United States
Environmental Protection
1=1 m m Agency
EPA/690/R-l 5/014F
Final
9-24-2015
Provisional Peer-Reviewed Toxicity Values for
2,4,4-Trimethylpentene
(CASRN 25167-70-8)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Jon Reid, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Paul Reinhart, PhD
National Center for Environmental Assessment, Washington, DC
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421 3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
li
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 5
HUMAN STUDIES 10
Oral Exposures 10
Inhalation Exposures 10
ANIMAL STUDIES 10
Oral Exposures 10
Inhalation Exposures 14
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 14
Genotoxicity 14
Acute Toxicity Studies 14
DERIVATION OI PROVISIONAL VALUES 16
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 16
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 16
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 17
APPENDIX A. SCREENING PROVISIONAL VALUES 18
APPENDIX B. DATA TABLES 25
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 29
APPENDIX D. REFERENCES 46
in
2,4,4-Trimethylpentene
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand white (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFS
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2,4,4-TRIMETHYLPENTENE (CASRN 25167-70-8)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
2,4,4-Trimethylpentene, CASRN 25167-70-8, is a mixture of isomers
2,4,4-trimethylpent-l-ene (CASRN 107-39-1) and 2,4,4-trimethylpent-2-ene
(CASRN 107-40-4); 75% consists of the 1-ene and 25% of the 2-ene. The mixture is commonly
referred to as "diisobutylene" (Sigma-Aldrich). In this document the mixture is referred to as
2,4,4-trimethylpentene which is the common usage. OECD (2008) also indicates that a synonym
is diisobutylene and indicates that the 1-ene is 70-80% and the 2-ene is 15—25% of the mixture.
This chemical is listed as a high-production-volume chemical by the Organisation for Economic
Cooperation and Development (OECD, 2004). It is primarily used as a chemical intermediate
for the production of other industrial chemicals, but also used as a solvent for paints, lacquers,
and varnishes (OECD, 2008). The annual production volume in 2002 was 40,000-50,000 tonnes
(metric) (OECD, 2008). There is an indication of bioaccumulation potential (OECD, 2008). The
empirical formula for 2,4,4-trimethylpentene is CxHu, (see Figure 1). A table of
physicochemical properties for a commercial mixture of 75% 2,4,4-trimethylpent-l-ene and 25%
2,4,4-trimethylpent-2-ene is provided below (see Table 1). The isomeric mixture has high vapor
pressure, indicating that it will predominantly exist in the atmosphere as a vapor (HSDB, 2002a,
b); further, for humans in the environment, the main intake route in regional scenario is
inhalation. The mixture is moderately soluble in water, but it is expected to display high
volatility from water surfaces (OECD, 2008).
Figure 1. 2,4,4-Trimethylpent-l-ene (Left) and
2,4,4-Trimethylpent-2-ene (Right) Structures
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Table 1. Physicochemical Properties of 2,4,4-Trimethylpentene (Mixture)
(CASRN 25167-70-8)a
Property (unit)
Value
Boiling point (°C)
97-107
Melting point (°C)
-101 to -106
Density (g/cm3)
0.72
Vapor pressure (mmHg at 38°C)
75-103 (100-137 hPa)
pH (unitless)
ND
Solubility in water (g/L at 20°C)
1.8 x 10-3b
Relative vapor density (air =1)
ND
K-ow
4.4 (OECD. 2008)
Molecular weight (g/mol)
112.22
aECB (2000)
bEU (2008)
ND = no data.
A summary of available toxicity values for 2,4,4-trimethylpentene from U.S. EPA and
other agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for
2,4,4-Trimethylpentene (Mixture) (CASRN 25167-70-8)
Sou rce/Parameterab
Value (applicability)
Notes
Reference
Noncancer
ACGIH
NV
NA
ACGIH (2015)
ATSDR
NV
NA
ATSDR (2015)
Cal/EPA
NV
NA
Cal/EPA (2014): Cal/EPA
(2015a): Cal/EPA (2015b)
NIOSH
NV
NA
NIOSH (2015)
OSHA
NV
NA
OSHA (2011); OSHA (2006)
IRIS
NV
NA
U.S. EPA (2015)
DWSHA
NV
NA
U.S. EPA (2012)
HEAST
NV
NA
U.S. EPA (2011a)
CARA HEEP
NV
NA
U.S. EPA (1994)
WHO
NV
NA
WHO (2015)
AIHA (WEEL)
75 ppm (344 mg/m3)
8-hr TWA
AIHA (2007)
Cancer
IRIS
NV
NA
U.S. EPA (2015)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012)
IARC
NV
NA
IARC (2013)
NTP
NV
NA
NTP (2014)
Cal/EPA
NV
NA
Cal/EPA (2015a): Cal/EPA
(2011): Cal/EPA (2015b)
ACGIH
NV
NA
ACGIH (2015)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; AIHA = American Industrial
Hygiene Association; ATSDR = Agency for Toxic Substances and Disease Registry; Cal/EPA = California
Environmental Protection Agency; CARA = Chemical Assessments and Related Activities; DWSHA = Drinking
Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables; HEEP = Health
and Environmental Effects Profile; IARC = Institute for Occupational Safety and Health; IRIS = Integrated Risk
Information Systems; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology
Program; OSHA = Occupational Safety and Health Administration; WHO = World Health Organization.
Parameters: TWA = time-weighted average; WEEL = workplace environmental exposure level.
NA = not applicable; NV = not available
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Literature searches were conducted in July 2013 covering years back to 1900 and in
March 2015 for studies relevant to the derivation of provisional toxicity values for
2,4,4-trimethylpentene, CASRN 25167-70-8. In March 2015 the literature search was updated
but no new relevant information was found. Searches were conducted using U.S. EPA's Health
and Environmental Research Online (HERO) database of scientific literature. HERO searches
the following databases: PubMed, TOXLINE (including TSCATS1), and Web of Science. The
following databases were searched outside of HERO for health-related values: ACGIH, ATSDR,
Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA Office of Water, U.S. EPA
TSCATS2/TSCATS8e, NIOSH, NTP, and OSHA.
REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide an overview of the relevant databases for
2,4,4-trimethylpentene and include all potentially relevant and repeated short-term-, subchronic-,
and chronic-duration studies. Principal studies are identified in bold. Reference can be made to
details provided in Tables 3A and 3B. The phrase "statistical significance," used throughout the
document, indicates ap-walue of < 0.05 unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for
2,4,4-Trimethylpentene (CASRN 25167-70-8)
Number of
Male/Female,
Strain, Species,
Study Type, Study
BMDL/
Category
Duration
Dosimetry3
Critical Effects
NO A EL1
BMCLa
LOAELa
Reference (comments)
Notesb
Human
1. Oral (mg/kg-d)a
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)a
Short-term
5 M/5 F, S-D CD-I
0, 100, 300,
Increased absolute
300
ND
1,000
Huntingdon Life
NPR
rat, gavage,
1,000
and relative
Sciences (1997b)
28 consecutive d.
liver-weights in both
ADD: 0,
sexes; increased
(increased kidney
100, 300,
serum albumin and
weights in male rats at
1,000
total protein levels in
1,000 mg/kg-d likely
males; increased
associated with a2u-g
absolute kidney
accumulation
weight in females,
(demonstrated in the
and decreased glucose
reproduction study); not
levels in females at
relevant to humans)
1,000 mg/kg-d
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Table 3A. Summary of Potentially Relevant Noncancer Data for
2,4,4-Trimethylpentene (CASRN 25167-70-8)
Number of
Male/Female,
Strain, Species,
Study Type, Study
BMDL/
Category
Duration
Dosimetry3
Critical Effects
NO A EL1
BMCLa
LOAELa
Reference (comments)
Notesb
Reproductive/
10 M/10 F, S-D
0,100,300,
Systemic: Increased
100 (systemic)
173 for
300 (systemic)
Huntingdon Life
PS; NPR
developmental
CD-I rat, gavage,
1,000
absolute and relative
increased
Sciences (1997a)
15 d prior to
liver weights in
relative
Swenbere and
pairing, through
ADD: 0,
males at 300 and
liver
Schoonhoven (2004)
mating, gestation,
100,300,
1,000 mg/kg-d;
weight in
and lactation to
1,000
increased absolute
males
LD 3 (44-46 doses
and relative liver
(nephrotoxicity in
for males;
weights in females at
male rats at
40-45 doses for
1,000 mg/kg-d;
>100 mg/kg-d
females). Parental
increased absolute
associated with a2u-g
animals were
and relative kidney
accumulation; not
treated for 6 wk
weight in females at
relevant to humans)
1,000 mg/kg-d; low
incidences of
minimal liver
pathology in both
sexes at
1,000 mg/kg-d;
Basophilic cortical
tubules at
100 mg/kg-d
Reproductive: No
1,000
ND
effects observed.
(reproductive)
(reproductive)
Developmental: No
1,000
ND
effects observed.
(developmental)
(developmental)
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Table 3A. Summary of Potentially Relevant Noncancer Data for
2,4,4-Trimethylpentene (CASRN 25167-70-8)
Category
Number of
Male/Female,
Strain, Species,
Study Type, Study
Duration
Dosimetry3
Critical Effects
NO A EL1
BMDL/
BMCLa
LOAELa
Reference (comments)
Notesb
ND
2. Inhalation (mg/m3)
aDosimetry: Values presented as adjusted daily dose (ADD) in mg/kg-day for oral exposure.
bNotes: NPR = not peer reviewed; PS = principal study.
Treatment/exposure duration: unless otherwise noted: short-term = repeated exposure for >24 hours <30 days (U.S. EPA. 20021: long-term (subchronic) = repeated
exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in typically used laboratory animal species) (U.S. EPA. 20021:
chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in typically used laboratory animal species) (U.S. EPA.
2002).
F = female(s); M = male(s); LD = Lactation Day; ND = not determined; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for 2,4,4-Trimethylpentene (CASRN 25167-70-8)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry
Critical Effects
BMDL/
BMCL
Reference
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
ND
2. Inhalation (mg/m3)
ND = no data.
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HUMAN STUDIES
Oral Exposures
No studies have been identified.
Inhalation Exposures
No studies have been identified.
ANIMAL STUDIES
Oral Exposures
Overview of Animal Oral Exposure Studies
Potentially relevant data for noncancer effects come from two unpublished gavage
studies conducted in rats: a 28-day study (Huntingdon Life Sciences. 1997b) and a 6-week
exposure study designed to evaluate reproductive effects as well as systemic effects on target
organs in parental animals (Swenberg and Schoonhoven, 2004; Huntingdon Life Sciences,
1997a). Note that the rats in the 28-day study were 35-42 days old at commencement of dosing
and were thus sexually mature during a large portion of the dosing period. The Huntingdon Life
Sciences studies (1997a. b) do not specify the relative amounts of the 1 -ene and 2-ene
components. Both these studies identified the liver and kidneys as target organs. No
treatment-related reproductive effects were observed in the 6-week study. In the 28-day study,
increased liver weight among rats at the highest dose tested (1,000 mg/kg-day) was associated
with variations in plasma protein (males) and glucose (females) concentrations in the absence of
corresponding pathological changes. Increased liver weights were also observed in male rats at
300 mg/kg-day and in both male and female rats at 1,000 mg/kg-day in the reproductive toxicity
study.
Increased kidney weight in the presence of renal lesions among male rats was observed in
the reproductive toxicity study at doses >100 mg/kg-day (Huntingdon Life Sciences. 1997a).
Females receiving 1,000 mg/kg-day in this study had slightly elevated kidney weights without
microscopic changes. Increased kidney weights were also observed in male rats at
1,000 mg/kg-day in the 28-day study, although no corresponding pathological changes were
observed (Huntingdon Life Sciences. 1997b). Immunohistochemistry staining using mouse
anti-alpha-2u-globulin monoclonal antibodies confirmed the accumulation of alpha 2u-globulin
(a2u-g), a low molecular weight protein almost exclusively produced by the male rat (Swenberg
and Schoonhoven. 2004). The significance of this is discussed in the derivation section of this
document.
Short-Term-Duration Studies
Huntingdon Life Sciences (1997b); gavage study, 4 weeks
Groups of 10 (35-42 days of age) Sprague-Dawley (S-D) CD-I rats (5/sex) received
daily doses of 2,4,4-trimethylpentene (50:50 mixture of 2 original batches of
2,4,4-trimethylpentene; purity 99.1%) at 0, 100, 300, or 1,000 mg/kg-day via gavage in maize oil
for 28 consecutive days. Survival and clinical signs were monitored twice daily, and body
weight and food consumption were measured weekly. Each animal was subjected to a functional
observation battery that evaluated open-field observations weekly (activity, alertness, behavior,
convulsions, defecations, exophthalmos, fur appearance, gait, grooming frequency, lacrimation,
palpebral closure, piloerection, posture, pupil size, ease of cage removal, respiration rate,
salivation, tremors, and amount of urination), and sensory reactivity tests (auditory pinna reflex,
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auditory startle reflex, body temperature, flexor responses, landing foot splay, pain response,
pupil closure response, reaction to handling, and righting reflex) followed by evaluations of grip
strength and motor activity administered during Week 4 of treatment. Each animal was starved
overnight following the neurobehavioral tests, and blood samples were collected for hematology
(packed cell volume [PCV], hemoglobin concentration [Hb], counts of red blood cells [RBCs],
total and differential white blood cells [WBCs], platelets, mean cell hemoglobin concentration
[MCHC], mean cell hemoglobin [MCH], mean cell volume [MCV], and prothrombin time [PT])
and serum chemistry (alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate
aminotransferase [AST], gamma-glutamyl transpeptidase [GGT], urea, glucose [GLUC],
cholesterol [CHOL], creatinine [CREA], plasma protein, albumin, albumin/globulin ration
[A/G], sodium, and potassium). At sacrifice, all animals were subjected to a detailed necropsy
that included gross pathology, organ weights (adrenals, brain, epididymides, heart, kidneys, liver,
spleen, testes, and thymus), histopathology, and collection of bone marrow samples for
evaluation of composition. The organ tissues evaluated microscopically for the control and
high-dose groups were adrenals, brain, caecum, colon, duodenum, epididymides, heart, ileum,
jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, sciatic nerve, spinal cord, spleen,
stomach, testes, thymus, thyroid, trachea, urinary bladder, and uterus with cervix. Organ tissue
evaluated from the low- and mid-dose groups consisted of kidneys, liver, and lungs only.
No deaths were observed in the study (Huntingdon Life Sciences. 1997b). Slight
increases in weight gain, food consumption, and food conversion efficiency were noted for
high-dose females. A slight increase in food intake was also observed among high-dose males
and a slightly increased body-weight gain, and food conversion efficiency was observed for
mid-dose females (see Table B-l). High-dose animals exhibited brown staining of the dorsal and
ventral fur (see Table B-l). Open-field observations showed that staining was first observed in
males during Week 2 and only at the end of treatment in females. Females in this dose group
also showed an ungroomed appearance. Salivation after administration, a common finding in
gavage studies, was observed on isolated occasions in some high-dose animals. It is unclear
whether the cause of the brown staining of the coat in high-dose animals was related to
salivation. No treatment-related changes were observed based on the sensory reactivity tests.
There were no statistically significant differences in grip strength or motor activity. Changes in
hematology and blood chemistry parameters are shown in Table B-l. Hematological changes
were restricted to slightly higher (statistically significant) MCH and MCV in high-dose females.
In the absence of other hematological changes and the absence of similar findings in males, the
toxicological significance of these effects is uncertain. Changes in blood chemistry parameters
included statistically significantly decreased plasma glucose concentrations in high-dose females,
and statistically significantly increased plasma protein and albumin concentrations in high-dose
males. Changes in plasma urea concentrations differed by sex, whereby statistically significant
increases were observed in high-dose males and statistically significant decreases were observed
in mid- and high-dose females. No treatment-related effects were observed on cellularity or
composition of the bone marrow. Organ weights are shown in Table B-2. The absolute and
relative liver weights were statistically and biologically significantly increased in high-dose
animals (46 and 44%, respectively, in males; 30 and 21%, respectively, in females) than in
controls. The absolute kidney weights were statistically (not females) and biologically
significantly increased in high-dose males (39%) and females (14%); relative kidney weights
were statistically and biologically significantly increased in mid-dose (not statistically
significant) and high dose males (11% and 35%, respectively) than in controls. No
corresponding changes in pathology were observed. A lowest-observed-adverse-effect level
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(LOAEL) of 1,000 mg/kg-day is identified for increased liver and kidney weights. The
no-observed-adverse-effect level (NOAEL) is 300 mg/kg-day.
Subchronic-Duration Studies
No studies have been identified.
Chronic-Duration Studies
No studies have been identified.
Reproductive/Developmental Studies
Huntingdon Life Sciences (1997a), Swenberg and Schoonhoven (2004)
Groups of 20 S-D CD-I rats (10/sex) received daily doses of 2,4,4-trimethylpentene
(50:50 mixture of 2 original batches of 2,4,4-trimethylpentene; purity 99.1%) at 0, 100, 300, or
1,000 mg/kg-day via gavage in maize oil for 15 days prior to mating. Treatment was continued
in females through mating, gestation, and lactation to Day 3 of lactation, and to termination
following approximately 6 weeks (between 44 and 46 dose administrations) of treatment in
males. Survival and clinical signs were monitored twice daily. Male body weights were
measured weekly; female body weights were measured weekly until mating was detected, and
then on Gestation Days (GDs) 0, 7, 14, and 20 and Lactation Days (LDs) 1 and 4. Food
consumption was recorded weekly until mating for both males and females, and then for females
on GDs 0-3, 4-6, 7-10, 11-13, 14-16, and 17-19 and LDs 1-3. Vaginal smears were taken
from females 10 days prior to mating to evaluate the regularity and duration of the estrus cycle.
Gestation lengths were calculated and the offspring evaluated for litter size, survival (number
live and dead), weight (live offspring only), sex ratio, clinical signs, and gross pathology. At
termination, all adult animals were subjected to a complete gross necropsy. The numbers of
corpora lutea and uterine implantation sites were recorded for all females. Organ weights
(epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus with cervix)
were measured, and histopathology was performed on all parental animals. The tissues
evaluated microscopically for abnormalities in the control and high-dose groups were
epididymides, kidneys, liver, ovaries, and testes. Tissues evaluated for abnormalities in the
low- and mid-dose groups were kidneys and liver only.
One high-dose female that displayed underactive behavior, hunched posture, piloerection,
slow respiration, brown-colored urine, and brown perigenital staining was sacrificed on LD 2
(Huntingdon Life Sciences. 1997a). Necropsy of this animal revealed signs of poor feeding, a
swollen liver, dark urinary bladder contents, and pale, mottled kidneys. Microscopic kidney
changes included moderate cortical tubule degeneration and moderate proteinaceous casts in the
collecting ducts. Similar signs or findings were not observed in other animals; thus, the study
authors considered the death of this animal as incidental. High-dose animals exhibited staining
of the dorsal and ventral fur (see Table B-3). Staining was first observed in males during
Week 2 and in females during Week 4. Transient salivation after administration was observed
among high-dose animals and as single occurrences for three mid-dose males and one low-dose
male. Salivation is a common finding in gavage studies, and it is unclear if the cause of the
brown staining of the coat in high-dose animals was related to salivation. No significant
treatment-related effects were observed on body weight gain, food consumption, estrous cycle
regularity or duration, mating performance, fertility, gestation length, parturition, or gestation
index. The numbers of corpora lutea, implantation sites, litter sizes, offspring survival, and
offspring body weights and weight gains during the first 4 days of age were not affected by
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treatment. One litter showed low offspring survival resulting from overnight flooding of the
cage on LDs 2-3 that was not treatment-related. Necropsy of offspring revealed no findings that
could clearly be ascribed to parental treatment to 2,4,4 trimethylpentene. Organ weights and
incidences of pathological findings in parent rats are shown in Table B-3. No significant
treatment-related effects were observed on reproductive organs of parental animals.
Absolute liver weights were statistically and biologically significantly increased in
mid- and high-dose males (17 and 61%, respectively) and high-dose females (22%); in addition,
the relative liver weights were also statistically and biologically significantly increased in
mid- and high-dose males (15 and 62%, respectively) and high-dose females (26%). Similarly,
absolute kidney weights were statistically and biologically significantly increased in mid- and
high-dose males (23 and 29%, respectively) and high-dose females (12%) than in controls;
relative kidney weights were statistically (not females) and biologically significantly increased in
mid- and high-dose males (21 and 29%, respectively) and high-dose females (17%).
Macroscopic evaluation of high-dose parental animals revealed swollen livers or liver lobes
among all males and four females, and enlarged kidneys among two males. Microscopic
evaluation of the livers from treated animals revealed centriacinar fatty vacuolation in two
high-dose females (one of these animals was the early decedent), as well as arteritis and biliary
fibrosis in one high-dose male, and focal inflammation with associated hepatocytic degeneration
in one high-dose male. Microscopic evaluation of the kidneys from treated animals revealed
basophilic cortical tubules in males of all treatment groups, as well as proteinaceous casts and
interstitial inflammatory cells in mid- and high-dose males. The incidence of basophilic cortical
tubules was statistically significantly increased at >100 mg/kg-day in male rats. The incidences
of proteinaceous casts and interstitial inflammatory were statistically significantly increased only
at 300 mg/kg-day in males.
Kidney sections from the rats evaluated in the Huntingdon Life Sciences (1997a) study
were subsequently analyzed by immunostaining using mouse anti-a2u-g monoclonal antibodies
for the presence of a2u-g (Swenberg and Schoonhoven. 2004) (see Table B-4). Renal lesions
ascribed to a2u-g-associated nephropathy with the involvement of hyaline droplet accumulation
are considered a species- and sex-specific effect for male rats (U.S. EPA. 1991). These findings
are discussed in more detail in the derivation section. Formalin-fixed kidney tissue samples
(from males [10/dose] at 0, 100, 300, and 1,000 mg/kg-day and females [10/dose] at 0 and
1,000 mg/kg-day) were blocked in paraffin and sectioned for a2u-g staining. Kidney sections
from male F344 rats gavaged with 150 mg/kg-day d-limonene for 4 days were also included in
the study as positive controls. All positive controls gave the expected response. None of the
kidney sections from female rats exhibited any positive staining for a2u-g. In contrast, all slides
from male animals exhibited some positive staining for a2u-g. Control males demonstrated
minimal staining with an average score of 1.2. Average scores increased for male rats with dose,
whereby low-dose males demonstrated mild staining (average score 2.2) and mid- and high-dose
males demonstrated moderate to severe staining (average scores of 3.6 and 3.8, respectively).
Most of the male mid- and high-dose rats exhibited severe staining with numerous specific positive
staining droplets. Tissue samples from all but two of the high-dose males exhibited positive
staining granular casts. Swenberg and Schoonhoven (2004) concluded that a2u-g disease was
present in the male rat kidneys.
The reproductive/developmental NOAEL for the Huntingdon Life Sciences (1997a)
study is 1,000 mg/kg-day, and no reproductive/developmental LOAEL is identified based on the
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lack of any significant treatment-related effects. The study authors of the Huntingdon Life
Sciences (1997a) study also identified a LOAEL of 100 mg/kg-day for nephrotoxic effects based
on the pathological renal changes observed in male rats at this dose level and higher. However,
based on the subsequent evaluation of all the relevant information (see details in Appendix A),
the observed renal lesions in male rats were considered to be a consequence of formation of
a2u-g, a known species-specific effect in male rats that is not predictive of similar effects in
humans. Thus, the systemic LOAEL for this study is 300 mg/kg-day based on statistically and
biologically significantly increased absolute and relative liver weights in males with a
corresponding NOAEL of 100 mg/kg-day
Secondary Source Publications
EU (2008). EU (2009)
This report summarizes relevant physical, chemical, and toxicological information on
2,4,4-trimethylpentene with an emphasis is on fate and transport and health and safety measures.
The Huntingdon Life Sciences (1997a, b) and Swenberg and Schoonhoven (2004) papers are
summarized but no tables are provided. There is a detailed discussion of a2u-g disease, which is
unique to the male rat kidney and not relevant to human toxicity. The authors agreed with
Swenberg and Schoonhoven (2004) that a2u-g disease is present in the Huntingdon Life
Sciences (1997a) study. A paper from the European Commission (SCHER. 2006) entitled
Opinion on Risk Assessment Report on 2,4,4-Trimethylpentene, Human Health Part, states
agreement with the opinions in EU (2008). However, the European Union (EU) authors did not
apply all of the criteria required in the EPA guidance for verification of a2u-g disease.
Inhalation Exposures
No studies examining the effects of 2,4,4-trimethylpentene in animals exposed via
inhalation have been identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Genotoxicity
A secondary source (EU. 2008) summarized the findings from two unpublished in vitro
mutagenicity studies that were not available to review at the time of preparing this PPRTV
assessment. 2,4,4-Trimethylpentene was negative for bacterial mutation in Salmonella
typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2uvrA in the
presence and absence of Aroclor-1254-induced rat liver S9 at concentrations ranging up to
5,000 |ig/plate [Shell (1996) as cited in EU (2008)1. Results were also negative in an assay for
chromosomal aberrations in human lymphocytes in the presence or absence of S9-mix [Shell
(1997) as cited in EU (2008)1.
Acute Toxicity Studies
EU (2008) also summarized the findings from three industry studies that evaluated the
acute toxicity of 2,4,4-trimethylpentene that were not available to review at the time of preparing
this PPRTV assessment.
Huntingdon Life Sciences (1996) as cited in EU (2008) administered commercial
2,4,4-trimethylpentene (purity 95.19%) to groups of rats (5/sex; strain not specified) at a dosage
of 2,000 mg/kg via gavage in maize oil as a single treatment. There were no effects on survival,
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clinical signs, or body-weight gain, and necropsy findings were unremarkable (endpoints not
specified).
Bayer AG (1972) as cited in EU (2008) treated groups of male Wistar rats (15/dose)
orally (not further specified) with a mixture of C8 olefins (approximately 75%
2,4,4-trimethylpentene-l and 15% 2,4,4-trimethylpentene-2) at 250, 500, 1,000, or 2,500 mg/kg
without inducing lethality at any dose. Additionally, a group of 30 male Wistar rats was treated
daily for 5 days with increasing doses of the same substance as follows: 200 mg/kg at Day 1,
300 mg/kg at Day 2, 450 mg/kg at Day 3, 675 mg/kg at Day 4, and 1,015 mg/kg at Day 5. There
were no mortalities over the 7-day observation period, but clinical signs (not specified) were
noted after Day 3.
Bayer AG (1972) as cited in EU (2008) exposed groups of Wistar rats
(20/sex/concentration) via whole-body inhalation to a mixture of C8 olefins (approximately 75%
2,4,4-trimethylpentene-l and 15% 2,4,4-trimethylpentene-2) as a mist at concentrations ranging
from 7.6-44.0 mg/L for 4 hours. Mortality was observed starting at concentrations of
approximately 21-25 mg/L; the median lethal concentration (LCso) values for male and female
rats were 31.5 and 30.0 mg/L, respectively. Subsequently, groups of Wistar rats (10/sex) were
exposed to the same substance at 21.6 mg/L for 4 hours/day on 5 consecutive days. Three males
and 2 females died within 24 hours. Clinical signs in the surviving animals included
convulsions, followed by sedation and respiratory distress.
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DERIVATION OF PROVISIONAL VALUES
The reproductive/developmental gavage study in rats by Huntingdon Life Sciences
(1997a) is considered inadequate for p-RfD derivation because it is a non-peer-reviewed and
unpublished report. However, the Huntingdon Life Sciences (1997a) study is suitable for the
derivation of screening toxicity values. Appendix A provides details on the screening subchronic
and chronic p-RfD.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No subchronic- or chronic-duration studies of humans or animals exposed to
2,4,4-trimethylpentene via inhalation were identified in the available literature, precluding
derivation of inhalation RfCs.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 4 identifies the cancer weight-of-evidence (WOE) descriptor for
2,4,4-trimethylpentene.
Table 4. Cancer Weight-of-Evidence Descriptor for 2,4,4-Trimethylpentene
Possible WOE Descriptor
Designation
Route of Entry
(oral, inhalation, or both)
Comments
"Carcinogenic to Humans "
NS
NA
There are no human data to
support this.
"Likely to Be Carcinogenic to
Humans "
NS
NA
There are no suitable animal
studies to support this.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
There are no suitable animal
studies to support this.
"Inadequate Information to
Assess Carcinogenic Potential"
Selected
Both
This descriptor is selected due to
the lack of any information on
the carcinogenicity of
2,4,4-trimethylpentene.
"Not Likely to Be Carcinogenic
to Humans "
NS
NA
There are no suitable animal
studies to support this.
NA = not applicable; NS = not selected
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DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
The lack of data on the carcinogenicity of 2,4,4-trimethylpentene precludes the derivation
of quantitative estimates for either oral (p-OSF) or inhalation (p-IUR) exposure (see Table 5).
Table 5. Summary of Cancer Values for 2,4,4-Trimethylpentene (CASRN 25167-70-8)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
NDr
p-IUR
NDr
NDr = not determined.
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APPENDIX A. SCREENING PROVISIONAL VALUES
SCREENING PROVISIONAL VALUES
For reasons noted in the main provisional peer-reviewed toxicity value (PPRTV)
document, it is inappropriate to derive provisional toxicity values for 2,4,4-trimethylpentene.
However, "screening" toxicity values may be developed from these studies which, under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in this Appendix and develops
"screening" values. Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty or lack of verification processes
associated with the derivation of an appendix screening toxicity value than for a value presented
in the body of the assessment. Questions or concerns about the appropriate use of screening
values should be directed to the Superfund Health Risk Technical Support Center. Table A-l
presents a summary of screening noncancer reference values.
Table A-l. Summary of Screening Noncancer Reference Values for 2,4,4-Trimethylpentene
(CASRN 25167-70-8)
Toxicity Type (units)
Species/Sex
Critical
Effect
p-Reference
Value
POD
Method
PODhed
UFc
Principal
Study
Screening subchronic
p-RfD
(mg/kg-d)
Rat/male
Increased
relative
liver weight
1 x KT1
BMDLio
BMDLiohed = 41.5
300
Huntingdon
Life Sciences
(1997a)
Screening chronic
p-RfD
(mg/kg-d)
Rat/male
Increased
relative
liver weight
1 x 10-2
BMDLio
BMDLiohed = 41.5
3,000
Huntingdon
Life Sciences
f 1997a)
Subchronic p-RfC
(mg/m3)
NDr
Chronic p-RfC (mg/m3)
NDr
NDr = not determined.
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Screening Subchronic Provisional RfD (Subchronic p-RfD)
The reproductive toxicity study in rats by Huntingdon Life Sciences (1997a) is selected
as the principal study for derivation of the screening subchronic provisional oral reference dose
(p-RfD). Increased relative liver weight in male rats is selected as the critical effect.
Justification for the Critical Effect
Increased relative liver weight was chosen as the critical effect for deriving the screening
subchronic p-RfD for 2,4,4-trimethylpentene because it is the most clearly identified effect
relevant to human health in the available information on oral effects from 2,4,4-trimethylpentene
exposure in rats. Increased liver weights (absolute and relative) were observed in rats at the
following lowest-observed-adverse-effect levels (LOAELs):
• 300 mg/kg-day (and no-observed-adverse-effect level [NOAEL] of
100 mg/kg-day) in male rats exposed via gavage for 6 weeks during a
reproductive toxicity study (Huntingdon Life Sciences. 1997a).
• 1,000 mg/kg-day (and NOAEL of 300 mg/kg-day) in female rats exposed via
gavage for approximately 6 weeks during a reproductive toxicity study
(Huntingdon Life Sciences. 1997a).
• 1,000 mg/kg-day (and NOAEL of 300 mg/kg-day) in male and female rats
exposed via gavage for 28 days (Huntingdon Life Sciences, 1997b).
Increases in liver weight (both absolute and relative) were relatively large in magnitude
and demonstrated a clear dose-response. Among male rats in the reproductive toxicity study, the
increases were >15% at the LOAEL of 300 mg/kg-day and >60% at 1,000 mg/kg-day. Increases
in liver weights among female rats at 1,000 mg/kg-day in this study were >20%. Additionally,
minimal histopathological changes were noted in a small number of animals at 1,000 mg/kg-day
that included arteritis and biliary fibrosis in one male, focal inflammation with associated
hepatocytic degeneration in one male, and centriacinar hepatocytic fatty vacuolation in two
females (one was an early decedent). Increases in liver weights (>20%) among rats in the 28-day
study were accompanied by changes in some serum chemistry parameters, including decreased
glucose in females and increased total protein and albumin levels in males, although
treatment-related pathological changes were not seen in the livers of treated rats in this study.
Justification of the Principal Study
The reproductive/developmental toxicity study of rats (Huntingdon Life Sciences. 1997a)
was selected as the principal study for the following reasons:
1) The study was performed in accordance with OECD Test Guideline 421 and
meets the standards of study design and performance with regard to numbers of
animals, examination of potential toxicity endpoints, and presentation of
information. Details of the study are provided in the "Review of Potentially
Relevant Data" section.
2) The study identified a LOAEL for increased liver weight in male rats that was
lower than the LOAEL for the same effect in the 28-day study (Huntingdon Life
Sciences. 1997b).
3) The study used a larger sample size (10/sex/dose) and a longer exposure duration
(6 weeks) than the 28-day study (sample size of 5/sex/dose).
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Discussion of Male Rat Kidney Findings
The only other target organ identified in the available studies was the kidney. Increases
in kidney weight and renal lesions were observed in male rats in the reproductive toxicity study
at doses >100 mg/kg-day of 2,4,4-trimethylpentene (Huntingdon Life Sciences. 1997a).
Increased kidney weights were also observed in male rats at >300 mg/kg-day in the 28-day
study, although no corresponding pathological changes were observed (Huntingdon Life
Sciences. 1997b). Kidneys of male and female rats from the 6-week study (Huntingdon Life
Sciences. 1997a) were examined by Swenberg and Schoonhoven (2004) for possible
alpha-2u-globulin (a2u-g) disease. None of the kidney sections from female rats exhibited any
positive staining for a2u-g. In contrast, all slides from male animals exhibited dose-dependent
increases in positive staining for a2u-g. Most of the male mid- and high-dose rats exhibited
severe a2u-g staining with numerous specific positive staining droplets. Tissue samples from all
but two of the high-dose males exhibited positive staining granular casts. Swenberg and
Schoonhoven (2004) concluded that a2u-g disease was present in the kidneys of male rats treated
with 2,4,4-trimethylpentene.
The EPA criteria for a2u-g verification requires observation of an increase in number and
size of hyaline droplets only in male kidneys, identification of the protein contained in the
hyaline droplets as a2u-g, and some observation of the sequelae of events (i.e., single cell
necrosis, exfoliation of epithelial cells into tubular lumen, and granular casts) in the development
of a2u-g disease. Verification of increase in the number and size of hyaline droplets only in
male kidneys was presented by Swenberg and Schoonhoven (2004) as were the immunological
test results for identification of a2u-g in the droplets (see Table B-4). Granular casts were
reported in the Huntingdon Life Sciences (1997a) 6-week study in males only with 7/10 and 2/10
rats at 300- and 1,000-mg/kg-day groups, respectively, as shown in Table B-3.
Additionally, basophilic cortical tubules were reported in the Huntingdon Life Sciences
(1997a) 6-week study at an occurrence of 0/10, 7/10, 9/10, and 7/10 at 0, 100-, 300-, and
1,000-mg/kg-day dose groups, respectively while being reported in only one female in the
100-mg/kg-day group (see Table B-3). According to Hard and Khan (2004). basophilic tubules,
along with thickened basement membranes, hyaline cast formation, and glomerulosclerosis are
histologic hallmarks of early chronic progressive nephropathy (CPN). Although CPN is
characterized as a disease of aging male rats, basophilic tubules may be observed in male rats as
young as 2-months-old. The authors indicate that basophilic tubules have a high rate of cell
proliferation and also that enlarged kidneys are often observed with terminal CPN. Thus, the
observed increase in kidney weight in male rats from the Huntingdon Life Sciences (1997a)
6-week study could be related to CPN. Hard and Khan (2004) report that CPN and a2u-g
disease are frequently observed together, and conclude that CPN is not relevant to humans under
these conditions.
Based on the information, the observed renal lesions and increased kidney weight in male
rats in the 6-week study (Huntingdon Life Sciences. 1997a) were considered to be a consequence
of formation of a2u-g, a known species-specific effect in male rats that is not predictive of
similar effects in humans.
Female kidney weight change was observed in both the 4-week and 6-week studies
(see Table B-2 and B-3) but is relatively small and occurred at higher doses vs. male kidneys and
no pathological change associated with a2u-g was observed in the female kidneys. EPA
guidance on alpha a2u-g, with regard to chemicals inducing a2u-g (CIGA), states ITJ.S. EPA
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(19911 page 4]: "In cases where nephrotoxicity was observed in mice or female rats, it was less
severe or qualitatively different from that in male rats and did not involve the spectrum of
discrete lesions associated with a2u-g accumulation in the male rat." This is consistent with the
observation in the female rats from the 4-week and 6-week studies. Thus, the findings related to
the female kidney weight change do not alter the conclusion that a2u-g disease is occurring in
the male rat.
Approach for Deriving the Screening Subchronic p-RfD
After discounting kidney effects in male rats as described above, the LOAEL of
300 mg/kg-day based on increased liver weights in male rats from the Huntingdon Life Sciences
(1997a) reproductive and developmental toxicity study is the lowest LOAEL of the available
studies of 2,4,4-trimethylpentene. The mean absolute and relative liver weights in male rats as
observed in the principal study were selected to derive potential point of departures (PODs) via
benchmark dose (BMD) modeling (see Table A-2). For comparative purposes, increased
absolute and relative kidney and liver weights in females were also selected for BMD modeling
(see Table A-2). Of the endpoints modeled, the lowest POD is a benchmark dose lower
confidence limit of 10 (BMDLio) of 41.5 mg/kg-day for increased relative liver weight in male
rats. Liver and kidney weight data from males and female rats from the 28-day study were not
modeled, due to the higher LOAEL of 1,000 mg/kg-day, smaller magnitude of change, smaller
sample size, and shorter exposure duration for that study.
Table A-2. Potential PODs in Rats Exposed via Gavage to
2,4,4-Trimethylpentene for 6 Weeks3
Endpoint
Animal PODa (mg/kg-d)
PODhed (mg/kg-d)
Huntingdon Life Sciences (1997a)
Increased absolute liver weight (M)
BMDLio = 175
42.0
Increased relative liver weight (M)
BMDLio = 173
41.5
Increased absolute liver weight (F)
BMDLio = 254
61.0
Increased relative liver weight (F)
BMDLio = 520
125
Increased absolute kidney weight (F)
NOAEL = 300
72.0
Increased relative kidney weight (F)
NOAEL = 300
72.0
aBMD modeling results are described in more detail in Appendix C.
'HED calculated by multiplying animal POD by a D AF of 0.24 for rats (U.S. EPA. 201 lb).
F = female; M = male.
Application of Dosimetric Adjustment Factor to Obtain a Human Equivalent Dose (HED)
In Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. LP A. 2011b). the U.S. EPA endorses a hierarchy of approaches to derive
human equivalent oral exposures from data from laboratory animal species, with the preferred
approach being physiologically based toxicokinetic modeling. Another approach may use
chemical-specific information, including what is known about the toxicokinetics and
toxicodynamics of the chemical, to derive chemical-specific adjustments. In lieu of
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chemical-specific information to derive human equivalent oral exposures, U.S. EPA endorses
body-weight scaling to the 3/4 power (i.e., BW3/4) as a default to extrapolate toxicologically
equivalent doses of orally administered agents from all laboratory animals to humans for the
purpose of deriving an RfD under certain exposure conditions. More specifically, the use of
BW3/4 scaling for deriving an RfD is recommended when the observed effects are associated
with the parent compound or a stable metabolite but not for portal-of-entry effects or
developmental endpoints. Following EPA guidance, the BMDLio obtained from modeling the
relative liver weight for male rats in the Huntingdon Life Sciences (1997a) study is converted to
a human equivalent dose (HED) through an application of a dosimetric adjustment factor (DAF)
derived as follows:
Relative liver weight in male rats is selected as the critical effect because it provides a
lower BMDL from the data set.
Using a BWa of 0.25 kg for rats and a standard BWh of 70 kg for humans, the resulting
DAF is 0.24 (U.S. EPA. 201 lb). Applying this DAF to the BMDL in obtained from modeling the
increased relative liver weight data for adult male rats yields a BMDLiohed as follows:
The screening subchronic p-RfD for 2,4,4-trimethylpentene for increased relative liver
weight in male rats, is derived as follows:
DAF = (BWa1/4 - BWh1/4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
BMDLiohed = BMDLio x DAF
= 173 mg/kg-day x 0.24
= 41.5 mg/kg-day
Screening Subchronic p-RfD
BMDLiohed ^ UFC
41.5 mg/kg-day -^300
1 x 10"1 mg/kg-day
Table A-3 summarizes the UFs for the screening subchronic p-RfD for
2,4,4-trimethylpentene.
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Table A-3. Uncertainty Factors for the Screening Subchronic p-RfD for
2,4,4-Trimethylpentene
UF
Value
Justification
UFa
3
A UFa of 3 is applied to account for remaining uncertainty such as the toxicodynamic
differences between rats and humans following oral 2,4,4-trimethylpentene exposure. The
calculation of the HED through application of a DAF accounted for the toxicokinetic
uncertainty as outlined in the EPA's Recommended Use of Body Weight4 as the Default
Method in Derivation of the Oral Reference Dose ('U.S. EPA. 20 lib).
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess variability of 2,4,4-trimethylpentene in humans.
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database; lack of
a 2-generation reproduction study or a comprehensive developmental study.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because the POD is based on a reproductive developmental toxicity
study whereby parental rats were exposed for 6 weeks.
UFC
300
Composite UF = UFA x UFH x UFD x UFL x UFS.
Derivation of Screening Chronic Provisional RfD (Chronic p-RfD)
There are no chronic-duration studies of humans or animals orally exposed to
2,4,4-trimethylpentene. In the absence of additional data, the subchronic p-RfD based on
increased relative liver weights in male rats following a 6-week exposure during a reproductive
developmental study (Huntingdon Life Sciences. 1997a) is used as the basis of a screening
chronic p-RfD.
The screening chronic p-RfD for 2,4,4-trimethylpentene, based on the subchronic
BMDLiohed for increased relative liver weight in male rats, is derived as follows:
Screening Chronic p-RfD = BMDLiohed ^ UFC
= 41.5 mg/kg-day ^ 3,000
= 1 x 10"2 mg/kg-day
Table A-4 summarizes the UFs for the screening chronic p-RfD for
2,4,4-trimethylpentene.
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Table A-4. Uncertainty Factors for the Screening Chronic p-RfD for
2,4,4-Trimethylpentene
UF
Value
Justification
UFa
3
A UFa of 3 is applied to account for remaining uncertainty such as the toxicodynamic
differences between rats and humans following oral 2,4,4-trimethylpentene exposure. The
calculation of the HED through application of a DAF accounted for the toxicokinetic
uncertainty as outlined in the EPA's Recommended Use of Body Weight4 as the Default
Method in Derivation of the Oral Reference Dose (TJ.S. EPA. 20 lib).
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess variability of 2,4,4-trimethylpentene in humans.
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database; lack of
suitable 2-generation reproduction study or comprehensive development study.
UFl
1
A UFl of 1 is applied because the POD is a BMDL, not a LOAEL.
UFS
10
A UFsof 10 is applied to account for the lack of chronic data for 2,4,4-trimethylpentene.
UFC
3,000
Composite UF = UFA x UFH x UFD x UFL x UFS.
24
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APPENDIX B. DATA TABLES
Table B-l. Selected Effects on Male and Female Sprague-Dawley CD-I Rats Exposed to
2,4,4-Trimethylpentene via Gavage for 4 Weeks3
Endpoint
Exposure Group (mg/kg-d)
0
100
300
1,000
Males
Number of animals
5
5
5
5
Body weight gain (g)
173 ± 24.4b
190 ±26.3
174 ± 22.2
180 ±33.6
Food consumption (g/animal)
678°
709
709
731
Food conversion efficiency (%)
25.5
26.8
24.5
24.6
Brown staining of fur
0/5 d
0/5
1/5
4/5
Ungroomed coat
0/5
0/5
0/5
0/5
MCH (pg)
19.5 ±0.6
19.8 ±0.6
19.9 ±0.7
19.7 ±0.5
MCV (fl)
56.2 ± 1.6
56.9 ± 1.5
57.0 ±2.4
56.7 ± 1.6
Plasma glucose (mmol/L)
6.0 ±0.8
5.6 ±0.5
4.9 ±0.4*
5.6 ±1.1
Plasma protein (g/L)
60 ±3
62 ±2
61 ± 1
65 ±4*
Albumin (g/L)
37 ±2
37 ± 1
37 ± 1
39 ±3*
Plasma urea (mmol/L)
3.6 ±0.5
4.0 ±0.5
3.8 ±0.4
4.8 ± 1.0*
Females
Number of animals
5
5
5
5
Body weight gain (g)
73 ± 13.6
63 ± 9.4
80 ± 12.5
89 ± 10.9
Food consumption (g/animal)
478
459
477
526
Food conversion efficiency (%)
15.3
13.7
16.8
16.9
Brown staining of fur
1/5
1/5
1/5
4/5
Ungroomed coat
0/5
0/5
0/5
4/5
MCH (pg)
19.1 ±0.4
19.5 ±0.6
19.5 ±0.3
19.9 ±0.4*
MCV (fl)
54.0 ±0.9
54.0 ± 1.6
54.5 ±0.6
56.1 ± 1.3*
Plasma glucose (mmol/L)
6.0 ±0.7
5.7 ±0.4
5.3 ±0.5
5.0 ±0.4**
Plasma protein (g/L)
62 ±3
64 ±4
62 ±2
65 ±2
Chemical albumin (g/L)
40 ±3
42 ±4
40 ± 1
41 ± 3
Plasma urea (mmol/L)
5.1 ±0.7
5.1 ± 1.2
3.9 ±0.4*
3.7 ±0.3**
"Huntingdon Life Sciences (1997b).
bMean± SD.
°Mean.
dNumber affected/number examined.
* Significantly different from controls (p < 0.05) based on Student's t-test, as reported by study authors.
**Significantly different from controls (p < 0.01) based on Student's t-test, as reported by study authors.
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Table B-2. Selected Effects on Organ Weights in Male and Female Sprague-Dawley CD
Rats Exposed to 2,4,4-Trimethylpentene via Gavage for 4 Weeks"
Endpoint
Exposure Group (mg/kg-d)
0
100
300
1,000
Males
Number of animals
5
5
5
5
Terminal body weight (g)
347.0 ±35.2b
353.9 ± 31.4
(+2.0%)
338.8 ±27.1
(-2.4%)
353.5 ±42.1
(+1.9%)
Absolute liver weight (g)
18.5 ±2.3
17.1 ±2.1
(-5.5%)
16.5 ± 1.7
(-11%)
27.1 ±3.2**
(+46%)**
Relative liver weight (%)
5.34 ±0.29
4.84 ±0.29
(-9.5%)
4.88 ±0.38
(-8.6%)
7.71 ±0.79**
(+44%)**
Absolute kidney weight (g)
2.63 ±0.21
2.84 ±0.35
(+8.0%)
2.87 ±0.27
(+9.1%)
3.65 ±0.67**
(+39%)**
Relative kidney weight (%)
0.761 ±0.059
0.802 ±0.041
(+5.4%)
0.846 ± 0.042
(+11%)
1.03 ±0.084**
(+35%)**
Females
Number of animals
5
5
5
5
Terminal body weight (g)
212.1 ± 11.8
204.5 ± 14.6
(-3.6%)
218.2 ± 17.1
(+2.9%)
232.3 ±21.1
(+9.5%)
Absolute liver weight (g)
9.9 ±0.8
9.9 ± 1.3
(0%)
10.1 ±0.8
(+2.0%)
12.9 ±0.8**
(+30%)**
Relative liver weight (%)
4.64 ±0.16
4.86 ±0.48
(+4.7%)
4.66 ±0.44
(+0.4%)
5.61 ±0.62*
(+21%)*
Absolute kidney weight (g)
1.75 ±0.22
1.82 ±0.24
(+4.0%)
1.88 ±0.16
(+7.4%)
2.00 ±0.18
(+14%)
Relative kidney weight (%)
0.822 ± 0.080
0.89 ±0.107
(+8.3%)
0.863 ±0.038
(+5.0%)
0.862 ± 0.088
(+4.9%)
"Huntingdon Life Sciences (1997b).
bMean± SD.
* Significantly different from controls (p < 0.05) based on Dunnett's test, as reported by study authors.
**Significantly different from controls (p < 0.01) based on Dunnett's test, as reported by study authors.
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Table B-3. Selected Effects on Parental Sprague-Dawley CD Rats Exposed to
2,4,4-Trimethylpentene via Gavage for Approximately 6 Weeks"
Endpoint
Exposure Group (mg/kg-d)
0
100
300
1,000
Males
Number of animals
10
10
10
10
Brown staining of fur
0/10b
0/10
1/10
6/10
Yellow staining of fur
0/10
0/10
0/10
10/10
Terminal body weight (g)
509.1 + 21.8C
519.9 ±52.9
(+2.1%)
517.6+16.6
(+1.7%)
506.5 + 27.5
(-0.5%)
Absolute liver weight of liver (g)
20.9 ±3.4
21.7 ±3.1
(+3.8%)
24.4+ 1.6*
(+17%)
33.7 + 3.1**
(+61%)
Relative liver weight (g)
4.10 ±0.60
4.17 ±0.34
(+1.7%)
4.72 + 0.31*
(+15%)
6.65 + 0.51**
(+62%)
Absolute kidney weight (g)
3.86 ±0.30
4.13±0.59
(+7.0%)
4.75 + 0.56**
(+23%)
4.97 + 0.45**
(+29%)
Relative kidney weight (g)
0.758 ±0.045
0.794 + 0.069
(+4.7%)
0.918 + 0.094**
(+21%)**
0.981 + 0.072**
(+29%)**
Basophilic cortical tubules
0/10
7/10**
9/10***
7/10**
Proteinaceous casts in collecting ducts
0/10
0/10
7/10**
2/10
Interstitial inflammatory cells
0/10
0/10
5/10*
1/10
Females
Number of animals
10
10
10
9
Brown staining of fur
0/10
2/10
1/10
4/10
Yellow staining of fur
0/10
0/10
1/10
6/10
Terminal body weight (g)
320.0 ± 19.9
316.4+15.9
(-1.1%)
336.2+ 15.8
(+5.1%)
311.9 + 33.5
(-2.5%)
Absolute liver weight (g)
17.4 ±2.0
16.8 ± 1.6
(+9.7%)
18.6 + 2.9
(+6.9%)
21.3 + 3.7**
(+22%)
Relative liver weight (g)
5.44 ±0.74
5.34 + 0.66
(-1.8%)
5.53 + 0.92
(+1.7%)
6.83 + 0.87**
(+26%)
Absolute kidney weight (g)
2.31 ±0.19
2.51 + 0.19
(+8.7%)
2.50 + 0.18
(+8.2%)
2.59 + 0.18**
(+12%)**
Relative kidney weight (g)
0.723 ±0.051
0.794+ 0.063t
(+9.8%)
0.745 + 0.056
(+3%)
0.845 + 0.151
(+17%)
Basophilic cortical tubules
0/10
1/10
0/10
0/9
Proteinaceous casts in collecting ducts
0/10
0/10
0/10
0/9
Interstitial inflammatory cells
0/10
0/10
0/10
0/9
"Huntingdon Life Sciences (1997a).
bNumber affected/number examined.
°Mean + SD.
* Significantly different from controls (p < 0.05) based on Dunnett's test, as reported by study authors.
**Significantly different from controls (p < 0.01) based on Dunnett's test, as reported by study authors.
***Significantly different from controls (p < 0.001) based on Dunnett's test, as reported by study authors.
Significantly different from controls (p < 0.05) using Behren's-Fisher's test, as reported by study authors.
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Table B-4. Immunohistochemical Staining of alpha 2u-globulin in Kidney Sections from
Parental Sprague-Dawley CD Rats Exposed to 2,4,4-Trimethylpentene via Gavage"
Endpoint
Exposure Group (mg/kg-d)
0
100
300
1,000
Males
Number of animals
10
10
10
10
Immunohistochemical gradingb
Negative
0/10°
0/10
0/10
0/10
Grade 1 (minimal)
8/10
0/10
0/10
0/10
Grade 2 (mild)
2/10
8/10
1/10
0/10
Grade 3 (moderate)
0/10
2/10
2/10
2/10
Grade 4 (strong)
0/10
0/10
7/10
8/10
Average grade
1.2
2.2
3.6
3.8
Females
Number of animals
10
10
10
9
Immunohistochemical grading
Negative
10/10
NA
NA
10/10
Grade 1 (minimal)
0/10
NA
NA
0/10
Grade 2 (mild)
0/10
NA
NA
0/10
Grade 3 (moderate)
0/10
NA
NA
0/10
Grade 4 (strong)
0/10
NA
NA
0/10
Average grade
0
NA
NA
0
"Swenberg and Schoonhoven (2004).
bGrading criteria for immunostaining for a2u-g. Specific staining of protein droplets enhanced the grading score by
0.2-0.4 depending on the number of immunostained droplets; Grade 4 indicates strong positive staining of protein,
protein droplets, and/or granular casts.
°Number affected/number examined.
NA = not applicable.
28
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE FOR CONTINUOUS DATA
The benchmark dose (BMD) modeling of liver and kidney weight data was conducted
with the U.S. EPA's Benchmark Dose Software (BMDS, Version 2.5). For these data, all
continuous models available within the software were fit using a benchmark response (BMR) of
10% increase from control mean for this endpoint. An adequate fit was judged based on the
goodness-of-fit p-value (p > 0.1), magnitude of the scaled residuals in the vicinity of the BMR,
and visual inspection of the model fit. In addition to these three criteria forjudging adequacy of
model fit, a determination was made as to whether the variance across dose groups was constant.
If a constant variance model was deemed appropriate based on the statistical test provided in
BMDS (i.e., Test 2), the final BMD results were estimated from a constant variance model. If
the test for homogeneity of variance was rejected (p < 0.1), the model was run again while
modeling the variance as a power function of the mean to account for this nonconstant variance.
If this nonconstant variance model did not adequately fit the variance data (i.e., Test 3',p< 0.1),
the data set was considered unsuitable for BMD modeling. Among all models providing
adequate fit, the lowest benchmark dose lower confidence limit (BMDL) was selected if the
BMDLs estimated from different models varied greater than threefold; otherwise, the BMDL
from the model with the lowest Akaike's Information Criterion (AIC) was selected as a potential
point of departure (POD) from which to derive a provisional oral reference dose (p-RfD).
For increased relative liver weight in male rats, all constant variance models provided
adequate fits to the variance and all, except the Exponential5 Model and the Hill Model,
provided adequate fit to the means (see Table C-l). For each data set, BMDLs from adequately
fitting models were sufficiently close, so the models with the lowest AIC were selected. The
Exponential Model was selected as the best fitting model for increased relative liver weight in
male rats.
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Table C-l. Relative Liver Weights in Male Rats Exposed by Gavage for 6 Weeks"
Model Name
BMD
BMDL
/7-Value
Test 2
/7-Value
Test 3
/7-Value
For Fit
AIC
Scaled
Residual
Exponential
191.636
173.174
0.1184
0.1184
0.7627
-20.49093
-0.5841
Exponential
205.769
173.294
0.1184
0.1184
0.4775
-18.52819
0.3146
Exponential4
151.146
131.817
0.1184
0.1184
0.2679
-17.80501
-0.58
Exponential
209.883
119.307
0.1184
0.1184
NDr
-16.69944
0.2358
Hill
239.276
123.061
0.1184
0.1184
NDr
-17.032725
-3.03 x 10-7
Linear
151.147
131.817
0.1184
0.1184
0.5413
-19.80505
-0.58
Polynomial
198.506
134.375
0.1184
0.1184
0.4714
-18.513931
-0.578
Polynomial
198.506
134.375
0.1184
0.1184
0.4714
-18.513931
-0.578
Power
209.772
135.081
0.1184
0.1184
0.5576
-18.688842
0.243
"Huntingdon Life Sciences (1997b)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NDr = not determined.
The BMDS output for the selected model (Exponential) follows.
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Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
1000
dose
11:43 12/15 2014
Figure D-l. Exponential! (Increased Relative Liver Weight, Male Rat)
Exponential Model. (Version: 1.9; Date: 01/29/2013)
Input Data File: M:/14 trimethylpentene PPRTV new/exp_Male rel
liver_Exp-ConstantVariance-BMR10-Up.(d)
Gnuplot Plotting File:
Mon Dec 15 11:43:08 2014
Exponential
BMDL
BMD
BMDS Model Run
The form of the response function by Model:
Model 2: Y[dose] = a * exp{sign * b * dose}
Model 3: Y[dose] = a * exp{sign * (b * dose)Ad}
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
Model 5: Y[dose] = a * [c-(c-l) * exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure = dose;
sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
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Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
MLE solution provided: Exact
Initial Parameter Values
Variable
lnalpha
rho(S)
a
b
c
d
Model 2
-1.67582
0
4.04479
0.000496862
0
1
(S)
Specified
Parameter Estimates
Variable
lnalpha
rho
a
b
c
d
Model 2
-1.66227
0
4.04423
0.00049735
0
1
Table of Stats From Input Data
Dose
0
100
300
1000
10
10
10
10
Obs Mean
4.1
4.17
4 .72
6. 65
Obs Std Dev
0.6
0.34
0.31
0.51
Dose
0
100
300
1000
Estimated Values of Interest
Est Mean Est Std Scaled Residual
4.044
4.25
4. 695
6. 65
0. 4356
0. 4356
0. 4356
0. 4356
0.4049
-0.5841
0.1816
-0.00113
Other models for which likelihoods are calculated:
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Model A1:
Model A2:
Model A3:
Model R:
Yij
= Mu(i) + e(i j )
Var{e(ij)}
= Sigma^2
Yij
= Mu(i) + e(i j )
Var{e(ij)}
= Sigma(i)^2
Yij
= Mu(i) + e(i j)
Var{e(ij)}
= exp(lalpha + log(mean(i)) * rho)
Yij
= Mu + e(i)
Var{e(ij)}
= Sigma^2
Model
A1
A2
A3
R
2
Likelihoods of Interest
Log (likelihood) DF
13.51636 5
16.44884 8
13.51636 5
-24.52685 2
13.24547 3
AIC
-17.03273
-16.89768
-17.03273
53.0537
-20.49093
Additive constant for all log-likelihoods = -36.76. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Test
1:
Test
2 :
Test
3:
Test
4 :
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio)
81. 95
5.8 65
5.8 65
0.5418
D. F.
6
3
3
2
p-value
< 0.0001
0.1184
0.1184
0.7627
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is greater than .1. A homogeneous
variance model appears to be appropriate here.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 4 is greater than .1. Model 2 seems
to adeguately describe the data.
Benchmark Dose Computations:
Specified Effect = 0.100000
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Risk Type = Relative deviation
Confidence Level = 0.950000
BMD = 191.63 6
BMDL = 173.174
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For increased absolute liver weight in male rats, all constant variance models provided
adequate fits to the variance and all, except the Exponential5 Model and the Hill Model,
provided adequate fit to the means (see Table C-2). For each data set, BMDLs from adequately
fitting models were sufficiently close, so the models with the lowest AIC were selected. The
Exponential and 3 Models were selected as the best fitting model for increased absolute liver
weight in male rats.
Table C-2. Absolute Liver Weights in Male Rats Exposed by Gavage for 6 Weeks"
Model Name
BMD
BMDL
77-Value
Test 2
/7-Value
Test 3
/7-Value
For Fit
AIC
Scaled
Residual
Exponential
199.012
175.156
0.1279
0.1279
0.9199
126.7712
-0.2621
Exponential
199.012
175.156
0.1279
0.1279
0.9199
126.7712
-0.2621
Exponential4
158.27
133.561
0.1279
0.1279
0.6581
128.8
-0.2521
Exponential
202.245
98.1559
0.1279
0.1279
NDr
130.6041
4.96 x 10-7
Hill
201.864
97.0004
0.1279
0.1279
NDr
130.60412
-3.44 x 10-7
Linear
158.288
133.578
0.1279
0.1279
0.9068
126.799776
-0.252
Polynomial
179.168
133.994
0.1279
0.1279
0.7563
128.700454
-0.25
Polynomial
179.167
133.994
0.1279
0.1279
0.7563
128.700454
-0.25
Power
186.682
134.14
0.1279
0.1279
0.8033
128.666187
-0.198
"Huntingdon Life Sciences (1997a)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NDr = not determined.
The BMDS output for the selected model (Exponential) follows.
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Exponential Model 2, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BMD
—I 1 1 1 1 1—
Exponential
35
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Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
MLE solution provided: Exact
Initial Parameter Values
Variable
lnalpha
rho(S)
a
b
c
d
Model 2
2.0151
0
20.8831
0.00048099
0
1
(S)
Specified
Parameter Estimates
Variable Model 2
lnalpha
rho
a
b
c
d
2.01928
0
20. 9021
0.000478918
0
1
Table of Stats From Input Data
Dose
0
100
300
1000
10
10
10
10
Obs Mean
20.9
21.7
24.4
33.7
Obs Std Dev
3.4
3.1
1.6
3.1
Dose
0
100
300
1000
Estimated Values of Interest
Est Mean Est Std Scaled Residual
20.9
21.93
24.13
33.74
2 .745
2 .745
2 .745
2 .745
-0.002405
-0.2621
0.3092
-0.0493
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Other models for which likelihoods are calculated:
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)
Model R: Yij = Mu + e(i)
Var{e(ij)} = Sigma^2
Likelihoods of Interest
Model Log(likelihood) DF AIC
A1 -60.30206 5 130.6041
A2 -57.45862 8 130.9172
A3 -60.30206 5 130.6041
R -90.17613 2 184.3523
2 -60.3856 3 126.7712
Additive constant for all log-likelihoods = -36.76. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio)
65.44
5 . 687
5 . 687
0.1671
D. F.
6
3
3
2
p-value
< 0.0001
0.1279
0.1279
0.9199
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is greater than .1. A homogeneous
variance model appears to be appropriate here.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 4 is greater than .1. Model 2 seems
to adeguately describe the data.
38
2,4,4-Trimethylpentene
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Benchmark Dose Computations:
Specified Effect = 0.100000
Risk Type = Relative deviation
Confidence Level = 0.950000
BMD = 199.012
BMDL = 175.15 6
39
2,4,4-Trimethylpentene
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For increased absolute liver weight in female rats, no constant variance models provided
adequate fit. Of the modeled variance models, all models provided adequate to the variance and
all models, except the Exponential5 Model and the Hill Model, provided adequate fit to the
means (see Table C-3). For each data set, BMDLs from adequately fitting models were
sufficiently close, so the models with the lowest AIC were selected. The Linear and Polynomials
Models were selected as the best fitting model for increased absolute liver weight in female rats.
Table C-3. Absolute Liver Weights in Female Rats Exposed by Gavage for 6 Weeks3
Model Name
BMD
BMDL
/7-Value
Test 2
77-Value
Test 3
/7-Value
For Fit
AIC
Scaled
Residual
Exponential
423.641
285.983
0.05349
0.7723
0.205
114.5728
0.4784
Exponential
423.641
285.983
0.05349
0.7723
0.205
114.5728
0.4784
Exponential
370.497
158.357
0.05349
0.7723
0.07942
116.4798
0.2959
Exponential
299.096
202.404
0.05349
0.7723
NDr
116.3688
-0.2949
Hill
298.828
193.177
0.05349
0.7723
NDr
116.368769
-0.295
Linear
396.663
254.484
0.05349
0.7723
0.2119
114.506154
0.391
Polynomial
396.663
254.484
0.05349
0.7723
0.2119
114.506154
0.391
Polynomial
396.663
254.484
0.05349
0.7723
0.2119
114.506154
0.391
Power
412.928
254.793
0.05349
0.7723
0.07895
116.489422
0.451
''Huntingdon I.if'c Sciences (1997a)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; ND = not determined.
The BMDS output for the selected model (Linear) follows.
40
2,4,4-Trimethylpentene
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09-24-2015
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Linear
24
22
20
18
16
BMDL
BMD
0
200
400
600
800
1000
dose
14:34 09/08 2015
Figure C-3. Absolute Liver Weights in Female Rats Exposed by Gavage for 6 Weeks
(Huntingdon Life Sciences, 1997a)
41
2,4,4-Trimethylpentene
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FINAL
09-24-2015
For increased relative liver weight in female rats, all constant variance models provided
adequate fits to the variance and all, except the Exponential5 Model and the Hill Model,
provided adequate fit to the means (see Table C-4). For each data set, BMDLs from adequately
fitting models were sufficiently close, so the models with the lowest AIC were selected. The
Polynomial Models were selected as the best fitting model for increased relative liver weight in
female rats.
Table C-4. Relative Liver Weights in Female Rats Exposed by Gavage for 6 Weeks"
Model Name
BMD
BMDL
/7-Value
Test 2
77-Value
Test 3
77-Value
For Fit
AIC
Scaled
Residual
Exponential
377.148
281.449
0.726
0.726
0.6043
24.59303
-0.6258
Exponential3
624.253
295.213
0.726
0.726
0.7296
25.70508
0.06
Exponential4
348.599
246.776
0.726
0.726
0.2613
26.84753
-0.7286
Exponential
613.743
223.092
0.726
0.726
NDr
27.70224
0.05497
Hill
337.118
223.824
0.726
0.726
NDr
27.672103
-2.38 x 10-6
Linear
348.6
246.777
0.726
0.726
0.5321
24.847527
-0.729
Polynomial
611.289
519.905
0.726
0.726
0.9433
23.702306
0.0489
Polynomial
611.289
519.905
0.726
0.726
0.9433
23.702306
0.0489
Power
613.746
266.529
0.726
0.726
0.7327
25.702242
0.055
aHimtingdon Life Sciences (1997a)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; ND = not determined.
The BMDS output for the selected model (Polynomial) follows.
42
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Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BM
Polynomial
7.5
6.5
5.5
BMDL
BMD
0
200
400
600
800
1000
dose
14:04 09/08 2015
Figure C-4. Relative Liver Weights in Female Rats Exposed by Gavage for 6 Weeks
43
2,4,4-Trimethylpentene
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FINAL
09-24-2015
For increased absolute kidney weight in female rats, no constant or modeled variance
models provided adequate fit to the data (see Table C-5).
Table C-5. Absolute Kidney Weights in Female Rats Exposed by Gavage for 6 Weeks"
Model Name
BMD
BMDL
/j-Valuc
Test 2
/7-Value
Test 3
/7-Value
For Fit
AIC
Scaled
Residual
Exponential
1,238.65
746.102
0.9958
0.9822
0.07833
-83.68289
-0.2535
Exponential
1,238.65
746.102
0.9958
0.9822
0.07833
-83.68289
-0.2535
Exponential4
234.103
0.324251
0.9958
0.9822
0.2679
-85.54913
-0.7826
Exponential
234.103
0.371548
0.9958
0.9822
0.2679
-85.54913
-0.7826
Hill
321.532
0.0059102
0.9958
0.9822
0.3609
-85.941752
-0.723
Linear
1,236.2
724.915
0.9958
0.9822
0.08074
-83.743478
-0.267
Polynomial
1,236.2
724.915
0.9958
0.9822
0.08074
-83.743478
-0.267
Polynomial
1,236.2
724.915
0.9958
0.9822
0.08074
-83.743478
-0.267
Power
1,236.2
724.915
0.9958
0.9822
0.08074
-83.743478
-0.267
''Huntingdon I.if'c Sciences (1997a)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose.
44
2,4,4-Trimethylpentene
-------
FINAL
09-24-2015
For increased relative kidney weight in female rats, no constant or modeled variance
models provided adequate fit to the data (see Table C-6).
Table C-6. Relative Kidney Weights in Female Rats Exposed by Gavage for 6 Weeks"
Model Name
BMD
BMDL
/7-Value
Test 2
/7-Value
Test 3
/7-Value
For Fit
AIC
Scaled
Residual
Exponential
315.596
217.829
<0.0001
<0.0001
NDr
-118.4772
-1.661
Exponential3
927.373
ND
<0.0001
<0.0001
NDr
-121.9865
-0.587
Exponential4
283.092
193.262
<0.0001
<0.0001
NDr
-115.6189
-1.719
Exponential
901.374
326.142
<0.0001
<0.0001
NDr
-119.9865
-0.587
Hill
893.088
328.212
<0.0001
0.3372
0.01048
-121.986497
-0.587
Linear
283.424
193.504
<0.0001
0.3372
0.001574
-117.628728
-1.72
Polynomial
550.47
411.155
<0.0001
0.3372
0.01085
-121.489747
-1.03
Polynomial
672.018
395.7
<0.0001
0.3372
0.02626
-123.258174
-0.585
Power
924.869
515.745
<0.0001
<0.0001
<0.0001
-123.986497
-0.587
"Huntingdon Life Sciences (1997a)
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NDr = not determined.
45
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09-24-2015
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