?/EPA
EPA/635/R-07/003
www.epa.gov/iris
TOXICOLOGICAL REVIEW
OF
2,2,4-TRIMETHYLPENTANE
(CAS No. 540-84-1)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
July 2007
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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CONTENTS—TOXICOLOGICAL REVIEW OF
2,2,4-TRIMETHYLPENTANE (CAS No. 540-84-1)
LIST OF TABLES v
LIST OF FIGURES v
LIST OF ABBREVIATIONS AND ACRONYMS vi
FOREWORD vii
AUTHORS, CONTRIBUTORS, AND REVIEWERS viii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 5
3.1. ABSORPTION 5
3.1.1. Oral 5
3.1.2. Inhalation 5
3.2. DISTRIBUTION 5
3.2.1. Oral 5
3.3. METABOLISM 6
3.3.1. Following Oral Administration 6
3.3.2. Following Inhalation Exposure 8
3.4. ELIMINATION 8
3.4.1. Following Oral Administration 8
3.4.2. Following Inhalation Exposure 8
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 9
4. HAZARD IDENTIFICATION 10
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS,
CLINICAL CONTROLS 10
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 10
4.2.1. Noncancer Toxicity 10
4.2.1.1. Oral Studies 10
4.2.1.2. Inhalation Studies 10
4.2.2. Cancer Bioassays 11
4.2.2.1. Initiation and Promotion Studies 11
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND
INHALATION 12
4.4. OTHER STUDIES 12
4.4.1. Acute and Short-Term Studies 12
4.4.1.1. Oral 12
4.4.1.2. Inhalation 17
4.4.1.3. Dermal 18
4.4.1.4. Ocular 18
4.4.2. Genotoxicity 18
4.4.2.1. Mutation and Chromosome Effects 18
4.4.2.2. DNA Damage 18
4.4.3. Cytotoxicity 19
iii
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4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS 19
4.5.1. Oral 19
4.5.2. Inhalation 19
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATION 20
4.6.1. Summary of Overall Weight of Evidence 20
4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 20
4.7. MODE-OF-ACTION INFORMATION 20
4.7.1. General Issues Concerning the Determination of a,2u-Globulin-Associated
Nephropathy 21
4.7.2. 2,2,4-Trimethylpentane and a,2u-Globulin-Associated Nephropathy 24
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 24
4.8.1. Possible Childhood Susceptibility 24
4.8.2. Possible Gender Differences 24
5. DOSE-RESPONSE ASSESSMENTS 25
5.1. Oral Reference Dose (RfD) 25
5.2. Inhalation Reference Concentration (RfC) 26
5.3. Cancer Assessment 26
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND
DOSE RESPONSE 27
6.1. Human Hazard Potential 27
6.2. Dose Response 28
7. REFERENCES 29
APPENDIX A. Summary of External Peer Review and Public Comments and Disposition 32
IV
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LIST OF TABLES
Table 2-1. Chemical and physical properties of 2,2,4-trimethylpentane.
Table 4-1. Summary of renal effects specific to male rats reported in
2,2,4-trimethylpentane studies 22
LIST OF FIGURES
Figure 3-1. Proposed pathway for 2,2,4-trimethylpentane metabolism 7
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LIST OF ABBREVIATIONS AND ACRONYMS
ACF atypical cell foci
ALT alanine aminotransferase
CASRN Chemical Abstracts Service registry number
CPN chronic progressive nephrosis
EPA U.S. Environmental Protection Agency
eq equivalents
GC/MS gas chromatography/mass spectrometry
GFR glomerular filtration rate
hsc73 heat shock cognate protein 73
IRIS Integrated Risk Information System
NAG N-acetyl-p-D-glucosaminidase
NBR NCI-black-Reiter
NZW New Zealand white
PC partition coefficient
po by mouth, oral(ly)
RCT renal cell tumor
RD50 50% depression in respiratory rate
RfC reference concentration
RfD reference dose
SCE sister chromatid exchange
SDH sorbitol dehydrogenase
TK thymidine kinase
TMP 2,2,4-trimethylpentane
UDS unscheduled DNA synthesis
UG unleaded gasoline
VOC volatile organic chemical
VI
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to
2,2,4-trimethylpentane. It is not intended to be a comprehensive treatise on the chemical or
toxicological nature of 2,2,4-trimethylpentane.
In Section 6, Major Conclusions in the Characterization of Hazard and Dose Response,
EPA has characterized its overall confidence in the quantitative and qualitative aspects of hazard
and dose response by addressing knowledge gaps, uncertainties, quality of data, and scientific
controversies. The discussion is intended to convey the limitations of the assessment and to aid
and guide the risk assessor in the ensuing steps of the risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
John Stanek, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
AUTHORS
Ms. Susan Goldhaber
Alpha-Gamma Technologies, Inc.
Raleigh, NC
Errol Zeiger, Ph.D.
Alpha-Gamma Technologies, Inc.
Raleigh, NC
Mr. Frank Stack
Alpha-Gamma Technologies, Inc.
Raleigh, NC
REVIEWERS
This document and the accompanying IRIS Summary have been peer reviewed by EPA
scientists and independent scientists external to EPA. Comments from all peer reviewers were
evaluated carefully and considered by the Agency during the finalization of this assessment.
During the finalization process, the IRIS Program Director achieved common understanding of
the assessment among the Office of Research and Development; Office of Air and Radiation;
Office of Prevention, Pesticides, and Toxic Substances; Office of Solid Waste and Emergency
Response; Office of Water; Office of Policy, Economics, and Innovation; Office of Children's
Health Protection; Office of Environmental Information; and EPA's regional offices.
Vlll
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INTERNAL EPA REVIEWERS
D. Charles Thompson, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Danielle Devoney, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Marion Hoyer, Ph.D.
Environmental Scientist
Office of Transportation and Air Quality
EXTERNAL PEER REVIEWERS
Susan Borghoff, Ph.D., DABT, Panel Chair
Integrated Laboratory Systems, Inc.
Deborah Barsotti, Ph.D.
MACTEC Engineering and Consulting
Lawrence Lash, Ph.D.
Wayne State University
Brian Short, Ph.D., DVM, DACVP
Allergan, Inc.
Summaries of the external peer reviewers' comments and public comments and the
disposition of their recommendations are provided in Appendix A.
IX
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1. INTRODUCTION
This document presents background information and justification for the Integrated Risk
Information System (IRIS) Summary of the hazard and dose-response assessment of
2,2,4-trimethylpentane. IRIS Summaries may include oral reference dose (RfD) and inhalation
reference concentration (RfC) values for chronic and less-than-lifetime exposure durations, and a
carcinogenicity assessment.
The RfD and RfC provide quantitative information for use in risk assessments for health
effects known or assumed to be produced through a nonlinear (possibly threshold) mode of
action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a daily exposure to the human population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
lifetime. The inhalation RfC (expressed in units of mg/m3) is analogous to the oral RfD, but
provides a continuous inhalation exposure estimate. The inhalation RfC considers toxic effects
for both the respiratory system (portal-of-entry) and for effects peripheral to the respiratory
system (extrarespiratory or systemic effects). Reference values may also be derived for acute
(<24 hours), short-term (up to 30 days), and subchronic (up to 10% of average lifetime) exposure
durations, all of which are derived based on an assumption of continuous exposure throughout
the duration specified.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure. The information includes a weight-of-evidence judgment of the likelihood that the
agent is a human carcinogen and the conditions under which the carcinogenic effects may be
expressed. Quantitative risk estimates are derived from the application of a low-dose
extrapolation procedure, and are presented in two ways to better facilitate their use. First, route-
specific risk values are presented. The "oral slope factor" is an upper bound on the estimate of
risk per mg/kg-day of oral exposure. Similarly, a "unit risk" is an upper bound on the estimate of
risk per unit of concentration, either per ug/L drinking water or per ug/m3 air breathed. Second,
the estimated concentration of the chemical substance in drinking water or air when associated
with cancer risks of 1 in 10,000, 1 in 100,000, or 1 in 1,000,000 is also provided.
Development of these hazard identification and dose-response assessments for
2,2,4-trimethylpentane has followed the general guidelines for risk assessment as set forth by the
National Research Council (1983). EPA guidelines and Risk Assessment Forum Technical
Panel Reports that were used in the development of this assessment include the following:
Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998a), Guidelines for Carcinogen
Risk Assessment (U.S. EPA, 2005a), Recommendations for and Documentation of Biological
Values for Use in Risk Assessment (U.S. EPA, 1988), Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994), Science
1
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Policy Council Handbook: Peer Review (U.S. EPA, 1998b, 2000a, 2006), Science Policy
Council Handbook: Risk Characterization (U.S. EPA, 2000b), and A Review of the Reference
Dose and Reference Concentration Processes (U.S. EPA, 2002).
The literature search strategy employed for this compound was based on the CASRN and
at least one common name. Any pertinent scientific information submitted by the public to the
IRIS Submission Desk was also considered in the development of this document. The relevant
literature was reviewed through September 2005.
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2. CHEMICAL AND PHYSICAL INFORMATION
2,2,4-Trimethylpentane is a hydrocarbon also known as isooctane, iso-octane,
isobutyltrimethylmethane, and TMP. Some relevant chemical and physical properties are listed
in Table 2-1.
Table 2-1. Chemical and physical properties of 2,2,4-trimethylpentane
CAS registry number
Empirical formula
Molecular weight
Vapor pressure
Vapor density
Boiling point
Melting point
Density/specific gravity
Solubilities
Viscosity
ppm Conversion
540-84-1
CgHjg
114.22
40.6mmHg(at21°C)
3.93
99.2°C
-107.4°C
0.69194 (at 20°C/4°C)
Practically insoluble in water; somewhat soluble in absolute alcohol;
soluble in benzene, toluene, xylene, chloroform, ether, carbon
disulfide, and carbon tetrachloride
<32 Saybolt Universal
1 ppm = 4.68 mg/m3; 1
Seconds
mg/m3 = 0.21 ppm
Source: NLM(2004).
2,2,4-Trimethylpentane is a colorless liquid with the odor of gasoline (NLM, 2004). It is
used primarily in the alkylation step of the reaction of isobutane and butylene in deriving high-
octane fuels. 2,2,4-Trimethylpentane is synthesized from the catalytic hydrogenation of
trimethylpentene with a nickel catalyst (C8Hi6 + H2 = C8Hi8) (API, 1985).
2,2,4-Trimethylpentane (isooctane) is one of two chemicals used in establishing the
octane rating for gasoline. The octane value is a number that reflects the resistance of a gasoline
mixture to knocking when used as fuel in an internal combustion engine. Pure
2,2,4-trimethylpentane has an octane value of 100, indicating that it burns without knocking,
whereas n-heptane has an octane value of 0 because it burns with considerable knocking (API,
1985). When testing the knock properties of gasoline, the octane number is the percentage of
2,2,4-trimethylpentane in the 2,2,4-trimethylpentane/n-heptane mixture that matches the knock
properties of the gasoline. For example, gasoline with an octane number of 89 has the same
knocking properties during combustion as a mixture comprised of 89% 2,2,4-trimethylpentane
and 11% n-heptane.
2,2,4-Trimethylpentane is released into the environment through the manufacture, use,
and disposal of products associated with the gasoline and petroleum industry. Automotive
exhaust and automotive evaporative emissions are important sources of atmospheric
2,2,4-trimethylpentane. Thus, the most probable route of exposure to the general population is
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by inhalation. When 2,2,4-trimethylpentane is released into water or onto dry or moist soil
surfaces, volatilization is expected to be the dominant removal process. Photolysis is not an
important removal process, as 2,2,4-trimethylpentane is transparent to wavelengths available in
sunlight. In subsurface soil, adsorption is expected to occur (NLM, 2004).
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3. TOXICOKINETICS
There are limited published studies on the toxicokinetics of 2,2,4-trimethylpentane. The
disposition of 2,2,4-trimethylpentane has been studied in rats after inhalation and oral exposures.
Blood:air partition coefficients (PCs) have been estimated by using reconstituted mixtures of
blood components from male rats.
3.1. ABSORPTION
3.1.1. Oral
Kloss et al. (1986) reported the disposition of radiolabeled 2,2,4-trimethylpentane in
F344 rats. Male and female rats (four/group) were administered [14C]-2,2,4-trimethylpentane
(500 mg/kg, by mouth [po]), and expired air, urine, and feces were collected for 72 hours.
Animals were sacrificed after 72 hours, and selected tissues were removed for radionuclide
analysis. Approximately 5% of the radioactivity was recovered in the feces, indicating high oral
absorption (approximately 95% of the administered dose). The majority of the radioactivity was
recovered in the expired air (43 to 48%) and urine (49 to 67%), while less than 1% of the
radioactive 2,2,4-trimethylpentane remained in the tissues of males and females.
3.1.2. Inhalation
Groups of four male F344/N rats were exposed to low (0.79 ppm or 3.7 mg/m3) and high
(385 ppm or 1800 mg/m3) concentrations of 2,2,4-trimethylpentane via nose-only inhalation for
2 hours (Dahl, 1989). Uptake rates were 0.307 and 96.7 |ig/kg-minute for the low and high
2,2,4-trimethylpentane concentrations, respectively. Based on these uptake rates, approximately
7 to 12% of the inspired 2,2,4-trimethylpentane was absorbed by the respiratory tract.
3.2. DISTRIBUTION
3.2.1. Oral
Kloss et al. (1986) reported the distribution of radiolabeled 2,2,4-trimethylpentane in
F344 rats. Male and female rats were administered [14C]-2,2,4-trimethylpentane (500 mg/kg,
po), and expired air, urine, and feces were collected for 72 hours. Animals (four/group) were
sacrificed after 72 hours and selected tissues (kidney, fat, liver, lung, heart, testis, and spleen)
were removed for radionuclide analysis. The majority of the radioactive 2,2,4-trimethylpentane
in male rats was found localized in the kidney, with minor amounts in peritoneal fat and liver.
Analysis revealed an approximate 8- to 10-fold higher level of 2,2,4-trimethylpentane in kidneys
of males than of females (1225 nmol equivalents [eq]/g wet tissue versus 157 nmol eq/g wet
tissue,/* < 0.05). The radioactivity found in the kidney was associated with the renal cortex.
Statistically significant differences between genders were not noted in the peritoneal fat and
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livers of males and females: 244 and 177 nmol eq/g wet tissue for males, and 336 and 193 nmol
eq/g wet tissue for females, respectively. Overall, these data indicate a marked difference in the
distribution of 2,2,4-trimethylpentane in male rats and, especially, in the male rat kidney, when
compared to female rats.
The results of Kloss et al. (1986) were essentially reproduced by Charbonneau et al.
(1987), who treated male and female F344 rats with 500 mg/kg (equivalent to 4.4 mmol/kg)
[14C]-2,2,4-trimethylpentane by gavage in a 48-hour disposition study. Tissue concentrations
were determined at 4, 8, 12, 24, and 48 hours after dosing. Peak tissue concentrations in the
kidney, liver, and plasma were reported to occur in males at 12 hours and in females at 8 hours
after dosing. The peak concentration in the male kidneys was twice that observed in females
(1252 versus 577 nmol eq/g wet weight,/? < 0.05). The peak tissue concentrations in the liver
and plasma were similar between males (1000 and 403 nmol eq/g wet weight, respectively) and
females (1163 and 317 nmol eq/g wet weight, respectively). In addition, in male rats, the kidney,
liver, and plasma concentrations of radiolabeled 2,2,4-trimethylpentane remained constant for 12
to 24 hours after dosing, whereas in female rats they declined rapidly 8 hours after dosing.
3.3. METABOLISM
3.3.1. Following Oral Administration
Charbonneau et al. (1987) proposed a metabolic pathway for 2,2,4-trimethylpentane
(Figure 3-1). This proposed pathway is based on urinary metabolites and oxidative reactions
common to hydrocarbons that are dependent on whether 2,2,4-trimethylpentane undergoes
oxidation at carbon 1, 4, or 5. In this study, male and female F344 rats were treated with a single
oral dose of [14C]-2,2,4-trimethylpentane (500 mg/kg; 2 uCi/mmol). Levels of radiolabeled
material in kidney, liver, and plasma were determined at 4, 8, 12, 24, and 48 hours after dosing.
Maximum concentrations of 2,2,4-trimethylpentane-derived radioactivity in kidney, liver, and
plasma of male rats were found after 12 hours (143, 114, and 46 mg eq/kg, respectively),
whereas maximum concentrations in females were found after 8 hours (66, 133, and 36 mg
eq/kg, respectively). The identification and quantitation of the urinary metabolites of
2,2,4-trimethylpentane showed that both male and female rats metabolized
2,2,4-trimethylpentane via the same pathway and at a similar rate. Female rats, however,
excreted more conjugates of 2,4,4-trimethyl-2-pentanol in urine than males. 2,4,4-Trimethyl-2-
pentanol was the major metabolite present in the male rat kidney but was absent from the female
rat kidney. In male F344 rats, renal a2u-globulin levels increased to 1.8 and 3.1 times control
values at 24 hours after 2,2,4-trimethylpentane treatments of 50 and 500 mg/kg (0.44 and
4.4 mmol/kg), respectively. The study also reported that two metabolites, 2,2,4-trimethyl-2-
pentanol and 2,4,4-trimethyl pentanoic acid, were detected in kidney homogenates of male, but
not female, rats at 8 to 24 hours after an oral dose of 500 mg/kg 2,2,4-trimethylpentane.
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CH,
CH,
CH,
X I \
1
HOH,C
4
CHi
H3C
2»2.4-trimethyi-1-pentan0i 2,4,4-trimethyl-2-pentanol
OHC
CH,
OH CH5OH
HOOC
'CH,
2,2,4-trimethyH-pentanoic acid
H3C'
CH2OH
2,4,4-trimethyi-1 -pentanoi
I
H3C
/K/\
CHO
CHO H3c' - -COOH
2,4,4-trimethyt-1 -pentanoic acid
HOOC"
'CH,OH
COOH
HOH2C
COOH
2,2,4-trifnethyl-5-hydroxy-
1-pentanoic acid
2,4,4-trirnethyl-2-hydroxyl-1
pentanoic acid
2,4,4-frimethyl-5-hydroxy-
1-pentanoic acid
Figure 3-1. Proposed pathway for 2,2,4-trimethylpentane metabolism.
*Indicates metabolites identified in male rat urine by Olson et al. (1985).
Source: Charbonneau et al. (1987).
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Olson et al. (1985) identified seven urinary products of 2,2,4-trimethylpentane
metabolism in a gavage study in which 2720 mg/kg 2,2,4-trimethylpentane was administered
every other day for 14 days to eight male F344 rats. Urinary metabolites were identified by
using a methylene chloride extract of the urine analyzed by gas-liquid chromatography, gas
chromatography/mass spectrometry (GC/MS), and thin-layer chromatography. The primary
metabolites identified in this study were trimethyl pentanols (2,2,4-trimethyl-l-pentanol,
2,4,4-trimethyl-l-pentanol, and 2,4,4-trimethyl-2-pentanol), pentanoic acids (2,4,4-trimethyl-l-
pentanoic acid and 2,4,4-trimethyl-2-hydroxyl-l-pentanoic acid), and hydroxypentanoic acids
(2,2,4-trimethyl-5-hydroxy-l-pentanoic acid and 2,4,4-trimethyl-5-hydroxy-l-pentanoic acid),
as indicated by asterisks in Figure 3-1.
3.3.2. Following Inhalation Exposure
Groups of four male F344/N rats were exposed to low (0.79 ppm or 3.7 mg/m3) and high
(385 ppm or 1800 mg/m3) concentrations of 14C-labeled 2,2,4-trimethylpentane via nose-only
inhalation for 2 hours (Dahl, 1989). Urine and feces were collected at 3, 6, 9, 18, 24, 30, 42, 54,
and 66 hours postexposure from rats exposed to each concentration and analyzed for the
presence of metabolites. The majority (73 to 79%) of recovered radioactivity was found in the
urine at each 2,2,4-trimethylpentane exposure concentration, but specific metabolites were not
identified. Elimination primarily by the urinary route continued throughout the 66-hour
postexposure observation period.
3.4. ELIMINATION
3.4.1. Following Oral Administration
Kloss et al. (1986) reported the disposition of radiolabeled 2,2,4-trimethylpentane in
F344 rats. Male and female rats were administered [14C]-2,2,4-trimethylpentane (500 mg/kg, po,
four/group), and expired air, urine, and feces were collected for 72 hours. Animals were
sacrificed after 72 hours and selected tissues were removed for radionuclide analysis. The total
recovered radioactivity exceeded 100% for both males (115%) and females (104%); the majority
of the radioactivity was recovered from urine (67% for males, 50% for females) and as expired
organic material (43% for males, 49% for females). A small amount (3.5% in males, 5% in
females) was recovered from the feces.
3.4.2. Following Inhalation Exposure
Groups of four male F344/N rats were exposed to low (0.79 ppm or 3.7 mg/m3) and high
(385 ppm or 1800 mg/m3) concentrations of [14C]-2,2,4-trimethylpentane via nose-only
inhalation for 2 hours (Dahl, 1989). Approximately 90% of the total amount of
2,2,4-trimethylpentane absorbed via inhalation was eliminated by 70 hours postexposure,
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approximately 60 to 70% of which was excreted via the urine at each exposure concentration.
Less than 10% was eliminated via feces or exhaled as carbon dioxide.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
No physiologically based pharmacokinetic or toxicokinetic models have been reported in
the literature for 2,2,4-trimethylpentane.
Beliveau and Krishnan (2000) used a water and lipid (oil) matrix as blood component
surrogates to estimate and compare rat blood:air PCs of some volatile organic chemicals (VOCs).
For 2,2,4-trimethylpentane, vial equilibration studies showed that the matrix:air PC for the
oil+water samples (mean, 2.88; SE, 0.5; n = 7) was similar to that of rat blood (mean, 1.92; SE,
0.4; n = 7). The authors concluded that, for some VOCs, knowledge of solubility in blood
components may be sufficient to determine the blood:air PC.
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS,
CLINICAL CONTROLS
No epidemiological studies, case reports, or clinical studies of 2,2,4-trimethylpentane in
humans were identified.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
4.2.1. Noncancer Toxicity
4.2.1.1. Oral Studies
No subchronic or chronic oral studies were identified for 2,2,4-trimethylpentane.
However, there have been a number of acute and short-term oral studies investigating the effects
of 2,2,4-trimethylpentane on the liver and kidney (see Section 4.4.1 for a discussion of these
studies).
4.2.1.2. Inhalation Studies
No chronic inhalation studies were identified for 2,2,4-trimethylpentane. Several acute
inhalation studies for 2,2,4-trimethylpentane were identified in the literature (see Section 4.4.1).
Only one subchronic inhalation study was identified.
Short et al. (1989a) exposed male and female F344 rats for 6 hours/day, 5 days/week to 0
(control) or 50 ppm (234 mg/m3) 2,2,4-trimethylpentane via inhalation to characterize the
pathogenesis of a,2u-globulin nephropathy. Body weight was the only other endpoint evaluated.
Male rats (three/group) were exposed for 3,10, 22, or 48 weeks and female rats (three/group)
were exposed for 3, 11, 23, or 50 weeks. The presence of a,2u-globulin in renal tissues was
measured by immunohistochemistry at each time point. Cell proliferation was determined by
measuring incorporation of thymidine administered during the last exposure week of each
exposure period. No significant differences were noted in body weights or in absolute or relative
kidney weights in male or female rats at any time point with or without treatment. A significant
increase in detectable a,2u-globulin in exposed males, but not females, was observed after
3 weeks of exposure, compared with controls (p < 0.05). This effect persisted throughout each
of the exposure periods and did not vary with time. In addition, the subcellular localization of
a2u-globulin corresponded with hyaline droplets in serial sections. Examination of the P2
segment of the proximal tubule of the kidney showed an increase of up to 11-fold in cell turnover
in male rats, starting at 3 weeks and persisting throughout the study. This proliferative response
closely paralleled the extent and severity of immunohistochemically detectable a,2u-globulin in
the ?2 segment. Neither cytotoxicity nor a,2U-globulin were noted in the PI or PS segments.
10
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Increased numbers of proximal tubules affected by chronic progressive nephrosis (CPN) were
noted in male rats exposed to 2,2,4-trimethylpentane for 22 (7.7 CPN foci/section) or
44 weeks (12.3 CPN foci/section), compared with controls (0.4 and 5.0 CPN foci/section,
respectively). These lesions contained highly proliferative epithelial cells. Control and exposed
female rats exhibited no evidence of a,2u-globulin-related nephropathy, increases in cell turnover,
or chronic nephrosis. In both control and exposed females, the number of CPN foci per kidney
section was 0.3 at 23 weeks and 1.3 at 50 weeks.
4.2.2. Cancer Bioassays
No cancer bioassays specific for 2,2,4-trimethylpentane were identified in the literature.
4.2.2.1. Initiation and Promotion Studies
Short et al. (1989b) used an initiation-promotion model to evaluate the potential
promoting and carcinogenic effects of both unleaded gasoline (UG) and 2,2,4-trimethylpentane.
In the promotion study, 28 to 30 rats/sex/concentration were exposed to 0, 10, 70, or 300 ppm
UG or 50 ppm (234 mg/m3) 2,2,4-trimethylpentane for 6 hours/day, 5 days/week by inhalation
for 59 to 61 weeks either alone (a promotion control group) or subsequent to a 2-week drinking
water exposure to an initiator, N-ethyl-N-hydroxyethylnitrosoamine (experimental
initiation/promotion group). In a shorter-term sequence reversal study, male rats only were
exposed to UG or 2,2,4-trimethylpentane for 24 weeks, followed by initiator exposure under the
same exposure conditions described above. Surviving animals for both the longer- and shorter-
term studies were sacrificed at 65 to 67 weeks after the start of the experiments, and the kidneys
were examined for incidence of atypical cell foci (ACF) or renal cell tumors (RCTs). No other
endpoints were evaluated. No RCTs were observed in the 2,2,4-trimethylpentane promotion
control group of either gender, whereas, in the initiation/promotion group, males had an
increased incidence of RCTs (4/29) compared with the initiator-only control group (1/29), while
females were not affected (1/29 in the initiator control group and 1/30 in the initiation/promotion
group). The incidence (4/29) observed in males was approximately double the 2/27 observed in
the highest exposure initiation/UG group (300 ppm). The sequence reversal study also showed
promoter-like results in males for both 2,2,4-trimethylpentane and UG in that the percentage of
rats affected with ACF was greater than that of concomitant controls. The incidence of ACF was
not elevated over that of controls in females in any experimental group. These results indicate
that, under the exposure conditions employed, UG and 2,2,4-trimethylpentane are both tumor
promoters in male, but not female, rats. Information on the relative contribution of
2,2,4-trimethylpentane to the UG response could not be determined from this study.
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4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
No reproductive or developmental studies were identified for 2,2,4-trimethylpentane.
4.4. OTHER STUDIES
4.4.1. Acute and Short-Term Studies
4.4.1.1. Oral
A number of acute and short-term oral studies investigating the effects of
2,2,4-trimethylpentane on the liver and kidney were found. Most of these studies involved oral
gavage administration of the chemical to rats at one or two doses for study durations ranging
from 1 day to 4 weeks, with subsequent examination of hepatic and renal tissues and renal
function.
In a 4-week study (API, 1985), male F344 rats were administered 2,2,4-trimethylpentane
via gavage in corn oil at a dose of 0.5 g/kg-day for 5 days/week. Controls were administered
corn oil. Animals were evaluated after either 2 or 4 weeks of exposure. After 2 weeks of
exposure, a significant decrease in glomerular filtration rate (GFR) as measured by inulin
clearance was seen in the treated animals compared with controls (0.60 ±0.13 versus
0.79 ± 0.21 mL/minute/100 g body weight,/? = 0.02). The reduction in GFR progressed, being
more significant at 4 weeks than at 2 weeks (0.41 ± 0.15 versus 0.94 ± 0.30 mL/minute/100 g
body weight, p = 0.001). Associated with this decline in GFR was a significant increase in the
urinary levels of the enzyme N-acetyl-p-D-glucosaminidase (NAG) in the treated animals
compared with controls at both 2 weeks (271 mU/24 hours [treated] versus 115 mU/24 hours
[controls], p < 0.05) and 4 weeks (231 mU/24 hours [treated] versus 139 mU/24 hours [controls],
p<0.05).
In another 4-week study (API, 1983), male F344 rats (10/group) were administered either
0.5 g/kg or 2.0 g/kg 2,2,4-trimethylpentane by gavage on 5 days/week. After sacrifice, the
kidneys were embedded in paraffin, sectioned, stained, and examined for histopathologic
evidence of hydrocarbon nephropathy, based on a grading criterion of characteristic lesions
(hyaline droplet change, regenerative epithelium, and tubular dilatation with granular material).
The three lesions were graded separately for each animal on a severity score of 1 to 4 and added
together, and the total scores from individual animals were averaged across each treatment
group. The average graded nephropathy severity scores were 7.6 (for the 0.5 g/kg dose group)
and 8.5 (for the 2.0 g/kg dose group) compared with a score of 2.9 for the saline-treated controls.
The scores in the treated groups were considered to be indicative of hydrocarbon nephropathy.
In a study by Short et al. (1986), groups of five male F344 rats were administered
0 (control), 50, 100, 200, or 500 mg/kg 2,2,4-trimethylpentane via gavage for 21 days, followed
by light microscopic characterization of renal lesions, as well as localization and quantitation of
sites of renal cell proliferation. At animal sacrifice on day 22, the livers and kidneys were
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sectioned, stained, and examined. Cell proliferation was measured by thymidine incorporation.
Upon examination, lesions in the proximal convoluted tubule of the kidney were found to consist
of accumulated protein droplets and crystalloid bodies, degeneration, necrosis, and foci of
regenerative epithelium, similar to lesions noted to be induced by other hydrocarbon compounds.
In the cell proliferation studies, the most notable increase (five- to sixfold) in the labeling index
was observed in the same portion of the ?2 segment of the proximal convoluted tubule that
contained severe crystalloid body accumulation, degeneration, and necrosis. No dose-response
relationships were noted for the various effects, except for the degree of granular cast formation
in the thin limb segments, which was less in the 50 mg/kg group, compared with the three other
dose groups. No histological changes were seen in the liver.
The effect of 2,2,4-trimethylpentane administration on a,2u-globulin accumulation in rat
kidneys was examined by Saito et al. (1992). Male Sprague-Dawley rats were treated with
50 mg/kg 2,2,4-trimethylpentane by gavage for 14 consecutive days. Control animals were
administered corn oil. In treated animals, there was a marked increase (approximately 450%
compared with controls) in the intensity of a protein band corresponding to a,2u-globulin as
measured by gel electrophoresis and immunoblot analysis. Band intensity for a,2u-globulin was
further increased in animals treated additionally with the cysteine protease inhibitors leupeptin
and E-64, indicating that proteases play an important role in the degradation of a,2u-globulin in
the male rat kidney.
Borghoff et al. (1992) administered 0.95, 3, 6, or 30 mg/kg 2,2,4-trimethylpentane by
gavage for 10 consecutive days to male F344 rats (five/group) to examine a,2u-globulin
nephropathy and renal cell proliferation. Control animals were administered corn oil. The
presence of a,2u-globulin was determined by immunohistochemistry, and cell proliferation was
measured by thymidine incorporation. Twenty-four hours after the final dose, protein droplet
accumulation, a,2u-globulin concentration, and cell replication were measured in the kidneys of
control and treated rats. Dose-related increases in protein droplet accumulation, a,2u-globulin
concentration, and cell proliferation were detected in the kidneys of rats exposed to
2,2,4-trimethylpentane. An approximate twofold increase in each of these effects was noted at
the highest dose tested, and the changes were found to be localized extensively in the ?2
segment. The magnitude of these effects was similar to that induced by exposure to UG. No
significant changes in the kidney-to-body weight ratios of the exposed animals were reported.
Lock et al. (1987a) administered 12 mmol/kg (-1370 mg/kg) 2,2,4-trimethylpentane by
gavage for 10 consecutive days to male and female Alderley Park rats (five/sex). Control
animals were administered corn oil. Significant increases (p < 0.05) in liver weight and liver-to-
body weight ratios in both treated males and females compared with controls and a small but
significant increase in kidney weight and kidney-to-body weight ratios in treated males only
were reported. The authors associated these effects with various findings, including selective
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cytochrome P-450 induction, oxidation of fatty acids, and proliferation of peroxisomes. In
treated males, no change in hepatic cytochrome P-450 or cytochrome bs content was seen,
although renal cytochrome P-450 was significantly elevated compared with controls (p < 0.05).
In treated females, increases in hepatic cytochrome P-450 and cytochrome bs contents were seen,
but no change in renal cytochrome P-450 was noted. In addition, 2,2,4-trimethylpentane induced
focal necrosis of the proximal tubules and a marked increase in the renal content of a2u-globulin
in male, but not female, rats.
The mechanism of uptake of a,2u-globulin in kidney lysosomes was investigated by
Cuervo et al. (1999). Adult male Wistar rats were administered 2,2,4-trimethylpentane (1 g/kg)
or corn oil vehicle via gavage for 7 consecutive days. After administration of the last dose,
animals were fasted for 20 hours prior to sacrifice. Intact kidney lysosomes were isolated from
treated and untreated rats, and their ability to take up a,2u-globulin was compared. In rats exposed
to 2,2,4-trimethylpentane, the specific lysosomal transport of a,2u-globulin was found to increase.
This direct transport of a,2u-globulin into lysosomes occurs in the presence of heat shock cognate
protein 73 (hsc73). These results suggest that the chemically induced accumulation of cytosolic
o,2u-globulin in lysosomes is mediated by an increased rate of direct uptake into lysosomes.
Dietrich and Swenberg (1991) investigated the hypothesis that the presence of
a,2U-globulin is essential for development of nephropathy in male rats exposed to
2,2,4-trimethylpentane by comparing the responses observed in male NCI-black-Reiter (NBR)
rats and male and female F344 rats. In other studies, NBR rats have been shown to not
synthesize a,2u-globulin or develop renal disease when exposed to decalin, a compound known to
induce a,2u-globulin nephropathy in other rat strains. The induction of a,2u-globulin nephropathy
in F344 male rats with lindane was used as a positive control, and this response was contrasted to
male NBR and female F344 rats treated with lindane. Five to seven 11-week-old male NBR rats
were treated with TMP (500 mg/kg-day) or lindane (10 mg/kg-day), and five 11-week-old male
and female F344 rats were treated with lindane (10 mg/kg-day) by oral gavage on 4 consecutive
days. NBR male and male and female F344 rats gavaged with corn oil were used as vehicle
controls. Perfusion-fixed kidneys were histopathologically analyzed for the presence of hyaline
droplets, and the presence of a,2u-globulin was determined immunohistochemically. Under
exposure conditions that clearly induce a,2u-globulin nephropathy in male F344 rats, no lesions,
hyaline droplets, or a,2u-globulin were detectable in TMP-treated or control male NBR and
female F344 rats.
A marked increase in urinary a,2u-globulin was noted in a study in which male Sprague-
Dawley rats received 171 mg/kg 2,2,4-trimethylpentane by gavage for 7 consecutive days (Saito
et al., 1996). This increase in urinary a,2u-globulin levels (measured on days 0, 1, 3, 5, and 7)
was associated with increased concentrations of renal o,2U-globulin and hyaline droplet
accumulation in renal proximal convoluted tubule epithelial cells.
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To study renal a,2u-globulin accumulation, male and female F344 rats (five/sex) were
orally administered 500 mg/kg 2,2,4-trimethylpentane per day for 5 days (Blumbach et al.,
2000). Control rats received corn oil alone. Increases in o,2U-globulin content and relative
kidney weight were observed in male, but not female, rats. In treated male rats, a,2u-globulin
accounted for 70% of the total renal cytosolic protein. This increase in a2u-globulin
accumulation was accompanied by the formation of protein droplets in the P2 segment of the
proximal tubules.
In a study to examine the localization of a,2u-globulin within protein droplets of the
kidney, four male F344 rats were gavaged once with 50 mg/kg 2,2,4-trimethylpentane (Burnett
et al., 1989). Male and female controls were administered corn oil. For analysis, the renal
tissues were embedded, sectioned, and stained, and the presence of a,2u-globulin was determined
by immunohistochemistry. Image analysis of selected P2 segments in treated and control rats
72 hours after treatment revealed a high correlation between subcellular localization of
o,2u-globulin and protein droplet deposition in the cytoplasm of P2 segment cells. Microscopic
evaluation also revealed subcellular localization of a,2u-globulin within lysosomes of P2 segment
cells. Quantitative morphometry of proximal tubule epithelium stained for a,2u-globulin
demonstrated a 1.5- to 2-fold increase in staining area of tubules from treated rats compared with
controls. Similar increases in protein droplets were also observed.
In another study by Lock et al. (1987b), male and female F344 rats were dosed by gavage
with a single dose of [3H]-2,2,4-trimethylpentane (4.4 mmol/kg or -500 mg/kg). The
concentration of the radiolabeled material in various subcellular fractions of the kidney and the
presence of a,2u-globulin were examined 24 and 72 hours after dosing by using column
chromatography and immunohistochemistry. The kidneys from male rats were observed to
contain more radiolabeled material compared with female rats at both time points. In addition, it
was observed that covalent binding of 2,2,4-trimethylpentane to kidney proteins did not occur.
However, GC/MS analysis and dialysis with and without sodium dodecyl sulfate of the low
molecular weight protein fraction from male rat kidneys showed reversible binding between a
metabolite of 2,2,4-trimethylpentane, identified as 2,4,4-trimethyl-2-pentanol, and o,2U-globulin.
This was the only metabolite detected to be bound to o,2U-globulin in the kidney.
Hyaline droplet accumulation was stimulated in the kidney of postpubertal male Alderley
Park rats 24 to 48 hours after a single oral dose of 12 or 24 mmol/kg (approximately 1370 or
2740 mg/kg) 2,2,4-trimethylpentane but had returned to normal after 7 days. No such effect was
observed in female or prepubertal male animals (Stonard et al., 1986). A dose-dependent
increase in the renal concentration of a,2u-globulin was also observed in postpubertal male rats
24 hours after single oral doses of 2,2,4-trimethylpentane over a range of 0.3 to 12 mmol/kg. In
male rats treated with 12 mmol/kg, a,2u-globulin staining in the ?2 segment was greater compared
with controls. No changes in urine volume, specific gravity, glucose, or NAG levels were
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observed in treated rats, indicating that renal tubular function was not impaired. None of the
effects noted in males were observed in female rats exposed to the same treatment regimen.
Carruthers et al. (1987) employed a similar study paradigm, wherein a single oral gavage dose
(2,2,4-trimethylpentane, 12 mmol/kg in corn oil, or vehicle alone) was administered to male CD
rats (180-250 g, eight/treatment group). Liver and kidney tissues were harvested, and plasma and
urine were collected 24 hours later. A significant increase in the level of a,2u-globulin in renal
tissues, but not in liver, plasma, or urine, was found. Cycloheximide pretreatment significantly
inhibited the accumulation of a,2u-globulin in the kidney in response to 2,2,4-trimethylpentane
treatment (2.2 versus 24.0 mg/g kidney, respectively), which suggests that at least part of the
increased accumulation in the kidney may be due to increased synthesis of a,2u-globulin in the
liver. However, Carruthers et al. (1987) described the results of a single experiment in a small
number of animals, and, thus, it is unclear how much significance can be attributed to the
reported findings.
Fowlie et al. (1987) exposed nine male Wistar rats to 2 mL/kg (-1.4 g/kg)
2,2,4-trimethylpentane by gavage for 2 to 3 days and examined liver and kidney effects. The
animals were examined on each day of exposure for changes in body weight, food and water
consumption, and urinary parameters. There were significant decreases in food and water
consumption and considerable weight loss in treated animals by day 2 that led to an early
cessation of the experiment. In the urine of treated rats, NAG activity was significantly
increased (p < 0.01) after day 2 and alkaline phosphatase activity was significantly increased on
day 1 (p < 0.01) and on day 2 (p < 0.05) compared with control animals. Urinary creatinine
levels were significantly decreased (p < 0.001) after day 1 compared with controls. These
changes in urinary parameters are consistent with renal toxicity. Six rats were sacrificed on the
second day and the remainder on the third day. At necropsy, there was a significant increase
(p < 0.001) in the relative weights of both the liver and kidneys of treated animals compared with
controls. Microscopic examination of the liver demonstrated centrilobular necrosis and
hydrophobic degeneration of hepatocytes. Microscopic examination of the kidneys revealed
hyaline droplet accumulation in the proximal tubules as well as tubule degeneration and
dilatation.
In a study by Loury et al. (1986), replicative DNA synthesis was significantly increased
(p < 0.05) in hepatocytes isolated from male F344 rats and from male and female B6C3Fi mice
treated with 500 mg 2,2,4-trimethylpentane/kg by gavage. This effect was seen in rats at
24 hours after exposure but not at shorter (2 and 12 hours) or longer (48 hours) times
postexposure. Mice were sampled only at 24 hours postexposure, and no other effects were
measured or reported in mice. The 500 mg/kg dose was based on pilot experiments that showed
this dose caused a maximum accumulation of hyaline droplets in the rat kidney. A significant
increase in mg DNA/gram liver but not in total liver DNA or liver weight was reported in rats
following administration of 2,2,4-trimethylpentane (100 mg/kg) by gavage for 11 days.
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Similar results were observed in mice in a study conducted by Standeven and
Goldsworthy (1994). Five to six female B6C3Fi mice were treated with 1000 mg/kg-day
2,2,4-trimethylpentane by intragastric intubation for 3 days (days 2-4) after implantation of a
bromodeoxyuridine osmotic pump on day 1. Control animals were administered 5 mL/kg corn
oil vehicle. Animals were sacrificed on day 5. Treatment with 2,2,4-trimethylpentane produced
a significant increase in the hepatocyte-labeling index and an increase in relative liver weight,
indicating a mitogenic effect on the liver. No changes in serum alanine aminotransferase (ALT)
or sorbitol dehydrogenase (SDH) activities were noted.
4.4.1.2. Inhalation
Swann et al. (1974) exposed Swiss mice (four/group, sex not reported) to
2,2,4-trimethylpentane for 5 minutes via inhalation at concentrations of 1000, 2000, 4000, 8000,
16,000, 32,000, 64,000, and 128,000 ppm (approximately 4700 to 600,000 mg/m3). At
16,000 ppm, sensory and motor irritation were observed throughout the exposure, and 1/4 of the
mice had sudden respiratory arrest. At 32,000 ppm, all of the mice stopped breathing within
4 minutes of the onset of exposure. No apparent anesthesia was noted at any exposure
concentration.
In another inhalation study (Exxon, 1987), male and female Sprague-Dawley rats, CD-I
mice, and Hartley guinea pigs (10/group) were exposed to 39,630 mg/m3 (8322 ppm)
2,2,4-trimethylpentane for up to 4 hours. No abnormal signs were seen in the rats after
15 minutes of exposure; however, after 20 minutes, convulsions were seen in most animals and
two rats died within 30 minutes of the onset of exposure. Convulsions, excessive lacrimation
and salivation, and labored breathing were reported in the surviving rats, and all rats were dead
within 55 minutes of the start of the exposure. In the mice, one animal died after 20 minutes and
all the mice were dead within 75 minutes of exposure. Death occurred in 8/10 guinea pigs
between 60 and 120 minutes of the onset of exposure. At necropsy, lung discoloration was
observed in all of the animals, and liver and kidney discoloration was observed in 2/10 of the
animals.
In a study of the potential toxicity of volatile emissions from indoor carpeting,
Stadler and Kennedy (1996) evaluated the sensory irritation potential of a number of VOCs that
were identified in carpet emissions, including 2,2,4-trimethylpentane. (The authors indicate
2,2,4-trimethylpentane to be known by the U.S. Environmental Protection Agency (EPA) to be
frequently detectable in carpet volatiles but do not otherwise elaborate on the specific reason for
its being present in carpets.) Toxicity was assessed by measuring the airborne concentration
required to elicit a 50% depression in respiratory rate (RD50) in Swiss-Webster mice, both for
carpet emission mixtures and for pure chemical vapors. The mice were first exposed to a
chemical vapor mixture emitted from a heated carpet sample for two exposures per day (each
exposure 1 hour in duration) for 2 days. Total VOCs emitted were analyzed, and general signs
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of toxicity, respiratory rate decreases, and breathing patterns of respiratory irritation in the mice
were noted. In a second set of experiments, 2,2,4-trimethylpentane was one of 11 identified
VOCs that were tested as pure chemical vapors (single 30-minute exposures).
2,2,4-Trimethylpentane, along with four other VOCs, exhibited RDso values that were each
greater than 1000 ppm, indicating that these VOCs are nonirritating chemicals when present
alone.
4.4.1.3. Dermal
In a dermal study (Exxon, 1987), doses of 0.2 g/kg and 3.15 g/kg 2,2,4-trimethylpentane
were applied to the abdominal area of New Zealand white (NZW) rabbits (four/group) for
24 hours with no mortality reported. At necropsy, in the low-dose group, one animal appeared to
be normal, three had dark livers, and two had mottled livers. In the high-dose group, four
animals had dark livers, two animals had mottled livers, and one had a pale kidney.
4.4.1.4. Ocular
Exxon (1987) also conducted an eye irritation study in NZW rabbits.
2,2,4-Trimethylpentane (0.1 mL, or -70 mg) was instilled into the conjunctival sac of one eye of
six rabbits. The ocular reactions were graded at 1 and 4 hours and at 1, 2, 3, 4, and 7 days after
instillation. The results showed that 2,2,4-trimethylpentane was nonirritating to the eye.
4.4.2. Genotoxicity
There are a few reports on testing of 2,2,4-trimethylpentane for genetic toxicity.
There are no available reports of testing for mutagenic activity in bacterial cells (e.g., the Ames
salmonella test) or for chromosome breaking activity in vitro or in vivo.
4.4.2.1. Mutation and Chromosome Effects
A human lymphoblastoid cell line, TK6, was treated with a saturated (5% v/v) solution of
2,2,4-trimethylpentane in cell culture medium for 3 hours in the presence and absence of rat liver
S9 fraction (Richardson et al., 1986). There were no detected increases in gene mutations at the
thymidine kinase (TK) locus or in sister chromatid exchanges (SCEs).
4.4.2.2. DNA Damage
McLaren et al. (1994) investigated the induction of DNA double-strand breaks and poly-
ADP-ribosylation in the renal cortex of male Wistar rats administered 12 mmol/kg
(-1370 mg/kg) of 2,2,4-trimethylpentane via gavage for 5 consecutive days. Treatment failed to
induce poly-ADP-ribosylation or a significant increase in DNA double-strand breaks in the renal
cortex. Unscheduled DNA synthesis (UDS) was not induced in isolated male F344 hepatocytes
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exposed to 2,2,4-trimethylpentane at final media concentrations of 0.33, 1.00, or 3.33% (high
dose may be cytotoxic) (Loury et al., 1986).
4.4.3. Cytotoxicity
Two- to three-day HeLa cell cultures containing 0.1 to 7.5% 2,2,4-trimethylpentane did
not exhibit altered cell growth or changes in viability or adenosine triphosphate/ADP content
(Forman et al., 1999). However, Loury et al. (1986) observed cytotoxicity in isolated male rat
hepatocytes exposed to a media concentration of 3.33% 2,2,4-trimethylpentane.
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS
4.5.1. Oral
A number of acute and short-term studies were identified in the literature. Overall, these
studies provide focused or limited information, as either they were designed to investigate only
endpoints specific to a,2u-globulin-associated nephropathy in male rats or found no other
significant 2,2,4-trimethylpentane-induced effects. The majority of noncancer effects induced by
2,2,4-trimethylpentane exposure were found to occur primarily in the kidneys of male rats, as the
majority of the studies examined only the kidney. The effects reported included altered renal
function, an increase in a,2u-globulin protein and hyaline droplet accumulation in the proximal
tubules of male rats, necrosis of the tubule epithelium, increased cell turnover, and foci of
regenerative epithelium (Blumbach et al., 2000; Saito et al., 1996, 1992; Borghoff et al., 1992;
Burnett et al., 1989; Lock et al., 1987a,b; Short et al., 1986; Stonard et al., 1986; API, 1985,
1983). No increases in a,2u-globulin protein and hyaline droplet accumulation in the proximal
tubules or in necrosis of the tubule epithelium were noted to occur in female rats (Blumbach
et al., 2000; Lock et al., 1987a,b).
Of the studies with sufficient 2,2,4-trimethylpentane-specific dose-effect information (see
Section 4.4.1), only two studies reported effects in organs other than kidney: Fowlie et al. (1987)
observed centrilobular necrosis and hydrophobic degeneration of hepatocytes induced by
2,2,4-trimethylpentane, and Lock et al. (1987a) observed increases in liver weight and liver-to-
body weight ratios in both treated males and females. The effects noted by Lock et al. (1987a)
were thought to result from an induction in cytochrome P-450 and peroxisome proliferation.
However, Short et al. (1986) found no significant histological changes in the liver.
4.5.2. Inhalation
Only one subchronic inhalation study was identified for 2,2,4-trimethylpentane
(Short et al., 1989a). In this study, male and female F344 rats were exposed for 3 to 50 weeks to
50 ppm (234 mg/m3) 2,2,4-trimethylpentane to characterize the pathogenesis of a,2u-globulin
nephropathy. Body weight was the only other endpoint to be evaluated. As observed in the oral
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studies, the notable effects in this study were limited to the male rat kidney and consisted of an
increase in a,2u-globulin protein and hyaline droplet accumulation in the ?2 segment of the
proximal tubules, necrosis of the tubule epithelium, sustained regenerative tubule cell
proliferation, and enhancement of CPN in male rats. Control and exposed female rats exhibited
no evidence of a2u-globulin-nephropathy, increases in cell turnover, or chronic nephrosis.
Otherwise, only three other inhalation studies were identified (Stadler and Kennedy,
1996; Exxon, 1987; Swann et al., 1974), and, as discussed above, these studies were designed to
assess only acute toxicity endpoints. Results from such studies are not amenable to use in the
development of chronic inhalation RfC values.
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER CHARACTERIZATION
4.6.1. Summary of Overall Weight of Evidence
In accordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a),
there is "inadequate information to assess carcinogenic potential" for 2,2,4-trimethylpentane.
No epidemiological studies in humans and no chronic bioassay studies are available that assess
the carcinogenic effects of 2,2,4-trimethylpentane. The majority of the reported studies
contribute information specifically related to the histopathological sequence of a,2u-globulin-
associated nephropathy. Thus, these studies did not examine any other tissues/organs except the
kidney. In comparing the tumor-promoting capability between 2,2,4-trimethylpentane and UG
(a mixture), Short et al. (1989b) showed that both agents had promoting potential in male but not
female rats. However, the results were not sufficiently descriptive to ascribe the portion of the
promoting potential of UG that could be attributable to 2,2,4-trimethylpentane. The few studies
available on its genotoxic potential were negative, as 2,2,4-trimethylpentane did not increase
mutations at the TK locus (Richardson et al., 1986), induce DNA double-strand breaks (McLaren
et al., 1994), or stimulate UDS (Loury et al., 1986).
4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence
No other studies or supportive information are available on the carcinogenic effects of
2,2,4-trimethylpentane.
4.7. MODE-OF-ACTION INFORMATION
A limited number of studies evaluating the effects of exposure to 2,2,4-trimethylpentane
by the oral or inhalation routes have been conducted, and no human studies are available. The
majority of these studies have been designed to specifically address and characterize the
involvement of a,2u-globulin in the renal toxicity observed in the male rat, with little or no
evaluation of other endpoints in tissues/organs other than the kidney or of other potential modes
of action.
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4.7.1. General Issues Concerning the Determination of ttiu-Globulin-Associated
Nephropathy
o,2u-Globulin is a member of a large superfamily of low-molecular-weight proteins and
was first characterized in male rat urine. It has been detected in various tissues and fluids of
most mammals, including humans. However, the particular isoform of a,2u-globulin commonly
detected in male rat urine appears to be largely specific for the male rat; moreover, the urine and
kidney concentrations detected in the mature male rat are several orders of magnitude greater
than in any other age, sex, or species tested (U.S. EPA, 1991).
The mode of action ascribed to a,2u-globulin-associated nephropathy is defined by a
progressive sequence of effects in the male rat kidney, often culminating in renal tumors. The
involvement of hyaline droplet accumulation in the early stages of nephropathy associated with
a,2u-globulin-binding chemicals is an important difference from the sequence of events observed
with classical carcinogens. The pathological changes that precede the proliferative sequence for
classical renal carcinogens also include early nephrotoxicity (e.g., cytotoxicity and cellular
necrosis) but no apparent hyaline droplet accumulation. Furthermore, the nephrotoxicity that can
ensue from hyaline droplet accumulation is novel because it is associated with excessive
o,2u-globulin accumulation. This a,2u-globulin accumulation is proposed to result from reduced
renal catabolism of the a2u-globulin chemical complex and is thought to initiate a sequence of
events leading to chronic proliferation of the renal tubule epithelium, as well as an exacerbation
of CPN. The histopathological sequence in mature male rats consists of the following (see Table
4-1 for a summary of this sequence specific for 2,2,4-trimethylpentane):
• Excessive accumulation of hyaline droplets containing a,2u-globulin in renal proximal
tubules
• Subsequent cytotoxicity and single-cell necrosis of the tubule epithelium
• Sustained regenerative tubule cell proliferation
• Development of intralumenal granular casts from sloughed cellular debris associated with
tubule dilatation and papillary mineralization
• Foci of tubule hyperplasia in the convoluted proximal tubules
• Renal tubule tumors
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Table 4-1. Summary of renal effects specific to male rats reported in 2,2,4-trimethylpentane studies
Study
(route, dose, duration)
Short etal. (1989a)
(inhalation, 50 ppm [234 mg/m3]
6 h/d, 5 d/w, 3-50 w)
API (1983)
(oral, 0.5 or 2.0 g/kg-d, 5 d/w, 4 w)
Short etal. (1986)
(oral, 50-500 mg/kg-d, 21 d)
Saito etal. (1992)
(oral, 50 mg/kg-d, 14 d)
Borghoff etal. (1992)
(oral, 0.95-30 mg/kg-d, 10 d)
Lock etal. (1987a)
(oral, 1370 mg/kg-d, 10 d)
Saito et al. (1996)
(oral, 17 1 mg/kg-d, 7 d)
Blumbach et al. (2000)
(oral, 500 mg/kg-d, 5 d)
Burnett etal. (1989)
(oral, 50 mg/kg, 1 d)
Lock etal. (1987b)
(oral, 500 mg/kg, 1 d)
Stonard etal. (1986)
(oral, 34-2740 mg/kg, 1 d)
Accumulation of
a2u-globulm
hyaline droplets
X
X
X
X
X
X
X
X
X
X
X
Cytotoxicity,
necrosis of tubule
epithelium
X
X
X
Sustained
regenerative tubule
cell proliferation
X
X
X
Intralumenal granular
casts and papillary
mineralization
X
X
Foci of tubule
hyperplasia
X
22
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In addition to this histopathological sequence, EPA (1991) provides more specific
guidance for evaluating chemically induced male rat renal tubule tumors for the purpose of risk
assessment. To determine the appropriateness of the data for use in risk assessment, chemicals
inducing renal tubule tumors in the male rat are examined in terms of three categories:
• The o,2u-globulin sequence of events accounts for the renal tumors.
• Other potential carcinogenic processes account for the renal tumors.
• The o,2u-globulin-associated events occur in the presence of other potential carcinogenic
processes, both of which result in renal tumors.
Therefore, it is important to determine whether the a,2u-globulin process is involved and, if so, to
what extent a,2u-globulin-associated events, rather than other processes, account for the tumor
increase.
Determination of these elements requires a substantial database of bioassay data not only
from male rats but also from female rats and mice, and such toxicity studies must demonstrate
whether or not a,2u-globulin processes are operative. In the absence of minimum information
demonstrating the involvement of a,2u-globulin processes, it should be assumed that any male rat
renal toxicity/tumors are relevant for risk assessment purposes. A technical report available from
EPA (1991) outlines the data necessary to determine the involvement of a,2u-globulin.
As outlined in the EPA Risk Assessment Forum Technical Panel report (U.S. EPA,
1991), the following information from adequately conducted studies of male rats is used for
demonstrating that the a,2u-globulin process may be a factor in any observed renal effects—an
affirmative response in each of the three categories is required. If data are lacking for any of the
criteria in any one category, the available renal toxicity data should be analyzed in accordance
with standard risk assessment principles. The three categories of information and criteria are as
follows:
• Increased number and size of hyaline droplets in the renal proximal tubule cells of
treated male rats. The abnormal accumulation of hyaline droplets in the ?2 segment
helps differentiate a,2u-globulin inducers from chemicals that produce renal tubule tumors
by other modes of action.
• Accumulating protein in the hyaline droplets is a2U-globulin. Hyaline droplet
accumulation is a nonspecific response to protein overload, and, thus, it is necessary to
demonstrate that the protein in the droplet is, in fact, a,2u-globulin.
• Additional aspects of the pathological sequence of lesions associated with a2U-globulin
nephropathy are present. Typical lesions include single-cell necrosis, exfoliation of
epithelial cells into the proximal tubular lumen, formation of granular casts, linear
mineralization of papillary tubules, and tubule hyperplasia. If the response is mild, not
all of these lesions may be observed. However, some elements consistent with the
pathological sequence must be demonstrated to be present. This pathological sequence is
23
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outlined above and in Table 4-1 for effects specifically demonstrated for
2,2,4-trimethylpentane.
4.7.2. 2,2,4-Trimethylpentane and (Xiu-Globiilin-Associated Nephropathy
A number of studies (see Sections 4.2.1.2 and 4.4.1) have demonstrated that oral or
inhalation exposures to 2,2,4-trimethylpentane result in renal effects in male rats in dosing
regimens ranging from a single dose to daily dosing for periods as long as 50 weeks (Blumbach
et al., 2000; Saito et al., 1996, 1992; Borghoff et al., 1992; Burnett et al., 1989; Short et al.,
1989a, 1986; Lock et al., 1987a,b; Stonard et al., 1986; API, 1983). The effects observed in
male rat kidney include an increase in protein in the proximal tubules, an increase in hyaline
droplets, the confirmed presence of a,2u-globulin, necrosis and degeneration of the tubule
epithelium, increased cell turnover/proliferation, foci of regenerative epithelium, and
exacerbation of chronic nephrosis. The significant effects noted in these studies provide
evidence that corresponds to the histopathological sequence for o,2U-globulin-associated
nephropathy in male rats (see Table 4-1), as well as the additional categorical criteria outlined
above. These effects have not been observed in female rats, mice, guinea pigs, dogs, or monkeys
(Alden, 1986). Taken together, this information provides strong evidence that the renal effects
induced by 2,2,4-trimethylpentane result from a mode of action associated with a,2u-globulin
accumulation.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.8.1. Possible Childhood Susceptibility
No studies are available on possible childhood susceptibility to 2,2,4-trimethylpentane.
4.8.2. Possible Gender Differences
No studies are available on possible gender differences in humans regarding
2,2,4-trimethylpentane. Overall, the available animal studies provide focused or limited
information on gender-specific differences, as either they were designed to investigate only
endpoints specific to a,2u-globulin-associated nephropathy in male rats or found no other
significant 2,2,4-trimethylpentane-induced effects (Blumbach et al., 2000; Saito et al., 1996,
1992; Borghoff et al., 1992; Burnett et al., 1989; Short et al., 1989a, 1986; Lock et al., 1987a,b;
Stonard et al., 1986; API, 1983). These a,2u-globulin-associated renal effects have not been
shown to occur in female rats (Blumbach et al., 2000; Lock et al., 1987a,b). These renal effects,
specific to the male rat, are not thought to be relevant to humans. No other studies are available
that indicate gender-specific effects.
24
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
No subchronic or chronic oral studies were identified that demonstrated a dose-response
effect that could be used to determine the noncarcinogenic risk for 2,2,4-trimethylpentane.
A number of acute and short-term studies were identified in the literature. Overall, these
studies provide focused or limited information, as either they were designed to investigate only
endpoints specific to a,2u-globulin-associated nephropathy in male rats or found no other
significant 2,2,4-trimethylpentane-induced effects. The majority of noncancer effects induced by
2,2,4-trimethylpentane exposure were found to occur primarily in the kidney of male rats as the
majority of the studies examined only the kidney. The effects reported include altered renal
function, an increase in a,2u-globulin protein and hyaline droplet accumulation in the proximal
tubules, necrosis of the tubule epithelium, increased cell turnover, and foci of regenerative
epithelium (Blumbach et al., 2000; Saito et al., 1996, 1992; Borghoff et al., 1992; Burnett et al.,
1989; Lock et al., 1987a,b; Short et al., 1986; Stonard et al., 1986; API, 1983). No increases in
o,2u-globulin protein and hyaline droplet accumulation in the proximal tubules or necrosis of the
tubule epithelium were noted to occur in female rats (Blumbach et al., 2000; Lock et al.,
1987a,b).
Detailed studies that contain sufficient dose-response and duration information on
2,2,4-trimethylpentane for endpoints other than nephropathy are currently lacking. Liver effects
were noted in two acute or short-term oral studies. Fowlie et al. (1987) observed centrilobular
necrosis and hydrophobic degeneration of hepatocytes induced by 2,2,4-trimethylpentane and
Lock et al. (1987a) observed increases in liver weight and liver-to-body weight ratios in both
treated males and females. The effects noted by Lock et al. (1987a) were thought to result from
an induction in cytochrome P-450 and peroxisome proliferation. Short et al. (1986) found no
significant histological changes in the liver.
As discussed above (see Sections 4.4.1, 4.5.1, and 4.7), the available studies provide
evidence that the kidney toxicity induced by 2,2,4-trimethylpentane in male rats is related to
o,2u-globulin accumulation in the proximal tubules. Because this response is specific to male
rats, as a matter of science policy, EPA (1991) has concluded that "if a chemical induces
o,2u-globulin accumulation in male rats, the associated nephropathy is not used as an endpoint for
determining noncarcinogenic hazard. Estimates of noncarcinogenic risk are based on other
endpoints." No other studies were considered suitable for the derivation of an RfD. Therefore,
an oral RfD was not derived. The previous IRIS assessment (dated 11/01/1991) did not include
an RfD derivation.
25
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5.2. INHALATION REFERENCE CONCENTRATION (RfC)
No subchronic or chronic inhalation studies were identified that demonstrate a dose-
response effect that could be used to determine the noncarcinogenic risk for
2,2,4-trimethylpentane.
Only one subchronic inhalation study was identified for 2,2,4-trimethylpentane
(Short et al., 1989a). In this study, male and female F344 rats were exposed for 3 to 50 weeks to
50 ppm 2,2,4-trimethylpentane to characterize the pathogenesis of a,2u-globulin-associated
nephropathy. Body weight was the only other endpoint evaluated. As observed in the oral
studies, the notable effects in this study were limited to the male rat kidney and consisted of an
increase in a,2u-globulin protein and hyaline droplet accumulation in the ?2 segment of the
proximal tubules, necrosis of the tubule epithelium, sustained regenerative tubule cell
proliferation, and enhancement of CPN in male rats. Control and exposed female rats exhibited
no evidence of a,2u-globulin-associated nephropathy, increases in cell turnover, or chronic
nephrosis.
In addition, three short-term inhalation studies (Stadler and Kennedy, 1996; Exxon, 1987;
Swann et al., 1974) were identified that addressed only limited endpoints (e.g., lethality and
irritancy) at high-exposure concentrations (>4600 mg/m3) and acute exposure durations (<4 hrs)
and, thus, are of limited value for chronic reference concentration assessment purposes.
As discussed above (see Sections 4.2.2, 4.5.2, and 4.7), the available studies provide
evidence that the kidney toxicity induced by 2,2,4-trimethylpentane in male rats is related to
o,2u-globulin accumulation in the proximal tubules. Because this response is specific to male
rats, as a matter of science policy, EPA (1991) has concluded that "if a chemical induces
a2u-globulin accumulation in male rats, the associated nephropathy is not used as an endpoint for
determining noncarcinogenic hazard. Estimates of noncarcinogenic risk are based on other
endpoints." No other studies were considered suitable for the derivation of the RfC. Therefore,
an inhalation RfC was not derived. The previous IRIS assessment (dated 11/01/1991) did not
include an RfC derivation.
5.3. CANCER ASSESSMENT
No studies are available on the carcinogenic effects of 2,2,4-trimethylpentane on which to
base a cancer assessment. This overall lack of information represents a data gap and does not
allow for a quantitative assessment of the carcinogenicity of 2,2,4-trimethylpentane.
26
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE
6.1. HUMAN HAZARD POTENTIAL
2,2,4-Trimethylpentane is a colorless liquid with the odor of gasoline (NLM, 2004). It is
used primarily in the alkylation step of the reaction of isobutane and butylene in deriving high-
octane fuels. 2,2,4-Trimethylpentane is synthesized from the catalytic hydrogenation of
trimethylpentene with a nickel catalyst (API, 1985). 2,2,4-Trimethylpentane is released to the
environment through the use and disposal of gasoline-associated products, and inhalation appears
to be the major route of exposure (NLM, 2004).
Epidemiological or poisoning case studies of 2,2,4-trimethylpentane in humans are not
available. Exposure of laboratory animals, including mice, rats, and guinea pigs, to high levels
(>8322 ppm) of 2,2,4-trimethylpentane via inhalation has resulted in death. There are few
reliable oral or inhalation studies that have evaluated the toxicity of 2,2,4-trimethylpentane
administered at lower, nonlethal levels in any species other than rats, the database being
dominated by studies in male rats characterizing a species- and gender-specific renal effect that
is not relevant to humans. Toxicity studies in other species and studies examining effects in
other tissues/organs except the kidney are lacking, which is considered a major deficiency in the
hazard identification process for 2,2,4-trimethylpentane.
Limited data are available on the absorption of 2,2,4-trimethylpentane. The available
studies in rats suggest that the chemical has high oral absorption and is distributed to the kidney,
fat, and liver, with higher concentrations detected in the kidneys of males compared with females
(Kloss et al., 1986). Based on the results from the inhalation study conducted by Dahl (1989),
absorption via the respiratory tract may be quite low (7 to 12% of inspired concentration). The
major metabolites identified in rats are trimethyl pentanols, pentanoic acids, and
hydroxypentanoic acids (Olson et al., 1985), with one study (Charbonneau et al., 1987) reporting
two metabolites, 2,2,4-trimethyl-2-pentanol and 2,2,4-trimethyl pentanoic acid, detected in the
liver of male, but not female, rats. Elimination of 2,2,4-trimethylpentane in rats occurs primarily
via the urine (60 to 70%), and less than 10% is eliminated via the feces (Dahl, 1989; Kloss et al.,
1986).
In addition, no subchronic or chronic oral animal studies are available for the chemical.
However, a number of acute and short-term oral studies were identified in the literature. The
majority of noncancer effects induced by 2,2,4-trimethylpentane exposure were found to occur in
the kidney of male rats and included altered renal function, an increase in a,2u-globulin protein
and hyaline droplet accumulation in the proximal tubules, necrosis of the tubule epithelium,
increased cell turnover, and foci of regenerative epithelium (Blumbach et al., 2000; Saito et al.,
1996, 1992; Borghoff et al., 1992; Burnett et al., 1989; Lock et al., 1987a,b; Short et al., 1986;
Stonard et al., 1986; API, 1983). No increases in a,2u-globulin protein and hyaline droplet
27
-------
accumulation in the proximal tubules, or necrosis of the tubule epithelium, were noted to occur
in female rats (Blumbach et al., 2000; Lock et al., 1987a,b). One acute study also reported
effects on the liver (Fowlie et al., 1987).
Chronic inhalation studies for 2,2,4-trimethylpentane are lacking. Only one subchronic
inhalation study was identified for 2,2,4-trimethylpentane (Short et al., 1989a). In this study,
male and female F344 rats were exposed for 3 to 50 weeks to 50 ppm (234 mg/m3)
2,2,4-trimethylpentane. As observed in the oral studies, the notable effects in this study were
limited to the male rat kidney and consisted of an increase in a,2u-globulin protein and hyaline
droplet accumulation in the ?2 segment of the proximal tubules, necrosis of the tubule
epithelium, sustained regenerative tubule cell proliferation, and enhancement of CPN in male
rats. Control and exposed female rats exhibited no evidence of a,2u-globulin nephropathy,
increases in cell turnover, or increase in the extent of chronic nephrosis.
No studies are available on the reproductive or developmental effects of
2,2,4-trimethylpentane. Limited genotoxicity data are available. The few studies available on its
genotoxic potential were negative, as 2,2,4-trimethylpentane did not increase mutations at the
TK locus, did not induce SCEs in a human lymphoblastoid cell line (Richardson et al., 1986) or
DNA double-strand breaks in male rat kidney (McLaren et al., 1994), and did not stimulate UDS
in isolated male rat or mouse hepatocytes (Loury et al., 1986).
One study (U.S. EPA, 1991) has reviewed the available data on a,2u-globulin-associated
nephropathy in rats and has concluded that this endpoint is not relevant to humans and, therefore,
should not be used to determine the noncarcinogenic hazard. In addition, most of the animal
studies of 2,2,4-trimethylpentane were designed to investigate only the renal effects and
o,2u-globulin nephropathy in male rats. Consequently, the limited focus of studies that have been
conducted with 2,2,4-trimethylpentane identifies a need for additional studies that examine a
more comprehensive set of toxicological endpoints.
6.2. DOSE RESPONSE
In the case of 2,2,4-trimethylpentane, there is currently a lack of studies presenting
sufficient dose-response and duration information from which to derive quantitative estimates of
noncancer or cancer risks. As described above, the available information demonstrates that the
renal effects induced by 2,2,4-trimethylpentane are primarily attributable to processes involving
a,2U-globulin. In addition, the available data are concordant with the histopathological sequence
and criteria outlined in the EPA (1991) guidance developed to address the issues concerning
o,2U-globulin-associated renal toxicity (nephropathy) and neoplasia. Consequently, in agreement
with EPA science policy, no quantitative estimates of noncancer risk were developed for
2,2,4-trimethylpentane.
28
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7. REFERENCES
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using reconstituted mixtures of blood components. Toxicol Lett 116:183-188.
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ethyl- and dimethyl methylphosphonate. Toxicol Sci 53:24-32.
Borghoff, SJ; Youtsey, NL; Swenberg, JA. (1992) A comparison of European high test gasoline and PS-6 unleaded
gasoline in their abilities to induce alpha2u-globulin nephropathy and renal cell proliferation. Toxicol Lett 63:21-33.
Burnett, VL; Short, BG; Swenberg, JA. (1989) Localization of alpha2u-globulin within protein droplets of male rat
kidney immunohistochemistry using perfusion-fixed GMA-embedded tissue sections. J Histochem Cytochem
37:813-818.
Carruthers, L; Reeves, K; Moses, P; et al. (1987) The role of a2u globulin synthesis in the production of renal hyaline
droplets by iso-octane. Biochem Pharmacol 36(16):2577-2580.
Charbonneau, M; Lock, EA; Strasser, J; et al. (1987) 2 2 4-Trimethylpentane-induced nephrotoxicity I. Metabolic
disposition of TMP in male and female Fischer 344 rats. Toxicol Appl Pharmacol 91:171-181.
Cuervo, AM; Hildebrand, H; Bomhard, EM; et al. (1999) Direct lysosomal uptake of a2-microglobulin contributes
to chemically induced nephropathy. Kidney Int 55:529-545.
Dahl, AR. (1989) The fate of inhaled octane and the nephrotoxicant, isooctane, in rats. Toxicol Appl Pharmacol
100:334-341.
Dietrich, DR; Swenberg, JA. (1991) NCI-black-Reiter (NBR) male rats fail to develop renal disease following
exposure to agents that induce a-2u-globulin (a2u) nephropathy. Fundam Appl Toxicol 16:749-762.
Exxon. (1987) Eight toxicity reports on 2,2,4-trimethyl pentane (isooctane) with attachments and cover letter dated
072987. Submitted under TSCA Section 8D; EPA Document No. 86-870000769; NTIS No. OTS0515208.
Forman, S; Kas, J; Fini, F; et al. (1999) The effect of different solvents on the ATP/ADP content and growth
properties of HeLa cells. J Biochem Mol Toxicol 13(l):ll-5.
Fowlie, AJ; Grasso, P; Bridges, JW. (1987) Renal and hepatic lesions induced by 2,2,4-trimethylpentane. J Appl
Toxicol 7:335-341.
Kloss, MW; Swenberg, J; Bus, JS. (1986) Sex-dependent differences in disposition of [14C-5]-
2,2,4-trimethylpentane in Fischer 344 rats with cover sheet. Submitted under TSCA Section 8D; EPA Document
No. 86-870001586; NTIS No. OTS0516167.
Lock, EA; Stoner, MD; Elcombe, CR. (1987a) The induction of omega and beta-oxidation of fatty acids and effect
on alpha 2u globulin content in the liver and kidney of rats administered 2,2,4-trimethylpentane. Xenobiotica
17:513-522.
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Lock, EA; Charbonneau, M; Strasser, J; et al. (1987b) 2,2,4-Trimethylpentane-induced nephrotoxicity II. The
reversible binding of a TMP metabolite to a renal protein fraction containing alpha-2u globulin. Toxicol Appl
Pharmacol 91:182-192.
Loury, DJ; Smith-Oliver, T; Strom, S; et al. (1986) Assessment of unscheduled and replicative DNA synthesis in
hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane. Toxicol Appl Pharmacol
85:11-23.
McLaren, J; Boulikas, T; Vamvakas, S. (1994) Induction of poly(ADP-ribosyl)ation in the kidney after in vivo
application of renal carcinogens. Toxicology 88:101-112.
NLM (National Library of Medicine). (2004) Isooctane. HSDB (Hazardous Substances Data Bank). National
Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD. Available online at
http://toxnet.nlm.nih.gov.
NRC (National Research Council). (1983) Risk assessment in the federal government: managing the process.
Washington, DC: National Academy Press.
Olson, CT; Yu, KO; Hobson, DW; et al. (1985) Identification of urinary metabolites of the nephrotoxic hydrocarbon
2,2,4-trimethylpentane in male rats. Biochem Biophys Res Commun 130:313-316.
Richardson, KA; Wilmer, JL; Smith-Simpson, D; et al. (1986) Assessment of the genotoxic potential of unleaded
gasoline and 2,2,4-trimethylpentane in human lymphoblasts in vitro. Toxicol Appl Pharmacol 82:316-322.
Saito, K; Kaneko, H; Isobe, N; et al. (1992) Differences in alpha2u-globulins increased in male rat kidneys following
treatment with several alpha2u-globulin accumulating agents: cysteine protease(s) play(s) an important role in
production of kidney-type-alpha2u-globulin. Toxicology 76:177-186.
Saito, K; Uwagawa, S; Kaneko, H; et al. (1996) Alpha2u-globulins in the urine of male rats: a reliable indicator for
alpha2u-globulin accumulation in the kidney. Toxicology 106:149-157.
Short, BG; Burnett, VL; Swenberg, JA. (1986) Histopathology and cell proliferation induced by
2,2,4-trimethylpentane in the male rat kidney. Toxicol Pathol 14:194-203.
Short, BG; Burnett, VL; Swenberg, JA. (1989a) Elevated proliferation of proximal tubule cells and localization of
accumulated alpha2u-globulin in F344 rats during chronic exposure to unleaded gasoline or 2,2,4-trimethylpentane.
Toxicol Appl Pharmacol 101:414-431.
Short, BG; Steinhagen, WH; Swenberg, JA. (1989b) Promoting effects of unleaded gasoline and
2,2,4-trimethylpentane on the development of atypical cell foci and renal tubular cell tumors in rats exposed to n-
ethyl-n-hydroxyethylnitrosamine. Cancer Res 49:6369-6378.
Stadler, JC; Kennedy, GL, Jr. (1996) Evaluation of the sensory irritation potential of volatile organic chemicals from
carpets alone and in combination. Food Chem Toxicol 11:1125-1130.
Standeven, AM; Goldsworthy, TL. (1994) Identification of hepatic mitogenic and cytochrome P-450-inducing
fractions of unleaded gasoline in B6C3F1 mice. J Toxicol Environmental Health 43: 213-224.
Stonard, MD; Phillips, PGN; Foster, JR; et al. (1986) Alpha2u-globulin: measurement in rat kidney following
administration of 2,2,4-trimethylpentane. Toxicology 41:161-168.
Swann, HE; Kwon, BK; Hogan, GK; et al. (1974) Acute inhalation toxicology of volatile hydrocarbons. Am Indust
HygAssocJ 35:511-518.
U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and documentation of biological values
for use in risk assessment. Environmental Criteria and Assessment Office, Office of Health and Environmental
Assessment, Cincinnati, OH; EPA/600/6-87/008. Available from the National Technical Information Service,
Springfield, VA; PB88-179874/AS, and online at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=34855.
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U.S. EPA. (1991) Alpha 2u-globulin: association with chemically induced renal toxicity and neoplasia in the male
rat. Risk Assessment Forum, Washington, DC; EPA/625/3-91/019F.
U.S. EPA. (1994) Methods for derivation of inhalation reference concentrations and application of inhalation
dosimetry. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH; EPA/600/8-90/066F. Available from the National Technical Information Service, Springfield, VA,
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Development, Washington, DC; EPA 100-B-98-001. Available from the National Technical Information Service,
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND
PUBLIC COMMENTS AND DISPOSITION
The Toxicological Review of2,2,4-Trimethylpentane has undergone internal review by
scientists within EPA, an interagency review, and an external panel peer review. The external
review was conducted in February 2007 in accordance with EPA guidance on peer review (U.S.
EPA, 2006). Comments made by the internal as well as interagency reviewers were addressed
prior to submitting the document for external review and are not part of this appendix. For the
external peer review, the reviewers were tasked with providing written answers to general
questions on the overall assessment and on chemical-specific charge questions, addressing key
scientific issues of the assessment. The charge questions, summary of reviewer comments, and
EPA's disposition of the comments are provided below. Editorial comments were considered
and incorporated into the document as appropriate and are not discussed further. EPA also
received comments from the public. A summary of public comments and EPA's responses are
also included.
Charge to External Reviewers
Question 1: Are there additional key published studies or publicly available scientific reports that
are missing from the draft document that might be useful for the discussion of the hazards of
2,2,4-trimethylpentane?
Question 2: No oral RfD has been derived in the current draft assessment. Has the rationale and
justification for not deriving an RfD been transparently described? Is the rationale scientifically
justified and appropriate?
Question 3: No inhalation RfC has been derived in the current draft assessment. Has the rationale
and justification for not deriving an RfC been transparently described? Is the rationale
scientifically justified and appropriate?
Question 4: Does the Toxicological Review provide sufficient information to support a
conclusion that there is a causal relationship between accumulation of a2u-globulin and the
pathology observed exclusively in the male rat kidney in response to 2,2,4-trimethylpentane
exposure?
Question 5: The majority of the studies available for 2,2,4-trimethylpentane were designed only
to investigate various aspects of a2u-globulin-induced nephropathy. Thus, data and information
on effects in target organ systems other than the kidney are limited in quantity and quality (e.g.,
liver). Has the available information on effects unrelated to a2u-globulin-associated nephropathy
been adequately and appropriately described?
Question 6: Has the appropriate cancer descriptor been chosen? Has the rationale and
justification for not deriving a quantitative cancer assessment been transparently described? Do
you agree with EPA's rationale, justification, and conclusion?
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Comments and Response
Question 1: Two reviewers thought the literature database was complete and could not identify
any additional literature. Two reviewers suggested additional studies from the literature that
would add support to the conclusions made and would perhaps add value but not change the
conclusions reached in the assessment.
Response: The suggested references (Cuervo et al., 1999; Standeven and Goldsworthy, 1994;
Dietrich and Swenberg, 1991) were reviewed and incorporated into the assessment in section
4.4.1.1, Other Studies, Acute and Short-term Studies, Oral. Specifically, these references added
information to support the mode-of-action information for TMP, and TMP-/TMP metabolite-
renal protein binding.
Question 2: All four reviewers felt that the studies available were not sufficient for use in
deriving an RfD, as they were mainly designed to address specific mode-of-action questions. The
reasoning, justification, and discussion presented for not deriving an RfD were transparently
described and scientifically appropriate.
Response: No response required.
Question 3: All four reviewers felt that the studies available were not sufficient for use in
deriving an RfC, as they were mainly designed to address specific mode-of-action questions. The
reasoning, justification, and discussion presented for not deriving an RfC were transparently
described and scientifically appropriate.
Response: No response required.
Question 4: All four reviewers felt that sufficient information was provided, documented, and
described to support the causal relationship between accumulation of a2U-globulin and the
pathology observed exclusively in the male rat kidney in response to 2,2,4-trimethylpentane
exposure. In addition, one reviewer provided an additional reference on chemical-a2U-globulin
binding to support the mode of action described.
Response: The reference cited was incorporated into the assessment in section 4.4.1.1, Other
Studies, Acute and Short-term Studies, Oral.
Question 5: All reviewers agreed that the limited information for the effects of TMP on other
organs was adequately described except for a paper by Standeven and Goldsworthy (1994)
describing liver mitogenic effects in mice. However, the inclusion of this study would not change
the conclusions reached in the assessment.
Response: Reference to this study was added to the assessment in section 4.4.1.1, Other Studies,
Acute and Short-term Studies, Oral, as part of the response to Question 1 to complete the
database.
Question 6: All reviewers agreed that the appropriate cancer descriptor was used and the analysis
and rationale to justify not assessing the cancer risk for TMP were transparently described.
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Response: No response required.
Comments from the Public
Comment: One reviewer agreed with and endorsed the assessment's conclusion on the renal
effects characterized for IMP. This reviewer also offered a few literature citations of studies
conducted for several isoparaffinic and hydrocarbon solvents that the reviewer believes could be
used to qualitatively inform the database and enhance the hazard characterization of TMP.
Response: While we agree that other information on similar chemicals could be used to enhance
the database and findings as a whole, those studies identified examined solvent mixtures that
may or may not be related to or contain TMP. An attempt was made in the assessment to include
some information on the mixture, unleaded gasoline, of which TMP is a component but only in
those studies in which TMP was examined separately. Even in those studies, there is great
difficulty in specifically ascribing the effects that directly result from TMP alone and not from
the mixture.
Comment: One reviewer had general comments concerning risk assessment practices, the period
of time to comment, animal testing, and a reference to thalidomide.
Response: No response required as no comments pertaining specifically to this assessment were
made.
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