populations or lifestages.
The focus of the analysis is to describe, if
possible, mechanistic pathways that lead to a
health effect. These pathways encompass:
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
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85
86
87
88
89
90
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Toxicokinetic processes of absorption,
distribution, metabolism, and
elimination that lead to the formation
of an active agent and its presence at
the site of initial biologic interaction.
Toxicodynamic processes that lead to a
health effect at this or another site
(also known as a mode of action}.
For each effect, the assessment discusses
the available information on its modes of
action and associated key events (key events
empirically observable, necessary
precursor steps or biologic markers of such
steps; mode of action being a series of key
; involving interaction with cells,
operational and anatomic changes, and
resulting in disease). Pertinent information
may also come from studies of metabolites or
of compounds that are structurally similar or
act through similar mechanisms.
Information on mode of action is not required
a conclusion that the agent is causally
related to an effect (U.S. EPA. 2005a. 52.5).
The assessment addresses several
questions about each hypothesized mode of
action fU.S. EPA. 2005a. 32.4.3.41.
Is the hypothesized mode of action
sufficiently supported in test animals?
Strong support for a key event being
necessary to a mode of action can come
from experimental challenge to the
hypothesized mode of action, in which
studies that suppress a key event observe
suppression of the effect Support for a
mode of action is meaningfully
strengthened by consistent results in
different experimental models, much
more so than by replicate experiments in
the same model. The assessment may
consider various aspects of causation in
addressing this question.
Is the hypothesized mode of action
relevant to humans? The assessment
reviews the key events to identify critical
similarities and differences between the
test animals and humans. Site
concordance is not assumed between
animals and humans, though it may hold
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1 for certain effects or modes of action.
2 Information suggesting quantitative
3 differences in doses where effects would
4 occur in animals or humans is considered
5 in the dose-response analysis. Current
6 levels of human exposure are not used to
7 rule out human relevance, as IRIS
8 assessments may be used in evaluating
9 new or unforeseen circumstances that
10 may entail higher exposures.
11 3) Which populations or lifestages can be
12 particularly susceptible to the
13 hypothesized mode of action? The
14 assessment reviews the key events to
15 identify populations and lifestages that
16 might be susceptible to their occurrence.
17 Quantitative differences may result in
18 separate toxicity values for susceptible
19 populations or lifestages.
20 The assessment discusses the likelihood
21 that an agent operates through multiple
22 modes of action. An uneven level of support
23 for different modes of action can reflect
24 disproportionate resources spent
25 investigating them fU.S. EPA. 2005a. 32.4.3.3).
26 It should be noted that in clinical reviews, the
27 credibility of a series of studies is reduced if
28 evidence is limited to studies funded by one
29 interested sector [Guyattetal.. 2008a).
30 For cancer, the assessment evaluates
31 evidence of a mutagenic mode of action to
32 guide extrapolation to lower doses and
33 consideration of susceptible lifestages. Key
34 data include the ability of the agent or a
35 metabolite to react with or bind to DNA,
36 positive results in multiple test systems, or
37 similar properties and structure-activity
38 relationships to mutagenic carcinogens [U.S.
39 EPA. 2005a.32.3.51.
40 5.5. Characterizing the overall weight
41 of the evidence
42 After evaluating the human, animal, and
43 mechanistic evidence pertinent to an effect,
44 the assessment answers the question: Does
45 the agent cause the adverse effect? [NRG,
46 2009. 1983). In doing this, the assessment
47 develops a narrative that integrates the
48 evidence pertinent to causation. To provide
49 clarity and consistency, the narrative includes
50 a standard hazard descriptor. For example, the
51 following standard descriptors combine
52 epidemiologic, experimental, and mechanistic
53 evidence of carcinogenicity [U.S. EPA. 2005a.
54 §2,5).
55 Carcinogenic to humans: There is convincing
56 epidemiologic evidence of a causal
57 association (that is, there is reasonable
58 confidence that the association cannot be
59 fully explained by chance, bias, or
60 confounding); or there is strong human
61 evidence of cancer or its precursors,
62 extensive animal evidence, identification
63 of key precursor events in animals, and
64 strong evidence that they are anticipated
65 to occur in humans.
66 Likely to be carcinogenic to humans: The
67 evidence demonstrates a potential hazard
68 to humans but does not meet the criteria
69 for carcinogenic. There may be a plausible
70 association in humans, multiple positive
71 results in animals, or a combination of
72 human, animal, or other experimental
73 evidence.
74 Suggestive evidence of carcinogenic
75 potential: The evidence raises concern for
76 effects in humans but is not sufficient for a
77 stronger conclusion. This descriptor
78 covers a range of evidence, from a positive
79 result in the only available study to a single
80 positive result in an extensive database
81 that includes negative results in other
82 species.
83 Inadequate information to assess
84 carcinogenic potential: No other
85 descriptors apply. Conflicting evidence can
86 be classified as inadequate information if
87 all positive results are opposed by
88 negative studies of equal quality in the
89 same sex and strain. Differing results,
90 however, can be classified as suggestive
91 evidence or as likely to be carcinogenic.
92 Not likely to be carcinogenic to humans:
93 There is robust evidence for concluding
94 that there is no basis for concern. There
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1 may be no effects in both sexes of at least
2 two appropriate animal species; positive
3 animal results and strong, consistent
4 evidence that each mode of action in
5 animals does not operate in humans; or
6 convincing evidence that effects are not
7 likely by a particular exposure route or
8 below a defined dose.
9 Multiple descriptors may be used if there
10 is evidence that carcinogenic effects differ by
11 dose range or exposure route [U.S. EPA. 2005a.
12 §2,5).
13 Another example of standard descriptors
14 comes from the EPA's Integrated Science
15 Assessments, which evaluate causation for the
16 effects of the criteria pollutants in ambient air
17 fU.S. EPA. 2010. 31.61.
18 Causal relationship: Sufficient evidence to
19 conclude that there is a causal
20 relationship. Observational studies cannot
21 be explained by plausible alternatives, or
22 they are supported by other lines of
23 evidence, for example, animal studies or
24 mechanistic information.
25 Likely to be a causal relationship: Sufficient
26 evidence that a causal relationship is
27 likely, but important uncertainties remain.
28 For example, observational studies show
29 an association but co-exposures are
30 difficult to address or other lines of
31 evidence are limited or inconsistent; or
32 multiple animal studies from different
33 laboratories demonstrate effects and
34 there are limited or no human data.
35 Suggestive of a causal relationship: At least
36 one high-quality epidemiologic study
37 shows an association but other studies are
38 inconsistent
39 Inadequate to infer a causal relationship:
40 The studies do not permit a conclusion
41 regarding the presence or absence of an
42 association.
43 Not likely to be a causal relationship: Several
44 adequate studies, covering the full range of
45 human exposure and considering
46 susceptible populations, are mutually
47 consistent in not showing an effect at any
48 level of exposure.
49 The EPA is investigating and may on a trial
50 basis use these or other standard descriptors
51 to characterize the overall weight of the
52 evidence for effects other than cancer.
53 6. Selecting studies for derivation of
54 toxicity values
55 For each effect where there is credible
56 evidence of an association with the agent, the
57 assessment derives toxicity values if there are
58 suitable epidemiologic or experimental data.
59 The decision to derive toxicity values may be
60 linked to the hazard descriptor.
61 Dose-response analysis requires quantitative
62 measures of dose and response. Then, other
63 factors being equal:
64 - Epidemiologic studies are preferred
65 over animal studies, if quantitative
66 measures of exposure are available
67 and effects can be attributed to the
68 agent.
69 - Among experimental animal models,
70 those that respond most like humans
71 are preferred, if the comparability of
72 response can be determined.
73 - Studies by a route of human
74 environmental exposure are
75 preferred, although a validated
76 toxicokinetic model can be used to
77 extrapolate across exposure routes.
78 - Studies of longer exposure duration
79 and follow-up are preferred, to
80 minimize uncertainty about whether
81 effects are representative of lifetime
82 exposure.
83 - Studies with multiple exposure levels
84 are preferred for their ability to
85 provide information about the shape
86 of the exposure-response curve.
87 - Studies with adequate power to detect
88 effects at lower exposure levels are
89 preferred, to minimize the extent of
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extrapolation to levels found in the
environment
3 Studies with non-monotonic exposure-
4 response relationships are not necessarily
5 excluded from the analysis. A diminished
6 effect at higher exposure levels may be
7 satisfactorily explained by factors such as
8 competing toxicity, saturation of absorption or
9 metabolism, exposure misclassification, or
10 selection bias.
11 If a large number of studies are suitable for
12 dose-response analysis, the assessment
13 considers the study characteristics in this
14 section to focus on the most informative data.
15 The assessment explains the reasons for not
16 analyzing other groups of studies. As a check
17 on the selection of studies for dose-response
18 analysis, the EPA asks peer reviewers to
19 identify studies that were not adequately
20 considered.
21 7. Deriving toxicity values
22 7.1. General framework for dose-
23 response analysis
24 The EPA uses a two-step approach that
25 distinguishes analysis of the observed dose-
26 response data from inferences about lower
27 doses fU.S. EPA. 2005a. §31
28 Within the observed range, the preferred
29 approach is to use modeling to incorporate a
30 wide range of data into the analysis. The
31 modeling yields a point of departure (an
32 exposure level near the lower end of the
33 observed range, without significant
34 extrapolation to lower doses) (Sections 7.2-
35 7.3).
36 Extrapolation to lower doses considers
37 what is known about the modes of action for
38 each effect (Sections 7.4-7.5). If response
39 estimates at lower doses are not required, an
40 alternative is to derive reference values, which
41 are calculated by applying factors to the point
42 of departure in order to account for sources of
43 uncertainty and variability (Section 7.6).
44 For a group of agents that induce an effect
45 through a common mode of action, the dose-
46 response analysis may derive a relative
47 potency factor for each agent A full dose-
48 response analysis is conducted for one well-
49 studied index chemical in the group, then the
50 potencies of other members are expressed in
51 relative terms based on relative toxic effects,
52 relative absorption or metabolic rates,
53 quantitative structure-activity relationships,
54 or receptor binding characteristics (U.S. EPA.
55 2005a. §3.2.6: 2000b. §4.4).
56 Increasingly, the EPA is basing toxicity
57 values on combined analyses of multiple data
58 sets or multiple responses. The EPA also
59 considers multiple dose-response approaches
60 if they can be supported by robust data.
61 7.2. Modeling dose to sites of biologic
62 effects
63 The preferred approach for analysis of
64 dose is toxicokinetic modeling because of its
65 ability to incorporate a wide range of data. The
66 preferred dose metric would refer to the
67 active agent at the site of its biologic effect or
68 to a close, reliable surrogate measure. The
69 active agent may be the administered chemical
70 or a metabolite. Confidence in the use of a
71 toxicokinetic model depends on the
72 robustness of its validation process and on the
73 results of sensitivity analyses (U.S. EPA.
74 2006a: 2005a. §3.1: 1994. §4.3).
75 Because toxicokinetic modeling can
76 require many parameters and more data than
77 are typically available, the EPA has developed
78 standard approaches that can be applied to
79 typical data sets. These standard approaches
80 also facilitate comparison across exposure
81 patterns and species.
82 - Intermittent study exposures are
83 standardized to a daily average over
84 the duration of exposure. For chronic
85 effects, daily exposures are averaged
86 over the lifespan. Exposures during a
87 critical period, however, are not
88 averaged over a longer duration (U.S.
89 EPA. 2005a. §3.1.1: 1991b.§3.2).
90 - Doses are standardized to equivalent
91 human terms to facilitate comparison
92 of results from different species.
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1 - Oral doses are scaled allometrically
2 using mg/kg3/4-day as the equivalent
3 dose metric across species. Allometric
4 scaling pertains to equivalence across
5 species, not across lifestages, and is
6 not used to scale doses from adult
7 humans or mature animals to infants
8 or children [U.S. EPA.
9 2011: 2005a. 33.1.3).
10 - Inhalation exposures are scaled using
11 dosimetry models that apply species-
12 specific physiologic and anatomic
13 factors and consider whether the
14 effect occurs at the site of first contact
15 or after systemic circulation [U.S. EPA.
16 2012a: 1994.331.
17 It can be informative to convert doses
18 across exposure routes. If this is done, the
19 assessment describes the underlying data,
20 algorithms, and assumptions [U.S. EPA.
21 2005a. 33.1.41.
22 In the absence of study-specific data on,
23 for example, intake rates or body weight, the
24 EPA has developed recommended values for
25 use in dose-response analysis [U.S. EPA.
26 19881
27 7.3. Modeling response in the range of
28 observation
29 Toxicodynamic ("biologically based")
30 modeling can incorporate data on biologic
31 processes leading to an effect. Such models
32 require sufficient data to ascertain a mode of
33 action and to quantitatively support model
34 parameters associated with its key events.
35 Because different models may provide
36 equivalent fits to the observed data but
37 diverge substantially at lower doses, critical
38 biologic parameters should be measured from
39 laboratory studies, not by model fitting.
40 Confidence in the use of a toxicodynamic
41 model depends on the robustness of its
42 validation process and on the results of
43 sensitivity analyses. Peer review of the
44 scientific basis and performance of a model is
45 essential [U.S. EPA. 2005a. §3.2.2).
46 Because toxicodynamic modeling can
47 require many parameters and more
48 knowledge and data than are typically
49 available, the EPA has developed a standard
50 set of empirical ("curve-fitting") models
51 (http://www.epa.gov/ncea/bmds/) that can
52 be applied to typical data sets, including those
53 that are nonlinear. The EPA has also developed
54 guidance on modeling dose-response data,
55 assessing model fit, selecting suitable models,
56 and reporting modeling results (U.S. EPA.
57 2012b). Additional judgment or alternative
58 analyses are used if the procedure fails to yield
59 reliable results, for example, if the fit is poor,
60 modeling may be restricted to the lower doses,
61 especially if there is competing toxicity at
62 higher doses (U.S. EPA. 2005a. §3.2.3).
63 Modeling is used to derive a point of
64 departure fU.S. EPA. 2012b: 2005a. 33.2.41.
65 (See Section 7.6 for alternatives if a point of
66 departure cannot be derived by modeling.):
67 - If linear extrapolation is used,
68 selection of a response level
69 corresponding to the point of
70 departure is not highly influential, so
71 standard values near the low end of
72 the observable range are generally
73 used (for example, 10% extra risk for
74 cancer bioassay data, 1% for
75 epidemiologic data, lower for rare
76 cancers).
77 - For nonlinear approaches, both
78 statistical and biologic considerations
79 are taken into account.
80 - For dichotomous data, a response level
81 of 10% extra risk is generally used for
82 minimally adverse effects, 5% or
83 lower for more severe effects.
84 - For continuous data, a response level
85 is ideally based on an established
86 definition of biologic significance. In
87 the absence of such definition, one
88 control standard deviation from the
89 control mean is often used for
90 minimally adverse effects, one-half
91 standard deviation for more severe
92 effects.
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1 The point of departure is the 95% lower
2 bound on the dose associated with the
3 selected response level.
4 7.4. Extrapolating to lower doses and
5 response levels
6 The purpose of extrapolating to lower
7 doses is to estimate responses at exposures
8 below the observed data. Low-dose
9 extrapolation, typically used for cancer data,
10 considers what is known about modes of
11 action [U.S. EPA. 2005a. 33.3.1 and 33.3.21.
12 1) If a biologically based model has been
13 developed and validated for the agent,
14 extrapolation may use the fitted model
15 below the observed range if significant
16 model uncertainty can be ruled out with
17 reasonable confidence.
18 2) Linear extrapolation is used if the dose-
19 response curve is expected to have a
20 linear component below the point of
21 departure. This includes:
22 - Agents or their metabolites that are
23 DNA-reactive and have direct
24 mutagenic activity.
25 - Agents or their metabolites for which
26 human exposures or body burdens are
27 near doses associated with key events
28 leading to an effect
29 Linear extrapolation is also used when
30 data are insufficient to establish mode of
31 action and when scientifically plausible.
32 The result of linear extrapolation is
33 described by an oral slope factor or an
34 inhalation unit risk, which is the slope of
35 the dose-response curve at lower doses or
36 concentrations, respectively.
37 3) Nonlinear models are used for
38 extrapolation if there are sufficient data to
39 ascertain the mode of action and to
40 conclude that it is not linear at lower
41 doses, and the agent does not demonstrate
42 mutagenic or other activity consistent
43 with linearity at lower doses. Nonlinear
44 approaches generally should not be used
45 in cases where mode of action has not
46 ascertained. If nonlinear extrapolation is
47 appropriate but no model is developed, an
48 alternative is to calculate reference values.
49 4) Both linear and nonlinear approaches may
50 be used if there a multiple modes of action.
51 For example, modeling to a low response
52 level can be useful for estimating the
53 response at doses where a high-dose mode
54 of action would be less important.
55 If linear extrapolation is used, the
56 assessment develops a candidate slope factor
57 or unit risk for each suitable data set. These
58 results are arrayed, using common dose
59 metrics, to show the distribution of relative
60 potency across various effects and
61 experimental systems. The assessment then
62 derives or selects an overall slope factor and
63 an overall unit risk for the agent, considering
64 the various dose-response analyses, the study
65 preferences discussed in Section 6, and the
66 possibility of basing a more robust result on
67 multiple data sets.
68 7.5. Considering susceptible
69 populations and lifestages
70 The assessment analyzes the available
71 information on populations and lifestages that
72 may be particularly susceptible to each effect.
73 A tiered approach is used [U.S. EPA.
74 2005a. 33.51.
75 1) If an epidemiologic or experimental study
76 reports quantitative results for a
77 susceptible population or lifestage, these
78 data are analyzed to derive separate
79 toxicity values for susceptible individuals.
80 2) If data on risk-related parameters allow
81 comparison of the general population and
82 susceptible individuals, these data are
83 used to adjust the general-population
84 toxicity values for application to
85 susceptible individuals.
86 3) In the absence of chemical-specific data,
87 the EPA has developed age-dependent
88 adjustment factors for early-life exposure
89 to potential carcinogens that have a
90 mutagenic mode of action. There is
91 evidence of early-life susceptibility to
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1 various carcinogenic agents, but most
2 epidemiologic studies and cancer
3 bioassays do not include early-life
4 exposure. To address the potential for
5 early-life susceptibility, the EPA
6 recommends fU.S. EPA. 2005b. 551
7 - 10-fold adjustment for exposures
8 before age 2 years.
9 - 3-fold adjustment for exposures
10 between ages 2 and 16 years.
11 7.6. Reference values and uncertainty
12 factors
13 An oral reference dose or an inhalation
14 reference concentration is an estimate of an
15 exposure (including in susceptible subgroups)
16 that is likely to be without an appreciable risk
17 of adverse health effects over a lifetime [U.S.
18 EPA. 2002. §4.2]. Reference values are
19 typically calculated for effects other than
20 cancer and for suspected carcinogens if a well
21 characterized mode of action indicates that a
22 necessary key event does not occur below a
23 specific dose. Reference values provide no
24 information about risks at higher exposure
25 levels.
26 The assessment characterizes effects that
27 form the basis for reference values as adverse,
28 considered to be adverse, or a precursor to an
29 adverse effect For developmental toxicity,
30 reproductive toxicity, and neurotoxicity there
31 is guidance on adverse effects and their
32 biologic markers [U.S. EPA,
33 1998b. 1996.1991b).
34 To account for uncertainty and variability
35 in the derivation of a lifetime human exposure
36 where adverse effects are not anticipated to
37 occur, reference values are calculated by
38 applying a series of uncertainty factors to the
39 point of departure. If a point of departure
40 cannot be derived by modeling, a no-
41 observed-adverse-effect level or a lowest-
42 observed-adverse-effect level is used instead.
43 The assessment discusses scientific
44 considerations involving several areas of
45 variability or uncertainty.
46 Human variation. The assessment accounts
47 for variation in susceptibility across the
48 human population and the possibility that
49 the available data may not be
50 representative of individuals who are
51 most susceptible to the effect A factor of
52 10 is generally used to account for this
53 variation. This factor is reduced only if the
54 point of departure is derived or adjusted
55 specifically for susceptible individuals
56 (not for a general population that includes
57 both susceptible and non-susceptible
58 individuals) (U.S. EPA.
59 2002.54.4.5: 1998b.54.2: 1996.54: 1994.
60 54.3.9.1: 1991b. 53.4).
61 Animal-to-human extrapolation. If animal
62 results are used to make inferences about
63 humans, the assessment adjusts for cross-
64 species differences. These may arise from
65 differences in toxicokinetics or
66 toxicodynamics. Accordingly, if the point
67 of departure is standardized to equivalent
68 human terms or is based on toxicokinetic
69 or dosimetry modeling, a factor of 101/2
70 (rounded to 3) is applied to account for the
71 remaining uncertainty involving
72 toxicokinetic and toxicodynamic
73 differences. If a biologically based model
74 adjusts fully for toxicokinetic and
75 toxicodynamic differences across species,
76 this factor is not used. In most other cases,
77 a factor of 10 is applied (U.S. EPA.
78 2011: 2002.54.4.5: 1998b. 54.2: 1996.54:
79 1994.54.3.9.1: 1991b. 53.41
80 Adverse-effect level to no-observed-
81 adverse-effect level. If a point of
82 departure is based on a lowest-observed-
83 adverse-effect level, the assessment must
84 infer a dose where such effects are not
85 expected. This can be a matter of great
86 uncertainty, especially if there is no
87 evidence available at lower doses. A factor
88 of 10 is applied to account for the
89 uncertainty in making this inference. A
90 factor other than 10 may be used,
91 depending on the magnitude and nature of
92 the response and the shape of the dose-
93 response curve (U.S. EPA.
94 2002.54.4.5: 1998b.54.2: 1996.54: 1994.
95 54.3.9.1: 1991b. 53.41
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1 Subchronic-to-chronic exposure. If a point
2 of departure is based on subchronic
3 studies, the assessment considers whether
4 lifetime exposure could have effects at
5 lower levels of exposure. A factor of 10 is
6 applied to account for the uncertainty in
7 using subchronic studies to make
8 inferences about lifetime exposure. This
9 factor may also be applied for
10 developmental or reproductive effects if
11 exposure covered less than the full critical
12 period. A factor other than 10 may be used,
13 depending on the duration of the studies
14 and the nature of the response [U.S. EPA,
15 2002.54.4.5: 1998b.54.2: 1994.34.3.9.11.
16 Incomplete database. If an incomplete
17 database raises concern that further
18 studies might identify a more sensitive
19 effect, organ system, or lifestage, the
20 assessment may apply a database
21 uncertainty factor [U.S. EPA.
22 2002.54.4.5: 1998b.54.2: 1996.54: 1994.
23 54.3.9.1: 1991b. 53.4). The size of the
24 factor depends on the nature of the
25 database deficiency. For example, the EPA
26 typically follows the suggestion that a
27 factor of 10 be applied if both a prenatal
28 toxicity study and a two-generation
29 reproduction study are missing and a
30 factor of 101/2 if either is missing (U.S.
31 EPA. 2002. §4.4.5).
32 In this way, the assessment derives
33 candidate values for each suitable data set and
34 effect that is credibly associated with the
35 agent These results are arrayed, using
36 common dose metrics, to show where effects
37 occur across a range of exposures [U.S. EPA.
38 1994.54.3.91
39 The assessment derives or selects an
40 organ- or system-specific reference value for
41 each organ or system affected by the agent.
42 The assessment explains the rationale for each
43 organ/system-specific reference value (based
44 on, for example, the highest quality studies,
45 the most sensitive outcome, or a clustering of
46 values). By providing these organ/system-
47 specific reference values, IRIS assessments
48 facilitate subsequent cumulative risk
49 assessments that consider the combined effect
50 of multiple agents acting at a common site or
51 through common mechanisms [NRG, 2009].
52 The assessment then selects an overall
53 reference dose and an overall reference
54 concentration for the agent to represent
55 lifetime human exposure levels where effects
56 are not anticipated to occur. This is generally
57 the most sensitive organ/system-specific
58 reference value, though consideration of study
59 quality and confidence in each value may lead
60 to a different selection.
61 7.7. Confidence and uncertainty in the
62 reference values
63 The assessment selects a standard
64 descriptor to characterize the level of
65 confidence in each reference value, based on
66 the likelihood that the value would change
67 with further testing. Confidence in reference
68 values is based on quality of the studies used
69 and completeness of the database, with more
70 weight given to the latter. The level of
71 confidence is increased for reference values
72 based on human data supported by animal
73 data fU.S. EPA. 1994. 54.3.9.21
74 High confidence: The reference value is not
75 likely to change with further testing,
76 except for mechanistic studies that might
77 affect the interpretation of prior test
78 results.
79 Medium confidence: This is a matter of
80 judgment, between high and low
81 confidence.
82 Low confidence: The reference value is
83 especially vulnerable to change with
84 further testing.
85 These criteria are consistent with
86 guidelines for systematic reviews that
87 evaluate the quality of evidence. These also
88 focus on whether further research would be
89 likely to change confidence in the estimate of
90 effect fGuyatt et al.. 2008bl
91 All assessments discuss the significant
92 uncertainties encountered in the analysis. The
93 EPA provides guidance on characterization of
94 uncertainty [U.S. EPA. 2005a. 53.6]. For
95 example, the discussion distinguishes model
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1 uncertainty (lack of knowledge aboutthe most 7 susceptibility or in exposures that modify the
2 appropriate experimental or analytic model) 8 effects of the agent).
3 and parameter uncertainty (lack of knowledge 9
4 about the parameters of a model). 10
5 Assessments also discuss human variation 11 August2013
6 (interpersonal differences in biologic
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EXECUTIVE SUMMARY
3 Summation of Occurrence and Health Effects
4 tert-Butanol does not occur naturally; it is produced by humans for multiple
5 purposes, such as a solvent for paints, a denaturant for ethanol and several other alcohols, a
6 dehydrating agent, and in the manufacture of flotation agents, fruit essences, and perfumes.
7 tert-Butanol also is a primary metabolite of methyl tert-butyl ether (MTBE) and ethyl tert-
8 butyl ether (ETBE). Exposure to tert-butanol primarily occurs through breathing air
9 containing tert-butanol vapors and consuming contaminated water or foods. Exposure can
10 also occur through direct skin contact.
11 Animal studies demonstrate that chronic oral exposure to tert-butanol is associated
12 with kidney and thyroid effects. Developmental effects (e.g., reduced fetal viability) have
13 been observed in short-term exposure to high levels of tert-butanol (via oral or inhalation
14 exposure) in animals. Neurodevelopmental effects also have been observed, but results
15 were inconsistent. No chronic inhalation exposure studies have been conducted. There is
16 suggestive evidence that tert-butanol is carcinogenic to humans based on renal tumors in
17 male rats and thyroid tumors in female mice.
18 Effects Other Than Cancer Observed Following Oral Exposure
19 Kidney effects are a potential human hazard of oral exposure to tert-butanol. Kidney toxicity
20 was observed in males and females in two strains of rats. Kidney weights were increased in male
21 and female rats after 13 weeks or 15 months of treatment. Histopathological examination in male
22 and female rats observed increased incidence or severity of nephropathy after 13 weeks of oral
23 exposure, increased severity of nephropathy after a 2-year oral exposure, and increased
24 transitional epithelial hyperplasia after 2 years of oral exposure. Additionally, increased
25 suppurative inflammation was noted in females after 2 years of oral exposure. In one strain of mice,
26 the only kidney effect observed was an increase in kidney weight (absolute or relative) in female
27 mice after 13 weeks, but no treatment-related histopathological lesions were reported in the
28 kidneys of male or female mice at 13 weeks or 2 years. A mode of action (MOA) analysis determined
29 that tert-butanol exposure induces a male rat-specific (X2u-globulin-associated nephropathy. tert-
30 Butanol, however, is a weak inducer of (X2u-globulin-nephropathy, and is not the sole process
31 contributing to renal tubule nephropathy. Chronic progressive nephropathy (CPN) may also be
32 involved in some of the noncancer effects, but the evidence is inconclusive. Endpoints specifically
33 related to either a2u-globulin-nephropathy or CPN were not considered for kidney hazard
34 identification. Changes in kidney weights, transitional epithelial hyperplasia, suppurative
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
inflammation, and severity and incidence of nephropathy, however, are considered to result from
tert-butanol exposure and are appropriate for identifying a hazard to the kidney.
There is suggestive evidence of developmental toxicity following tert-butanol exposure.
Developmental effects include increased fetal loss, decreased fetal body weight, and increased
skeletal variations. At this time, no conclusions were drawn in regard to reproductive system
toxicity. There is inadequate information at this time to draw conclusions regarding
neurodevelopmental toxicity, liver, and urinary bladder toxicity.
Oral Reference Dose (RfD) for Effects Other Than Cancer
Kidney toxicity, represented by kidney transitional epithelial hyperplasia, was chosen as the
basis for the overall oral reference dose (RfD) (see Table ES-1). The chronic study by NTP T19951
and the observed kidney effects were used to derive the RfD. The endpoint of transitional epithelial
hyperplasia was selected as the critical effect because it was observed in both rat sexes
consistently, it is a specific and sensitive indicator of kidney toxicity, and was induced in a dose-
responsive manner. Benchmark dose (BMD) modeling was used to derive the benchmark dose
lower confidence limit (BMDLio%) of 16 mg/kg-day. The BMDL was converted to a human
equivalent dose using body weight3/4 scaling, and the value of 3.84 mg/kg-day was used as the
point of departure (POD) for RfD derivation (U.S. EPA. 2011).
The overall RfD was calculated by dividing the POD for kidney transitional epithelial
hyperplasia by a composite uncertainty factor (UF) of 30 to account for the extrapolation from
animals to humans (3) and for interindividual differences in human susceptibility (10).
Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol
Hazard
Kidney
Overall RfD
Basis
Transitional epithelial
hyperplasia
Kidney
Point of
departure*
(mg/kg-day)
3.8
3.8
UF
30
30
Chronic RfD
(mg/kg-day)
1 x 10'1
1 x ID'1
Study
exposure
description
Chronic
Chronic
Confidence
High
High
22 *HED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
23 Effects Other Than Cancer Observed Following Inhalation Exposure
24 Kidney effects are a potential human hazard of inhalation exposure to tert-butanol.
25 Although no effects were observed in mice, kidney weights were increased in male and female rats
26 following 13 weeks of inhalation exposure. In addition, nephropathy severity increased in male
27 rats. No human studies are available to evaluate the effects of inhalation exposure. As discussed
28 above for oral effects, endpoints specifically related to either (X2uglobulin nephropathy or CPN were
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
not considered for kidney hazard identification. Changes in kidney weights and severity of
nephropathy, however, are considered a result of tert-butanol exposure and are appropriate for
identifying a hazard to the kidney.
Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
Kidney toxicity, represented by transitional epithelial hyperplasia, was chosen as the basis
for the inhalation reference concentration (RfC) (see Table ES-2). Although endpoints from a route-
specific study were considered, the availability of a physiologically based pharmacokinetic (PBPK)
model for tert-butanol in rats [modified by Salazar etal. (2015]] allowed for more specific and
sensitive equivalent inhalation PODs derived from a route-to-route extrapolation from the PODs of
the oral NTP (1995) study. The POD adjusted for the human equivalent concentration (HEC) was
26.1 mg/m3 based on transitional epithelial hyperplasia.
The RfC was calculated by dividing the POD by a composite UF of 30 to account for
toxicodynamic differences between animals and humans (3) and interindividual differences in
human susceptibility (10).
Table ES-2. Organ/system-specific RfCs and overall RfC for tert-butanol
Hazard
Kidney
Overall RfC
Basis
Transitional epithelial
hyperplasia
Kidney
Point of
departure*
(mg/m3)
26.1
26.1
UF
30
30
Chronic RfC
(mg/m3)
9 x 10'1
9 x ID'1
Study exposure
description
Chronic
Chronic
Confidence
High
High
16
17
18
19
20
21
22
23
*Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
tert-butanol as continuous oral exposure at the BMDL
Evidence of Human Carcinogenicity
Under the EPA's cancer guidelines (U.S. EPA, 2005a], there is suggestive evidence of
carcinogenic potential for tert-butanol. tert-Butanol induced kidney tumors in male (but not female)
rats and thyroid tumors (primarily benign) in male and female mice following long-term
administration in drinking water (NTP, 1995). The potential for carcinogenicity applies to all routes
of human exposure.
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Toxicological Review of tert-ButyI Alcohol
1 Quantitative Estimate of Carcinogenic Risk from Oral Exposure
2 A quantitative estimate of carcinogenic potential from oral exposure to tert-butanol was
3 based on the increased incidence of thyroid follicular cell adenomas in female B6C3Fi mice, and
4 thyroid follicular cell adenomas and carcinomas in male B6C3Fi mice [NTP, 1995]. The study
5 included histological examinations for tumors in many different tissues, contained three exposure
6 levels and controls, contained adequate numbers of animals per dose group (~50/sex/group),
7 treated animals for up to 2 years, and included detailed reporting of methods and results.
8 Although tert-butanol was considered to have "suggestive evidence of carcinogenic
9 potential," the NTP study was well conducted and quantitative analysis could be useful for
10 providing a sense of the magnitude of potential carcinogenic risk [U.S. EPA. 2005a). A slope factor
11 was derived for thyroid tumors in female and male mice. The modeled tert-butanol POD was scaled
12 to HEDs according to EPA guidance by converting the BMDLio on the basis of (body weight)3/4
13 scaling [U.S. EPA. 2011. 2005a). Using linear extrapolation from the BMDLio, a human equivalent
14 oral slope factor was derived (slope factor = 0.1/BMDLio). The resulting oral slope factor is 5 x 1Q-*
15 per mg/kg-day.
16 Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
17 No chronic inhalation exposure studies to tert-butanol are available. Lifetime exposure to
18 tert-butanol has been associated with increased renal tubule adenomas and carcinomas as well as
19 thyroid follicular cell adenomas and carcinomas. As stated above, the rat kidney tumors are
20 unsuitable for quantitative analysis as there is not enough data to determine the relative
21 contribution of azu-globulin nephropathy and other processes to the overall kidney tumor
22 response. Although the mouse thyroid tumors served as the basis for the oral slope factor, route-to-
23 route extrapolation is not possible for these thyroid effects in mice because the only PBPK model
24 available is for rats. Therefore, no quantitative estimate of carcinogenic risk could be determined
25 for inhalation exposure.
26 Susceptible Populations and Lifestages for Cancer and Noncancer
27 In vitro studies suggest that cytochrome P-450 (CYP450) (Cederbaum etal., 1983:
28 Cederbaum and Cohen, 1980], plays a role in the metabolism of tert-butanol. No studies, however,
29 have identified the specific CYPs responsible for the biotransformation of tert-butanol. Various
30 CYPs are under-expressed in the mouse fetus and neonate (Lee etal.. 2011] and decreased in older
31 mice (Lee etal., 2011] and rats (Lee etal., 2008). Decreased ability to detoxify and transport tert-
32 butanol out of the body could result in increased susceptibility to tert-butanol.
33 With regard to cancer, differences in lifestage sensitivity to chemically induced thyroid
34 carcinogenesis are unknown (U.S. EPA. 1998a]. An increased incidence of thyroid tumors was
35 identified in mice after tert-butanol exposure, and human studies have demonstrated that children
36 are more sensitive than adults are to thyroid carcinogenesis resulting from ionizing radiation.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-ButyI Alcohol
1 Collectively, there is little evidence on tert-butanol itself to identify any populations or lifestages
2 that may be especially susceptible.
3 Key Issues Addressed in Assessment
4 An evaluation of whether tert-butanol caused (X2u-globulin-associated nephropathy was
5 performed. The presence of (X2u-globulin in the hyaline droplets was confirmed in male rats by
6 (X2u-globulin immunohistochemical staining. Linear mineralization and tubular hyperplasia were
7 reported in male rats, although only in the chronic study. Other subsequent steps in the
8 pathological sequence, including necrosis, exfoliation, and granular casts, either were absent or
9 inconsistently observed across subchronic or chronic studies. None of these effects occurred in
10 female rats or in either sex of mice, although these endpoints were less frequently evaluated in
11 these models. Evidence implies an a2u-globulin MOA is operative, although it is relatively weak in
12 response to tert-butanol and is not solely responsible for the renal tubule nephropathy observed in
13 male rats. CPN also is instrumental in renal tubule nephropathy, in both male and female rats.
14 Several other effects in the kidney unrelated to (X2u-globulin or CPN, however, were observed in
15 female or male rats [U.S. EPA. 1991a). including suppurative inflammation in female rats,
16 transitional epithelial hyperplasia in male and female rats, and increased kidney weights in both
17 sexes of rats [NTP. 1997.1995). These specific effects are considered the result of tert-butanol
18 exposure and therefore, relevant to humans.
19 Concerning cancer, (X2u-globulin accumulation is indicated as relatively weak in response to
20 tert-butanol exposure and not the sole mechanism responsible for the renal tubule carcinogenicity
21 observed in male rats. Although CPN and other effects induced by both (X2u-globulin processes and
22 tert-butanol play a role in renal tubule nephropathy, the evidence indicates that CPN does not
23 induce the renal tubule tumors associated with tert-butanol exposure in male rats, suggesting that
24 other, unknown processes contribute to renal tumor development Based on this analysis of
25 available MOA data, these renal tumors are considered relevant to humans [U.S. EPA. 1991a).
26 In addition, an increase in the incidence of thyroid follicular cell adenomas was observed in
27 male and female mice in a 2-year drinking water study [NTP. 1995]. Thyroid follicular cell
28 hyperplasia was considered a preneoplastic effect associated with the thyroid tumors, and the
29 incidences of follicular cell hyperplasias were elevated in both male and female B6C3Fi mice
30 following exposure. U.S. EPA [1998a] describes the procedures the Agency uses in evaluating
31 potential human cancer hazard and dose-response assessments for chemicals that are animal
32 thyroid carcinogens. The available database is inadequate in four of the five required areas [U.S.
33 EPA. 1998a]. suggesting that an antithyroid MOA is not operating in mouse thyroid follicular cell
34 tumorigenesis. No other MOAs for thyroid tumors were identified, and the mouse thyroid tumors
35 are considered relevant to humans [U.S. EPA. 2005a. 1998a].
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Toxicological Review of tert-ButyI Alcohol
LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
4 A literature search and screening strategy were used to identify literature characterizing
5 the health effects of tert-butanol. This strategy consisted of a broad search of online scientific
6 databases and other sources to identify all potentially pertinent studies. In subsequent steps,
7 references were screened to exclude papers not pertinent to an assessment of the health effects of
8 tert-butanol, and remaining references were sorted into categories for further evaluation. This
9 section describes the literature search and screening strategy in detail.
10 The chemical-specific search was conducted in four online scientific databases, including
11 PubMed, Web of Science, Toxline, and TSCATS through May 2015, using the keywords and limits
12 described in Table LS-1. The overall literature search approach is shown graphically in Figure LS-1.
13 An additional seven citations were obtained using additional search strategies described in Table
14 LS-2. After electronically eliminating duplicates from the citations retrieved through these
15 databases, 2,648 unique citations were identified.
16 The resulting 2,648 citations were screened for pertinence and separated into categories as
17 presented in Figure LS-1 using the title and either abstract or full text, or both, to examine the
18 health effects of tert-butanol exposure. The inclusion and exclusion criteria used to screen the
19 references and identify sources of health effects data are provided in Table LS-3.
20 • 12 references were identified as "Sources of Health Effects Data" and were considered for
21 data extraction to evidence tables and exposure-response arrays.
22 • 200 references were identified as "Sources of Mechanistic and Toxicokinetic Data" and
23 "Sources of Supplementary Health Effects Data"; these included 39 studies describing
24 physiologically based pharmacokinetic (PBPK) models and other toxicokinetic information,
25 73 studies providing genotoxicity and other mechanistic information, 1 human case report,
26 74 irrelevant exposure paradigms (including acute, dermal, eye irritation, and injection
27 studies), 6 preliminary toxicity studies, and 7 physical dependency studies. Information
28 from these studies was not extracted into evidence tables; however, these studies were
29 considered as support for assessing tert-butanol health effects, for example, evaluation of
30 mode of action and extrapolation of experimental animal findings to humans. Additionally,
31 although still considered sources of health effects information, studies investigating the
32 effects of acute and direct chemical exposures are generally less pertinent for characterizing
33 health hazards associated with chronic oral and inhalation exposure. Therefore, information
34 from these studies was not considered for extraction into evidence tables. Nevertheless,
35 these studies were still evaluated as possible sources of supplementary health effects
36 information.
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Toxicological Review of tert-ButyI Alcohol
1 • 63 references were identified as "Secondary Literature and Sources of Contextual
2 Information" (e.g., reviews and other agency assessments); these references were retained
3 as additional resources for development of the Toxicological Review.
4 • 2,373 references were identified as not being pertinent (not on topic) to an evaluation of
5 the health effects of tert-butanol and were excluded from further consideration (see Figure
6 LS-1 for exclusion categories and Table LS-3 for exclusion criteria). For example, health
7 effect studies of gasoline and tert-butanol mixtures were not considered pertinent to the
8 assessment because the separate effects of the gasoline or other chemical components could
9 not be determined. Retrieving a large number of references that are not on topic is a
10 consequence of applying an initial search strategy designed to cast a wide net and to
11 minimize the possibility of missing potentially relevant health effects data.
12 The complete list of references and the sorting of these materials can be found on the tert-
13 butanol project page of the HERO website at
14 https://hero.epa.gov/index.cfm/project/page/project id/1543.
15 Selection of Studies for Inclusion in Evidence Tables
16 To summarize the important information systematically from the primary health effects
17 studies in the tert-butanol database, evidence tables were constructed in a standardized tabular
18 format as recommended by NRG (2011). Studies were arranged in evidence tables by effect, species,
19 duration, and design, and not by quality. Of the studies that were retained after the literature search
20 and screen, 12 studies were identified as "Sources of Health Effects Data" and were considered for
21 extraction into evidence tables for hazard identification in Chapter 1. Initial review found two
22 references fCirvello etal.. 1995: Lindamoodetal.. 19921 to be publications of the NTP T19951 data
23 prior to the release of the final National Toxicology Program (NTP) report One publication
24 (Takahashietal.. 1993) in the "Supplementary Studies" category also was based on data from the
25 NTP report The interim publications and the final NTP report differed. The finalized NTP (1995)
26 report was considered the more complete and accurate presentation of the data; therefore, this
27 report was included in evidence tables and Cirvello et al. (1995), Takahashietal. (1993), and
28 Lindamoodetal. (1992) were not Data from the remaining 10 references in the "Sources of Health
29 Effects Data" category were extracted into evidence tables.
30 Supplementary studies that contain pertinent information for the toxicological review and
31 augment hazard identification conclusions, such as genotoxic and mechanistic studies, studies
32 describing the kinetics and disposition of tert-butanol absorption and metabolism, pilot studies,
33 and one case report were not included in the evidence tables. Short-term and acute studies
34 (including an 18-day study and a 14-day study by NTP) using oral and inhalation exposures were
35 performed primarily in rats and did not differ qualitatively from the results of the longer studies
36 (i.e., >30-day exposure studies). These were grouped as supplementary studies, however, because
37 the database of chronic and subchronic rodent studies was considered sufficient for evaluating
38 chronic health effects of tert-butanol exposure. Additionally, studies of effects from chronic
39 exposure are most pertinent to lifetime human exposure (i.e., the primary characterization
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Toxicological Review of tert-Butyl Alcohol
1 provided by IRIS assessments) and are the focus of this assessment. Such supplementary studies
2 may be discussed in the narrative sections of Chapter 1 and are described in sections such as the
3 "Mode of Action Analysis" to augment the discussion or presented in appendices, if they provide
4 additional information.
Database Searches
(See Table LS-1 for keywords and limits)
PubMed
n = 1,741
Web of Science
n = 489
Toxline
(incl. TSCATS)
n = 964
TSCATS 2
n = 2
(After duplicates removed electronically)
n=2,641
Additional Search Strategies
(See Table LS-2 for methods and results)
n = 7
Combined Dataset
(After all duplicates removed)
n = 2,648
Manual Screening for Pertinence
(Title/Abstract/Full Text)
Excluded/Not on Topic (n = 2,310)
62 Abstract only/comment/society
abstracts
87 Biodegradation/environmental fate
85 Chemical analysis/fuel chemistry
1,286 Other chemical/non t-butanol
87 Method of detection/exposure and
biological monitoring
703 Methodology/solvent
Secondary Literature and Sources of
Contextual Information (n = 126}
41 Not relevant species/matrix (e.g.,
amphibians, fish)
14 QSAR
8 Mixtures
37 Reviews/editorials
13 Other agency assessments
13 Book chapter/section
Supporting Studies
Sources of Health Effects Data (n = 12)
0 Human health effects studies
12 Animal studies
Sources of Supporting Health Effects Data
{n = 88)
1 Human case reports
74 Not relevant exposure paradigms (e.g.,
dermal, eye irritation, acute)
6 Preliminary data
7 Physical dependency studies
Sources of Mechanistic and Toxicokinetic
Data (n = 112)
39 PBPK/ADME
22 Genotoxicity
51 Other mechanistic studies
5
6
Figure LS-1. Summary of literature search and screening process for
tert-butanol.
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Toxicological Review of tert-ButyI Alcohol
Table LS-1. Details of the search strategy employed for tert-butanol
Database
(Search Date)
Keywords
Limits
PubMed
(12/20/2012)
(4/17/2014)
(5/13/2015)
tert-butanol OR 75-65-0[rn] OR "t-
butyl hydroxide" OR "2-methyl-2-
propanol" OR "trimethyl carbinol"
OR "t-butyl alcohol" OR tert-butanol
OR "tert-butyl alcohol" OR tert-butyl
alcohol[mesh]
None
Web of Science
(12/20/2012)
(4/17/2014)
(5/13/2015)
Topic = (tert-butanol OR 75-65-0 OR
"t-butyl hydroxide" OR "2-methyl-2-
propanol" OR "trimethyl carbinol"
OR "t-butyl alcohol" OR "tert-
butanol" OR "tert-butyl alcohol")
Refined by: Research Areas = (cell biology OR
respiratory system OR microscopy OR biochemistry
molecular biology OR gastroenterology OR hepatology
OR public environmental occupational health OR
oncology OR physiology OR cardiovascular system
cardiology OR toxicology OR life sciences biomedicine
other topics OR hematology OR pathology OR
neurosciences neurology OR developmental biology)
Toxline (includes
TSCATS)
(1/11/2013)
(4/17/2014)
(5/13/2015)
tert-butanol OR 75-65-0 [rn] OR t-
butyl hydroxide OR 2-methyl-2-
propanol OR trimethyl carbinol OR t-
butyl alcohol OR tert-butanol OR
tert-butyl alcohol OR tert-butyl
alcohol
Not PubMed
TSCATS2
(1/4/2013)
(4/17/2014)
(5/13/2015)
75-65-0
None
Table LS-2. Summary of additional search strategies for tert-butanol
Approach used
Manual search of
citations from
reviews
Manual search of
citations from
reviews conducted
Source(s)
Review article: McGregor (2010).
Tert/o/y-butanol: A toxicological
review. Crit RevToxicol 40(8): 697-
727.
Review article: Chen (2005). Amended
final report of the safety assessment
of t-butyl alcohol as used in
cosmetics. Int J Toxicol 24(2): 1-20.
IPCS (1987a). Butanols: Four isomers:
1-butanol, 2-butanol, tert-butanol,
isobutanol [WHO EHC]. Geneva,
Date
performed
1/2013
1/2013
1/2013
Number of additional references
identified
5
2
None
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Toxicological Review of tert-ButyI Alcohol
Approach used
by other
international and
federal agencies
Source(s)
Switzerland: World Health
Organization.
OSHA (1992). Occupational safety and
health guideline for te/t-butyl alcohol.
Cincinnati, OH: Occupational Safety
and Health Administration.
Date
performed
1/2013
Number of additional references
identified
None
Table LS-3. Inclusion-exclusion criteria
Inclusion criteria
Exclusion criteria
Population
• Humans
• Standard mammalian animal models,
including rat, mouse, rabbit, guinea pig,
monkey, dog
• Ecological species*
• Nonmammalian species*
Exposure
• Exposure is to te/t-butanol
• Exposure is measured in an
environmental medium (e.g., air, water,
diet)
• Exposure via oral, inhalation, or dermal
routes
Study population is not exposed to te/t-butanol
Exposure to a mixture only (e.g., gasoline containing
te/t-butanol)
Exposure via injection (e.g., intravenous)
Exposure pattern less relevant to chronic health
effects (e.g., acute)
Outcome
Study includes a measure of one or
more health effect endpoints, including
effects on the nervous, musculoskeletal,
cardiovascular, immune, hematological,
endocrine, respiratory, urinary, and
gastrointestinal systems; reproduction;
development; liver; kidney; eyes; skin;
and cancer
Physical dependency studies where
withdrawal symptoms were evaluated
after removal of te/t-butanol treatment
Other
Not on topic, including:
• Abstract only, editorial comments were not
considered further because study was not
potentially relevant
• Bioremediation, biodegradation, or environmental
fate of te/t-butanol, including evaluation of
wastewater treatment technologies and methods
for remediation of contaminated water and soil
• Chemical, physical, or fuel chemistry studies
• Analytical methods for measuring/detecting/
remotely sensing te/t-butanol
• Use of te/t-butanol as a solvent or methodology for
testing unrelated to te/t-butanol
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of tert-ButyI Alcohol
Inclusion criteria
Exclusion criteria
• Not chemical specific: Studies that do not involve
testing of te/t-butanol
• Foreign language studies that were not considered
further because, based on title or abstract, judged
not potentially relevant
• QSAR studies
*Studies that met this exclusion criterion were not considered a source of health effects data or supplementary
health effects data/mechanistic and toxicokinetic data, but were considered as sources of contextual
information.
1 Database Evaluation
2 For this draft assessment, 12 references reported on experimental animal studies that
3 comprised the primary sources of health effects data; no studies were identified that evaluated
4 humans exposed to tert-butanol (e.g., cohort studies, ecological studies). The animal studies were
5 evaluated using the study quality considerations outlined in the Preamble, considering aspects of
6 design, conduct, or reporting that could affect the interpretation of results, overall contribution to
7 the synthesis of evidence, and determination of hazard potential as noted in various EPA guidance
8 documents [U.S. EPA. 2005a. 1998b. 1996.1991b). The objective was to identify the stronger, more
9 informative studies based on a uniform evaluation of quality characteristics across studies of
10 similar design. As stated in the Preamble, studies were evaluated to identify the suitability of the
11 study based on:
12 • Study design
13 • Nature of the assay and validity for its intended purpose
14 • Characterization of the nature and extent of impurities and contaminants of tert-butanol
15 administered, if applicable
16 • Characterization of dose and dosing regimen (including age at exposure) and their
17 adequacy to elicit adverse effects, including latent effects
18 • Sample sizes and statistical power to detect dose-related differences or trends
19 • Ascertainment of survival, vital signs, disease or effects, and cause of death
20 • Control of other variables that could influence the occurrence of effects
21 Additionally, several general considerations, presented in Table LS-4, were used in
22 evaluating the animal studies. Much of the key information for conducting this evaluation can be
23 determined based on study methods and how the study results were reported. Importantly, the
24 evaluation at this stage does not consider the direction or magnitude of any reported effects.
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Toxicological Review of tert-Butyl Alcohol
1 EPA considered statistical tests to evaluate whether the observations might be due to
2 chance. The standard for determining statistical significance of a response is a trend test or
3 comparison of outcomes in the exposed groups against those of concurrent controls. Studies that
4 did not report statistical testing were identified and, when appropriate, statistical tests were
5 conducted by EPA.
6 Information on study features related to this evaluation is reported in evidence tables and
7 documented in the synthesis of evidence. Discussion of study strengths and limitations were
8 included in the text where relevant If EPA's interpretation of a study differs from that of the study
9 authors, the draft assessment discusses the basis for the difference.
10 Experimental Animal Studies
11 The experimental animal studies, comprised entirely of studies performed in rats and mice,
12 were associated with drinking water, oral gavage, liquid diets (i.e., maltose/dextrin), and inhalation
13 exposures to tert-butanol. With the exception of neurodevelopmental studies, these sources were
14 conducted according to Organisation for Economic Co-operation and Development Good
15 Laboratory Practice (GLP) guidelines, presented extensive histopathological data, or clearly
16 presented their methodology; thus, these studies are considered high quality. These studies include
17 2-year bioassays using oral exposures in rats and mice; two subchronic drinking water studies in
18 rats and one in mice; an inhalation subchronic study in rats and mice; a reevaluation of the NTP
19 [1995] rat data; two oral developmental studies; two inhalation developmental studies; and a
20 single one-generation reproductive study that also evaluates other systemic effects (Table LS-5). A
21 more detailed discussion of any methodological concerns that were identified precedes each
22 endpoint evaluated in the hazard identification section. Overall, the experimental animal studies of
23 tert-butanol involving repeated oral or inhalation exposure were considered to be of acceptable
24 quality, and whether yielding positive, negative, or null results, were considered in assessing the
25 evidence for health effects associated with chronic exposure to tert-butanol.
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Table LS-4. Considerations for evaluation of experimental animal studies
Methodological
feature
Test animal
Experimental design
Exposure
Endpoint evaluation
Results presentation
Considerations
(relevant information extracted into evidence tables)
Suitability of the species, strain, sex, and source of the test animals
Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and
duration of exposure (e.g., hr/day, day/week); timing of endpoint evaluations; and
sample size and experimental unit (e.g., animals, dams, litters)
Characterization of test article source, composition, purity, and stability; suitability of the
control (e.g., vehicle control); documentation of exposure techniques (e.g., route,
chamber type, gavage volume); verification of exposure levels (e.g., consideration of
homogeneity, stability, analytical methods)
Suitability of specific methods for assessing the endpoint(s) of interest
Data presentation for endpoint(s) of interest (including measures of variability) and for
other relevant endpoints needed for results interpretation (e.g., maternal toxicity,
decrements in body weight relative to organ weight)
Table LS-5. Summary of experimental animal database
Study category
Chronic
Subchronic
Reproductive
Developmental
Neurodevelopmental
Study duration, species/strain, and administration method
2-year study in F344 rats (drinking water) NTP (1995)
2-year study in B6C3Fi mice (drinking water) NTP (1995)
13-week study in B6C3Fi mice (drinking water) NTP (1995)
13-week study in F344 rats (drinking water) NTP (1995)
13-week study in F344 rats (inhalation) NTP (1997)
13-week study in B6C3Fi mice (inhalation) NTP (1997)
10-week study in Wistar rats (drinking water) Acharya et al. (1997), Acharya et
One-generation reproductive toxicity study in Sprague-Dawley rats (gavage) L^
Chemical Co. (2004)
al. (1995)
ondell
Developmental study (GD 6-20) in Swiss Webster mice (diet) Daniel and Evans (1982)
Developmental study (GD 6-18) in CBA/J mice (drinking water) Faulkner et al. (1989)
Developmental study (GD 6-18) in C57BL/6J mice (drinking water) Faulkner et
Developmental study (GD 1-19) in Sprague-Dawley rats (inhalation) Nelson et
al. (1989)
al. (1989)
Neurodevelopmental study (GD 6-20) in Swiss Webster mice (diet) Daniel and Evans
(1982)
Neurodevelopmental study (GD 1-19) in Sprague-Dawley rats (inhalation) Nelson et al.
(1991)
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1. HAZARD IDENTIFICATION
2 1.1. Overview of Chemical Properties and Toxicokinetics
3 1.1.1. Chemical Properties
4 tert-Butanol is a white crystalline solid or colorless, highly flammable liquid (above 25.7°C)
5 with a camphor-like odor [NIOSH. 2005: IPCS. 1987a). tert-Butanol contains a hydroxyl chemical
6 functional group; is miscible with alcohol, ether, and other organic solvents; and is soluble in water
7 [IPCS. 1987a). Selected chemical and physical properties of tert-butanol are presented in Table 1-1.
Table 1-1. Physicochemical properties and chemical identity of tert-butanol
Characteristic
Chemical name
Synonyms/Trade names
Chemical formula
CASRN
Molecular weight
Melting point
Boiling point
Vapor pressure
Density/Specific gravity
Flashpoint
Water solubility at 25°C
Octanol/Water Partition
Coefficient (Log Kow )
Henry's Law Constant
Odor threshold
Conversion factors
Information
tert-Butanol
t-Butyl alcohol; tert-Butanol; tert-Butyl alcohol; t-
Butyl hydroxide; 1,1-Dimethylethanol; NCI-C55367;
2-Methyl-2-propanol; tertiary Butanol; Trimethyl
carbinol; Trimethyl methanol; t-butyl alcohol; TBA
C4HioO
75-65-0
74.12 ^^^ ^S
25.7°C
82.41°C
40.7 mm Hg @ 25°C
0.78581
11°C (closed cup)
1 x 10s mg/L
0.35
9.05 x 10"6 atm-m3/mole
219 mg/m3
1 ppm = 3.031 mg/m3
1 mg/m3 - 0.324 ppm
Reference
HSDB (2007)
HSDB (2007)
IPCS (1987b)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
HSDB (2007)
IPCS (1987b)
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Toxicological Review of tert-ButyI Alcohol
Characteristic
Chemical structure
Information
C
HO
(
;HS
mi
*Hg
Reference
HSDB (2007)
1 1.1.2. Toxicokinetics
2 tert-Butanol is rapidly absorbed following exposure by oral and inhalation routes (see
3 Appendix B, Section B.I.I). Studies in experimental animals indicate that 99% of the compound was
4 absorbed after oral administration. Comparable blood levels of tert-butanol and its metabolites
5 have also been observed after acute oral or inhalation exposures in rats [ARCO. 1983]. In another
6 study [Faulkner etal., 1989], blood concentrations indicated that absorption was complete at 1.5
7 hours following oral gavage doses of tert-butanol in female mice.
8 tert-Butanol is distributed throughout the body following oral, inhalation, and i.v. exposures
9 [Poet etal.. 1997: Faulkner etal.. 1989: ARCO. 1983]. Following exposure to tert-butanol in rats,
10 tert-butanol was found in kidney, liver, and blood, with male rats retaining more tert-butanol than
11 female rats [Williams and Borghoff. 2001].
12 A general metabolic scheme for tert-butanol, illustrating the biotransformation in rats and
13 humans, is shown in Figure 1-1 (see Appendix B.I.3].
14 Human data on the excretion of tert-butanol comes from studies of methyl tert-butyl ether
15 (MTBE) and ethyl tert-butyl ether (ETBE] fNihlenetal.. 1998a. b). The half-life of tert-butanol in
16 urine following MTBE exposure was 8.1 ± 2.0 hours (average of the 90.1- and 757-mg/m3 MTBE
17 doses]; the half-life of tert-butanol in urine following ETBE exposure was 7.9 ±2.7 hours (average
18 of 104- and 210-mg/m3 ETBE doses]. These studies reported urinary levels of tert-butanol (not
19 including downstream metabolites] to be less than 1% of administered MTBE or ETBE
20 concentrations (Nihlenetal., 1998a, b]. Ambergetal. (2000] also observed a similar half-life of
21 9.8 ± 1.4 hours after human exposure to ETBE of 170 mg/m3. The half-life for tert-butanol in rat
22 urine was 4.6 ± 1.4 hours at ETBE levels of 170 mg/m3.
23 A more detailed summary of tert-butanol toxicokinetics is provided in Appendix B,
24 Section B.I.
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of tert-ButyI Alcohol
CH3
I
glucuronide-O — — CH3
CH3
t-butyl glucuronide
rats, humans
_
HO-
[O]
-CH,
CH3
HO——CH3
CH3
t-butanol
rats
CYP450
rats,
humans
QU OH
2-methyl-1 ,2-propanediol
CH,
2-hydroxyisobutyric acid
formaldehyde
oA
CH,
acetone
-CH,
CH3
t-butyl sulfate
1 Source: NSF International (2003), ATSDR (1996), Bernauer et al. (1998), Amberg et al. (1999),
2 and Cederbaum and Cohen (1980).
3 Figure 1-1. Biotransformation of tert-butanol in rats and humans.
4 1.1.3. Description of Toxicokinetic Models
5 No physiologically based pharmacokinetic (PBPK) models have been developed specifically
6 for administration of tert-butanol. Some models have been used to study tert-butanol as the
7 primary metabolite after oral or inhalation exposure to MTBE or ETBE. The most recent models for
8 MTBE oral and inhalation exposure include a component for the binding of tert-butanol to
9 (X2u-globulin [Borghoff etal., 2010: Leavens and Borghoff, 2009]. A more-detailed summary of the
10 toxicokinetic models is provided in Appendix B, Section B.I.5.
11 A PBPK model for tert-butanol was modified by adapting previous models for MTBE and
12 tert-butanol [Leavens and Borghoff, 2009: Blancato etal., 2007]. The addition of a sequestered
13 blood compartment for tert-butanol substantially improved the model fit. The alternative
14 modification of changing to diffusion-limited distribution between blood and tissues also improved
15 the model fit, but was considered less biologically plausible. Physiological parameters and partition
16 coefficients were obtained from published measurements. The rate constants for tert-butanol
17 metabolism and elimination were from a published PBPK model of MTBE with a tert-butanol
18 subcompartment [Blancato etal.. 2007]. Additional model parameters were estimated by
19 calibrating to data sets for i.v., oral, and inhalation exposures as well as repeated dosing studies for
20 tert-butanol. Overall, the model produced acceptable fits to multiple rat time-course datasets of
21 tert-butanol blood levels following either inhalation or oral gavage exposures.
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1 1.1.4. Chemicals Extensively Metabolized to tert-Butanol
2 tert-Butanol is a metabolite of other compounds, including ETBE, MTBE, and tert-butyl
3 acetate. Some of the toxicological effects observed in these compounds are attributed to tert-
4 butanol. There are no assessments by national or international health agencies for ETBE. Animal
5 studies demonstrate that chronic exposure to ETBE is associated with noncancer kidney effects,
6 including increased kidney weights in male and female rats accompanied by increased chronic
7 progressive nephropathy (CPN), urothelial hyperplasia (in males), and increased blood
8 concentrations of total cholesterol, blood urea nitrogen, and creatinine [Saito etal.. 2013: Suzuki et
9 al.. 2012). In these studies, increased liver weight and centrilobular hypertrophy also were
10 observed in male and female rats exposed to ETBE. Liver adenomas and carcinomas were increased
11 in male rats following 2-year inhalation exposure [Saito etal., 2013].
12 In 1996, the U.S. Agency for Toxic Substances and Disease Registry's (ATSDR) Toxicological
13 Profile for MTBE [ATSDR. 1996] identified cancer effect levels of MTBE based on data on
14 carcinogenicity in animals. ATSDR reported that inhalation exposure was associated with kidney
15 cancer in rats and liver cancer in mice. ATSDR concluded that oral exposure to MTBE might cause
16 liver and kidney damage, and nervous system effects in rats and mice. The chronic inhalation
17 minimal risk level was derived based on incidence and severity of chronic progressive nephropathy
18 in female rats [ATSDR. 1996]. In 1997, EPA's Office of Water concluded that MTBE is carcinogenic
19 to animals and poses a potential carcinogenic potential to humans based on an increased incidence
20 of Leydig cell adenomas of the testes, kidney tumors, lymphomas, and leukemia in exposed rats
21 [U.S. EPA. 1997]. In 1998, the International Agency for Research on Cancer (IARC] found "limited
22 evidence" of MTBE carcinogenicity in animals and placed MTBE in Group 3 (i.e., not classifiable as
23 to carcinogenicity in humans] (IARC. 1999]. IARC reported that oral exposure in rats resulted in
24 testicular tumors in males and lymphomas and leukemias (combined] in females; inhalation
25 exposure in male rats resulted in renal tubule adenomas; and inhalation exposure in female mice
26 resulted in hepatocellular adenomas (IARC. 1999].
27 No assessments by national or international agencies or chronic studies for tert-butyl
28 acetate are available.
29 1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
30 1.2.1. Kidney Effects
31 Synthesis of Effects in Kidney
32 This section reviews the studies that investigated whether subchronic or chronic exposure
33 to tert-butanol can affect kidneys in humans or animals. The database examining kidney effects
34 following tert-butanol exposure contains eight studies from 5 references performed in rats or mice
35 (Lyondell Chemical Co.. 2004: Acharyaetal.. 1997: NTP. 1997: Acharya etal.. 1995: NTP. 1995]. and
36 a reevaluation of the rat data from NTP (1995]. published by Hard etal. (2011]: no human data are
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-ButyI Alcohol
1 available. Studies using short-term and acute exposures that examined kidney effects are not
2 included in the evidence tables; they are discussed in the text, however, if they provide data to
3 inform mode of action (MOA) or hazard identification. tert-Butanol exposure resulted in kidney
4 effects after both oral (drinking water) and inhalation exposure in both sexes of rats (Table 1-1,
5 Table 1-2, Figure 1-1, and Figure 1-2); studies are arranged in the evidence tables first by effect,
6 then by route, and then duration.
7 The design, conduct, and reporting of each study were reviewed, and each study was
8 considered adequate to provide information pertinent to this assessment. Interpretation of non-
9 neoplastic kidney endpoints in rats, however, is somewhat complicated by the common occurrence
10 of age-related, spontaneous lesions characteristic of chronic progressive nephropathy (CPN) (NTP.
11 2015: HardetaL 2013: MelnicketaL 2012: U.S. EPA.
12 1991a]:http://ntp.niehs.nih.gov/nnl/urinary/kidney/necp/index.htm]. CPN is more severe in male
13 rats than in females and is particularly common in the Sprague-Dawley and Fischer 344 strains.
14 Dietary and hormonal factors play a role in modifying CPN, although the etiology is largely
15 unknown (see further discussion below).
16 Kidney weight. Changes in kidney weight (absolute and relative to body weight) were
17 observed in male and female F344 rats following exposures of 13 weeks (oral and inhalation) (NTP.
18 1997) and 15 months (oral) (NTP. 1995). Lyondell Chemical Co. (2004) also reported increases in
19 absolute and relative kidney weight in Sprague-Dawley rats administered tert-butanol orally for
20 approximately 10 weeks (tabular data presented in Supplemental Information to this Toxicological
21 Review). Changes were observed in both male and female rats, which exhibited strong dose-related
22 increases in absolute kidney weight (Spearman's rank coefficient > 0.78) following either oral or
23 inhalation exposures (Figure 1-3). Of the oral (Figure 1-4 and inhalation (Figure 1-5) mouse
24 studies, only inhalation exposure in female mice induced a strong dose-related increase
25 (Spearman's rank coefficient = 0.9) in absolute kidney weights.
26 Measures of relative, as opposed to absolute, organ weight are sometimes preferred
27 because they account for changes in body weight that might influence changes in organ weight
28 (Bailey et al.. 2004). although potential impact should be evaluated. For tert-butanol, body weight in
29 exposed animals noticeably decreased at the high doses relative to controls in the oral 13-week and
30 2-year studies NTP (1995). In this case, the decreased body weight of the animals affects the
31 relative kidney weight measures, resulting in an artificial exaggeration of changes. Thus, absolute
32 weight was determined the more reliable measure of kidney weight change for this assessment
33 Additionally, a recent analysis indicates that increased absolute, but not relative, subchronic kidney
34 weights are significantly correlated with chemically induced histopathological findings in the
35 kidney in chronic and subchronic studies (Craig etal.. 2014). Although relative and absolute kidney
36 weight data are both presented in exposure-response arrays (and in evidence tables in
37 Supplemental Information), the absolute measures were considered more informative for
38 determining tert-butanol hazard potential.
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1 Kidney histopathology. Treatment-related histopathological changes were observed in the
2 kidneys of male and female F344 rats following 13-week and 2-year oral exposures [NTP, 1995]
3 and male F344 rats following a 13-week inhalation exposure [NTP. 1997}. Similarly, male Wistar
4 rats exposed for approximately 10 weeks exhibited an increase in histopathological kidney lesions
5 [Acharya et al.. 1997: Acharya et al.. 1995). B6C3Fi mice, however, did not exhibit histopathological
6 changes when exposed for 13 weeks and 2 years via the oral route [NTP. 1995] and 13 weeks via
7 the inhalation route [NTP, 1997]. More specific details on the effects observed in rats, reported by
8 NTP [1997.1995] and Acharya etal. [1997]: [1995] are described below.
9 Nephropathy and severity of nephropathy were reported in male and female rats in the
10 13-week oral studies [NTP. 1995]. The nephropathy was characterized as "...a spontaneous
11 background lesion...typically consisting] of scattered renal tubules lined by basophilic
12 regenerating tubule epithelium." [NTP. 1995]. NTP [1995] noted that the increase in severity of
13 nephropathy was related to tert-butanol and "characterized by an increase in the number and size
14 of foci of regeneration." The severity of nephropathy increased, compared with controls, in the
15 13-week male rats, which exhibited nephropathy in 94% of all exposed animals and 70% of
16 controls. Conversely, lesion severity was unchanged in the females, although nephropathy
17 incidence significantly increased with tert-butanol exposure. In the 13-week inhalation study [NTP.
18 1997], nephropathy was present in all but two male rats, including controls. NTP [1997]
19 characterized the reported chronic nephropathy in control male rats as "1 to 3 scattered foci of
20 regenerative tubules per kidney section. Regenerative foci were characterized by tubules with
21 cytoplasmic basophilia, increased nuclear/cytoplasmic ratio, and occasionally thickened basement
22 membranes and intraluminal protein casts." In exposed groups, the severity generally increased
23 from minimal to mild with increasing dose as "evidenced by an increased number of foci." No
24 treatment-related kidney histopathology was reported in the female rats exposed through
25 inhalation [NTP. 1997].
26 In the 2-year oral study by NTP [1995]. nephropathy was reported at 15 months and 2
27 years. The NTP [1995] characterization of nephropathy following chronic exposure included
28 multiple lesions: "thickened tubule and glomerular basement membranes, basophilic foci of
29 regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell
30 aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis." At 15
31 months, male and female rats (30/30 treated; 10/10 controls] had nephropathy, and the severity
32 scores ranged from minimal to mild. At 2 years, male and female rats (149/150 treated; 49/50
33 controls] also had nephropathy, and although the severity was moderate in the control males and
34 minimal to mild in the control females, severity increased with tert-butanol exposure in both sexes
35 [NTP. 1995].
36 The lesions collectively described by NTP [1997,1995] as nephropathy and noted to be
37 common spontaneous lesions in rats, are consistent with CPN. The effects characterized as CPN are
38 related to age and not considered histopathological manifestations of chemically induced toxicity
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Toxicological Review of tert-ButyI Alcohol
1 [see U.S. EPA [1991a]. p. 35 for further details and a list of the typical observable histopathological
2 features of CPN]. These lesions, however, are frequently exacerbated by chemical treatment [NTP,
3 1997}. as evidenced by the dose-related increases in severity of the nephropathy compared to
4 female and male rat controls. The chemical-related changes in nephropathy severity are included in
5 the consideration of hazard potential.
6 NTP [1995] observed other kidney lesions, described as being associated with nephropathy
7 but diagnosed separately. Renal mineralization is defined by NTP [1995] as "focal mineral deposits
8 primarily at the corticomedullary junction." This mineralization is distinct from linear
9 mineralization, which is considered a lesion characteristic of azu-globulin nephropathy (for further
10 discussion of this particular lesion, see Mode of Action Analysis—Kidney Effects]. The mineralization
11 is characterized as distinct linear deposits along radiating medullary collecting ducts. An increased
12 incidence of linear mineralization was limited to exposed males in the 2-year oral study [NTP.
13 1995].
14 Renal mineralization was observed in essentially all female rats at all reported treatment
15 durations. A dose-related, increased incidence of mineralization was reported in male rats at the
16 13-week, 15-month, and 2-year oral evaluations [NTP. 1995]. NTP [1995] describes focal,
17 medullary mineralization as being associated with CPN but notes that focal mineralization is
18 "usually more prominent in untreated females than in untreated males," which is consistent with
19 the widespread appearance of this lesion in females. This description, however, is inconsistent with
20 the observation in this and other databases that age-related nephropathy (i.e., CPN] is generally
21 more prevalent and more severe in male rats compared to females [U.S. EPA. 1991a]. The
22 association of mineralization with CPN is unclear, considering the lack of spontaneous lesions in the
23 control and low-dose groups of 13-week males and the dose-response relationships the tert-
24 butanol-exposed males exhibited in the 13-week [NTP. 1997.1995] and 2-year studies [NTP.
25 1995]. Furthermore, due to the overwhelming presence of mineralization in the control and treated
26 female rats, the contribution, if any, of tert-butanol to the formation of this lesion in females could
27 not be determined. Thus, the mineralization could be related to both aging of the animals and tert-
28 butanol exposure.
29 Two other histological kidney lesions observed in male and female rats are suppurative
30 inflammation and transitional epithelial hyperplasia. These lesions were observed in the 2-year
31 oral NTP [1995] study. Although NTP [1995] describes these lesions as related to the nephropathy
32 (characterized above as common and spontaneous, and considered CPN], that suppurative
33 inflammation and transitional epithelial hyperplasia exhibited incidence patterns different from
34 those reported for nephropathy is notable. Incidence of suppurative inflammation in female rats
35 was low in the control group and increased with dose, with incidences >24% in the two highest
36 dose groups, compared with controls. In comparison, 20% of the control males exhibited
37 suppurative inflammation, and the changes in incidence were not dose related (incidences ranging
38 from 18 to 36%]. The data for males suggest that CPN plays a role in the induction of suppurative
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1 inflammation; considering the responses in the females, however, the effect appears to be
2 predominantly treatment related. Suppurative inflammation was not observed in the animals of the
3 13-week oral fNTP. 19951 or inhalation study fNTP. 19971 which both reported nephropathy (as
4 CPN), providing further support that this lesion is not specifically related to the nephropathy.
5 Transitional epithelial hyperplasia was observed in both male and female rats exposed
6 orally [NTP. 1995). In the control males, 50% of the animals exhibited transitional epithelial
7 hyperplasia and the incidence and severity increased with dose. Only the mid- and high-dose
8 females, however, exhibited dose-related increases in incidence and severity of transitional
9 epithelial hyperplasia; this lesion was not reported in the control or low-dose females. NTP [1995]
10 described transitional epithelial hyperplasia as increased layers of the transitional epithelial lining
11 of the renal pelvis; study authors noted no progression of this hyperplastic lesion to neoplasia. The
12 relatively high background in male controls (i.e., 50%) suggests some potential influence, other
13 than tert-butanol treatment, on this effect The absence of this effect in female control and low-dose
14 animals and the dose-related increases in both males and females, however, indicate that similar to
15 the suppurative inflammation, the transitional epithelial hyperplasia is predominantly treatment
16 related. Transitional epithelial hyperplasia should not be confused with another lesion noted at the
17 2-year evaluation, renal tubule hyperplasia, which was considered preneoplastic (for further details
18 regarding this type of hyperplasia, see the discussion under kidney tumors below).
19 Additional histopathological changes, including increased tubular degeneration,
20 degeneration of the basement membrane of the Bowman's capsule, diffused glomeruli, and
21 glomerular vacuolation were noted in a 10-week study in male Wistar rats (Acharyaetal.. 1997:
22 Acharyaetal., 1995). A decrease in glutathione in the kidney accompanied these changes, which the
23 study authors noted as potentially indicative of oxidative damage. Acharyaetal. (1997): Acharya et
24 al. (1995) used one dose and a control group and did not report incidences. The increased tubule
25 degeneration and glomerular vacuolation could be characterized as tubular atrophy and glomerular
26 hyalinization, respectively, consistent with CPN; however, without quantitative information,
27 examining the differences between the control and treated animals to determine if CPN plays a role
28 in development of these effects is not possible. Although based on the noted appearance of the
29 effects in the treated animals compared with controls, the effects likely are treatment related.
30 Serum or urinary biomarkers informative of kidney toxicity were not measured in the
31 studies discussed above. Some changes occurred in urinalysis parameters (e.g., decreased urine
32 volume and increased specific gravity), accompanied by reduced water consumption, and thus
33 might not be related to an effect of kidney function (NTP. 1995).
34 Kidney tumors. The kidney is also a target organ for cancer effects (Table 1-3, Figure 1-1).
35 Male F344 rats had an increased incidence of combined renal tubule adenomas or carcinomas in
36 the 2-year oral bioassay (Hardetal.. 2011: NTP. 1995). The increase in tumors from control was
37 similar in the low- and high-dose groups and highest in the mid-dose group. Overall, tumor
38 increases were statistically significant in trend testing, which accounted for mortality (p < 0.018).
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Toxicological Review of tert-ButyI Alcohol
1 Mortality increased with increasing exposure (p = 0.001); increased mortality alone, however, does
2 not account for the highest tumor incidence occurring at the middle dose.
3 Increases in incidence and severity of renal tubule hyperplasia also were observed in male
4 rats. NTP [1995] stated that "[t]he pathogenesis of proliferative lesions of renal tubule epithelium is
5 generally considered to follow a progression from hyperplasia to adenoma to carcinoma [Hard.
6 1986]." Similarly, EPA considered the renal tubule hyperplasia to be a preneoplastic effect
7 associated with the renal tubule tumors. Renal tubule hyperplasia was found in one high-dose
8 female [NTP, 1995]: no increase in severity was observed. This effect in females, which was not
9 considered toxicologically significant, is not discussed further. Two renal tubular adenocarcinomas
10 in male mice also were reported [NTP. 1995]. one each in the low- and high-dose groups, but were
11 not considered by NTP to be "biologically noteworthy changes"; thus the tumors in mice are not
12 discussed further.
13 A Pathology Working Group, sponsored by Lyondell Chemical Company, reevaluated the
14 kidney changes in the NTP 2-year study to determine if additional histopathological changes could
15 be identified to inform the MOA for renal tubule tumor development [Hardetal., 2011]. In all cases,
16 working group members were blinded to treatment groups, and used guidelines published by Hard
17 and Wolf T19991 and refinements reported by (Hard and Seely. 2006): Hard and Seely f20051 and
18 Hard (2008). The group's report and analysis by Hardetal. (2011) confirmed the NTP findings of
19 renal tubule hyperplasia and renal tubule tumors in male rats at 2 years. In particular, they
20 reported similar overall tumor incidences in the exposed groups. Hardetal. (2011). however,
21 reported fewer renal tubule adenomas and carcinomas in the control group than in the original NTP
22 study. As a result, all treated groups had statistically significant increases in renal tubule adenomas
23 and carcinomas (combined) when compared to controls. Additionally, Hardetal. (2011) considered
24 fewer tumors to be carcinomas than did the original NTP study. Results of both NTP (1995) and the
25 reanalysis by Hardetal. (2011) are included in Table 1-3 and Figure 1-1.
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Toxicological Review of tert-Butyl Alcohol
50
s> 40 -
I
o
^ 30H
0
;S 10-
<
o -
Male rats
Female rats
rho= 0.78 (all)
rho= 0.89 (oral)
rho= 0.80 (inhalation)
rho= 0.78 (all)
rho= 0.72 (oral)
rho= 0.9 (inhalation)
10
100 1000
tert-butanol blood concentration (mg/l)
•
o
Oral exposure
Inhalation exposure
3
4
5
6
7
Figure 1-2. Comparison of absolute kidney weight change in male and female
rats across oral and inhalation exposure based on internal blood
concentration. Spearman rank correlation coefficient (rho) was calculated to
evaluate the direction of a monotonic association (e.g., positive value =
positive association) and the strength of association.
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Toxicological Review of tert-ButyI Alcohol
Male mice
Female mice
3>
12 -
10 -
4 -
2 -
rho=-0.1
rho= 0.9
2000 4000 6000
Administered dose (mg/kg-day)
8000 0 2000 4000 6000 8000 10000 12000
Administered dose (mg/kg-day)
1
2
3
4
5
6
7
8
9
10
11
-10 -
-12
Figure 1-3. Comparison of absolute kidney weight change in male and female
mice following oral exposure based on administered concentration. Spearman
rank correlation coefficient (rho) was calculated to evaluate the direction of a
monotonic association (e.g., positive value = positive association) and the
strength of association.
Male mice
Female mice
mo= 0.2
rho=-0.1
1000 2000 3000 4000 5000 6000
Administered dose (mg/m3)
1000 2000 3000 4000 5000 6000 7000
Administered dose (mg/m3)
Figure 1-4. Comparison of absolute kidney weight change in male and female
mice following inhalation exposure based on administered concentration.
Spearman rank correlation coefficient (rho) was calculated to evaluate the
direction of a monotonic association (e.g., positive value = positive
association) and the strength of association.
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Toxicological Review of tert-ButyI Alcohol
Table 1-2. Changes in kidney histopathology in animals following exposure to
tert-butanol
Reference and study design
Results
Acharyaetal. (1997)
Acharyaetal. (1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or
575 mg/kg-d
10 weeks
1" tubular degeneration, degeneration of the basement membrane of the
Bowman's capsule, diffused glomeruli, and glomerular vacuolation (no
incidences reported)
4, kidney glutathione (~40%)*
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5, 10, 20,
or 40 mg/mL)
M: 0, 230, 490, 840, 1,520,
3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560,
3,620a mg/kg-d
13 weeks
Incidence (severity):
Males
Females
Dose Minerali- Nephro- Dose Minerali- Nephro-
(mg/kg-d) zation^ pathy£ (mg/kg-d) zation^ pathy£
0
230
490
0/10 7/10 (1.0)
0/10
10/10
(1.6*)
2/10 (1.5) 10/10
(2.6*)
0
290
590
10/10 (1.7) 2/10 (1.0)
10/10 (2.0) 3/10 (1.0)
840
10/10 (2.0) 5/10 (1.0)
850 10/10 (2.0) 7/10* (1.0)
1,560 10/10 (2.0) 8/10* (1.0)
3,610a 4/10*(1.0) 7/10(1.1) 3,620a 6/10(1.2) 7/10* (1.0)
8/10* (1.4) 10/10
(2.7*)
1,520 4/10* (1.0) 10/10
(2.6*)
NTP (1995)
B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20,
or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940,
8,210a mg/kgd
F: 0, 500, 820, 1,660, 6,430,
ll,620a mg/kgd
13 weeks
Study authors indicated no treatment-related changes in kidney
histopathology (histopathological data not provided for the 13-week study)
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 months interim)
Drinking water (0, 1.25, 2.5, 5, 10
mg/mL)
M: 0, 90, 200, 420a mg/kg-d
F: 0, 180, 330, 650a mg/kg-d
2 years
NTP (1995)
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5, 10, or 20
mg/mL)
M: 0, 540, 1,040, or 2,070a
mg/kg-d
Results
Incidence (severity):
Males
Dose
(mg/kg-d)
0
90
200
420a
Dose
(mg/kg-d)
0
90
200
420a
Females
Dose
(mg/kg-d)
0
180
330
650a
Dose
(mg/kg-d)
0
180
330
650a
No treatment-related
Mineralization^
(interim)
1/10 (1.0)
2/10 (1.0)
5/10 (1.8)
9/10* (2.3)
Transitional
epithelial
hyperplasia
25/50 (1.7)
32/50 (1.7)
36/50* (2.0)
40/50* (2.1)
Mineralization^
Interim
10/10 (2.8)
10/10 (2.9)
10/10 (2.9)
10/10 (2.8)
Transitional
epithelial
hyperplasia
0/50
0/50
3/50 (1.0)
17/50*(1.4)
changes in kidney
Mineralization^ n
(terminal)
26/50 (1.0)
28/50 (1.1)
35/50 (1.3)
48/50* (2.2)
Nephropathy£
severity
3.0
3.1
3.1
3.3*
Mineralization^
Terminal
49/50 (2.6)
50/50 (2.6)
50/50 (2.7)
50/50 (2.9)
Nephropathy£
severity
1.6
1.9*
2.3*
2.9*
related histopathology
Lin 63 r
mineralization^
(terminal)
0/50
5/50* (1.0)
24/50* (1.2)
46/50* (1.7)
nflammation
[suppurative)
incidence
2/50
3/50
13/50*
17/50*
observed
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Results
F: 0, 510,1,020, or 2,110 mg/kg-
d
2 years
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation analytical
concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,
1,643, 3,273 or 6,368 mg/m3)
(dynamic whole-body chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist
Ultrasonic spray nozzle
nebulizer), analytical
concentration and method were
reported
Male
Concentration
(mg/m3-)
0
406
824
1,643
3,273
6,368
Incidence of
chronic
nephropathy-
9/10
8/10
9/10
10/10
10/10
10/10
Average severity
of chronic
nephropathy
1.0
1.4
1.4
1.6
1.9
2.0
Females: no treatment-related changes in kidney related histopathology
observed
Severity categories: 1= minimal, 2= mild. No results from statistical tests
reported
NTP (1997)
No treatment-related changes in kidney related histopathology observed
B6C3Fi mouse; 10/sex/treatment
Inhalation analytical
concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,
1,643, 3,273 or 6,368 mg/m3)
(dynamic whole-body chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist
Ultrasonic spray nozzle
nebulizer), analytical
concentration and method were
reported
1
2
3
4
5
* Statistically significant p < 0.05 as determined by the study authors.
a The high-dose group had an increase in mortality.
b Mineralization defined in NTP (1995) as focal mineral deposits, primarily at the corticomedullary junction. Linear
mineralization was defined as foci of distinct linear deposits along radiating medullary collecting ducts; linear
mineralization not observed in female rats.
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Toxicological Review of tert-Butyl Alcohol
1
2
3
4
5
6
7
8
c Nephropathy defined in NTP (1995) as lesions including thickened tubule and glomerular basement membranes,
basophilic foci of regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell
aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis.
d Nephropathy characterized in NTP (1997) as scattered foci of regenerative tubules (with cytoplasmic basophilia,
increased nuclear/cytoplasmic ratio, and occasionally thickened basement membranes and intraluminal protein
casts).
Note: Conversions from drinking water concentrations to mg/kg-day performed by study authors.
Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
9
10
Table 1-3. Changes in kidney tumors in animals following exposure to
tert-butanol
Reference and study design
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 months)
Drinking water (0, 1.25, 2.5, 5, or
10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0, 180, 330, or 650a mg/kg-d
2 years
^^
Results
Male
Dose
(mg/kg-d)
0
90
200
420a
Dose
(mg/kg-d)
0
90
200
420a
Female
Dose
(mg/kg-d)
0
180
330
650a
Renal tubule
hyperplasia
(standard and
extended
evaluation
combined)
14/50 (2.3)
20/50 (2.3)
17/50 (2.2)
25/50* (2.8)
Renal tubule
carcinoma
0/50
2/50
1/50
1/50
Renal tubule
hyperplasia
0/50
0/50
0/50
1/50 (1.0)
Renal tubule
adenoma (single)
7/50
7/50
10/50
10/50
Renal tubule
adenoma (single
or multiple) or
carcinoma
8/50
13/50
19/50*
13/50
Renal tubule
adenoma (single)
0/50
0/50
0/50
0/50
Renal tubule
adenoma
(multiple)
1/50
4/50
9/50*
3/50
Renal tubule
adenoma
(multiple)
0/50
0/50
0/50
0/50
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Hard etal. (2011)
Reanalysis of the slides from
male rats (all slides in controls
and high-dose groups of males
and females, and slides from all
other males with renal tumors) in
the NTP (1995) study (see above)
NTP (1995)
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5, 10, or
20 mg/mL)
M: 0, 540, 1,040, or 2,070a
mg/kg-d
F: 0, 510, 1,020, or 2,110 mg/kg-
d
2 years
Results
Dose
(mg/kg-d)
Renal tubule
adenoma (single
Renal tubule or multiple) or
carcinoma carcinoma
0 0/50 0/50
180 0/50 0/50
330 0/50 0/50
650a 0/50 0/50
Based on standard and extended evaluations (combined). Results
include the animals sacrificed at 15 months.
Male
Dose
(mg/kg-d)
0
90
200
420
Renal tubule Renal tubule
adenoma adenoma Renal tubule
(single) (multiple) carcinoma
3/50 1/50 0/50
9/50 3/50 1/50
9/50 9/50 0/50
9/50 3/50 1/50
do not
Renal tubule
adenoma
(single or
multiple) or
carcinoma
4/50
13/50*
18/50*
12/50*
No increases in kidney-related tumors. Two renal tubule adenocarcinomas,
one in the low-dose and one in the high-dose groups, were observed in male
mice. These tumors were not considered treatment related.
^L
1
2
3
4
* Statistically significant p < 0.05 as determined by the study authors.
a The high-dose group had an increase in mortality.
Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
x = exposures at which all animals were dead and unable to be examined for the endpoint
Kidney Absolute weight; M Rat; Reproductive (C]
Weight Relative weight; M Rat; Reproductive (C]
Absolute weight; F Rat; Reproductive [C]
Relative weight; F Rat; Reproductive fC]
Absolute weight; M Rat; 13wk (D]
Relative weight; M Rat; 13wk [D]
Absolute weight; F Rat; 13wk CD]
Relative weight; F Rat; 13wk CD]
Absolute weight; M Mouse; 13wk CD]
Relative weight; M Mouse; 13wk CD]
Absolute weight; F Mouse; 13wk (D]
Relative weight; F Mouse; 13wk CD]
Absolute weight; M Rat; 15mo CD]
Relative weight; M Rat; 15mo CD]
Absolute weight; F Rat; 15mo CD]
Relative weight; F Rat; 15mo (D]
Kidnev Decreased glutathione; M Rat; lOwk (A]
Histopathology Inflammation; F Rat; 2yr(D]
Ncphropathy severity; M Rat; 13wk (D)
Nephropathy incidence; F Rat; 13wk CD]
Mineralization; M Rat; 13wk [D]
Mineralization; F Rat; 13wk (D]
Nephropathy severity; M Rat; 2yr CD]
Nephropathy severity; F Rat; 2yr (D]
Linear mineralization; M Rat; 2yr CD)
Interim/terminal mineralization; M Rat; 2yrfD]
Interim/terminal mineralization; F Rat; 2yr (D]
Transitional epithelium hyperplasia; M Rat; 2yr (D]
Transitional epithelium hyperplasia; F Rat; 2yr (D]
Renal tubular hyperplasia; M Rat; 2yr (D]
Renal tubule hyperplasia; F Rat; 2yr CD]
Kidnev ^ena' tubular adenoma or carcinoma; M Rat; 2yr CD]
Tumors Renal tubular adenoma or carcinoma; M Rat; 2yr CB]
Renal tubular adenomaor carcinoma; F Rat; 2yr CD]
Renal tubular adenoma or carcinoma; M Mouse; 2yr (D)
Renal tubular adenoma or carcinoma; F Mouse; 2yr [D]
E
E
E
•
E
E
E
E
•
Dn
1 D D
1 • •
•
D-«-«
BDH
Qn •
) D •
1 • •
1 D •
B-B-0
1 • •
D D •
1 D •
Q-B-O
1 • D
1 • •
D-B-FJ
B-E
B-E
]
]
Bn
3-D
3-E
-
10 100 1,000 10,000 100,000
Dose (mg/kg-day)
1
2
3
4
Sources: (A) Acharya et al. (1997); (1995); (B) Hard etal. (2011)*; (C) Lyondell Chemical Co. (2004) (D) NTP
(1995); *reanalysis of NTP (1995).
Figure 1-5. Exposure response array for kidney effects following oral exposure
to tert-butanol.
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
Absolute weight; M Rat
Relative weight; M Rat
Absolute weight; F Rat
Relative weight; F Rat
tf^
4
Absolute/relative weight; M Mouse
Absolute weight; F Mouse
Relative weight; F Mouse -
Qi— i
Qi— i
BB •
B|— i i— i
100 1,000 10,000
Concentration (mg/m3)
Source: NTP (1997).
Figure 1-6. Exposure-response array of kidney effects following inhalation
exposure to tert-butanol (13-week studies, no chronic studies available).
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Toxicological Review of tert-Butyl Alcohol
1 Mode of Action Analysis—Kidney Effects
2 a) a?,,-Globulin-Associated Renal Tubule Nephropathy and Carcinogenicity
3 One disease process to consider when interpreting kidney effects in rats is related to the
4 accumulation of (X2u-globulin protein. (X2u-Globulin, a member of a large superfamily of low-
5 molecular-weight proteins, was first characterized in male rat urine. Such proteins have been
6 detected in various tissues and fluids of most mammals (including humans), but the particular
7 isoform of (X2u-globulin commonly detected in male rat urine is considered specific to that sex and
8 species. Exposure to chemicals that induce a2u-globulin accumulation can initiate a sequence of
9 histopathological events leading to kidney tumorigenesis. Because a2u-globulin-associated renal
10 tubule nephropathy and carcinogenicity occurring in male rats are presumed not to be relevant for
11 assessing human health hazards [U.S. EPA. 1991a). evaluating the data to determine if a2u-globulin
12 plays a role is important. The role of (X2u-globulin accumulation in the development of renal tubule
13 nephropathy and carcinogenicity observed following tert-butanol exposure was evaluated using the
14 U.S. EPA[1991a] Risk Assessment Forum Technical panel report, Alpha2u-Globulin: Association with
15 Chemically Induced Renal Toxicity and Neoplasia in the Male Rat. This report provides specific
16 guidance for evaluating renal tubule tumors in male rats that are related to chemical exposure for
17 the purpose of risk assessment, based on an examination of the potential involvement of
18 (X2u-globulin accumulation.
19 Studies in the tert-butanol database evaluated and reported effects on the kidney, providing
20 some evidence to evaluate this MOA. Additionally, several studies were identified that specifically
21 evaluated the role of (X2u-globulin in tert-butanol-induced renal tubule nephropathy and
22 carcinogenicity fBorghoffetal.. 2001: Williams and Borghoff. 2001: TakahashietaL 19931 Because
23 the evidence reported in these studies is specific to a2u-globulin accumulation, it is presented in this
24 section; it was not included in the animal evidence tables in the previous section.
25 The hypothesized sequence of (X2u-globulin renal tubule nephropathy, as described by U.S.
26 EPA [1991a]. is as follows. Chemicals that induce (X2u-globulin accumulation do so rapidly.
27 a2u-Globulin accumulating in hyaline droplets is deposited in the S2 (P2) segment of the proximal
28 tubule within 24 hours of exposure. Hyaline droplets are a normal constitutive feature of the
29 mature male rat kidney; they are particularly evident in the S2 (P2) segment of the proximal tubule
30 and contain (X2u-globulin [U.S. EPA. 1991a). Abnormal increases in hyaline droplets have more than
31 one etiology and can be associated with the accumulation of different proteins. As hyaline droplet
32 deposition continues, single-cell necrosis occurs in the S2 (P2) segment, which leads to exfoliation
33 of these cells into the tubule lumen within 5 days of chemical exposure. In response to the cell loss,
34 cell proliferation occurs in the S2 (P2) segment after 3 weeks and continues for the duration of the
35 exposure. After 2 or 3 weeks of exposure, the cell debris accumulates in the S3 (P3) segment of the
36 proximal tubule to form granular casts. Continued chemical exposure for 3 to 12 months leads to
37 the formation of calcium hydroxyapatite in the papillae which results in linear mineralization. After
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Toxicological Review of tert-ButyI Alcohol
1 1 or more years of chemical exposure, these lesions can result in the induction of renal tubule
2 adenomas and carcinomas (Figure 1-7).
3 U.S. EPA(1991a) identified two questions that must be addressed to determine the extent
4 to which a2u-globulin-mediated processes induce renal tubule nephropathy and carcinogenicity.
5 First, whether the a2u-globulin process is occurring in male rats and is involved in renal tubule
6 tumor development must be determined. Second, whether the renal effects in male rats exposed to
7 tert-butanol are solely due to the (X2u-globulin process also must be determined.
8 U.S. EPA (1991a) stated the criteria for answering the first question in the affirmative are as
9 follows:
10 1) hyaline droplets are increased in size and number in treated male rats,
11 2) the protein in the hyaline droplets in treated male rats is (X2uglobulin (i.e.,
12 immunohistochemical evidence), and
13 3) several (but not necessarily all) additional steps in the pathological sequence appear in
14 treated male rats as a function of time, dose, and progressively increasing severity
15 consistent with the understanding of the underlying biology, as described above, and
16 illustrated in Figure 1-7.
17 The available data relevant to this first question are summarized in Table 1-4, Figures 1-8
18 and 1-9, and are evaluated below.
19
4 k
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Toxicological Review of tert-ButyI Alcohol
Male rat liver
Male rat kidney
Synthesis of a2u-globulin
^< TBA binding
Resorption of poorly digestible
protein-chemical complex
1
Hyaline droplet accumulation
within lysosomes
Cell death and exfoliation
> 1 days
1 - 150 days
5 days - 48 weeks
3-48 weeks
> 12 months
> 12 months
1
2
3
4
5
6
7
8
9
10
Figure 1-7. Temporal pathogenesis of a2u-globulin-associated nephropathy in
male rats. a2U-Globulin synthesized in the livers of male rats is delivered to the
kidney, where it can accumulate in hyaline droplets and be retained by epithelial
cells lining the S2 (P2) segment of the proximal tubules. Renal pathogenesis
following continued tert-butanol exposure and increasing droplet accumulation can
progress step-wise from increasing epithelial cell damage, death and dysfunction
leading to the formation of granular casts in the corticomedullary junction, linear
mineralization of the renal papillae, and carcinogenesis of the renal tubular
epithelium. Adapted from Swenberg and Lehman-McKeeman [1999] and U.S. EPA
C1991a1.
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Toxicological Review of tert-ButyI Alcohol
1 Table 1-4. Summary of data on the ct2u -globulin process in male rats exposed
2 to tert-butanol
Duration Dose Results Comments Reference
1) Hyaline droplets are increased in size and number
10 d (inhalation) 0, 758,1,364, 5,304 + stat sig at 5,304 mg/m3; Borghoff et al. (2001)
mg/m3 stat sig trend
13 wk (inhalation) 0,3,273,6,368 mg/m3 - NTP (1997)a
13 wk (oral) 0,230,490,840, (+) observed in all but NTP (1995)
1,520, 3,610 mg/kg-d highest dose group
2) The protein in the hyaline droplets is
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Toxicological Review of tert-ButyI Alcohol
Duration
Dose
d) Linear mineralization of tubules in the
13 wk (oral)
2 yr (oral)
0, 230, 490, 840,
1,520, 3,610 mg/kg-d
0, 90, 200, 420
mg/kg-d
Results Comments
renal papilla
+; (+) all doses stat sig
Reference
NTP (1995); Hard et al.
(2011)c
NTP (1995); Hard et al.
(2011)d
e) Foci of tub ular hyperplasia
2 yr (oral)
0, 90, 200, 420
mg/kg-d
+ stat sig trend at all
doses; stat sig at 420
mg/kg-d
NTP (1995)
1
2
3
4
5
6
7
8
9
10
+ = Statistically significant change reported in one or more treated groups.
(+) = Effect was reported in one or more treated groups, but statistics not reported.
- = No statistically significant change reported in any of the treated groups.
a NTP (1997) did not observe any effects consistent with cuu-globulin nephropathy.
b Precursors to granular casts reported.
c Reanalysis of hematoxylin and eosin-stained kidney sections from all male control and 1,520 mg/kg-d groups, as
well as a representative sample of kidney sections stained with Mallory Heidenhain stain, from the 13-wk study
from NTP (1995).
d Reanalysis of slides for all males in the control and 420 mg/kg-day dose group and all animals with renal tubule
tumors from 2-vr NTP (1995). Protein casts reported, not granular casts.
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
x = exposures at which all animals were dead and unable to be examined for the endpoint
• = exposures at which effect was observed but statistics not reported
size/number
Identification of a2u- Williams and Borghoff [2001);
globulin in hyaline j 2 hr after singie dose
droplets
Acharya et al. [1997); 10 wk
Cytotoxicity/single-cell
necrosis of tubule epithelium;
epithelial cell exfoliation
Acharya et al. (1997); 10 wk
Tubule cell
proliferation
Granular
casts/tubule
dilation
NTP [1995); Hard et al. [2011); 2 yr
t
Linear papillary
mineralization
Foci of
tubular NTP 11995); 2 yr
hyperplasia
B
m
•
•
•
D
B Q
• y
* Hard et al. [2011] reported presence of "precursor
granular casts"
"NTP(1995) 13-wkstudy reported kidney
mineralization but not linear mineralization
10
100 1,000
Dose (mg/kg-day)
10,000
1
2
3
4
5
*Hard et al. (2011) reported presence of "precursor granular casts."
**NTP (1995) 13-wk study reported kidney mineralization but not linear mineralization.
Figure 1-8. Exposure-response array for effects potentially associated with
a2u-globulin renal tubule nephropathy and tumors in male rats after oral
exposure to tert-butanol.
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
Borghoffetal. (2001)-10d -
t Hyaline
droplet
size/number
NTP (1997) - 13 wks -
Identification
ofo2u-
globulinin Borghoff ctal. (2001)-10d
hyaline
droplets
Tubule cell Borghoff ct al. (2001) - lOd -
proliferation
•
BB
• H
100 1,000 10,t
Exposure Concentration (mg/m3)
Figure 1-9. Exposure-response array for effects potentially associated with
«2u-globulin renal tubule nephropathy and tumors in male rats after
inhalation exposure to tert-butanol.
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Toxicological Review of tert-ButyI Alcohol
1 Question One: Is the a2uglobulin process occurring in male rats exposed to tert-butanol?
2 (1) The first criterion to consider is whether hyaline droplets are increased in size and
3 number in male rats. As noted above, the excessive accumulation of hyaline droplets can appear
4 quickly, within 1 or 2 days, and persist throughout chronic exposures, although the severity begins
5 to decline around 5 months [U.S. EPA. 1991a]. A statistically significant positive trend in the
6 accumulation of large protein droplets with crystalloid protein structures was observed in kidneys
7 of male rats exposed to inhalation concentrations of 758,1,364, and5,304mg/m3 tert-butanol for 6
8 hr/day for 10 days [Borghoff etal.. 2001]. These droplets were small and minimally present in
9 control male rats and were not observed in female rats. Similarly, data from the 13-week NTP oral
10 study [NTP. 1995: Takahashi etal.. 1993: Lindamoodetal.. 1992] demonstrated an increase in the
11 accumulation of hyaline droplets. The lowest dose of 230 mg/kg-day had minimal hyaline droplet
12 formation compared to controls, although the next three doses (490, 840, and 1,520 mg/kg-day]
13 had a higher accumulation of droplets with angular, crystalline structures that was similar in
14 incidence and severity among these dose groups. No droplets were observed in female rats or in
15 mice.
16 NTP [1997]. however, found no difference between the control and treatment groups
17 stained for hyaline droplet formation in male rats exposed to 0, 3,273, or 6,368 mg/m3 tert-butanol
18 via inhalation for 13 weeks; in fact, this study did not report any other lesions that could be
19 specifically associated with (X2u-globulin nephropathy in male rats. These results from NTP [1997],
20 which are inconsistent with the findings of both Borghoff etal. [2001] and NTP [1995]. do not
21 appear to be due to differences in dose. Comparison of the oral and inhalation studies on the basis
22 of tert-butanol blood concentration (see Supplemental Information] showed that a 13-week
23 exposure in the range of the NTP [1995] doses of 490-840 mg/kg-day leads to the same average
24 blood concentration as 6-hr/day, 5 day/week inhalation exposures to 3,273-6,368 mg/m3. The
25 absence of similar histopathological findings in the 13-week inhalation NTP [1997] study compared
26 to those reported in the two oral studies is not understood, but might be indicative of the strength
27 of tert-butanol to induce, consistently, (X2u-globulin nephropathy. The results from the two other
28 studies [Borghoff etal.. 2001: NTP. 1995] indicate that hyaline droplets increase in size and number
29 in male rats following tert-butanol exposures. Therefore, the available data are sufficient to fulfill
30 the first criterion that hyaline droplets are increased in size and number in male rats.
31 (2] The second criterion to consider is whether the protein in the hyaline droplets in male
32 rats is (X2u-globulin. Accumulated hyaline droplets with an (X2u-globulin etiology can be confirmed
33 by using immunohistochemistry to identify the (X2u-globulin protein. Two short-term studies
34 measured (X2u-globulin immunoreactivity in the hyaline droplets of the renal proximal tubular
35 epithelium [Borghoff etal.. 2001: Williams and Borghoff. 2001]. Following 10 days of inhalation
36 exposure, Borghoff etal. [2001] did not observe an exposure-related increase in a2u-globulin using
37 immunohistochemical staining. When using an enzyme-linked immunosorbent assay [ELISA], a
38 more sensitive method of detecting a2u-globulin, however, a statistically significant positive
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Toxicological Review oftert-Butyl Alcohol
1 correlation of (X2u-globulin concentration with dose of tert-butanol (determined by correlating with
2 cell proliferation labeling indices) was observed, with accumulation of a2u-globulin protein
3 statistically significant by pairwise comparison only in the highest dose group. No positive staining
4 for a2u-globulin was observed in exposed female rats. In a follow-up study, Williams and Borghoff
5 [2001] used a single gavage dose of 500 mg/kg [selected on the basis of results by NTP [1995] for
6 induction of hyaline droplet accumulation], and reported a statistically significantly higher renal
7 concentration of (X2u-globulin (by ELISA) in treated male rats than in controls 12 hours after
8 exposure. Further, equilibrium dialysis methods determined that the binding of tert-butanol to
9 (X2u-globulin was reversible. These data indicate the presence of azu-globulin in tert-butanol-treated
10 male rats, although requiring a more sensitive method of detection of azu-globulin than is typically
11 used could indicate that tert-butanol is not a strong inducer of (X2u-globulin accumulation.
12 Therefore, the available data are sufficient to fulfill the second criterion for (X2u-globulin present in
13 the hyaline droplets, but suggest weak induction of (X2u-globulin by tert-butanol.
14 (3) The third criterion considered is whether several (but not necessarily all) additional
15 events in the histopathological sequence associated with (X2u-globulin nephropathy appear in male
16 rats in a manner consistent with the understanding of (X2u-globulin pathogenesis. Evidence of
17 cytotoxicity and single-cell necrosis of the tubule epithelium subsequent to the excessive
18 accumulation of hyaline droplets, with exfoliation of degenerate epithelial cells, should be
19 observable after five days of continuous exposure, peaking at 19 days [reviewed in U.S. EPA
20 (1991a)]. The formation and accumulation of granular casts from the exfoliated cellular debris
21 would follow, causing tubule dilation at the junction of the S3 (P3) segment of the proximal tubule
22 and the descending thin loop of Henle, and the commencement of compensatory cell proliferation
23 within the S2 (P2) segment, both occurring after three weeks of continuous exposure. Following
24 chronic exposures, this regenerative proliferation could result in focal tubular hyperplasia, and
25 eventually progress to renal adenoma and carcinoma (Figure 1-7).
26 Several of these steps were observed following tert-butanol exposure in male rats, most
27 notably linear papillary mineralization and foci of tubular hyperplasia, consistent with the expected
28 disease progression. Some lack of consistency and dose-related concordance, however, was evident
29 across the remaining steps in the histopathological sequence. First, the accumulation of hyaline
30 droplets and the concentrations of (X2u-globulin in the hyaline droplets at doses that induced
31 significant tumor formation in male rats were not significant Next, necrosis or cytotoxicity was
32 absent and only precursors to granular casts at stages well within the expected timeframe of
33 detectability were present Finally, a 13-week inhalation study found no evidence of a2u-globulin
34 nephropathy (NTP. 1997). despite evaluating exposure concentrations predicted to result in similar
35 blood tert-butanol levels as for the 13-week oral study (NTP. 1995). which reported increases in
36 droplet accumulation and sustained regenerative tubule cell proliferation. A detailed evaluation
37 and analysis of all the evidence relevant to this criterion follows.
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Toxicological Review of tert-Butyl Alcohol
1 Detailed evaluation of the available evidence supporting the third criterion
2 Single cell death and exfoliation into the renal tubules was inconsistently observed. Single
3 cell death or necrosis was not associated with tert-butanol exposure in male rat kidneys after 10 or
4 13 weeks [Acharyaetal., 1997: NTP, 1995]. Acharya et al. [1997] reported degeneration of renal
5 tubules, one pathological consequence of single cell necrosis, however, in male rats exposed to tert-
6 butanol in drinking water for 10 weeks. As renal tubule epithelial cell death and epithelial
7 degeneration should occur as early as 5 days post exposure and persist for up to 48 weeks
8 [Swenberg and Lehman-McKeeman. 1999: Short etal.. 1989]. the lack of consistency in these
9 observations could be the result of both weak induction of a2u-globulin and a lack of later
10 examinations.
11 a. Sustained regenerative cell proliferation also was not observed. Acharya et al. [1997]
12 did not observe tert-butanol-induced proliferation following 10 weeks of oral exposure,
13 but renal tubule proliferation was observed following another chemical exposure
14 (trichloroacetic acid] in the same study. Therefore, the inference is that tert-butanol
15 treatment did not induce regenerative tubule cell proliferation in male rats from this
16 study. Borghoff etal. [2001] reported a dose-related increase in epithelial cell
17 proliferation within the proximal tubule as measured by BrdU labeling indices in all
18 male rats exposed to tert-butanol via inhalation for 10 days. The study did not report
19 cytotoxicity, however, which, combined with the early time point makes unlikely that
20 the cell proliferation was compensatory. NTP [1995] also observed increased cell
21 proliferation in the renal tubule epithelium following 13-week oral exposures in male
22 rats [only male rats were studied in the retrospective analysis by Takahashi et al.
23 [1993] reported in NTP [1995]]. Proliferation was elevated at 840-1,520 mg/kg-day, a
24 range higher than the single 575-mg/kg-day dose eliciting no such proliferative effect
25 [Acharya et al.. 1997]. as described above. NTP [1995] reported, however, that no
26 necrosis was observed, suggesting the proliferation was not regenerative.
27 b. Granular cast formation was not observed, although one study noted precursors to cast
28 formation. NTP [1995] did not observe the formation of granular casts or tubular
29 dilation; however, Hard etal. [2011] reanalyzed the 13-week oral NTP data from male
30 rats treated with 0 or 1,520 mg/kg-day and identified precursors to granular casts in
31 5/10 animals in the treated group. The significance of these granular cast precursors,
32 described as sporadic basophilic tubules containing cellular debris, is unknown, because
33 13 weeks of exposure is within the expected timeframe of frank formation and
34 accumulation of granular casts (>3 weeks]. Granular cast formation, however, might not
35 be significantly elevated with weak inducers of (X2u-globulin [Short etal., 1986], which is
36 consistent with the reported difficulty in measuring (X2u-globulin in hyaline droplets
37 associated with tert-butanol exposure.
38 c. Linear mineralization of tubules within the renal papillae was consistently observed in
39 male rats. This lesion typically appears at chronic time points, occurring after exposures
40 of 3 months up to 2 years [U.S. EPA. 1991a]. Consistent with this description, 2-year oral
41 exposure to tert-butanol induced a dose-related increase in linear mineralization, but
42 not following 13-week exposure [[NTP. 1995]: Table 1-2].
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1 d. Renal tubule hyperplasia was observed in the only available 2-year study. Renal tubule
2 hyperplasia is the preneoplastic lesion associated with a2u-globulin nephropathy in
3 chronic exposures that leads to renal tubule tumors [U.S. EPA, 1991a]. A dose-related
4 increase in renal tubule hyperplasia was observed in male rats following 2-year oral
5 exposures [NTP. 1995]. By comparison, renal tubule hyperplasia was observed in only
6 one high-dose female.
7 The progression of histopathological lesions for (X2u-globulin nephropathy is predicated on
8 the initial response of excessive hyaline droplet accumulation (containing (X2u-globulin) leading to
9 cell necrosis and cytotoxicity, which in turn cause the accumulation of granular casts, linear
10 mineralization, and tubular hyperplasia. Therefore, observations of temporal and dose-response
11 concordance for these effects are informative for drawing conclusions on causation.
12 As mentioned above, most steps in the sequence of a2u-globulin nephropathy are observed
13 at the expected time points following exposure to tert-butanol. Accumulation of hyaline droplets
14 was observed early, at 12 hours following a single bolus exposure [Williams and Borghoff. 2001).
15 and at 10 days [Borghoff etal.. 2001] or 13 weeks [NTP. 1995] following continuous exposure;
16 (X2u-Globulin was identified as the protein in these droplets [Borghoff etal., 2001: Williams and
17 Borghoff. 2001]. Lack of necrosis and exfoliation might be due to the weak induction of (X2u-globulin
18 and a lack of later examinations. Granular cast formation was not reported by any of the available
19 studies, which could also indicate weak (X2u-globulin induction. Regenerative cell proliferation,
20 which was not observed, is discussed in more detail below. Observations of the subsequent linear
21 mineralization of tubules and focal tubular hyperplasia fall within the expected timeframe of the
22 appearance of these lesions. Overall, no explicit inconsistencies are present in the temporal
23 appearance of the histopathological lesions associated with (X2u-globulin nephropathy; however, the
24 dataset would be bolstered by measurements at additional time points to lend strength to the MOA
25 evaluation.
26 Inconsistencies do occur in the dose-response among lesions associated with the
27 a2u-globulin nephropathy progression. Hyaline droplets were induced in the proximal tubule of all
28 surviving male rats in the 13-week NTP oral study [NTP. 1995: Takahashietal.. 1993: Lindamood
29 etal.. 1992]. although the incidence at the lowest dose was minimal, while the incidence at the
30 three higher doses was more prominent These results are discordant with the tumor results, given
31 that all treated groups of male rats in the NTP 2-year oral bioassay had increased kidney tumor
32 incidence, including the lowest dose of 90 mg/kg-day [according to the reanalysis by Hard et al.
33 [2011]]. This lowest dose was less than the 230 mg/kg-day in the 13-week oral study that had only
34 minimal hyaline droplet formation. Furthermore, although the incidence of renal tubule
35 hyperplasia had a dose-related increase [NTP. 1995]. a corresponding dose-related increase in the
36 severity of tubular hyperplasia did not result Severity of tubule hyperplasia was increased only at
37 the highest dose, which was not consistent with renal tumor incidence.
38 Although the histopathological sequence has data gaps, such as the lack of observable
39 necrosis or cytotoxicity or granular casts at stages within the timeframe of detectability, overall, a
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1 sufficient number of steps (e.g., linear papillary mineralization, foci of tubular hyperplasia) were
2 observed to fulfill the third criterion.
3 Summary and Conclusions for Question One:
4 Oral exposure to male F344 rats resulted in an increased incidence of renal tubule tumors in
5 a 2-year oral bioassay [Hardetal., 2011: NTP, 1995]. Several histopathological observations in
6 exposed male rats were consistent with an a2u-globulin MOA. This evidence includes the increased
7 size and number of hyaline droplets and the accumulated (X2u-globulin protein in the hyaline
8 droplets. Additionally, several subsequent steps in the histopathological sequence were observed.
9 Overall, available data are sufficient for all three required criteria, suggesting that the a2u-globulin
10 process is operative. Although the evidence indicates a role for a2u-globulin accumulation in the
11 etiology of kidney tumors induced by exposure to tert-butanol in male rats, that tert-butanol is a
12 weak inducer of a2u-globulin is plausible, considering the limited histopathological observations
13 and uncertainty regarding the temporal and dose concordance of the lesions.
14 Question Two: Are the renal effects in male rats exposed to tert-butanol solely due to the a2U-globulin
15 process?
16 If the a2u-globulin process is operative, U.S. EPA [1991a] identifies a second question that
17 must be answered regarding whether the renal effects are (a) solely due to the a2u-globulin
18 process, (b) a combination of the a2u-globulin process and other carcinogenic processes, or (c)
19 primarily due to other processes. U.S. EPA[1991a] states that additional data can help inform
20 whether the a2u-globulin process is the sole contributor to renal tubule tumor development in male
21 rats. These additional considerations are highlighted and discussed, where possible, in detail below.
22 Hypothesis-testing of the a2U-globulin sequence of effects and structure-activity relationships
23 that might suggest the chemical belongs in a different class of suspected carcinogens: No data are
24 available to evaluate these considerations.
25 Biochemical information regarding binding of the chemical to the a2U-globulin protein:
26 Williams and Borghoff [2001] report that tert-butanol reversibly and noncovalently binds to
27 a2u-globulin in the kidneys of male rats. This provides additional support to the involvement of the
28 a2u-globulin process.
29 Presence of sustained cell replication in the S2 (P2] segment of the renal tubule at doses
30 used in the cancer bioassay and a dose-related increase in hyperplasia of the renal tubule:
31 Sustained cell division in the proximal tubule of the male rat is consistent with, although not
32 specific to, the a2u-globulin process. Cell proliferation was observed in two studies [13-week, NTP
33 [1995] and 10-day, Borghoff etal. [2001]] but whether the proliferation was compensatory is
34 unknown, as cytotoxicity was not observed in these studies. Although the data do not support
35 sustained cell division occurring subsequent to cytotoxic cell death, renal tubule hyperplasia in
36 male rats was reported after 2 years of exposure [NTP, 1995]. Thus, although some evidence of
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1 sustained cell replication is available, it does not specifically support (X2u-globulin protein
2 accumulation.
3 Covalent binding to DMA or other macromolecules, suggesting another process leading to
4 tumors andgenotoxicity (a2u-globulin-inducers are essentially nongenotoxic): One study [Yuanetal.,
5 2007] observed a dose-related increase in tert-butanol-DNA adducts in liver, kidney, and lung of
6 mice administered a single low dose of tert-butanol (<1 mg/kg) in saline via gavage (see Appendix
7 B.3 in Supplemental Information for further details). An extremely sensitive method of detection
8 was used (accelerator mass spectrometry), but the DNA adduct species were not identified, and no
9 validation of these results has been identified in the literature. The few studies available to assess
10 the genotoxic potential of tert-butanol primarily are negative, although a few studies report DNA
11 damage induced by oxidative stress. DNA damage induced by oxidative stress is consistent with the
12 decreased levels of glutathione in male rat kidneys reported by Acharyaetal. (1995) after 10 weeks
13 of tert-butanol exposure. This type of genetic damage would not necessarily preclude a role for
14 (X2u-globulin, but not enough information is available to determine whether oxidative stress could
15 initiate or promote kidney tumors in concert with (X2u-globulin accumulation in male rat kidneys.
16 Nephrotoxicity not associated with the a2u-globulin process or CPN, suggesting the possibility
17 of other processes leading to renal tubule nephrotoxicity and carcinogenicity: Nephropathy reported
18 in the 13-week oral and inhalation and 2-year oral studies was considered CPN, but these effects
19 were exacerbated by treatment with tert-butanol. At 13 weeks (NTP. 1997.1995] and 2 years (NTP.
20 1995], oral and inhalation exposure increased the severity of nephropathy in male rats (NTP,
21 1995]. Similarly, the severity of nephropathy was increased in females at 2 years, but only the
22 incidence of nephropathy was increased in females following a 13-week oral exposure (NTP. 1995].
23 Increased incidences of suppurative inflammation and kidney transitional epithelial hyperplasia
24 were observed in female rats orally exposed to tert-butanol for 2 years. Although NTP (1995]
25 characterized these endpoints as associated with CPN, the low background incidence in the controls
26 combined with the dose-related increase in incidences indicate that these effects were not related
27 to an age-associated, spontaneous induction of nephropathy. At 2 years, the male rats also exhibited
28 dose-related increases in focal mineralization and transitional epithelial hyperplasia, although the
29 background incidence in the controls was high (i.e., approximately 50%] (NTP. 1995]. Neither
30 endpoint in males can be attributed to CPN or (X2u-globulin.
31 Kidney weights also were increased in male and female rats in the 13-week oral and
32 inhalation evaluations (NTP. 1997.1995] and 15-month oral evaluation (NTP. 1995]. The dose-
33 related increases observed in both male and female rats suggest that the kidney weight changes are
34 indicative of treatment-related molecular processes primarily unrelated to either (X2u-globulin
35 protein accumulation or CPN. The exacerbation of CPN and the appearance of kidney effects in
36 female (i.e., suppurative inflammation, transitional epithelial hyperplasia] and male rats (i.e., focal
37 mineralization, transitional epithelial hyperplasia] that are not attributed to CPN or (X2u-globulin
38 indicate that tert-butanol induces renal tubule nephrotoxicity partially independently of
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1 (X2u-globulin. The evidence that other processes might be responsible for the renal tubule
2 nephrotoxicity thereby decreases the likelihood that a2u-globulin accumulation is solely
3 responsible for the renal tubule tumors.
4 Positive tubule tumor responses in female rats and other species implying that a2u-globulin-
5 related processes alone do not account for the renal tubule tumor response: No increase in renal
6 tubule tumor incidence was reported in tert-butanol-exposed female rats or mice compared with
7 concurrent controls. Renal tubule tumors were observed only in male rats, providing support for an
8 (X2u-globulin process in tumor development.
9 Summary and Conclusions for Question Two:
10 Although the evidence suggests that tert-butanol induces a2u-globulin nephropathy, the
11 data indicate that tert-butanol is a weak inducer of a2u-globulin and that this process is not solely
12 responsible for the renal tubule nephropathy and carcinogenicity observed in male rats. The lack of
13 compensatory cell proliferation in male rats and evidence of nephrotoxicity in female rats suggest
14 that other processes, in addition to the (X2u-globulin process, are operating. Furthermore, the
15 accumulation of hyaline droplets and the induction of renal tubule hyperplasia were affected at
16 higher doses compared to those inducing renal tubule tumors. Collectively, these data suggest that
17 tert-butanol induces the (X2u-globulin pathway at high doses (>420 mg/kg-day), which results in
18 tumor formation. Other, unknown pathways, however, could be operative at lower doses
19 (<420 mg/kg-day), which contribute to renal tumor induction.
20 b) Chronic Progressive Nephropathy and Renal Carcinogenicity
21 There is scientific disagreement regarding the extent to which CPN can be characterized as
22 a carcinogenic MOA suitable for analysis under the EPA's cancer guidelines. Proponents of CPN as
23 an MOA have developed an evolving series of empirical criteria for attributing renal tubule tumors
24 to CPN. Hard and Khan [2004] proposed criteria for concluding that a chemical is associated with
25 renal tubule tumors through an interaction with CPN. Hard etal. [2013] slightly revised and
26 restated their criteria for considering exacerbation of CPN as an MOA for renal tubule tumors in
27 rats. Table 1-5 lists these sets of proposed empirical criteria for attributing renal tubule tumors to
28 CPN.
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Table 1-5. Proposed empirical criteria for attributing renal tumors to CPN
• First and foremost, the chemical must have been
shown to exacerbate CPN to very advanced
stages of severity, especially end-stage kidney
disease, in comparison to control rats in a 2-year
carcinogenicity study.
• The tumors should occur in very low incidence
and, for the most part, be minimal grade lesions
conforming to small adenomas or lesions
borderline between atypical tubule hyperplasia
(ATM) and adenoma.
• Such tumors should be associated only with the
highest grades of CPN severity.
• The tumors and any precursor foci of ATM must
be restricted to CPN-affected parenchyma and
are usually observed only toward the end of the
2-year studies.
• Careful microscopic examination of renal
parenchyma not involved in the CPN process
should reveal no evidence of compound-induced
cellular injury or other changes that would
suggest alternative modes of action.
Source: Hard and Khan (2004)
• Lack of genotoxic activity based on overall
evaluation of in vitro and in vivo data.
• Tumor incidence is low, usually <10%.
• Tumors are found toward the end of 2-year
studies.
• Lesions are usually ATM or adenomas (carcinomas
can occasionally occur).
• Chemical exacerbates CPN to most advanced
stages, including end-stage kidney disease.
• ATM and tumors occur in rats with advanced CPN
and in CPN-affected tissue.
• Absence of cytotoxicity in CPN-unaffected
tubules, in rats with lower grades of CPN, and in
subchronic studies.
Source: Hard et al. (2013)
2 Hardetal. [2013] maintain knowing the detailed etiology or underlying mechanism for CPN
3 is not necessary. Instead, identifying increased CPN with its associated increase in tubule cell
4 proliferation as the key event is adequate. Nonetheless, Hardetal. [2013] also postulated a
5 sequence of key events for renal tumorigenesis involving exacerbation of CPN:
6 • Exposure to chemical (usually at high concentrations];
7 • Metabolic activation (if necessary];
8 • Exacerbated CPN, including increased number of rats with end-stage renal disease;
9 • Increased tubule cell proliferation because more kidney is damaged due to CPN
10 exacerbation;
11 • Hyperplasia; and
12 • Adenoma (infrequently carcinoma].
13 In contrast to Hardetal. [2013]: Hard and Khan [2004].Melnicketal. [2013]: Melnick et al.
14 [2012] concluded, based on an analysis of 60 NTP studies, no consistent association exists between
15 exacerbated CPN and the incidence of renal tubule tumors in rats. Without a consistent association
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1 and an understanding of its key events, they maintain that determining the human relevance of
2 processes that might be occurring in rats is not possible. An earlier analysis of 2 8 NTP studies
3 [Seely etal.. 2002] found a slight but statistically significant increase in CPN severity in animals
4 with renal tubule tumors, without determining that this relationship is causal. They suggested that
5 the number of tumors due to chemically exacerbated CPN would be few.
6 Evaluation of the MOA Proposed by Hard etal. (2013)
7 Setting aside the question of whether CPN is [Hard etal.. 2013: Hard and Khan. 2004] or is
8 not [Melnick et al.. 2013: Melnicketal.. 2012] an MOA suitable for analysis, this section provides an
9 analysis of the mechanistic data pertinent to CPN. EPA's cancer guidelines [U.S. EPA, 2005a] define
10 a framework for judging whether available data support a hypothesized MOA; the analysis in this
11 section follows the structure presented in the cancer guidelines.
12 Description of the hypothesized MOA. Under the EPA framework, toxicokinetic studies are
13 important for identifying the active agent, but toxicokinetic events per se are not key events of an
14 MOA. Thus, the EPA analysis of the MOA proposed by Hard etal. [2013] begins with
15 (1] exacerbated CPN, including increased number of rats with end-stage renal disease, and
16 proceeds via (2] increased tubule cell proliferation, (3] hyperplasia, and (4] adenoma, or
17 infrequently, carcinoma.
18 Strength, consistency, specificity of association. The relationship between exacerbated CPN
19 and renal tumors is moderate to strong in male rats in the NTP [1995] study. According to the NTP
20 [1995] analysis, the mean CPN grades (same as "severity of nephropathy" reported by NTP]
21 presented on a scale 1-4 for male rats with renal tumors were 3.5, 3.6, 3.7, and 3.4 at doses 0,1.25,
22 2.5, and 5 mg/mL. The mean CPN grades for male rats without renal tumors were 2.9, 2.8, 2.8, and
23 3.2 for the same dose groups. The reanalysis of the NTP data by Hard etal. [2011] yielded similar
24 numbers. The relationship between CPN and renal tumors, however, is neither consistent nor
25 specific in the NTP [1995] study: No female rats developed renal tumors regardless of the presence
26 of relatively low-grade or relatively high-grade CPN. For example, in female rats surviving more
27 than 700 days, the mean CPN grades were 1.7 and 3.2 at doses of 0 and 10 mg/mL, respectively, but
28 no tumors developed in either group.
29 Dose-response concordance. The dose-response relationships for CPN, renal tubule
30 hyperplasia, and renal tubule tumors somewhat differ. According to the NTP [1995] analysis, at
31 doses of 0,1.25, 2.5, and 5 mg/mL, the mean CPN grades for all male rats were 3.0, 3.1, 3.1, and 3.3;
32 the incidences of renal tubule hyperplasia (standard and extended evaluation combined] were
33 14/50, 20/50,17/50, and 25/50; and the incidences of renal tubule adenomas or carcinomas were
34 8/50,13/50,19/50, and 13/50 (Table 1-3]. The reanalysis by Hard etal. [2011] reported similar
35 tumor incidences (4/50,13/50,18/50, and 12/50], except that four fewer rats in the controls and
36 one fewer rat in the group exposed to 2.5 mg/mL had tumors. The lower control incidence
37 observed in this reanalysis accentuates the differences in these dose-response relationships. In
38 examining the various lesions at the mid-dose—the dose with the greatest increase in renal tubule
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1 tumors in male rats—a minor increase (14/50 in controls versus 17/50 in the mid-dose group) in
2 renal tubule hyperplasia incidence was observed, with a marginal change in CPN severity (i.e.,
3 group average of 3.0 to 3.1). That a minor increase in hyperplasia and marginal increase in CPN
4 severity would be associated with significant tumor induction seems inconsistent Furthermore,
5 CPN severity is nearly as great in the female rats, yet no females developed tumors, as noted above.
6 Temporal relationship. The severity of CPN progressed over time. According to the NTP
7 (1995) analysis, the mean CPN grades in the 13-week study of male rats were 1.0,1.6, 2.6, 2.7, 2.6,
8 and 1.1 at doses of 0, 2.5, 5,10, 20, and 40 mg/mL. At the 15-month interim evaluation of the 2-year
9 study, the mean CPN grades were 2.4, 2.8, 2.7, and 2.6 at doses of 0,1.25, 2.5, and 5 mg/mL and at
10 2 years, increased to 3.0, 3.1, 3.1, and 3.3. Similarly, the severity of neoplastic lesions increased at
11 the end of life. At the 15-month interim evaluation, only two rats had developed renal tubule
12 hyperplasia and one other had a renal tubule adenoma; at 2 years, the incidences of these two
13 lesions were much higher in all dose groups (see previous paragraph). These results are consistent
14 with CPN as an age-related disease and with hyperplasia and tumors appearing near the end of life.
15 Biological plausibility and coherence. In general, the relationship between exacerbated CPN
16 and renal tubule tumors in male rats appears plausible and coherent. Some patterns in the dose-
17 response relationships for CPN, hyperplasia, and tumors are discrepant. Perhaps more importantly,
18 the patterns also are discrepant for the relationships between CPN grades and renal tubule tumors
19 in male and female rats. In addition, the increased incidences in renal tubule tumors in all exposed
20 male rats exceed the 10% criterion proposed by Hardetal. (2013) (Table 1-5), even more so when
21 making comparisons with the lower control tumor incidence from the Hardetal. (2011) reanalysis.
22 Conclusions about the hypothesized CPN-related MOA
23 As recommended by EPA's cancer guidelines (U.S. EPA, 2005a), conclusions about the
24 hypothesized MOA can be clarified by answering three questions presented below.
25 (a) Is the hypothesized MOA sufficiently supported in the test animals? Exacerbated CPN
26 leading to renal tubule tumors in male rats late in life appears to have some support There is lack
27 of consistency, however, between males and females and in the dose-response relationships
28 between CPN, hyperplasia, and adenomas. These inconsistencies make difficult attributing all renal
29 tumors to either CPN or to (X2u-globulin-related nephropathy (see previous section on a2U-globulin),
30 raising the likelihood of another, yet unspecified MOA.
31 (b) Is the hypothesized MOA relevant to humans? There is scientific disagreement on this
32 question. Hard etal. (2013): Hard et al. (2009)cite several differences in pathology between rat CPN
33 and human nephropathies in their arguments that CPN-related renal tumors in rats are not relevant
34 to humans. On the other hand, Melnicketal. (2013): Melnicketal. (2012) argue that the etiology of
35 CPN and the mechanisms for its exacerbation by chemicals are unknown and fail to meet
36 fundamental principles for defining an MOA and for evaluating human relevance. This issue is
37 unresolved.
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1 (c) Which populations or lifestages can be particularly susceptible to the hypothesized MOA?
2 There are no indications of a human population or lifestage that is especially susceptible to tumors
3 induced through exacerbated CPN.
4 In summary, considering discrepant patterns in the dose-response relationships for CPN,
5 hyperplasia, and renal tubule tumors and the lack of relationships between CPN grades and renal
6 tubule tumors in male and female rats, together with the lack of a generally accepted MOA for CPN,
7 the renal tubule tumors in rats cannot be attributed to CPN.
8 This position can be reconciled with that of Melnicketal. [2013]: Melnicketal. [2012], who
9 argued against dismissing renal tubule tumors in rats that can be related to exacerbated CPN. It also
10 can be reconciled with Hardetal. [2013]. who, while maintaining that these tumors are not
11 relevant to humans, also allow that there is no generally accepted MOA for CPN akin to that for ct2U-
12 globulin-related nephropathy. Hardetal. [2013] made this statement after reporting on the
13 collective experience of national and international health agencies worldwide with the use of CPN
14 as an MOA. Of 21 substances that exacerbated CPN and caused renal tumors, most were multisite
15 carcinogens, and other tumor sites contributed to the evaluations. Only two assessments explicitly
16 considered CPN as a renal tumor mechanism. One was the assessment of ethylbenzene by the
17 German Federal Institute for Occupational Safety and Health, in which the agency concluded that
18 the kidney tumors were associated with the high, strain-specific incidence of CPN that is unknown
19 for humans [as discussed in Hardetal. [2013]]. The other was the IRIS assessment of
20 tetrahydrofuran, for which EPA found the evidence insufficient to conclude that the kidney tumors
21 are mediated solely by the hypothesized MOAs [U.S. EPA. 2012d]. Hardetal. [2013] attributed
22 these different conclusions to either different data for the two chemicals or the lack of a generally
23 accepted MOA akin to (X2u-globulin-related nephropathy.
24 Relevant to this last point, IARC [1999] developed a consensus statement that listed
25 considerations for evaluating (X2u-globulin-related nephropathy in rats, which was based on the
26 work of 22 scientists, including three who were co-authors of Hardetal. [2013] and two who were
27 co-authors of Melnicketal. [2013]: Melnicketal. [2012]. A similar broad-based consensus that
28 defines a sequence of key events for exacerbated CPN, distinguishes it more clearly from a2u-
29 globulin-related nephropathy, and evaluates its relevance to humans would be helpful in advancing
30 the understanding of these issues.
31 Overall Con elusions on MOA for Kidn ey Effects
32 tert-Butanol increases (X2uglobulin deposition and hyaline droplet accumulation in male rat
33 kidneys, as well as several of the subsequent steps in that pathological sequence. These data
34 provide sufficient evidence (albeit minimal] that the (X2uglobulin process is operating, although
35 based on further analysis this chemical appears to be a weak inducer of a2uglobulin-nephropathy
36 and this induction is not the sole contributor to renal tubule nephropathy and carcinogenicity. CPN
37 and the exacerbation of CPN (likely due to both a2u-globulin and tert-butanol] play a role in renal
38 tubule nephropathy. Although CPN was indicated in the induction of renal tubule nephropathy, the
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1 available evidence indicates that it does not induce the renal tubule tumors in male rats.
2 Additionally, several endpoints indicate renal tubule nephrotoxicity and increased kidney weights
3 related to tert-butanol exposure cannot be explained by the a2uglobulin or CPN processes.
4 Collectively, the evidence indicates other, unknown processes contribute to renal tubule
5 nephrotoxicity and carcinogenicity.
6 Integration of kidney effects
7 Kidney effects (increases in nephropathy, severity of nephropathy, hyaline droplets, linear
8 mineralization, suppurative inflammation, transitional epithelial hyperplasia, mineralization, and
9 kidney weight) were observed, predominantly in male and female rats across the multiple tert-
10 butanol studies. The available evidence indicates that multiple processes induce the noncancer
11 kidney effects. The group of lesions generally reported as "nephropathy," is related to CPN. Because
12 this disease is considered to be spontaneous and age-related in rats, the endpoints associated with
13 CPN would not be relevant to humans for purposes of hazard identification. Additionally, two
14 endpoints in male rats (hyaline droplets, linear mineralization) are components of the (X2u-globulin
15 process. U.S. EPA(1991a) states that if the (X2u-globulin process is occurring in male rats, the renal
16 tubule effects associated with this process in male rats would not be relevant to humans for
17 purposes of hazard identification. In cases such as these, the characterization of human health
18 hazard for noncancer kidney toxicity would rely on effects not specifically associated with CPN or
19 the a2u-globulin-process in male rats.
20 Several other noncancer endpoints resulted from tert-butanol exposure and are appropriate
21 for consideration of a kidney hazard, specifically: suppurative inflammation in female rats,
22 transitional epithelial hyperplasia in male and female rats, severity of nephropathy in male and
23 female rats, incidence of nephropathy in female rats, incidence of mineralization in male rats, and
24 increased kidney weights in rats but not mice. Based on dose-related increases in these noncancer
25 endpoints in rats, kidney effects are a potential human hazard of tert-butanol exposure. The hazard
26 and dose-response conclusions regarding these noncancer endpoints associated with tert-butanol
27 exposure are discussed further in Section 1.3.1.
28 The carcinogenic effects observed following tert-butanol exposure include increased
29 incidences of renal tubule hyperplasia (considered a preneoplastic effect) and tumors in male rats.
30 EPA concluded that the three criteria were met to indicate that an (X2u-globulin process is operating.
31 Because renal tubule tumors in male rats did not arise solely due to the (X2u-globulin process and
32 some of the tumors are attributable to other carcinogenic processes, such tumors remain relevant
This document is a draft for review purposes only and does not constitute Agency policy.
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1 for purposes of hazard identification U.S. EPA (1991a).3 The hazard and dose-response conclusions
2 regarding the renal tubule hyperplasia and tumors associated with tert-butanol exposure are
3 further discussed as partof the overall weight of evidence for carcinogenicity in Section 1.3.2.
4 1.2.2. Thyroid Effects
5 Synthesis of Effects in Thyroid
6 The database on thyroid effects following tert-butanol exposure contains no human data,
7 two oral subchronic and two oral chronic studies (one of each duration in rats and in mice) [NTP.
8 1995], and two inhalation subchronic studies (one in rats and one in mice) [NTP, 1997}. Studies
9 employing short-term and acute exposures that examined thyroid effects are not included in the
10 evidence table; they are discussed, however, in the text if they provide data informative of MOA or
11 hazard identification. No gross thyroid effects were reported in the 13-week evaluations of mice or
12 rats following oral or inhalation exposure [NTP. 1997.1995). and therefore subchronic studies
13 were not included in the evidence table. The two available chronic studies are arranged in the
14 evidence table by effect and then by species. The design, conduct, and reporting of each study were
15 reviewed, each study was considered adequate to provide information pertinent to this assessment
16 (Table 1-6 and Figure 1-10).
17 Thyroid effects, specifically follicular cell hyperplasia and adenomas, were observed in mice
18 of both sexes after 2 years of oral exposure via drinking water (NTP, 1995). NTP (1995) noted that
19 "[proliferation of thyroid gland follicular cells is generally considered to follow a progression from
20 hyperplasia to adenoma and carcinoma." Similarly, EPA considered the thyroid follicular cell
21 hyperplasia to be a preneoplastic effect associated with the thyroid tumors. Both male and female
22 mice exhibited a dose-related increase in the incidence of hyperplasia, and the average severity
23 across all dose groups was minimal to mild with scores ranging from 1.2 to 2.2 (out of 4). Increased
24 incidence of adenomas were also observed in the tert-butanol-treated mice, with the only
25 carcinoma observed in high-dose males. No treatment-related thyroid effects were reported in rats
26 of either sex following 2 years of oral exposure (NTP. 1995).
27 Although the tumor response in male mice showed a statistically significant increasing
28 trend (Cochran-Armitage trend test, p = 0.041) (analysis performed by EPA using the mortality-
29 adjusted rates), the response was non-monotonic, with a slightly lower response at the high-dose
3 When the
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Toxicological Review of tert-ButyI Alcohol
1 level than at the mid-dose level. The reason for the non-monotonicity is unclear, although it could
2 be related to the increased mortality in the high-dose group (17/60 animals survived compared
3 with 27/60 animals in the control group). The decreased survival of male mice might have affected
4 the thyroid tumor incidences because animals could have died before tumors could develop. High
5 mortality in the high-dose group occurred before tumors appeared; about 40% of the high-dose
6 males died before the first tumor (a carcinoma) appeared in this group at week 83. By comparison,
7 only ~10% of the control group had died by this time, and the single tumor in the control group
8 was observed at study termination. Mortality in the exposed female mice was similar to controls.
9
10
Table 1-6. Evidence pertaining to thyroid effects in animals following oral
exposure to tert-butanol
Reference and study design
Follicular cell hyperplasia
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Drinking water (0, 1.25, 2.5, 5, or 10
mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0, 180, 330, or 650a mg/kg-d
2 years
NTP (1995)
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5, 10, or 20 mg/mL)
M: 0, 540, 1,040, or 2,070a mg/kg-d
F: 0, 510, 1,020, or 2,110 mg/kg-d
2 years
Follicular cell tumors
NTP (1995)
Incidence15
Males
Dose
(mg/kg-d)
0
90
200
420a
Incidence (severity)
Males
Dose
(mg/kg-d)
0
540
1,040
2,070 a
Incidence15
Result
Follicular cell
hyperplasia
3/50
0/49
0/50
0/50
Follicular cell
hyperplasia
5/60 (1.2)
18/59* (1.6)
15/59* (1.4)
18/57* (2.1)
S
Females
Dose
(mg/kg-d)
0
180
330
650 a
Females
Dose
(mg/kg-d)
0
510
1,020
2,110
Follicular cell
hyperplasia
0/50
0/50
0/50
0/50
Follicular cell
hyperplasia
19/58 (1.8)
28/60 (1.9)
33/59* (1.7)
47/59* (2.2)
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Reference and study design
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Drinking water (0, 1.25, 2.5, 5, or 10
mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0, 180, 330, or 650a mg/kg-d
2 years
NTP (1995)
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5, 10, or 20 mg/mL)
M: 0, 540, 1,040, or 2,070a mg/kg-d
F: 0, 510, 1,020, or 2,110 mg/kg-d
2 years
A
^
Results
Dose (mg/kg-d'
Male
0
90
200
420 a
Female
0
180
330
650 a
Incidence
Follicular cell
adenoma
2/50
0/49
0/50
0/50
1/50
0/50
1/50
0/50
Dose Follicular cell Follicular cell
(mg/kg-d)
Male
0
540
1,040
2,070 a
Female
0
510
1,020
2,110
adenoma carcinoma
1/60 0/60
0/59 0/59
4/59 0/59
1/57 17^7
2/58 0/58
3/60 0/60
2/59 0/59
9/59* 0/59
Follicular cell
carcinoma
2/50
0/49
0/50
0/50
1/50
0/50
1/50
0/50
Follicular cell
adenoma or
carcinoma
(mortality
adjusted rates)-^
1/60 (3.6%)
0/59 (0.0%)
4/59 (10.1%)
2/57 (8.7%)
2/58 (5.6%)
3/60 (8.6%)
2/59 (4.9%)
9/59* (19.6%)
Animals
surviving to
study
termination
27/60
36/60
34/60
17/60
36/60
35/60
41/60
42/60
1 aThere was a significant decrease in survival in the high-dose group.
2 bResults do not include the animals sacrificed at 15 months.
3 cMortality-adjusted rates were not calculated by study authors for follicular cell carcinoma. The mortality-adjusted rates for the
4 incidence of adenomas are the same as the combined rates, with the exception of the male high-dose group, where the rate
5 for adenomas alone was 5.9%.
6 dCochran-Armitage trend test was applied to mortality-adjusted thyroid tumor incidences, by applying the NTP adjusted rates
7 to the observed numbers of tumors to estimate the effective number at risk in each group. For male mice, p = 0.041; for
8 female mice, p = 0.028.* Statistically significant p < 0.05 as determined by the study authors.
9 Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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Toxicological Review of tert-ButyI Alcohol
2
3
4
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
Hyperplasia; M mouse -
Hyperplasia; F mouse -
NONCANCER
Hyperplasia; M ral
Hyperplasia; F ral -
Adenoma; M mouse
Adenoma; F mouse
CANCER
Adenoma; M rat
Adenoma; F rat
10
Source: NTP (1995)
D
-B B
Q B B
B \1
B B
-B B
B B B
100 1,000
Dose (mg/kg-day)
Figure 1-10. Exposure-response array of thyroid follicular cell effects
following chronic oral exposure to tert-butanol. (Note: Only one carcinoma
was observed in male mice at the high-dose group.)
10,000
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Toxicological Review of tert-ButyI Alcohol
1 Mode of Action Analysis—Thyroid Effects
2 The MOA responsible for tert-butanol-induced thyroid effects has not been the subject of
3 much study. One hypothesis is that tert-butanol increases liver metabolism of thyroid hormones,
4 triggering a compensatory increase in pituitary thyroid-stimulating hormone (TSH) production.
5 Such sustained increases in TSH could induce elevated thyroid follicular cell proliferation and
6 hyperplasia and lead to follicular cell adenoma and carcinoma, that, an antithyroid MOA, as
7 identified in U.S. EPA's guidance on the assessment of thyroid follicular cell tumors [U.S. EPA.
8 1998a].
9 To determine if the thyroid follicular cell tumors result from a chemically induced
10 antithyroid MOA, U.S. EPA[1998a] requires that the available database demonstrate: (1) increases
11 in thyroid cell growth, (2) thyroid and pituitary hormone changes consistent with the antithyroid
12 MOA, (3) site(s) of the antithyroid action, (4) dose correlation among the various effects, and (5)
13 reversibility of effects in the early stages of disruption. The available evidence pertaining to each of
14 these aspects of antithyroid activity following tert-butanol exposure is discussed below.
15 1] Increases in cell growth [required]
16 U.S. EPA[1998a] considers increased absolute or relative thyroid weights, histological
17 indicators of cellular hypertrophy and hyperplasia, DNA labeling, and other measurements (e.g., Ki-
18 67 or proliferating cell nuclear antigen expression) to be indicators of increased cell growth. Only a
19 few studies [NTP. 1997.1995] have evaluated the thyroid by routine histological examination
20 following tert-butanol exposure, and none investigated specific molecular endpoints. None of the
21 available long-term studies measured thyroid weight in mice, likely due to the technical limitations
22 involved, and no thyroid effects were attributed to tert-butanol exposure in rats treated up to 2
23 years [NTP. 1997.1995]. Although the short-term female mouse study by Blancketal. [2010]
24 stated that thyroids were weighed, no results were reported.
25 An increase in thyroid follicular cell hyperplasia was observed in both female and male mice
26 after a 2-year drinking water exposure to tertbutanol- [NTP. 1995]. The increase was dose
27 dependent in female mice with a slight increase in severity in the highest dose, while male mice
28 experienced a similar magnitude of hyperplasia induction at all doses, with increased severity at
29 the highest dose [NTP. 1995]. Thyroid follicular cell hyperplasia was not observed in any mouse
30 study with less than 2 years of exposure: no treatment-related histological alterations in the thyroid
31 of tert-butanol-treated (2 or 20 mg/mL] female mice after 3 or 14 days of drinking water exposure
32 [Blancketal.. 2010] were reported, in male or female mice after 13 weeks of drinking water
33 exposure [NTP. 1995] or in male or female mice following 18-day or 13-week inhalation studies
34 [NTP. 1997]. The observation of increased hyperplasia in male and female mice after 2 years of
35 exposure is sufficient evidence to support increased thyroid cell growth.
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Toxicological Review of tert-ButyI Alcohol
1 2] Changes in thyroid and relevant pituitary hormones [required]
2 Evidence of hormonal changes, including decreases in thyroxine (14) and triiodothyronine
3 [Ts] and increases in TSH, are required to demonstrate a disruption in the thyroid-pituitary
4 signaling axis [U.S. EPA, 1998a]. Blancketal. [2010] evaluated serum thyroid hormones in mice
5 after 3 or 14 days of exposure to tert-butanol. No tert-butanol-related effects were observed in Ts,
6 T4, or TSH levels after 3 days, and although both T3 and T4 levels were significantly decreased
7 approximately 10-20% after 14 days of treatment with tert-butanol, TSH levels remained
8 unaffected. Similar results were reported with the positive control [phenobarbital]. The limited
9 evidence available from this single study suggests that although T3 and T4 levels were decreased
10 after 14 days, this perturbation likely was not in excess of the range of homeostatic regulation in
11 female B6C3Fi mice and thus not likely to induce compensatory thyroid follicular cell proliferation.
12 Multiple lines of evidence support this observation: [1] TSH levels were unaffected, indicating that
13 the decrease in Ts and T4 levels was not severe enough to stimulate increased TSH secretion by the
14 pituitary; [2] thyroid hyperplasia was not induced in this study, or any others exposing mice for
15 2.5-13 weeks, suggesting that thyroid proliferation was either not induced by the hormone
16 fluctuations or that any follicular cell proliferation during this period was too slight to be detected
17 by routine histopathological examination; [3] the maximal decrease in Ts or T4 hormone levels
18 induced by tert-butanol exposure after 14 days (i.e., ~20%] was well within the range of fluctuation
19 in Ts and T4 hormone levels reported to occur between the 3- and 14-day control groups [15-40%;
20 [Blancketal.. 2010]]. Although the lower Ts and T4 levels following tert-butanol were later
21 attributed by the study authors to an increase in liver metabolism (see next section], they could in
22 fact be due to a decrease in thyroid hormone production, resulting from some, as of yet,
23 uninvestigated molecular interactions of tert-butanol in the thyroid, pituitary, or hypothalamus.
24 The absence of information regarding thyroid hormone levels in male mice and lack of
25 molecular studies evaluating exposures >2 weeks in female mice are significant deficiencies in the
26 available database. Together, although small decreases in some thyroid hormone levels have been
27 reported in female mice, the available evidence is inadequate to determine if tert-butanol
28 negatively affects the pituitary-thyroid signaling axis in female mice; furthermore, no evidence was
29 available to evaluate this effect in male mice.
30 3] Site[s] of antithyroid action [required]
31 The thyroid and liver are two of several potential sites of antithyroid action, with the liver
32 the most common site of action, where increased microsomal enzyme activity could enhance
33 thyroid hormone metabolism and removal (U.S. EPA. 1998a]. Rats are thought to be more sensitive
34 than mice to this aspect of antithyroid activity (Rogues etal.. 2013: Qatanani et al.. 2 005: U.S. EPA.
35 1998a]: however, rats exposed to tert-butanol for 2 years did not exhibit treatment-related thyroid
36 effects, while mice did. Typically, chronic induction of liver microsomal enzyme activity resulting
37 from repeated chemical exposure would manifest some manner of liver histopathology, such as
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Toxicological Review of tert-ButyI Alcohol
1 hepatocellular hypertrophy or hyperplasia [U.S. EPA. 1998a: NTP. 1995]. In a 14-day mechanistic
2 investigation, tert-butanol had no effect on liver weight when compared to the control group, but
3 centrilobular hepatocellular hypertrophy was reported in 2/5 livers from high-dose mice (versus
4 0/6 in control and 0/5 in low-dose mice [Blancketal., 2010]. Relative liver weights increased in
5 male and female mice after 13 weeks of oral exposure [NTP. 1995] to higher doses than those
6 evaluated by Blancketal. [2010]. although absolute liver weight measurements in treated animals
7 showed little change from controls suggesting that the relative measures could have been related to
8 decreases in body weight rather than specific liver effects. Relative (and absolute] liver weights
9 were increased in female mice (only] after 13 weeks of inhalation exposure at the two highest
10 concentrations (NTP. 1997]: liver weight was not reported in mice orally exposed for 2 years (NTP.
11 1995]. No increase in mouse hepatocellular hypertrophic or hyperplastic histopathology was
12 reported following 2.5 weeks to 2 years of exposure (NTP. 1997.1995]. In fact, the only liver
13 pathology associated with tert-butanol exposure in these studies was an increase in fatty liver in
14 male mice in the high-dose group after 2 years of oral exposure (NTP. 1995]. Although increased
15 fatty liver could indicate some non-specific metabolic alteration, the absence of a similar treatment-
16 related effect in livers from female mice, which were sensitive to both thyroid follicular cell
17 hyperplasia and tumor induction, suggests that it might not be related to the thyroid tumorigenesis.
18 One study evaluated liver enzyme expression and found highly dose-responsive induction
19 of a single phase I cytochrome p450 enzyme (CYP2B10] following 14 days of tert-butanol exposure
20 in female mice, with much smaller increases in the expression of another phase I enzyme CYP2B9,
21 and the phase II thyroid hormone-metabolizing enzyme, sulfotransferase 1A1 [(SULT1A1; Blanck et
22 al. (2010]]. CYP2B enzyme induction is commonly used as an indication of constitutive androstane
23 receptor (CAR] activation; CAR can induce expression of a wide range of hepatic enzymes, including
24 several CYPs along with thyroid hormone-metabolizing sulfotransferases (Rogues etal.. 2013]. The
25 only thyroid hormone-metabolizing enzyme induced by tert-butanol, however, was SULT1A1,
26 which has been reported to be inducible in a CAR-independent manner in mice (Oatanani etal..
27 2005]. Based on alterations in hepatic phase I and phase II enzyme activities and gene expression,
28 the above data suggest a possible role for increased thyroid hormone clearance in the liver
29 following repeated tert-butanol exposure; however, the expression changes in these few enzymes
30 are not supported by any liver histopathological effects in mice exposed for longer durations, so
31 whether this enzyme induction is transient, or simply insufficient to induce liver pathology after >2
32 weeks of exposure, is unknown. No evidence is available to evaluate the potential for intrathyroidal
33 or any other extrahepatic effects in female mice or for any of these molecular endpoints in male
34 mice; therefore, the available evidence is inadequate to determine if major site(s] of antithyroid
35 action are affected.
36 4] Dose correlation (required]
37 Confidence in the disruption of the thyroid-pituitary function is enhanced when dose
38 correlation is present among the hormone levels producing various changes in thyroid
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Toxicological Review of tert-ButyI Alcohol
1 histopathology, including thyroid tumors [U.S. EPA. 1998a]. Furthermore, if thyroid hormone levels
2 were affected by liver enzyme induction, confidence would be increased by a concordance among
3 liver effects, thyroid hormone levels, and thyroid pathology. Thyroid hormone levels were
4 evaluated only in female mice exposed to tert-butanol; after 2 weeks of exposure, both T4 and T3
5 were decreased with both doses (2 and 20 mg/L), and TSH was unaffected at either dose [Blanck et
6 al.. 2010). Liver expression of CYP2B10 was increased in a dose-responsive manner, while
7 SULT1A1 mRNA was induced by 20-30% at both doses [Blanck etal.. 2010]. As described above,
8 induction of liver microsomal enzyme activity would manifest some manner of liver histopathology
9 [Maronpotetal.. 2010: U.S. EPA. 1998a: NTP. 1995). Toxicol Pathol 38:776-795), and consistent
10 with this expected association, centrilobular hepatocellular hypertrophy was reported in 2/5 high-
11 dose mice exposed for 2 weeks [Blanck etal., 2010]. No liver histopathology, however, was
12 attributed to tert-butanol exposure in female mice exposed for 2.5 weeks to 2 years to comparable
13 tert-butanol concentrations [NTP. 1997.1995]. Although liver enzyme levels and activity were not
14 specifically evaluated following subchronic to chronic exposure, the lack of liver pathology suggests
15 a comparable lack of enzyme induction. Conversely, no histopathological alterations were reported
16 in the thyroids of female mice after 2 weeks of oral exposure at doses that elevated some liver
17 enzyme levels [Blanck etal.. 2010].
18 Following 2 years of oral exposure, both follicular cell hyperplasia and follicular cell tumor
19 incidence was increased in mice despite a lack of treatment-related liver pathology [NTP. 1995]
20 (Table 1-6]. Any associations relating hormone changes to thyroid pathology or liver enzyme
21 induction are limited due to the inadequate database (described above]; the available evidence
22 suggests little concordance among reports of liver, pituitary, and thyroid effects in female mice, and
23 no evidence was available to evaluate these associations in male mice.
24 5] Reversibility [required]
25 Chemicals acting via an antithyroid MOA have effects (e.g., increased TSH levels, thyroid
26 follicular cell proliferation] that are reversible after cessation of treatment [U.S. EPA. 1998a].
27 Although increased TSH levels have not been demonstrated following tert-butanol exposure,
28 thyroid follicular cell proliferation was observed following chronic exposure. As no studies have
29 evaluated changes in thyroid hormones or thyroid histopathology after cessation of tert-butanol
30 treatment, however, the available evidence is inadequate to evaluate reversibility of these effects.
31 In summary, the available database sufficiently supports only [1] increases in thyroid cell
32 growth. The existing data are inadequate to evaluate [2] thyroid and pituitary hormone changes
33 consistent with the antithyroid MOA, (3] site(s] of the antithyroid action, or (5] reversibility of
34 effects in the early stages of disruption. Although these inadequacies also limit the evaluation of [4]
35 dose correlation among the various effects, the available evidence suggests that little correlation
36 exists among reported thyroid, pituitary, and liver endpoints. Together, the database is inadequate
37 to determine if an antithyroid MOA is operating in mice. In the absence of information to indicate
38 otherwise, the thyroid tumors observed in mice are considered relevant to humans.
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Toxicological Review of tert-ButyI Alcohol
1 Integration of thyroid effects
2 The thyroid endpoints reported following chronic exposure to tert-butanol include
3 increases in follicular cell hyperplasia and tumors in male and female mice. As discussed above, due
4 to inadequacies in four of the five required areas [U.S. EPA, 1998a], the evidence is inadequate to
5 determine if an antithyroid MOA is operating in mice; therefore, the MOA(s) for thyroid
6 tumorigenesis has not been identified. EPA considers the thyroid follicular cell hyperplasia to be an
7 early event in the neoplastic progression of thyroid follicular cell tumors, and no other noncancer
8 effects on the thyroid were observed. Thus, the hazard and dose-response conclusions regarding
9 the thyroid follicular cell hyperplasia and tumors associated with tert-butanol exposure are
10 discussed as partof the overall weight of evidence for carcinogenicity in Section 1.3.2.
11 1.2.3. Developmental Effects
12 Synthesis of effects related to development
13 Four studies evaluated developmental effects [three oral or inhalation developmental
14 studies [Faulkner etal.. 1989: Nelson etal.. 1989: Daniel and Evans. 1982] and a one-generation,
15 oral reproductive study [Lyondell Chemical Co., 2004]] in animals exposed to tert-butanol via liquid
16 diet (i.e., maltose/dextrin], oral gavage, or inhalation. No developmental epidemiology studies are
17 available for tert-butanol. The animal studies are arranged in the evidence tables by species, strain,
18 and route of exposure. The design, conduct, and reporting of each study were reviewed, and each
19 study was considered adequate to provide information pertinent to this assessment. One study was
20 considered less informative, Faulkner et al. [1989]. because it did not provide sufficient information
21 on the dams to determine if fetal effects occurred due to maternal toxicity.
22 Developmental effects of tert-butanol observed after oral exposure (liquid diets or gavage]
23 in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increased
24 number of resorptions, decreased numbers of neonates per litter] and decreased fetal body weight
25 (Lyondell Chemical Co.. 2004: Faulkner etal.. 1989: Daniel and Evans. 1982]. Daniel and Evans
26 (1982] also observed decreases in body weight gain during post-natal days (PNDs] 2-10; data
27 suggest, however, that this effect might be due to altered maternal behavior or nutritional status. In
28 addition, a single dose study reported a small increase in the incidence of variations of the skull or
29 sternebrae in two mouse strains (Faulkner etal., 1989]. Although variations in skeletal
30 development were noted in the study, no malformations were reported. Similar developmental
31 effects were observed after whole-body inhalation exposure in Sprague-Dawley rats for 7
32 hours/day on gestation days (CDs] 1-19 (Nelson etal., 1989]. Fetal effects included dose-related
33 reductions in body weight in male and female fetuses and higher incidence of skeletal variations
34 when analyzed based on individual fetuses (but not on a per litter basis].
35 In these studies, fetal effects are generally observed at doses that cause toxicity in the dams
36 as measured by clinical signs (e.g., decreased body weight gain, food consumption] (Table 1-7;
37 Figure 1-11; Figure 1-12]. As stated in the Guidelines for Developmental Toxicity Risk Assessment
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Toxicological Review of tert-ButyI Alcohol
1 [U.S. EPA. 1991b]. "an integrated evaluation must be performed considering all maternal and
2 developmental endpoints." "[W]hen adverse developmental effects are produced only at doses that
3 cause minimal maternal toxicity; in these cases, the developmental effects are still considered to
4 represent developmental toxicity and should not be discounted." Although, at doses of "excessive
5 maternal toxicity...information on developmental effects may be difficult to interpret and of limited
6 value." In considering the fetal and maternal toxicity data following tert-butanol exposure, the
7 severity of the maternal effects were minimal and therefore the developmental effects in the fetuses
8 should not be discounted [U.S. EPA, 1991b]. The observed fetal effects occurred, however, at doses
9 resulting in maternal toxicity across all available studies. Therefore, whether the fetal effects are
10 directly related to tert-butanol treatment or are secondary to maternal toxicity remains unclear.
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Table 1-7. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol
Reference and study design
Results
Lyondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
FO males: 9 weeks beginning 4 weeks prior
to mating
FO females: 4 weeks prior to mating
through PND21
Fl Males and Females: 7 weeks
(throughout gestation and lactation; 1 male
and 1 female from each litter was dosed
directly from PND 21-28)
Response relative to control
Dose
(mg/kg-d) 0 64 160 400 1000
Maternal effects
Body weight gain GD 0-20
0 -3-40 -16*
Food consumption GD 0-20
0 00+40
Body weight gain PND 1-21
0 +3 -10 +3 +100*
Food consumption LD1-14
0 -2-60 -16
Live pups/litter response relative to control
0 -9 -11 -7 -33*
Dams dosed with 400 or 1000 mg/kg-d showed CNS effects (e.g.. ataxia. lethargy)
which were undetectable by 4-weeks of exposure in animals exposed to 400
mg/kg-d but not those in the higher dose group.
Fl effects
Viability index (pup survival to PND4)
96.4% 98.7% 98.2% 99.4% 74.1%*
Lactation index (pup survival to PND21)
100% 100% 100% 99.2% 98.8%
Sex ratio (% males)
54.4 52.3 50.9 53.4 52.1
Pup weight/litter PND 1 relative to control (%)
0 +6 +4 +7 -10
Pup weight PND 28 relative to control (%)
M: 0 +200 -12*
F: 0 0-4 -2 -8
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Daniel and Evans (1982)
Swiss Webster (Cox) mouse; 15 pregnant
dams/treatment
Liquid diet (0, 0.5, 0.75, 1.0%, w/v)
0 (isocaloric amounts of maltose/dextrin)
3,324, 4,879, 6,677 mg/kg-d
GD6-20
Faulkner etal. (1989)
CBA/J mouse; 7 pregnant females in
control, 12 pregnant females in treated
Gavage (10.5 mmoles/kg twice a day);
0 (tap water), 1,556 mg/kg-d
GD6-18
Results
No statistical analysis was conducted on any of these data
Maternal
Percent change compared to control:
Number of litters
Dose Food consumption Body weight (% pregnant
(mg/kg-d) (mean g/animal/day) gain dams)
000 11(77%)
3,324 +2 -3 12(80%)
4,879 -3 -19 8(53%)
6,677 -4 -20 7(47%)
^H
Authors note that lower food consumption in higher tert-butanol dose groups
reflects problems with pair feeding and maternal sedation.
Fetal
Percent change compared to control:
Dose Number of Fetal body weight
(mg/kg-d) neonates/litter on PND 2
000
3,324 -1 -7
4,879 -29 -19
6,677 -49 -38
Number of stillborn also increased with dose (3, 6, 14, and 20, respectively), but
the number of stillborn per litter was not provided. The high dose also caused a
delay in eye opening and a lag in weight gain during PND 2-10 (information was
only provided in text or figures)
Maternal results not reported.
Fetal
Percent change compared to control: Incidence:
Live
Dose fetuses/ Fetal Sternebral Skull
(mg/kg-d) Resorptions/litter litter weight variations variations
0 000 4/28 1/28
1,556 +118* -41* -4 7/30 3/30
Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (10 resorptions/66 implants in controls, 37/94
implants in treated) increased (p < 0.05)
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Faulkner etal. (1989)
C57BL/6J mouse; 5 pregnant females in
controls, 9 pregnant females treated
Gavage (10.5 mmoles/kg twice a day)
0 (tap water), 1,556 mg/kg-d
GD6-18
Nelson etal. (1989)
Sprague-Dawley rat; 15 pregnant
dams/treatment
Inhalation analytical concentration: 0,
2,200, 3,510, 5,030 ppm (0, 6,669, 10,640,
15,248 mg/m3), (dynamic whole body
chamber)
7hr/d
GD 1-19
Results
Maternal results not reported.
Fetal
Percent change compared to control: Incidence:
Live
Dose fetuses/ Fetal Sternebral Skull
(mg/kg-d) Resorptions/litter litter weight variations variations
0 000 5/21 1/21
1,556 +428* -58* -4 9/16 7/16
Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (4 resorptions/44 implants in controls, 38/68
implants in treated) increased (p < 0.05)
Maternal: Unsteady gait (no statistical tests reported), dose-dependent •]/ in
body weight gain (results presented in figure only), dose-dependent •]/ in food
consumption ranging from 7-36% depending on dose and time
Fetal
Percent change compared to control (mean ± standard error):
Dose Number of live Resorptions
(mg/m3-) fetuses/litter per litter
0 0(13±2) 0(1.1+1.2)
6,669 0(13±4) +9(1.2±1.1)
10,640 +15 (15±2) -18 (0.9±1.0)
15,248 +8(14±2) 0(1.1±0.9)
Percent change compared to
control: Incidence:
Skeletal Skeletal
Dose Fetal weight Fetal weight variation variation
(mg/m-) (males) (females) by litter by fetus
000 10/15 18/96
6,669 -9* -9* 14/17 35/104
10,640 -12* -13* 14/14 53/103*
15,248 -32* -31* 12/12 76/83*
Skeletal variation by litter refers to the number of variations observed in the
number of litters examined. Skeletal variation by fetus refers to the number of
variations observed in the total number of fetuses examined. Fetuses are not
categorized by litter.
1
2
3
4
5
* Statistically significant p < 0.05 as determined by study authors. Conversions from diet concentrations to mg/kg-d
performed by study authors. Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
Note: Percentage change compared to control = (treated value - control value) -f control value x 100.
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
DEVELOPMENTAL
i Maternal body weight gain
(CD 0-20]; F rat (C)
TMaternal body weight gain
(LD 1-21); F rat (C)
•iNumberof live pups per litter; M+F
rat(C)
-IViability index; M+F rat(C)
Lactation index; M+F rat (C)
Sex ratio; M+F rat (C)
-I Pup weight per litter
[PNDl);M+Frat(C)
JPup weight per litter
(PND28);Mrat(C)
IPup weight per litter
(PND28);Frat(C)
-1 Maternal body weight gain; F
mouse (A) *
-INumberof neonates/Iitter, fetal
body weight; M+F mouse (A)*
TNumberof resorptions per litter;
M+F mouse (B)
INumberof live fetuses per litter;
M+F mouse (B)
-IFetal weight; M+F mouse (B)
Skeletal variations; M+F mouse (B)
E
,
,
Q-B-B
m-m-m
•
D
10
100 1,000
Dose (mg/kg-day)
10,000
3
4
5
6
* Study authors did not conduct statistical analysis on these endpoints, but results are determined by EPA
to be biologically significant.
Sources: (A) Daniel and Evans (1982); (B) Faulkner et al. (1989); Lyondell Chemical Co. (2004)
Figure 1-11. Exposure-response array of developmental effects following oral
exposure to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
DEVELOPMENTAL
Number of live fetuses per litter; M+F rat
(Nelson etal., 1989)
Number of resorpn'ons per litter; M+F rat
(Nelson etal., 1989)
^Fetal weight; M rat
(Nelson etal., 1989)
iFetal weight; F rat
(Nelson etal., 1989)
Skeletal variation by litter; M+F rat
(Nelson etal., 1989)
Skeletal variation by fetus; M+F rat
(Nelson etal., 1989)
D B—B
D B—B
1,000 10,000
Exposure Concentration (rng/m3)
100,000
Figure 1-12. Exposure-response array of developmental effects following
inhalation exposure to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol
1 Integration of developmental effects
2 There is suggestive evidence of developmental effects associated with tert-butanol
3 exposure. Exposure to tert-butanol during gestation resulted in increased fetal loss, decreased fetal
4 body weight, and increases in skeletal variations in exposed offspring. Dams had body weight losses
5 or gains (or both), decreased food consumption, and clinical signs of intoxication at the same doses
6 of tert-butanol causing fetal effects. Therefore, determining whether tert-butanol exposure results
7 in specific developmental toxicity or whether the fetal effects are due to maternal toxicity is
8 difficult The observed maternal effects are minimal, however, and thus, the developmental effects
9 observed in the fetuses are not discounted as being secondary to maternal toxicity [U.S. EPA.
10 1991b) and the evidence is considered suggestive of developmental toxicity.
11 1.2.4. Neurodevelopmental Effects
12 Synthesis of effects related to neurodevelopment
13 Three studies evaluated neurodevelopmental effects [Nelson etal.. 1991: Daniel and Evans.
14 1982.)[one in male rats; one in female rats] following tert-butanol exposure via liquid diet (i.e.,
15 maltose/dextrin) or inhalation. No epidemiology studies on neurodevelopment are available. The
16 animal studies evaluating neurodevelopmental effects of tert-butanol contain study design
17 limitations. Daniel and Evans (1982) had a small number of animals per treatment group, lacked
18 comparison of treatment-related effects to controls for all endpoints investigated, and did not use
19 long-term neurodevelopmental testing. The two studies by Nelson etal. (1991) evaluated
20 neurodevelopmental effects after either paternal or maternal exposure but did not run the
21 exposures concurrently or provide exposure methods to indicate the studies were conducted
22 similarly. The studies are arranged in the evidence tables by species and sex.
23 Various neurodevelopmental effects have been observed in the available studies. These
24 include changes in rotarod performance following oral or inhalation exposures and decreases in
25 open field behavior and cliff avoidance following oral exposure, and reduced time hanging on wire
26 after inhalation exposure during gestation (Table 1-8).
27 Rotarod performance
28 Inconsistent results were observed across studies. Although Daniel and Evans (1982) found
29 decreased rotarod performance in mouse pups of dams orally exposed during gestation, Nelson et
30 al. (1991) observed an increase in rotarod performance in rat pups of dams exposed via inhalation
31 during gestation.
32 Neurochemical measurements
33 Biochemical or physiological changes in the brain of offspring exposed during gestation or
34 early in the postnatal period were examined in one study. In this study, Nelson etal. (1991)
35 reported statistically significant changes in neurochemical measurements in the brain in offspring
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Toxicological Review of tert-ButyI Alcohol
1 of both dams exposed via inhalation during gestation and treated adult males mated with untreated
2 dams. The strength of these results is compromised, however, because the two concentrations
3 tested (in both experiments) were not run concurrently, and only data on statistically significant
4 effects were reported. Therefore, comparison across doses or trend analysis for the effects is not
5 feasible.
6 Physiological and psychomotor development
7 Daniel and Evans [1982] cross-fostered half the mouse pups born to treated mothers with
8 untreated surrogate females to test the effects of maternal nutrition and behavioral factors on the
9 pups' physiological and psychomotor development Results indicated that pups fostered to control
10 dams performed significantly better than those maintained with treated dams (Table l-8][Daniel
11 and Evans. 1982). Data suggest that neurodevelopmental effects were not solely due to in utero
12 exposure to tert-butanol [Daniel and Evans, 1982]. Interpretation of these results is limited,
13 however, as the neurodevelopmental data were presented only in figures and could not be
14 compared with controls.
15
16
Table 1-8. Evidence pertaining to neurodevelopmental effects in animals
following exposure to tert-butanol
Reference and study design
Results
Daniel and Evans (1982)
Swiss Webster (Cox) mouse; 15 pregnant
dams/treatment
Liquid diet (0, 0.5, 0.75, or 1.0%, w/v); GD6-20;
after birth half the pups were nursed with their
treated dams and the other half were fostered
by untreated dams who recently gave birth
0 (isocaloric amounts of maltose/dextrin),
3,324, 4,879, or 6,677 mg/kg-d
• a dose-dependent increase in righting reflex time, with more time
needed in animals maintained with maternal dams
• a dose-dependent decrease in open field behavior, with less
activity in pups maintained with maternal dams
• a dose-dependent decrease in rotarod performance with the
pups from maternal dams having lower performances
• a dose-dependent decrease in the amount of time the pups were
able to avoid a cliff, with animals maintained with their maternal
dams having less avoidance time
Nelson etal.(1991)
Sprague-Dawley rat; 15 pregnant
dams/treatment
Inhalation analytical concentration: 0, 6,000, or
12,000 mg/m3; (dynamic whole body chamber)
7hr/d
GD 1-19
Data were not presented specifically by dose nor were any tables or figures
of the data provided
Maternal toxicity was noted by decreased food consumption and body
weight gains
Results in offspring
• increase in rotarod performance in high-dose group (16 versus 26
revolutions/min for controls and 12,000 mg/m3 animals,
respectively)
• decreased time held on wire in the performance ascent test in the
low-dose group (16 sec versus 10 sec for controls and 1,750
mg/m3 animals, respectively)
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Results
The following differences in neurochemical measurements in the brain
between control and treated offspring were observed,
• 53% decrease in norepinephrine in the cerebellum at
12,000 mg/m3
• 57% decrease in met-enkephalin in the cerebrum at
12,000 mg/m3 and 83% decrease at 6,000 mg/m3
• 61% decrease in P-endorphin in the cerebellum at 12,000 mg/m3
• 67% decrease in serotonin in the midbrain at 6,000 mg/m3
Nelson et al. (1991)
Adult male Sprague-Dawley rats (18/treatment)
mated to untreated females
Inhalation analytical concentration: 0, 6,000, or
12,000 mg/m3; (dynamic whole body chamber)
7 hr/d for 6 wk
Data were not presented specifically by dose nor were any tables or figures
of the data provided
Results (generally only specified as paternally treated versus controls) in
offspring indicate
• increase in rotarod performance (16 versus 20 revolutions/min
for controls and 12,000 mg/m3 animals, respectively)
• decreased time in open field (less time to reach the outer circle of
the field, 210 sec versus 115 seconds for controls and 12,000
mg/m3 animals, respectively)
The following differences in neurochemical measurements in the brain
between control and treated offspring were observed
• 39% decrease in norepinephrine in the cerebellum at
12,000 mg/m3
• 40% decrease in met-enkephalin in the cerebrum at
12,000 mg/m3 and 75% decrease at 6,000 mg/m3
• 71% decrease in P-endorphin in the cerebellum at 12,000 mg/m3
• 47% decrease in serotonin in the midbrain at 6,000 mg/m3
1 * Statistically significant p < 0.05 as determined by study authors.
2
3 Note: Conversions from diet concentrations to mg/kg-d performed by study authors.
4 Percentage change compared to control = (treated value - control value) 4- control value x 100.
5 Mechanistic Evidence
6 No mechanistic evidence is available for reproductive, developmental, or
7 neurodevelopmental effects.
8 Integration of neurodevelopmental effects
9 Neurodevelopmental effects, including decreased brain weight, changes in brain
10 biochemistry, and changes in behavioral performances, have been observed. Each study evaluating
11 neurodevelopmental effects, however, had limitations in study design, reporting, or both. In
12 addition, results were not always consistent between studies or across dose. At this time, there is
13 inadequate information to draw conclusions regarding neurodevelopmental toxicity.
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1 1.2.5. Reproductive Effects
2 Synthesis of effects related to reproduction
3 Several studies evaluated reproductive effects [a one-generation, oral reproductive study
4 [Lyondell Chemical Co.. 2004] and subchronic evaluations in rats and mice following oral and
5 inhalation exposure [NTP. 1997.1995]] in animals exposed to tert-butanol via oral gavage, drinking
6 water, or inhalation for >63 days. The studies are arranged in the evidence tables by sex, route of
7 exposure, duration of exposure, and species. The collection of studies evaluating reproductive
8 effects of tert-butanol is limited by the absence of two-generation reproductive oral or inhalation
9 studies and by having no human studies on reproduction. The design, conduct, and reporting of
10 each study were reviewed, and each study was considered adequate to provide information
11 pertinent to this assessment.
12 Reproductive endpoints, such as reproductive organ weights, estrous cycle length, and
13 sperm effects were examined following either oral or inhalation exposure [Lyondell Chemical Co..
14 2004: NTP. 1997.1995] (Table l-9;Figure 1-13; Figure 1-14]. In males, the only significant effect
15 observed was a slight decrease in sperm motility for FO males treated with 1000 mg/kg-day of tert-
16 butanol [Lyondell Chemical Co.. 2004]. No significant changes in sperm motility were reported
17 following oral exposure in other rat studies or via inhalation exposure in mice or rats. In addition,
18 the reduced motility in treated animals falls within the range of historical control data and,
19 therefore, its biological significance is uncertain. In female B6C3Fi mice, estrous cycle length was
20 increased 28% following oral exposure to 11,620 mg/kg-day [NTP. 1995]. No significant changes in
21 estrous cycle length were observed following oral exposure in rats, or inhalation exposure in mice
22 or rats.
23
24
Table 1-9. Evidence pertaining to reproductive effects in animals following
exposure to tert-butanol
Reference and study design
Results
Male reproductive effects
Lvondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64, 160, 400, or 1,000 mg/kg-d
FO males: 9 weeks beginning 4 weeks prior to
mating
PND21
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
13 weeks
FO reproductive effects
Sperm motility (only control and high-dose groups examined)
0: 94% 1000: 91%*
No other significant effect on weights of male reproductive organs or sperm
observed
No significant effect on weights of male reproductive organs or sperm
observed
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Toxicological Review of tert-ButyI Alcohol
Reference and study design
Results
NTP (1995)
B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
13 weeks
No significant effect on weights of male reproductive organs or sperm
observed
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation analytical concentration: 0,134, 272,
542, 1,080, or 2,101 ppm (0, 406, 824, 1,643,
3,273 or 6,368 mg/m3) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3)
NTP (1997)
B6C3Fi mouse; 10/sex/treatment
Inhalation analytical concentration: 0,134, 272,
542, 1,080, or 2,101 ppm (0, 406, 824, 1,643,
3,273 or 6,368 mg/m3) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3)
Female reproductive effects
Lyondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
FO females: 4 weeks prior to mating through
PND21
Pregnancy index
91.7% 91.7% 100% 100% 91.7%
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
No significant effect on female estrous cycle (0, -2, -4, 0, +8 % change
relative to control)
NTP (1995)
B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
T" length of estrous cycle
Response relative to control: 0, +5, +5, +5, +6, +28*%
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Reference and study design
Results
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation analytical concentration: 0,134, 272,
542, 1,080, or 2,101 ppm (0, 406, 824, 1,643,
3,273 or 6,368 mg/m3) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on female estrous cycle (0, -4, +2, +4 % change relative
to control)
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3)
NTP (1997)
B6C3Fi mouse; 10/sex/treatment
Inhalation analytical concentration: 0,134, 272,
542, 1,080, or 2,101 ppm (0, 406, 824, 1,643,
3,273 or 6,368 mg/m3) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on female estrous cycle (0, -3, -9, -5 % change relative
to control)
Evaluations were only performed for concentrations >542 ppm
(1,643 mg/m3)
1 * Statistically significant p < 0.05 as determined by the study authors.
2 Notes: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
3 Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
4 Percentage change compared to control = (treated value - control value) 4- control value x 100
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Toxicological Review of tert-ButyI Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
REPRODUCTIVE EFFECTS
/[ale reproductive effects
Reproductive organs or sperm; M
rat (A]
Reproductive organs or sperm; M
rat(B)
Reproductive organs or sperm; M
mouse (B)
emale reproductive effects
Estrous cycle length; F rat (B)
10
100 1,000 10,000
Dose (mg/kg-day)
100,000
2
3
Sources: (A) Lyondell Chemical Co. (2004); (B) NTP (1995).
Figure 1-13. Exposure-response array of reproductive effects following oral
exposure to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol
• = exposures at which the endpoint was reported statistically significant by study authors
D = exposures at which the endpoint was reported not statistically significant by study authors
REPRODUCTIVE EFFECTS
Male reproductive effects
Reproductive organs or sperm; M rat
(NTP, 1997]
Reproductive organs or sperm; M mouse
(NTP, 1997)
Female reproductive effects
Estrous cycle; F rat (NTP, 1997)
Estrous cycle; F mouse (NTP, 1997)
1,000
10,000
Exposure Concentration (mg/m:i)
1
2
Figure 1-14. Exposure-response array of reproductive effects following
inhalation exposure to tert-butanol.
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Toxicological Review of tert-ButyI Alcohol
1 Integration of reproductive effects
2 At this time, no conclusions are drawn in regard to reproductive toxicity. The database is
3 limited to a one-generation study [Lyondell Chemical Co.. 2004: NTP. 1995). No two-generation
4 reproductive studies are available that evaluate oral or inhalation exposure. In males, the only
5 observed effect was a slight decrease in sperm motility for FO males in the highest dose group of
6 rats treated with tert-butanol. This effect was not observed, however, in other studies with orally
7 treated rats and mice or in rats exposed via inhalation. In females, NTP [1995] reported an
8 increased length of the estrous cycle in the highest dose group of orally exposed mice. This effect
9 was not observed in similarly treated rats or in mice and rats exposed via inhalation.
10 1.2.6. Other Toxicological Effects
11 Effects other than those related to kidney, thyroid, reproductive, developmental, and
12 neurodevelopmental toxicity were observed in some of the available rodent studies; these include
13 liver and urinary bladder effects. Due to a lack of consistency in the liver effects and minimal to
14 mild effects with a lack of progression in urinary bladder, however, inadequate information is
15 available to draw conclusions regarding liver or urinary bladder toxicity at this time.
16 Additionally, central nervous system (CNS) effects similar to those caused by ethanol
17 (animals appearing intoxicated and having withdrawal symptoms after cessation of oral or
18 inhalation exposure) were observed. Due to study quality concerns (e.g., lack of data reporting,
19 small number of animals per treatment group), however, adequate information to assess CNS
20 toxicity is unavailable at this time. For more information on these other toxicological effects, see
21 Appendix B.3.
22 1.3. INTEGRATION AND EVALUATION
23 1.3.1. Effects Other Than Cancer
24 Kidney effects were identified as a potential human hazard of tert-butanol exposure based
25 on several endpoints, including suppurative inflammation in female rats, transitional epithelial
26 hyperplasia in male and female rats, severity of nephropathy in male and female rats, incidences of
27 nephropathy in female rats, mineralization in male rats, and increased kidney weights in both male
28 and female rats. These effects are similar to the kidney effects observed with ETBE exposure (e.g.,
29 CPN and urothelial hyperplasia) and MTBE (e.g., CPN and mineralization) fATSDR. 19961
30 Several effects were observed in the kidneys of rats. Based on mechanistic evidence
31 indicating that an a2u-globulin-related process is operating in male rats (Hardetal., 2011: Cirvello
32 etal., 1995: NTP, 1995: Lindamoodetal., 1992), any kidney effects associated with a2U-globulin
33 nephropathy are not considered relevant for human hazard identification. In addition, CPN played a
34 role in the renal tubule nephropathy observed following tert-butanol exposure, and effects
35 associated with such nephropathy are not considered relevant for human hazard identification.
36 Although increases in severity (males and females) or incidence (females) of nephropathy were
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1 related to tert-butanol exposure and could have arisen from chemical-specific processes
2 independent from CPN, the association of these effects with CPN makes this measure less suitable
3 for dose-response analysis, and therefore these effects were not considered for the derivation of
4 reference values. Furthermore, some uncertainty exists regarding whether mineralization is also
5 associated with CPN in male rats; due to this uncertainty, and because other kidney effects were
6 identified as being associated with tert-butanol exposure and yet independent from CPN,
7 mineralization in male rats was not considered for dose-response analysis. The remaining effects
8 (suppurative inflammation, transitional epithelial hyperplasia, and increased kidney weights) are
9 considered the result of tert-butanol exposure and relevant to human hazard characterization.
10 These effects therefore are suitable for consideration for dose-response analysis and derivation of
11 reference values, in Section 2.
12 There is suggestive evidence of developmental effects associated with tert-butanol
13 exposure. Increased fetal loss, decreased fetal body weight, and increases in skeletal variations in
14 exposed offspring were observed following exposure to relatively high doses of tert-butanol during
15 gestation.. These effects are similar to the developmental effects observed with MTBE exposure
16 (e.g., decreased fetal body weight and increases in skeletal variations) [ATSDR. 1996).
17 No mechanistic evidence is available for developmental effects of tert-butanol. Although the
18 evidence is suggestive of developmental toxicity, due to the uncertainty as to whether fetal effects
19 were due to direct effects of tert-butanol or indirect effects of maternal toxicity and the lack of
20 consistency across some endpoints, developmental effects were not considered for dose-response
21 analysis and derivation of reference values in Section 2. Furthermore, no adverse effects were
22 reported in one- and two-generation reproductive/developmental studies on ETBE [Gaoua. 2004a.
23 b), providing further support for the lack of evidence supporting reproductive or developmental
24 effects as possible human hazards following tert-butanol exposure.
25 At this time, there is inadequate information to draw conclusions regarding
26 neurodevelopmental effects as a human hazard of tert-butanol exposure. Although
27 neurodevelopmental effects have been observed, the studies had limitations in design or reporting,
28 or both, and results were inconsistent between studies and across dose groups. No mechanistic
29 evidence is available to inform the MOA for neurodevelopmental effects of tert-butanol. These
30 effects were not considered further for dose-response analysis and derivation of reference values.
31 At this time, no conclusions are drawn regarding reproductive effects as a human hazard of
32 tert-butanol exposure. The only reproductive effect observed due to tert-butanol exposure was
33 increased length of estrous cycle [NTP. 1995) in the highest dose group of orally exposed mice, and
34 this effect was not observed in orally exposed rats or in mice and rats exposed via inhalation.
35 Further, the database was limited and contained only two oral exposure studies and one subchronic
36 inhalation study. No mechanistic or MOA information is available for reproductive effects of tert-
37 butanol. These effects were not considered further for dose-response analysis and derivation of
38 reference values.
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1 At this time, there is inadequate information to draw conclusions regarding liver or urinary
2 bladder toxicity due to lack of consistency of effects and minimal/mild effects showing a lack of
3 progression, respectively. No mechanistic evidence is available for these effects. The liver and
4 urinary bladder effects were not considered further for dose-response analysis and the derivation
5 of reference values.
6 1.3.2. Carcinogenicity
7 Summary of evidence
8 In F344/N rats, administration of tert-butanol in drinking water increased the incidence of
9 renal tubule tumors, mostly adenomas, in males; no renal tumors in females were reported [Hard et
10 al.. 2011: NTP. 1995). As discussed in Section 1.2.1, some of these tumors might be associated with
11 a2u-globulin nephropathy, an MOA considered specific to the male rat [U.S. EPA, 1991a]. Evidence
12 in support of this hypothesized MOA includes the accumulation of hyaline droplets in renal tubule
13 cells, the presence of (X2u-globulin in the hyaline droplets, and additional aspects associated with
14 (X2u-globulin nephropathy, including linear papillary mineralization and foci of tubular hyperplasia.
15 Other evidence, however, is not supportive: The accumulation of hyaline droplets was minimal;
16 concentrations of (X2u-globulin were low at doses that induced tumors; and no significant necrosis
17 or cytotoxicity was associated with compensatory regenerative proliferation or induction of
18 granular casts observed within a timeframe consistent with (X2u-globulin-mediated nephropathy.
19 Renal tumors also could be associated with chronic progressive nephropathy, but the data on CPN
20 are not coherent: Dose-response relationships for CPN, renal tubule hyperplasia, and renal tubule
21 tumors were different; in addition, CPN was nearly as severe in female rats as in male rats, yet no
22 female rats developed renal tumors. Thus, some renal tumors may be attributable to a2u-globulin
23 nephropathy and some to other, yet unspecified, processes. Taken together, and according to EPA's
24 guidance on renal tumors in male rats [U.S. EPA. 1991a]. renal tumors induced by tert-butanol are
25 relevant for human hazard identification.
26 InB6C3Fi mice, administration of tert-butanol in drinking water increased the incidence of
27 thyroid follicular cell adenomas in females, and adenomas or carcinomas (only one carcinoma
28 observed) in males [NTP, 1995], as discussed in Section 1.2.2. According to EPA's thyroid tumor
29 guidance [U.S. EPA, 1998a], chemicals that produce thyroid tumors in rodents might pose a
30 carcinogenic hazard to humans.
31 In addition, as mentioned in Section 1.1.4, tert-butanol is a primary metabolite of MTBE and
32 of ETBE, two compounds tested in rats and mice that could provide supplementary information on
33 the carcinogenicity of tert-butanol. For MTBE, the most recent cancer evaluation by a national or
34 international health agency is from IARC [1999]. IARC reported that oral gavage exposure in
35 Sprague-Dawley rats resulted in testicular tumors in males and lymphomas and leukemias
36 (combined] in females; inhalation exposure in male and female F344 rats resulted in renal tubule
37 adenomas in males; and inhalation exposure in male and female CD-I mice resulted in
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1 hepatocellular adenomas in females [IARC. 1999]. For ETBE, a draft IRIS assessment under
2 development concurrently with this assessment reports that inhalation exposure in male and
3 female F344 rats resulted in hepatocellular tumors, mostly adenomas, in males; no significant
4 tumor increases were reported for 2-year studies by drinking water exposure in male and female
5 F344 rats or by oral gavage in male and female Sprague-Dawley rats.
6 Integration of evidence
7 This evidence leads to consideration of two hazard descriptors under EPA's cancer
8 guidelines [U.S. EPA. 2005a]. The descriptor likely to be carcinogenic to humans is appropriate when
9 the evidence is "adequate to demonstrate carcinogenic potential to humans" but does not support
10 the descriptor carcinogenic to humans. One example from the cancer guidelines is "an agent that has
11 tested positive in animal experiments in more than one species, sex, strain, site, or exposure route,
12 with or without evidence of carcinogenicity in humans." tert-Butanol matches the conditions of this
13 example, having increased tumor incidences in two species, in both sexes, and at two sites.
14 Alternatively, the descriptor suggestive evidence of carcinogenic potential is appropriate
15 when the evidence raises "a concern for potential carcinogenic effects in humans" but is not
16 sufficient for a stronger conclusion. The results for tert-butanol raise a concern for cancer but none
17 of the effects is particularly strong. The kidney tumors resulted, in part, from an MOA that is specific
18 to male rats, while no kidney tumors occurred in female rats. The thyroid tumors induced in male
19 and female mice were almost entirely benign. In addition, while MTBE was also associated with
20 male rat kidney tumorigenesis, there is little coherence of results between tert-butanol and ETBE
21 associated tumorigenesis in rats. MTBE or ETBE effects following chronic oral exposure in mice
22 have not been investigated, however, so no evidence exists to evaluate the coherence of the thyroid
23 tumorigenesis observed following tert-butanol exposure in B6C3Fi mice.
24 These considerations, interpreted in light of the cancer guidelines, support the conclusion
25 that there is suggestive evidence of carcinogenic potential for tert-butanol. Although increased tumor
26 incidences were reported for two species, two sexes, and two sites, none of the tumor responses
27 was strong or coherent with the results for ETBE, and this was decisive in selecting a hazard
28 descriptor.
29 The descriptor suggestive evidence of carcinogenic potential applies to all routes of human
30 exposure. Oral administration of tert-butanol to rats and mice induced tumors at sites beyond the
31 point of initial contact, and inhalation exposure for 13 weeks resulted in absorption and
32 distribution of tert-butanol into the systemic circulation, as discussed in Section 1.2.1. According to
33 the cancer guidelines, this information provides sufficient basis to apply the cancer descriptor
34 developed from oral studies to other exposure routes.
35 Biological considerations for dose-response analysis
36 Regarding hazards to bring forward to Section 2 for dose-response analysis, EPA's guidance
37 on renal tumors in male rats [U.S. EPA. 1991a] advises that unless the relative contribution of
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1 (X2u-globulin nephropathy and other process can be determined, dose-response analysis should not
2 be performed. As discussed in Section 1.2.1, the available data do not allow such determination, and
3 so an analysis of kidney tumors does not appear in Section 2.
4 EPA's guidance on thyroid tumors and EPA's cancer guidelines [U.S. EPA, 1998a] advises
5 that for thyroid tumors resulting from thyroid-pituitary disruption, dose-response analysis should
6 use nonlinear extrapolation, in the absence of MOA information to indicate otherwise. As discussed
7 in Section 1.2.2, increases in thyroid follicular cell hyperplasia in male and female mice provide
8 partial support for thyroid-pituitary disruption. Other necessary data on tert-butanol, however, are
9 not adequate or are not supportive. There is little correlation among thyroid, pituitary, and liver
10 effects in female mice, and no data are available to evaluate the potential for antithyroid effects in
11 male mice. Data are not adequate to conclude that thyroid hormone changes exceed the range of
12 homeostatic regulation or to evaluate effects on extrahepatic sites involved in thyroid-pituitary
13 disruption. Also, no data are available to evaluate reversibility of effects upon cessation of exposure.
14 Thus, according to EPA's thyroid tumor guidance, concluding that the thyroid tumors result from
15 thyroid-pituitary disruption is premature, and dose-response analysis should use linear
16 extrapolation. The data are well suited to dose-response analysis, coming from an NTP study that
17 tested multiple dose levels.
18 1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
19 No chemical-specific data that would allow for the identification of populations with
20 increased susceptibility to tert-butanol exposure exist In vitro studies have implicated the liver
21 microsomal mixed function oxidase (MFO) system, namely CYP450 [Cederbaum etal.. 1983:
22 Cederbaum and Cohen. 1980]. as playing a role in the metabolism of tert-butanol. No studies,
23 however, have identified the specific CYPs responsible for the biotransformation of tert-butanol.
24 Pharmacokinetic differences among the fetus, newborns, children, and the aged might alter
25 responses to chemicals compared to adults, resulting in differences in health effects. In the
26 presence of environmental chemicals, metabolic homeostasis is maintained by the liver's ability to
27 detoxify and eliminate xenobiotics. This process is accomplished, in part, by the expression of
28 xenobiotic metabolizing enzymes and transporters (XMETs), which metabolize and transport
29 xenobiotics and determine whether exposure will result in altered responses. The expression of
30 XMETs, including various CYPs, has been found to be underexpressed in the mouse fetus and
31 neonate [Lee etal.. 2011] and decreased in older mice [Lee etal.. 2011] and rats [Lee etal.. 2008].
32 Decreased ability to detoxify and transport tert-butanol out of the body could result in increased
33 susceptibility to tert-butanol in the young and old.
34 In regard to cancer, although children are more sensitive than adults to thyroid
35 carcinogenesis resulting from ionizing radiation, relative differences in lifestage sensitivity to
36 chemically induced thyroid carcinogenesis are unknown [U.S. EPA, 1998a]. In addition, the data on
37 tert-butanol mutagenicity are inconclusive.
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1 Collectively, there is little evidence on tert-butanol itself to identify susceptible populations
2 or lifestages.
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2. DOSE-RESPONSE ANALYSIS
2 2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
3 The reference dose (RfD, expressed in units of mg/kg-day) is defined as an estimate (with
4 uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population
5 (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects
6 during a lifetime. The RfD can be derived from a no-observed-adverse-effect level (NOAEL), lowest-
7 observed-adverse-effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL),
8 with uncertainty factors (UFs) generally applied to reflect limitations of the data used.
9 2.1.1. Identification of Studies and Effects for Dose-Response Analysis
10 EPA identified kidney effects as a potential human hazard of tert-butanol exposure (see
11 Section 1.2.1). Studies within this effect category were evaluated using general study quality
12 characteristics [as discussed in Section 6 of the Preamble; see also U.S. EPA (2002)] to help inform
13 the selection of studies from which to derive toxicity values. No other hazards were identified for
14 further for consideration in the derivation of reference values.
15 Human studies are preferred over animal studies when quantitative measures of exposure
16 are reported and the reported effects are determined to be associated with exposure. No human
17 occupational or epidemiological studies of oral exposure to tert-butanol, however, are available.
18 Animal studies were evaluated to determine which studies provided: (1) the most relevant
19 routes and durations of exposure, (2) multiple exposure levels to provide information about the
20 shape of the dose-response curve, and (3) power to detect effects atlow exposure levels. The
21 database for tert-butanol includes both chronic and subchronic studies showing effects in the
22 kidney that are suitable for deriving reference values.
23 Kidney Toxicity
24 EPA identified kidney effects as a potential human hazard of tert-butanol-induced toxicity
25 based on findings in male and female rats (summarized in Section 1.3.1). Kidney toxicity was
26 observed across multiple chronic, subchronic, and short-term studies following oral and inhalation
27 exposure. Kidney effects observed after chronic exposure, such as suppurative inflammation and
28 transitional epithelial hyperplasia, may impact the ability of the kidney to filter waste. Observed
29 changes in kidney weight could also indicate toxic effects in the kidney. For the oral tert-butanol
30 database, there are several studies available that evaluated these kidney effects. Lyondell Chemical
31 Co. (2004) conducted a reproductive study in Sprague-Dawley rats that was of shorter duration,
32 and reported changes in kidney weight but did not examine changes in histopathology. NTP
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1 conducted a 2-year drinking water study [NTP. 1995] in F344 rats that evaluated multiple doses in
2 both males and females, and reported on all three endpoints highlighted above. NTP [1995] was
3 identified as most suitable for dose-response assessment considering the study duration,
4 comprehensive reporting of outcomes, and multiple doses tested.
5 In the NTP [1995] 2-year drinking water study, male F344 rats were exposed to
6 approximate doses of 0, 90, 200, or 420 mg/kg-day; female F344 rats were exposed to approximate
7 doses of 0,180, 330, or 650 mg/kg-day. Reduced body weights and survival were observed and
8 reflected in some of the effects. Kidney effects, including changes in organ weight, histopathology,
9 or both, were observed in both sexes of rats after 13 weeks, 15 months, and 2 years of treatment
10 [NTP. 1995]. Specific endpoints chosen for dose-response analysis were absolute kidney weight
11 (observed in males and females], kidney suppurative inflammation (observed in females], and
12 kidney transitional epithelial hyperplasia (observed in males and females]. For absolute kidney
13 weight, data from 15 months was selected as described in Section 1.2.1; for the other endpoints,
14 data at the longest duration of 2 years were selected.
15 2.1.2. Methods of Analysis
16 No biologically based dose-response models are available for tert-butanol. In this situation,
17 EPA evaluates a range of dose-response models thought to be consistent with underlying biological
18 processes to determine how best to empirically model the dose-response relationship in the range
19 of the observed data. The models in EPA's Benchmark Dose Software (BMDS] were applied.
20 Consistent with EPA's Benchmark Dose Technical Guidance [U.S. EPA. 2012b). the BMD and the
21 BMDL are estimated using a benchmark response [BMR] to represent a minimal, biologically
22 significant level of change. In the absence of information regarding the level of change that is
23 considered biologically significant, a BMR of 1 standard deviation from the control mean for
24 continuous data or a BMR of 10% extra risk for dichotomous data is used to estimate the BMD and
25 BMDL, and also to facilitate a consistent basis of comparison across endpoints, studies, and
26 assessments. Endpoint-specific BMRs, where feasible, are described further below. When modeling
27 was feasible, the estimated BMDLs were used as points of departure (PODs]; the PODs are
28 summarized in Table 2-1. Further details including the modeling output and graphical results for
29 the model selected for each endpoint can be found in Appendix C of the Supplemental Information
30 to this Toxicological Review.
31 Kidney weights were analyzed as absolute weights rather than relative to body weight In
32 general, absolute and relative kidney weight data can both be considered appropriate endpoints for
33 analysis [Bailey etal.. 2004]. In the NTP [1995] 2-year drinking water study, body weight in
34 exposed animals noticeably decreased relative to controls at the 15-month interim sacrifice (see
35 Table 1-1], but this decrease in body weight impacted the measure of relative kidney weight
36 resulting in an exaggeration of the kidney weight change. There was greater confidence in the
37 absolute kidney weight measure; thus, it was considered more appropriate for dose-response
38 analysis, and changes in relative kidney weights were not analyzed. A 10% relative change from
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1 control was used as a BMR for absolute kidney weight by analogy with a 10% change in body
2 weight as an indicator of toxicity. A BMR of 10% extra risk was considered appropriate for the
3 quantal data on incidences of kidney suppurative inflammation and kidney transitional epithelial
4 hyperplasia.
5 Human equivalent doses (HEDs) for oral exposures were derived from the PODs according
6 to the hierarchy of approaches outlined in EPA's Recommended Use of Body Weight3/4 as the Default
7 Method in Derivation of the Oral Reference Dose [U.S. EPA, 2011]. The preferred approach is
8 physiologically based toxicokinetic modeling (PBPK). Other approaches include using chemical-
9 specific information in the absence of a complete PBPK model. As discussed in Appendix B of the
10 Supplemental Information, human PBPK models for inhalation of ETBE or inhalation and dermal
11 exposure to MTBE have been published, which include tert-butanol submodels. A validated human
12 PBPK model for tert-butanol, however, is not available for extrapolating doses from animals to
13 humans. In lieu of either chemical-specific models or data to inform the derivation of human
14 equivalent oral exposures, body weight scaling to the % power (i.e., BW3/4) is applied to extrapolate
15 toxicologically equivalent doses of orally administered agents from adult laboratory animals to
16 adult humans for the purpose of deriving an oral RfD.
17 Consistent with EPA guidance (U.S. EPA. 2011). the PODs estimated based on effects in adult
18 animals were converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
19 as follows:
%
20 DAF = (BWa1/* / BWh1/4),
21 where
22 BWa = animal body weight
23 BWh = human body weight
24 Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA. 1988).
25 the resulting DAF is 0.24 for rats. Applying this DAF to the POD identified for effects in adult rats
26 yields a PODHED as follows (see Table 2-1):
27 PODHED = Laboratory animal dose (mg/kg-day) x DAF
28 Table 2-1 summarizes all PODs and the sequence of calculations leading to the derivation of
29 a human-equivalent POD for each endpoint discussed above.
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Table 2-1. Summary of derivations of points of departure following oral
exposure for up to 2 years
Endpointand
reference
Species/
sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODADjb(m
g/kg-d)
Kidney
Increased absolute
kidney weight at 15
months
NTP (1995)
Increased absolute
kidney weight at 15
months
NTP (1995)
Kidney inflammation
(suppurative)
NTP (1995)
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/M
Rat/F
Rat/F
Rat/M
Rat/F
Linear
(constant
variance)
Exponential
(M4)
(constant
variance)
Log-probit
Log-logistic
Multistage,
3-degree
10%
10%
10%
10%
10%
657
164
254
30
412
296
91
200
16
339
296
91
200
16
339
PODHEDc(mg/
kg-d)
71
22
48
3.84
81.4
3
4
5
6
8
9
10
11
12
13
14
15
16
17
18
aFor modeling details, see Appendix C in Supplemental Information.
bFor studies in which animals were not dosed daily, EPA would adjust administered doses to calculate the TWA
daily doses prior to BMD modeling. However, this adjustment was not required for the NTP (1995) study.
CHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
NA= not applicable
2.1.3. Derivation of Candidate Values
Consistent with EPA's A Review of the Reference Dose and Reference Concentration Processes
[[U.S. EPA. 2002]: Section 4.4.5], also described in the Preamble, five possible areas of uncertainty
and variability were considered when determining the application of UFs to the PODs presented in
Table 2-1. An explanation follows:
An intraspecies uncertainty factor, UFn, of 10 was applied to all PODs to account for
potential differences in toxicokinetics and toxicodynamics in the absence of information on the
variability of response in the human population following oral exposure to tert-butanol [U.S. EPA.
2002).
An interspecies uncertainty factor, UFA, of 3 (10°5 = 3.16, rounded to 3) was applied to all
PODs because BW3/4 scaling was used to extrapolate oral doses from laboratory animals to humans.
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1 Although BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and
2 toxicodynamic processes, some residual uncertainty in the extrapolation remains. In the absence of
3 chemical-specific data to quantify this uncertainty, EPA's BW3/4 guidance [U.S. EPA. 2011]
4 recommends use of an uncertainty factor of 3.
5 A subchronic to chronic uncertainty factor, UFS, of 1 was applied to all PODs because the
6 endpoints were all observed following chronic exposure.
7 A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied to all PODs derived because the
8 current approach is to address this factor as one of the considerations in selecting a BMR for
9 benchmark dose modeling. In this case, BMRs of a 10% relative change in absolute kidney weight, a
10 10% extra risk of kidney suppurative inflammation, and a 10% extra risk of transitional cell
11 hyperplasia were selected assuming they represent minimal biologically significant response levels.
12 A database uncertainty factor, UF, of 1 was applied to all PODs. The tert-butanol oral toxicity
13 database includes chronic and subchronic toxicity studies in rats and mice [Acharyaetal.. 1997:
14 Acharyaetal.. 1995: NTP. 1995] and developmental toxicity studies in rats and mice [Lyondell
15 Chemical Co.. 2004: Faulkner et al.. 1989: Daniel and Evans. 1982]. In the developmental studies, no
16 effects were observed at exposure levels below 1000 mg/kg-day, and effects observed at
17 >1000 mg/kg-day were accompanied by evidence of maternal toxicity. These exposure levels are
18 much higher than the PODs for kidney effects, suggesting developmental toxicity is not as sensitive
19 an endpoint as kidney effects. No immunotoxicity or multigenerational reproductive studies are
20 available for tert-butanol. Studies on ETBE, which is rapidly metabolized to systemically available
21 tert-butanol, are informative for consideration of the gaps in the tert-butanol oral database. The
22 database for ETBE does not indicate immunotoxicity [Bantonetal.. 2011: Lietal.. 2011]. suggesting
23 immune system effects would not be a sensitive target for tert-butanol. No adverse effects were
24 reported in one- and two-generation reproductive/developmental studies on ETBE [Gaoua. 2004a.
25 b], indicating that reproductive/developmental effects would not be a sensitive target for tert-
26 butanol. Additionally, a one-generation, reproductive toxicity study in rats from a Toxic Substances
27 Control Act submission [Lyondell Chemical Co.. 2004] is available for tert-butanol. This study did
28 not observe reproductive effects. Although the oral toxicity database for tert-butanol has some
29 gaps, the available data on tert-butanol, informed by the data on ETBE, do not suggest that
30 additional studies would lead to identification of a more sensitive endpoint or a lower POD.
31 Therefore, a database UFD of 1 was applied.
32 Figure 2-1 presents graphically the candidate values, UFs, and PODHED values, with each bar
33 corresponding to one data set described in Tables 2-1 and 2-2.
34 Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each POD
35 to derive a candidate value for each data set, preliminary to the derivation of the organ/system-
36 specific RfDs. These candidate values are considered individually in the selection of a
37 representative oral reference value for a specific hazard and subsequent overall RfD for tert-
38 butanol. Figure 2-1 presents graphically the candidate values, UFs, and PODHED values, with each
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1 bar corresponding to one data set described in Tables 2-1 and 2-2.
Table 2-2. Effects and corresponding derivation of candidate values
Endpoint and reference
PODhHED
(mg/kg-d)
POD type
UFA
UFH
UFL
UFs
UFD
Composite
UF
Candidate
value
(mg/kg-d)
Kidney
Increased absolute kidney weight;
male rat at 15 months
NTP (1995)
Increased absolute kidney weight;
female rat at 15 months
NTP (1995)
Kidney inflammation (suppurative);
female rat NTP (1995)
Kidney transitional epithelial
hyperplasia; male rat
NTP (1995)
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
71
22
48
3.8
81
BMDLio%
BMDLio%
BMDLio%
BMDLio%
BMDLio%
^
3
3
3
3
3
10
10
10
10
10
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
30
30
30
30
30
2x10°
7 x 10'1
2x10°
1 x 10'1
3x10°
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T Absolute kidney
weight; male rat
(NTP, 1995)
T Absolute kidney
weight; female rat
(NTP, 1995)
Kidney inflammation;
female rat (NTP, 1995]
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995)
0.1
10
100
Candidate RfD
PODHED
Composite UF
mg/kg-day
Figure 2-1. Candidate values with corresponding POD and composite UF. Each
bar corresponds to one data set described in Table 2-1 and Table 2-2.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2.1.4. Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. Organ or system-specific RfDs are useful for subsequent cumulative risk assessments that
consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For tert-butanol, candidate values were for several different kidney effects in both sexes,
spanning a range from 1 x lO1 to 3 x 10° mg/kg-day, for an overall 30-fold range. To estimate an
exposure level below which kidney toxicity from tert-butanol exposure is not expected to occur, the
RfD for increased incidence of transitional epithelial hyperplasia in male rats (1 x 10'1 mg/kg-day)
was selected as the kidney-specific reference dose for tert-butanol. Unlike kidney suppurative
inflammation, this effect was observed in both sexes, with males appearing to be more sensitive
than females. Additionally, this indicator of kidney toxicity is more specific and more sensitive than
the relatively non-specific endpoint of absolute kidney weight changes. Confidence in this kidney-
specific RfD is high. The PODs are based on benchmark dose modeling, and the candidate values are
derived from a well-conducted long-term study, involving a sufficient number of animals per group,
including both sexes, and assessing a wide range of kidney endpoints.
Table 2-3. Organ/system-specific RfDs and overall RfD for tert-butanol
Effect
Kidney
Overall RfD
Basis
Incidence of transitional
epithelial hyperplasia (NTP
(1995)
Kidney
RfD (mg/kg-day)
1 x 10'1
1 x 1C'1
Study exposure
description
Chronic
Chronic
Confidence
High
High
18
19
20
21
22
23
24
25
26
27
28
29
2.1.5. Selection of the Overall Reference Dose
For tert-butanol, only kidney effects were identified as a hazard and carried forward for
dose-response analysis; thus only one organ/system-specific reference dose was derived.
Therefore, the kidney specific RfD of (1 x 1Q-1 mg/kg-day) is the overall RfD for tert-butanol. This
value is based on increased incidence of transitional epithelial hyperplasia in male rats exposed to
tert-butanol.
The overall reference dose is derived to be protective of all types of effects for a given
duration of exposure and is intended to protect the population as a whole, including potentially
susceptible subgroups [U.S. EPA. 2002). Decisions concerning averaging exposures over time for
comparison with the RfD should consider the types of toxicological effects and specific lifestages of
concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages
could lead to an appreciable risk, even if average levels over the full exposure duration were less
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1 than or equal to the RfD. In the case of tert-butanol, there is potential for early lifestage
2 susceptibility to tert-butanol exposure as discussed in Section 1.3.3.
3 2.1.6. Confidence Statement
4 A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
5 the overall database, and the RfD, as described in Section 4.3.9.2 of EPA's Methods for Derivation of
6 Inhalation Reference Concentrations and Application of Inhalation Dosimetry [U.S. EPA, 1994]. The
7 overall confidence in this RfD is high. Confidence in the principal study [NTP. 1995] is high. This
8 study was well conducted, complied with Food and Drug Administration (FDA] Good Laboratory
9 Practice (GLP] regulations, involved a sufficient number of animals per dose group (including both
10 sexes], and assessed a wide range of tissues and endpoints. Although the toxicity database for tert-
11 butanol has some gaps, they are informed by the data on ETBE, a parent compound of tert-butanol.
12 Therefore, the confidence in the database is high. Reflecting high confidence in the principal study
13 and high confidence in the database, confidence in the RfD is high.
14 2.1.7. Previous IRIS Assessment
15 No previous oral assessment for tert-butanol is available in IRIS.
is 2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
17 THAN CANCER
18 The inhalation RfC (expressed in units of mg/m3] is defined as an estimate (with
19 uncertainty spanning perhaps an order of magnitude] of a continuous inhalation exposure to the
20 human population (including sensitive subgroups] that is likely to be without an appreciable risk of
21 deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower
22 bound on the benchmark concentration (BMCL], with UFs generally applied to reflect limitations of
23 the data used.
24 2.2.1. Identification of Studies and Effects for Dose-Response Analysis
25 As for oral exposure, EPA identified kidney effects as a potential human hazard of tert-
26 butanol inhalation exposure (summarized in Section 1.3.1]. No chronic inhalation study for tert-
27 butanol is available; there is only one 13-week study in rats and mice (NTP. 1997]. Sufficient data
28 were available to modify and utilize a PBPK model in rats for both oral and inhalation exposure in
29 order to perform a route-to-route extrapolation, so rat studies from both routes of exposure were
30 considered for dose-response analysis.
31 The database for tert-butanol includes oral and inhalation studies and data sets that are
32 potentially suitable for use in deriving inhalation reference values. Specifically, effects associated
33 with tert-butanol exposure in animals include observations of organ weight and histological
34 changes in the kidney in chronic and subchronic studies in male and female rats.
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1 Kidney Toxicity
2 EPA identified kidney effects as a potential human hazard of tert-butanol exposure based on
3 findings of organ weight changes and histopathology primarily in male rats. These findings were
4 observed across multiple chronic, subchronic, and short-term studies following oral and inhalation
5 exposure. The subchronic NTP [1997] inhalation study is the only route-specific study available,
6 and was carried forward for further analysis. For oral studies considered for route-to-route
7 extrapolation, see Section 2.1.1 for a summary of considerations for selecting oral studies for dose-
8 response analysis. Overall, the NTP 2-year drinking water study NTP [1995] was identified as the
9 study most suitable for dose-response assessment, given the study duration, comprehensive
10 reporting of outcomes, use of multiple species tested, multiple doses tested, and availability of a
11 PBPK model for route-to-route extrapolation. This study was discussed previously in Section 2.1.1
12 as part of the derivation of the oral reference dose, so is not reviewed here again. The NTP [1997]
13 subchronic inhalation study shares many strengths with the 2-year drinking water study [NTP,
14 1995], and is described in more detail below.
15 NTP [1997] was a well-designed subchronic study that evaluated the effect of tert-butanol
16 exposure on multiple species at multiple inhalation doses. Absolute kidney weights were elevated
17 [10-11%] in male rats exposed at >3,273 mg/m3; relative kidney weights were elevated [~9%] in
18 males at >3,273 mg/m3 and in females at 6,368 mg/m3. Male rats exhibited an increase in the
19 severity of chronic nephropathy (characterized as number of foci of regenerative tubules]. Few
20 endpoints were available for consideration in the subchronic inhalation study, but changes in
21 kidney weights also were observed in the oral studies, such as the NTP [1995] 2-year drinking
22 water study.
23 2.2.2. Methods of Analysis
24 No biologically based dose-response models are available for tert-butanol. In this situation,
25 EPA evaluates a range of dose-response models considered consistent with underlying biological
26 processes to determine how best to model the dose-response relationship empirically in the range
27 of the observed data. Consistent with this approach, all models available in EPA's BMDS were
28 evaluated. Consistent with EPA's Benchmark Dose Technical Guidance [U.S. EPA. 2012b], the
29 benchmark dose or concentration [BMD/C] and the 95% lower confidence limit on the BMD/C
30 [BMD/CL] were estimated using a BMR of 10% change from the control mean for absolute kidney
31 weight changes (as described in Section 2.1.2]. As noted in Section 2.1.2., a BMR of 10% extra risk
32 was considered appropriate for the quantal data on incidences of kidney suppurative inflammation
33 and kidney transitional epithelial hyperplasia. The estimated BMD/CLs were used as PODs. Where
34 dose-response modeling was not feasible, NOAELs or LOAELs were identified and summarized in
35 Table 2-4. Further details, including the modeling output and graphical results for the best-fit
36 model for each endpoint, can be found in Appendix C of the Supplemental Information.
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1 PODsfrom Inhalation Studies
2 Because the RfC is applicable to a continuous lifetime human exposure but derived from
3 animal studies featuring intermittent exposure, EPA guidance [U.S. EPA. 1994] provides
4 mechanisms for (1] adjusting experimental exposure concentrations to a value reflecting
5 continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC)
6 from the animal exposure data. The former employs an inverse concentration-time relationship to
7 derive a health-protective duration adjustment to time weight the intermittent exposures used in
8 the studies. The modeled benchmark concentration from the inhalation study [NTP, 1997] was
9 adjusted to reflect a continuous exposure by multiplying it by (6 hours per day] 4- (24 hours per
10 day] and (5 days per week] 4 (7 days per week] as follows:
11 BMCLADj = BMCL (mg/m3] x (6 4 24] x (5 4 7]
12 = BMCL (mg/m3] x (0.1786]
13 The RfC methodology provides a mechanism for deriving an HEC from the duration-
14 adjusted POD (BMCLADj] determined from the animal data. The approach takes into account the
15 extra-respiratory nature of the toxicological responses and accommodates species differences by
16 considering blood:air partition coefficients for tert-butanol in the laboratory animal (rat or mouse]
17 and humans. According to the RfC guidelines (U.S. EPA. 1994]. tert-butanol is a Category 3 gas
18 because extra-respiratory effects were observed. Kaneko etal. (2000] measured a blood:gas
19 partition coefficient [(Hb/g]A] of531 ± 102 for tert-butanol in the male Wistar rat, while Borghoff et
20 al. (1996] measured a value of 481 ± 29 in male F344 rats. Ablood:gas partition coefficient
21 [(Hb/g]n] of 462 was reported for tert-butanol in humans (Nihlen et al.. 1995]. The calculation
22 (Hb/g]A 4 (Hb/g]H was used to calculate a blood:gas partition coefficient ratio to apply to the
23 delivered concentration. Because F344 rats were used in the study, the blood:gas partition
24 coefficient for F344 rats was used. Thus, the calculation was 481 4 462 = 1.04. Therefore, a ratio of
25 1.04 was used to calculate the HEC. This allowed a BMCLHEc to be derived as follows:
26 BMCL.HEC = BMCLADj (mg/m3] x (interspecies conversion]
27 = BMCLADj (mg/m3] x (481 4 462]
28 = BMCLADj (mg/m3] x (1.04]
29 Table 2-4 summarizes the sequence of calculations leading to the derivation of a human-
30 equivalent POD for each inhalation data set discussed above.
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Toxicological Review of tert-ButyI Alcohol
Table 2-4. Summary of derivation of PODs following inhalation exposure
Endpoint and
reference
Species/
Sex
Model"
BMR
BMCb
(mg/m3)
BMCLb
(mg/m3)
PODADjb
(mg/m3)
PODHEcc(
mg/m3)
Kidney
Increased absolute
kidney weight
NTP (1997)
Increased absolute
kidney weight
NTP (1997)
Male F344
rats
Female F344
rats
Hill
No model
selectedd
10%
10%
1931
~
1705
~
304
1137
304
1137
2 aFor modeling details, see Appendix C in Supplemental Information.
3 bBMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day /
4 24 hr) x (days exposed per week / 7 days).
5 CPODHEC calculated by adjusting the PODAoj by the DAF (=1.0, rounded from 1.04) for a Category 3 gas (U.S. EPA,
6 1994).
7 dBMD modeling failed to calculate a BMD value successfully (see Appendix C); POD calculated from no-observed
8 adverse effect level (NOAEL) of 6368 mg/m3.
9 PODs from oral studies - use ofPBPK model for route-to-route extrapolation
10 A PBPK model for tert-butanol in rats has been modified, as described in Appendix B of the
11 Supplemental Information. Using this model, route-to-route extrapolation of the oral BMDLs to
12 derive inhalation PODs was performed as follows. First, the internal dose in the rat at each oral
13 BMDL (assuming continuous exposure) was estimated using the PBPK model, to derive an "internal
14 dose BMDL." Then, the inhalation air concentration (again, assuming continuous exposure) that led
15 to the same internal dose in the rat was estimated using the PBPK model. The resulting BMCL was
16 then converted to a human equivalent concentration POD (PODHEc) using the methodology
17 previously described in "PODs from inhalation studies":
18 BMCL.HEC = BMCLADj (mg/m3) x (interspecies conversion)
19 = BMCLADj (mg/m3) x (481 -H 462)
20 = BMCLADj (mg/m3) x (1.04)
21 A critical decision in the route-to-route extrapolation is selection of the internal dose metric
22 that establishes "equivalent" oral and inhalation exposures. For tert-butanol-induced kidney effects,
23 the two options are the concentration of tert-butanol in blood and rate of tert-butanol metabolism.
24 Note that using the kidney concentration of tert-butanol will lead to the same route-to-route
25 extrapolation relationship as tert-butanol in blood because the distribution from blood to kidney is
26 independent of route. Data are not available that suggest that metabolites of tert-butanol mediate
27 its renal toxicity. Without evidence that suggests otherwise, tert-butanol is assumed the active
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Toxicological Review of tert-ButyI Alcohol
1 toxicological agent. Therefore, the concentration of tert-butanol in blood was selected as the dose
2 metric.
3 Table 2-5 summarizes the sequence of calculations leading to the derivation of a human-
4 equivalent inhalation POD from each oral data set discussed above.
Table 2-5. Summary of derivation of inhalation points of departure derived
from route-to-route extrapolation from oral exposures
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal dose3
(mg/L)
Equivalent
PODHEcb
(mg/m3)
Kidney
Mean absolute kidney weight
at 15 months NTP (1995)
Mean absolute kidney weight
at 15 months NTP (1995)
Kidney inflammation
(suppurative) NTP (1995)
Kidney transitional epithelial
hyperplasia
NTP (1995)
Kidney transitional epithelial
hyperplasia
NTP (1995)
Rat/M
Rat/F
Rat/F
Rat/M
10%
10%
10%
10%
Rat/F
10%
296
91
200
16
339
22.4
4.76
12.6
0.745
27.9
551
155
359
26.1
638
7 a Average blood concentration of tert-butanol under continuous oral exposure at the BMDL
8 b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
9 tert-butanol as continuous oral exposure at the BMDL.
10 2.2.3. Derivation of Candidate Values
11 In EPA's A Review of the Reference Dose and Reference Concentration Processes [[U.S. EPA,
12 2002]: Section 4.4.5], also described in the Preamble, five possible areas of uncertainty and
13 variability were considered. Several PODs for the candidate inhalation values were derived using a
14 route-to-route extrapolation from the PODs estimated from the chronic oral toxicity study in rats
15 [NTP. 1995] in the derivation of the oral RfD (Section 2.1]. With the exception of the subchronic
16 inhalation [NTP. 1997] study, the uncertainty factors (UFs] selected and applied to PODs derived
17 from the chronic oral [NTP, 1995] study for route-to-route extrapolation are the same as those for
18 the RfD for tert-butanol (see Section 2.1.3). The model used to perform this route-to-route
19 extrapolation is a well-characterized model considered appropriate for the purposes of this
20 assessment One source of uncertainty regarding the route-to-route extrapolation is the assumption
21 of that 100% of inhaled tert-butanol reaches the gas-exchange region, that is, 100% of the inhaled
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Toxicological Review of tert-ButyI Alcohol
1 tert-butanol could be absorbed and distributed to the rest of the body in rats. If not all of the
2 compound is bioavailable for the rat, a lower blood concentration would be expected compared to
3 the current estimate, and thus, a higher RfC would be calculated.
4 For the PODs derived from the subchronic inhalation [NTP. 1997] study, a UFS of 10 was
5 applied to account for extrapolation from subchronic to chronic duration.
6 Table 2-6 is a continuation of Table 2-4 and Table 2-5, and summarizes the application of
7 UFs to each POD to derive a candidate value for each data set. The candidate values presented in the
8 table below are preliminary to the derivation of the organ/system-specific reference values. These
9 candidate values are considered individually in the selection of a representative inhalation
10 reference value for a specific hazard and subsequent overall RfC for tert-butanol.
11 Figure 2-2 presents graphically the candidate values, UFs, and PODnEc values, with each bar
12 corresponding to one data set described in Tables 2-4, 2-5, and 2-6.
13
Table 2-6. Effects and corresponding derivation of candidate values
Endpoint (sex and species) and
reference
PODHEC3
(mg/m3)
POD
type
UFA
UFH
UFL
UFs
UFD
Composite
UF
Candidate
value
(mg/m3)
Kidney
Increased absolute kidney weight
at 13 weeks; male rat
NTP (1997)
Increased absolute kidney weight
at 13 weeks; female rat
NTP (1997)
Increased absolute kidney weight
at 15 months; male rat
NTP (1995)
Increased absolute kidney weight
at 15 months; female rat
NTP (1995)
Kidney inflammation
(suppurative); female rat
NTP (1995)
Kidney transitional epithelial
hyperplasia; male rat
NTP (1995)
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
304
1137
551
155
359
26.1
638
BMCLio%
NOAEL
BMCLio%
BMCLio%
BMCLio%
BMCLio%
BMCLio%
3
3
3
3
3
3
3
10
10
10
10
10
10
10
1
1
1
1
1
1
1
10
10
1
1
1
1
1
1
1
1
1
1
1
1
300
300
30
30
30
30
30
1x10°
4x10°
2 x 101 *
5 x 10° *
1 x 101 *
9 x 10'1 *
2 x 101 *
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Toxicological Review of tert-ButyI Alcohol
1 These candidate values are derived using route-to-route extrapolated PODs based on NTP's chronic drinking
2 water study.
TAbsolute kidney weight;
male rat [NTP, 1997]
TAbsolute kidney weight;
female rat (NTP, 1997]
TAbsolute kidney weight;
male rat (NTP, 1995]
tAbsolute kidney weight;
female rat (NTP, 1995]
Kidney inflammation;
female rat (NTP, 1995]
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 1995]
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995]
Candidate RfC
PODHEC
Composite UF
0.1
10 100
mg/m3
1000 10000
Figure 2-2. Candidate RfC values with corresponding POD and composite UF.
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Toxicological Review of tert-ButyI Alcohol
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.2.4. Derivation of Organ/System-Specific Reference Concentrations
Table 2-7 distills the candidate values from Table 2-6 into a single value for the kidney.
Organ- or system-specific reference values can be useful for subsequent cumulative risk
assessments that consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For the derivation of candidate values, whether PODs from the subchronic inhalation study
of NTP [1997] would provide a better basis than the route-to-route extrapolated PODs based on the
chronic oral study of NTP [1995] must be considered. Candidate values were derived for increased
kidney weight observed in the subchronic inhalation study [NTP, 1997] and several kidney effects
observed in the chronic oral study [NTP. 1995] in both sexes of rat, spanning a range from 9 x lO1
to 2 x 101 mg/m3, for an overall 20-fold range. To estimate an exposure level below which kidney
toxicity from tert-butanol exposure is not expected to occur, the RfC for increased incidence of
transitional epithelial hyperplasia in male rats (9 x 1Q-1 mg/m3] was selected as the kidney-specific
RfC for tert-butanol, consistent with the selection of the kidney-specific RfD (see Section 2.1.4). As
discussed in Section 2.1.4, unlike kidney suppurative inflammation, this effect was observed in both
sexes, with males appearing to be more sensitive than females. Additionally, it is based on a longer
(chronic) duration and a more specific and sensitive indicator of kidney toxicity than the relatively
non-specific endpoint of kidney weight change. Confidence in this kidney-specific RfC is high. The
PODs are based on BMD modeling, and the candidate values are derived from a well-conducted
study, involving a sufficient number of animals per group, including both sexes, assessing a wide
range of kidney endpoints, and availability of a PBPK model for route-to-route extrapolation.
Table 2-7. Organ/system-specific RfCs and overall RfC for tert-butanol
Effect
Kidney
Overall RfC
Basis
Incidence of transitional
epithelial hyperplasia (NTP,
1995)
Kidney
RfC
(mg/m3)
9 x 10'1
9 x 1C'1
Study exposure
description
Chronic
Chronic
Confidence
High
High
23
24
25
26
27
28
29
2.2.5. Selection of the Overall Reference Concentration
For tert-butanol, kidney effects were identified as the primary hazard; thus, a single
organ-/system-specific RfC was derived. The kidney-specific RfC of 9 x 1Q-1 mg/m3 is selected as
the overall RfC, representing an estimated exposure level below which deleterious effects from
tert-butanol exposure are not expected to occur.
The overall RfC is derived to be protective of all types of effects for a given duration of
exposure and is intended to protect the population as a whole, including potentially susceptible
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Toxicological Review of tert-ButyI Alcohol
1 subgroups [U.S. EPA. 2002]. Decisions concerning averaging exposures over time for comparison
2 with the RfC should consider the types of toxicological effects and specific lifestages of concern.
3 Fluctuations in exposure levels that result in elevated exposures during these lifestages could
4 potentially lead to an appreciable risk, even if average levels over the full exposure duration were
5 less than or equal to the RfC. In the case of tert-butanol, there is potential for early lifestage
6 susceptibility to tert-butanol exposure as discussed in Section 1.3.3.
7 2.2.6. Confidence Statement
8 A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
9 the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
10 Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry [U.S. EPA.
11 1994). A PBPK model was utilized to perform a route-route extrapolation to determine a POD for
12 the derivation of the RfC from the NTP [1995] oral study and corresponding critical effect.
13 Confidence in the principal study [NTP. 1995] is high. This study was well-conducted, complied
14 with FDA GLP regulations, involved a sufficient number of animals per group (including both
15 sexes], and assessed a wide range of tissues and endpoints. Although there are some gaps in the
16 toxicity database for tert-butanol, these areas are informed by the data on ETBE, a parent
17 compound of tert-butanol. Therefore, the confidence in the database is high. Reflecting high
18 confidence in the principal study, high confidence in the database, and minimal uncertainty
19 surrounding the application of the modified PBPK model for the purposes of a route-to-route
20 extrapolation, the overall confidence in the RfC for tert-butanol is high.
21 2.2.7. Previous IRIS Assessment
22 No previous inhalation assessment for tert-butanol is available in IRIS.
23 2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration
24 The following discussion identifies uncertainties associated with the RfD and RfC for
25 tert-butanol. To derive the RfD, the UF approach [U.S. EPA. 2000a. 1994] was applied to a POD
26 based on kidney toxicity in rats treated chronically. UFs were applied to the POD to account for
27 extrapolating from an animal bioassay to human exposure, and the likely existence of a diverse
28 human population of varying susceptibilities. These extrapolations are carried out with default
29 approaches given the lack of data to inform individual steps. To derive the RfC, this same approach
30 was applied, but a PBPK model was used to extrapolate from oral to inhalation exposure.
31 The database for tert-butanol contains no human data on adverse health effects from
32 subchronic or chronic exposure, and the PODs were calculated from data on the effects of tert-
33 butanol reported by studies in rats. The database for tert-butanol exposure includes one lifetime
34 bioassay, several reproductive/developmental studies, and several subchronic oral studies.
35 Although the database is adequate for reference value derivation, there is uncertainty
36 associated with the lack of a comprehensive multigeneration reproductive toxicity study.
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1 Additionally, only subchronic and short-term inhalation studies have been conducted, and no
2 chronic inhalation studies are available. Developmental studies identified significant increases in
3 fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed
4 offspring or pups. However, effects were not always consistent across exposure routes, and
5 maternal toxicity was present whenever developmental effects were observed.
6 The toxicokinetic and toxicodynamic differences for tert-butanol between the animal
7 species in which the POD was derived and humans are unknown. The tert-butanol database lacks
8 an adequate model that would inform potential interspecies differences (A limited data set exists
9 for tert-butanol appearing as a metabolite from ETBE exposure in humans, but none for direct
10 exposure to tert-butanol.) Generally, it was found that rats appear more susceptible than mice, and
11 males appear more susceptible than females to tert-butanol toxicity. However, the underlying
12 mechanistic basis of these apparent differences is not understood. Most importantly, it is unknown
13 which animal species and/or sexes may be more comparable to humans.
14 Another uncertainty to consider relates to the MOA analysis conducted for the kidney
15 effects. The assessment concluded that tert-butanol is a weak inducer of (X2u-globulin which is
16 operative in male kidney tumors; therefore, noncancer effects related to (X2u-globulin were
17 considered not relevant for hazard identification and, therefore, not suitable for dose response
18 consideration. If this conclusion was incorrect and the noncancer effects characterized in this
19 assessment as being related to a2u-globulin were relevant to humans, then the RfD and RfC values
20 could be underestimating toxicity. Similarly, the renal effects characterized as CPN and dismissed as
21 not being treatment related, if considered relevant, would likewise contribute to the hazard
22 potential and dose-response analysis for the kidney-specific RfD and RfC.
23 2.3. ORAL SLOPE FACTOR FOR CANCER
24 The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per mg/kg day
25 of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in mg/kg-day) to
26 estimate the lifetime cancer risk.
27 2.3.1. Analysis of Carcinogenicity Data
28 As noted in Section 1.3.2, that there is "suggestive evidence of carcinogenic potential" for
29 tert-butanol. The Guidelines for Carcinogen Risk Assessment [U.S. EPA. 2005a] state:
30 When there is suggestive evidence, the Agency generally would not attempt a dose-
31 response assessment, as the nature of the data generally would not support one; however
32 when the evidence includes a well-conducted study, quantitative analysis may be useful for
33 some purposes, for example, providing a sense of the magnitude and uncertainty of
34 potential risks, ranking potential hazards, or setting research priorities.
35 No human data relevant to an evaluation of the carcinogenicity of tert-butanol were
36 available. The cancer descriptor was based on the 2-year drinking water study in rats and mice by
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1 [NTP. 1995]. which reported renal tumors in male rats and thyroid tumors in both male and female
2 mice. This study was considered suitable for dose-response analysis. It was conducted in
3 accordance with FDA GLP regulations, and all aspects were subjected to retrospective quality
4 assurance audits. The study included histological examinations for tumors in many different
5 tissues, contained three exposure levels and controls, contained adequate numbers of animals per
6 dose group (~50/sex/group], treated animals for up to 2 years, and included detailed reporting of
7 methods and results. Additionally, the renal tumors were reexamined by a Pathology Working
8 Group [Hard etal.. 2011].
9 Based on a mode of action analysis, it was concluded that the (X2u-globulin process was at
10 least partially responsible for the male rat renal tumors, in addition to other, unknown, processes.
11 Because the relative contribution of each process to tumor formation cannot be determined [U.S.
12 EPA. 1991a]. the male rat renal tumors are not considered suitable for quantitative analysis.
13 Conversely, the mouse thyroid tumors are suitable for dose-response analysis and unit risk
14 estimation, as described in Section 1.3.2.
15 2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
16 The EPA Guidelines for Carcinogen Risk Assessment [U.S. EPA, 2005a] recommend that the
17 method used to characterize and quantify cancer risk from a chemical be determined by what is
18 known about the MOA of the carcinogen and the shape of the cancer dose-response curve. EPA
19 uses a two-step approach that distinguishes analysis of the observed dose-response data from
20 inferences about lower doses [U.S. EPA, 2005a]. Within the observed range, the preferred approach
21 is to use modeling to incorporate a wide range of data into the analysis, such as through a
22 biologically based model, if supported by substantial data. Without a biologically based model, as in
23 the case of tert-butanol, a standard model is used to curve-fit the data and estimate a POD. EPA uses
24 the multistage model in IRIS dose-response analyses for cancer [Gehlhaus etal.. 2011] because it
25 parallels the multistage carcinogenic process and fits a broad array of dose-response patterns.
26 The second step, extrapolation to lower exposures from the POD, considers what is known
27 about the modes of action for each effect As above, a biologically based model is preferred [U.S.
28 EPA, 2005a]. Otherwise, linear low-dose extrapolation is recommended if the MOA of
29 carcinogenicity is mutagenic or has not been established [U.S. EPA, 2005a]. For tert-butanol, the
30 mode(s] of carcinogenic action for thyroid follicular cell tumors has not been established (see
31 Section 1.3.2]. Therefore, linear low-dose extrapolation was used to estimate human carcinogenic
32 risk.
33 The dose-response modeling used administered dose because a PBPK model to characterize
34 internal dosimetry in mice was not available. For the analysis of male mice thyroid tumors, the
35 incidence data were adjusted to account for the increased mortality in high-dose male mice, relative
36 to the other groups, that reduced the number of mice at risk for developing tumors. The Poly-3
37 method [Bailer and Portier, 1988] was used to estimate the number at risk of developing tumors,
38 by weighting the length of time each animal was on study (details in Appendix C of the
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Toxicological Review of tert-Butyl Alcohol
1 Supplemental Information). This method was not applied to the female mice data because a
2 difference in survival with increasing exposure was not appreciable and only one tumor, in the
3 high-dose group, occurred before study termination.
4 The data modeled and other details of the modeling are provided in Appendix C. The BMDs
5 and BMDLs recommended for each data set are summarized in Table 2-8. The modeled tert-butanol
6 PODs were scaled to HEDs according to EPA guidance (U.S. EPA. 2011. 2005al In particular, the
7 BMDL was converted to an HED by assuming that doses in animals and humans are toxicologically
8 equivalent when scaled by body weight raised to the 3/4 power. Standard body weights of 0.025 kg
9 for mice and 70 kg for humans were used (U.S. EPA. 1988]. The following formula was used for the
10 conversion of oral BMDL to oral HED for mouse endpoints:
11 HED in mg/kgday- = (BMDL in mg/kgday-) x (animal body weight/70)1/4
12 = (BMDL in mg/kgday-) x 0.14
13 PODs for estimating low-dose risk were identified at doses at the lower end of the observed
14 data, corresponding to 10% extra risk in female mice and 5% extra risk in male mice.
15 2.3.3. Derivation of the Oral Slope Factor
16 The PODs estimated for each tumor data set are summarized in Table 2-8. The lifetime oral
17 cancer slope factor for humans is defined as the slope of the line from the lower 95% bound on the
18 exposure atthe POD to the control response (slope factor = BMR/BMDLBMR = 0.1/BMDLio). This
19 slope represents a plausible upper bound on the true population average risk. Using linear
20 extrapolation from the BMDLio, human equivalent oral slope factors were derived for male and
21 female mice and are listed in Table 2-8.
22 The oral slope factor based on the incidence of thyroid follicular cell adenomas in female
23 mice was 5 x 104 per mg/kg-day. Despite high mortality in high-dose male mice, estimating slope
24 factors using the poly-3 method was feasible for addressing competing risks. Whether using the full
25 data set (including the only thyroid follicular cell carcinoma observed at the highest dose) or
26 omitting the high-dose group altogether (under the assumption that mortality in this group was too
27 extensive to interpret the results), oral slope factors based on the incidence of thyroid follicular cell
28 adenomas or carcinomas in male mice were similar when rounded to one significant digit—5 x 1Q-4
29 per mg/kg-day or 6 x 1Q-4 per mg/kg-day, respectively.
30 The recommended slope factor for lifetime oral exposure to tert-butanol is
31 5 x KM per mg/kg-day, based on the thyroid follicular cell adenoma or carcinoma response in
32 male or female B6C3Fi mice. This slope factor should not be used with exposures exceeding
33 1400 mg/kg-day, the highest POD from the two data sets, because above this level the cancer risk
34 might not increase linearly with exposure. The slope of the linear extrapolation from the central
35 estimate BMDioHED derived from the female mouse data set is 0.1/[0.14 x (2002 mg/kg-day)] =
36 4 x IQ-4 per mg/kg-day.
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Toxicological Review of tert-Butyl Alcohol
Table 2-8. Summary of the oral slope factor derivation
Tumor
Thyroid follicular
cell adenoma
Thyroid follicular
cell adenoma or
carcinoma
Species/ sex
B6C3Fi
mouse/Female
B6C3Fi
mouse/Male
Selected
model
3° Multistage
All dose
groups: 1°
Multistage
High dose
omitted: 2°
Multistage
BMR
10%
5%c
5%c
BMD
(mg/kg-
d)
2002
1788
1028
POD=
BMDL
(mg/kg-
d)
1437
787
644
BMDLHEDa
(mg/kg-d)
201
110
90
Slope factor15
(mg/kg-day)
i
5 x 10'4
5 x 10'4
6 x 10'4
2 aHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
3 bHuman equivalent slope factor = 0.1/BMDLioHEo; see Appendix C of the Supplemental Information for details of
4 modeling results.
5 °Because the observed responses were <10%, a BMR of 5% was used to represent the observed response range for
6 low-dose extrapolation; human equivalent slope factor = 0.05/BMDLsHED.
7 2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
8 There is uncertainty when extrapolating data from animals to estimate potential cancer
9 risks to human populations from exposure to tert-butanol.
10 Table 2-9 summarizes several uncertainties that could affect the oral slope factor. There are
11 no other chronic studies to replicate these findings or that examined other animal models, no data
12 in humans to confirm a cancer response in general or the specific tumors observed in the NTP
13 [1995] bioassay, and no other data (e.g., MOA) to support alternative approaches for deriving the
14 oral slope factor.
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Toxicological Review of tert-ButyI Alcohol
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor
for tert-butanol
Consideration and
impact on cancer risk value
Decision
Justification
Selection of tumor type and
relevance to humans:
Mouse thyroid tumors are the basis
for estimating human cancer risk, as
the fraction of rat kidney tumors not
attributed to the male rat specific
o2u.-globulin process could not be
determined. Alternatively,
quantifying rat kidney tumors could
T* slope factor to 1 x 10'2 mg/kg-day
(see Appendix C, Supplemental
Information)
Thyroid tumors in female
and male mice were
selected U.S. EPA(1998a),
U.S. EPA(1991a).
MOA data suggested that mouse thyroid
tumors were relevant to humans.
Quantitation of thyroid tumors in male mice
was impacted only slightly by high mortality
in the high-dose group, and supports the
estimate based on female mice.
Selection of data set:
No other studies are available.
NTP (1995), oral (drinking
water) study, was selected
to derive cancer risks for
humans.
NTP (1995), the only chronic bioassay
available, was a well-conducted study.
Additional bioassays might add support to
the findings, facilitate determination of what
fraction of kidney tumors are not attributable
to a2u.-globulin process, or provide results
for different (possibly lower) doses, which
would affect (possibly increase) the oral
slope factor.
Selection of dose metric:
Alternatives could 4, or 1
factor
Used administered dose.
slope
For mice, PBPK-estimated internal doses
could impact the OSF value for thyroid
tumors if the carcinogenic moiety is not
proportional to administered dose, but no
PBPK model was available, and no
information is available to suggest if any
metabolites elicit carcinogenic effects.
Interspecies extrapolation of
dosimetry and risk:
Alternatives could 4, or ^ slope
factor (e.g., 3.5-fold 4, [scaling by
body weight] or ^ 2-fold [scaling by
BW2/3])
The default approach of
body weight374 was used.
No data to suggest an alternative approach
for tert-butanol. Because the dose metric
was not an area under the curve, BW3/4
scaling was used to calculate equivalent
cumulative exposures for estimating
equivalent human risks. Although the true
human correspondence is unknown, this
overall approach is expected neither to over-
or underestimate human equivalent risks.
Dose-response modeling:
Alternatives could 4, or 1"
factor
slope
Used multistage dose-
response model to derive a
BMDand BMDL
No biologically based models for tert-butanol
were available. The multistage model has
biological support and is the model most
consistently used in EPA cancer assessments.
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Toxicological Review of tert-ButyI Alcohol
Consideration and
impact on cancer risk value
Decision
Justification
Low-dose extrapolation:
4, cancer risk estimate would be
expected with the application of
nonlinear low-dose extrapolation
Linear extrapolation of risk
in low-dose region used
U.S. EPA(1998a).
Linear low-dose extrapolation for agents
without a known MOA is supported (U.S.
EPA, 2005a) and recommended for rodent
thyroid tumors arising from an unknown
MOA (U.S. EPA, 1998a).
Statistical uncertainty at POD:
4> oral slope factor 1.4-fold if BMD
used as the POD rather than BMDL
BMDL (preferred approach
for calculating slope factor)
Limited size of bioassay results in sampling
variability; lower bound is 95% Cl on
administered exposure at 10% extra risk of
thyroid tumors.
Sensitive subpopulations:
1" oral slope factor to unknown
extent
No sensitive populations
have been identified.
No chemical-specific data are available to
determine the range of human
toxicodynamic variability or sensitivity,
including the susceptibility of children.
Because determination of a mutagenic MOA
has not been made, an age-specific
adjustment factor is not applied.
1 2.3.5. Previous IRIS Assessment: Oral Slope Factor
2 No previous cancer assessment for tert-butanol is available in IRIS.
3 2.4. INHALATION UNIT RISK FOR CANCER
4 The carcinogenicity assessment provides information on the carcinogenic hazard potential
5 of the substance in question, and quantitative estimates of risk from oral and inhalation exposure
6 may be derived. Quantitative risk estimates may be derived from the application of a low-dose
7 extrapolation procedure. If derived, the inhalation unit risk (IUR) is a plausible upper bound on the
8 estimate of risk per [J.g/m3 air breathed.
9 No chronic inhalation exposure studies to tert-butanol are available. Lifetime oral exposure
10 has been associated with increased renal tubule adenomas and carcinoma in male F344 rats,
11 increased thyroid follicular cell adenomas in female B6C3Fi mice, and increased thyroid follicular
12 cell adenomas and carcinomas in male B6C3Fi mice. Because only a rat PBPK model exists,
13 however, route-to-route extrapolation cannot be performed for thyroid tumors in mice at this time.
14 The NTP [1995] drinking water study in rats and mice was the only chronic bioassay available for
15 dose-response analysis. Still, the rat PBPK model and kidney tumors from the NTP [1995] drinking
16 water study were not used for route-to-route extrapolation because enough information to
17 determine the relative contribution of (X2u-globulin nephropathy and other processes to the overall
18 renal tumor response [U.S. EPA. 1991a] is not available. Alternatively, if kidney tumors were
19 considered acceptable for quantitation, then route-to-route extrapolation could be conducted to
20 calculate an IUR (see Appendix C in Supplemental Information].
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Toxicological Review of tert-ButyI Alcohol
1 2.4.1. Previous IRIS Assessment: Inhalation Unit Risk
2 An inhalation cancer assessment for tert-butanol was not previously available on IRIS.
3 2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
4 As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
5 Carcinogens [U.S. EPA. 2005b]. either default or chemical-specific age-dependent adjustment
6 factors (ADAFs) are recommended to account for early-life exposure to carcinogens that act
7 through a mutagenic MOA Because chemical-specific lifestage susceptibility data for cancer are not
8 available, and because the MOA for tert-butanol carcinogenicity is not known (see Section 1.3.2),
9 application of ADAFs is not recommended.
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Toxicological Review of tert-Butyl Alcohol
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Toxicological Review of tert-ButyI Alcohol
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48 Williams, & Wilkins.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-ButyI Alcohol
1 (EPA/600/8-90/066F). Washington, DC: U.S. Environmental Protection Agency, Office of
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49 Environmental Protection Agency, Office of Research and Development, National Center for
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Toxicological Review of tert-ButyI Alcohol
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40
This document is a draft for review purposes only and does not constitute Agency policy.
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