#ti	United States
kS^laMJIjk Environmental Protectio
m mAgency
EPA/690/R-13/003F
Final
6-05-2013
Provisional Peer-Reviewed Toxicity Values for
Butylated Hydroxytoluene (BHT)
(CASRN 128-37-0)
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
J. Phillip Kaiser, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Ghazi Dannan, PhD
National Center for Environmental Assessment, Washington, DC
Q. Jay Zhao, PhD, MPH, DABT
National Center for Environmental Assessment, Cincinnati, OH
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).
l

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	4
HUMAN STUDIES	13
Oral Exposures	13
Short-term-duration Studies	13
Sub chronic-duration Studies	13
Chronic-duration Studies	13
Developmental and Reproductive Studies	14
Other Studies	14
Inhalation Exposures	14
Other Exposures	14
ANIMAL STUDIES	14
Oral Exposures	14
Short-term Studies	14
Sub chronic-duration Studies	17
Chronic-duration Studies	19
Developmental and Reproductive Studies	29
Other Studies	39
Inhalation Exposures	39
Other Exposures	39
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	39
DERIVATION 01 PROVISIONAL VALUES	48
DERIVATION OF ORAL REFERENCE CONCENTRATIONS	48
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	48
Derivation of Chronic Provisional RfD (Chronic p-RfD)	52
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	56
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR	56
Cancer Weight-of-Evidence Descriptor	56
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	57
Derivation of Provisional Oral Slope Factor (p-OSF)	57
Derivation of Provisional Inhalation Unit Risk (p-IUR)	61
APPENDIX A. PROVISIONAL SCREENING VALUES	62
APPENDIX B. DATA TABLES	63
APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE CHRONIC p-RfD
AND p-OSF	99
APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE
ORAL SLOPE FACTOR	114
APPENDIX E. REFERENCES	121
li

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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
111

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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
BUTYLATED HYDROXYTOLUENE (CASRN 128-37-0)
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 flittp://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 (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.
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
Butylated hydroxytoluene (BHT) is a phenol that is a colorless solid at room temperature
(OECD SIDS, 2002). The empirical formula for BHT is C15H24O (see Figure 1). A table of
physicochemical properties is provided below (see Table 1).
Figure 1. Butylated Hydroxytoluene Structure
Table 1. Physicochemical Properties of Butylated Hydroxytoluene (CASRN 128-37-0)a
Property (unit)
Value
Boiling point (°C)
265
Melting point (°C)
70
"3
Density (g/cm )
1.03
Vapor pressure (Pa at 20°C)
1.1
pH (unitless)
Not available
Solubility in water (mg/L at 20-25°C)
0.6-1.1
Relative vapor density (air =1)
Not available
Molecular weight (g/mol)
220.35
aOECD SIDS (2002).
No Reference Dose (RfD), Reference Concentration (RfC), or cancer assessment for
BHT is included on the IRIS database (U.S. EPA, 2010). The Drinking Water Standards and
Health Advisories List does not report values for BHT (U.S. EPA, 2006). The Health Effects
Assessment Summary Tables (HEAST; U.S. EPA, 2010) does not report any RfD or RfC values.
The Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1991, 1994a) does
not provide a Health and Environmental Effects Profile (HEEP) for BHT. The toxicity of BHT
has not been reviewed by the Agency for Toxic Substances and Disease Registry (ATSDR,
2008). The World Health Organization (WHO, 1996) has reviewed the toxicity of BHT and
reports an acceptable daily intake (ADI) of 0-0.3 mg/kg-body weight (bw). The California
Environmental Protection Agency (CalEPA, 2008) has not derived toxicity values for exposure
to BHT. The American Conference of Governmental Industrial Hygienists (ACGIH, 2008) has
derived a threshold limit value (TLV; 8-hour time-weighted average) of 2 mg/m3. The National
Institute of Occupational Safety and Health (NIOSH, 2011) has derived a recommended
exposure limit (REL; 10-hour time-weighted average) of 10 mg/m3. No occupational exposure
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limits are currently available from the Occupational Safety and Health Administration (OSHA,
2006).
The HEAST (U.S. EPA, 2010) has not reported an EPA (1986) cancer weight-of-
evidence classification for BHT. The ACGIH (2008) classifies BHT as "Not Classifiable as a
Human Carcinogen." The International Agency for Research on Cancer (IARC, 2010) classifies
BHT as having "limited evidence" to determine carcinogenicity to animals and states that "no
evaluation could be made of the carcinogenicity of butylated hydroxytoluene in humans" (IARC,
1986). BHT is not included in the 12th Report on Carcinogens (NTP, 2011). CalEPA (2008) has
not prepared a quantitative estimate of carcinogenic potential for BHT.
BHT has an extensive literature database due to its wide use (3529 references). In order
to develop a thorough list of relevant studies, the following methodology was employed.
Literature searches, using the Chemical Abstracts Service (CAS) registry number, were
conducted on sources published from 1900 through July 2011, for studies potentially relevant to
the derivation of provisional toxicity values for BHT, CAS No. 128-37-0. This search resulted in
the identification of 3529 references. Comprehensive toxicity reviews of BHT by OECD SIDS
(2002) and WHO (1996) revealed 19 studies pertinent to PPRTV toxicity value derivation. A
secondary literature search of studies after 2002 was conducted, and all relevant literature was
requested and incorporated into the PPRTV document (39 studies). Due to the equivocal nature
of the literature on the carcinogenic potential of BHT, a focused genotoxicity search was
conducted to support the carcinogenicity assessment. Studies cited by Williams et al. (1999) in a
comprehensive genotoxicity review were requested for evaluation and review (15 studies). An
additional search for genotoxicity data published after 1999 was conducted, and all relevant
studies were retrieved for evaluation and reviewed (9 studies). Any study mentioned in any of
the retrieved articles that seemed relevant to the derivation of a PPRTV value was requested and
reviewed. In total, 82 documents were retrieved and reviewed for this PPRTV document.
Searches were conducted using EPA's Health and Environmental Research Online
(HERO) database of scientific literature. HERO searches the following databases: AGRICOLA;
American Chemical Society; BioOne; Cochrane Library; DOE: Energy Information
Administration, Information Bridge, and Energy Citations Database; EBSCO: Academic Search
Complete; GeoRef Preview; GPO: Government Printing Office; Informaworld; IngentaConnect;
J-STAGE: Japan Science & Technology; JSTOR: Mathematics & Statistics and Life Sciences;
NSCEP/NEPIS (EPA publications available through the National Service Center for
Environmental Publications [NSCEP] and National Environmental Publications Internet Site
[NEPIS] database); PubMed: MEDLINE and CANCERLIT databases; SAGE; Science Direct;
Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network): ANEUPL, CCRIS,
ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX,
HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search, NIOSH, NTIS,
PESTAB, PPBIB, RISKLINE, TRI, and TSCATS; Virtual Health Library; Web of Science
(searches Current Content database among others); World Health Organization; and Worldwide
Science. Given the large quantity of data available for BHT, a selection of all appropriate well-
conducted relevant studies for incorporation into the PPRTV document were reviewed and
incorporated into the PPRTV document on BHT. The following databases outside of HERO
were searched for relevant health information: ACGIH, ATSDR, CalEPA, EPA IRIS, EPA
HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
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REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 2 provides an overview of the database for BHT and includes all potentially
relevant repeated short-term-, subchronic-, and chronic-duration studies. The phrase, "statistical
significance" used throughout the document, indicates ap-value of <0.05.
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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Human
1. Oral (mg/kg-day)a
Acute
0/2 human, oral, single
dose
4 and 80 g
Severe epigastric cramping; nausea;
vomiting; neurological disorders
N/D
N/D
N/D
Grogan (1986);
Shlian and
Goldstone (1986)

Subchronic
None
Chronic
None
Developmental
and
Reproductive
None
Carcinogenic
2035 men and women,
diet, ~6.3 years
351 ng/day on
average
No association found between
exposure and stomach cancer
incidence
N/D
N/D
N/D
Botterweck et al.
(2000)

Other
2/0 patients, dermal
bandages, unknown
Unknown
Eczema and skin sensitivity
N/D
N/D
N/D
Dissanayake and
Powell (1989)

Other
1336 men and women,
dermal patch test, 2 days,
3 days or 1 week
Unknown
Patch test results: negative
N/D
N/D
N/D
Flyvholm and
Menne (1990)

2. Inhalation (mg/m3)a
Subchronic
None
Chronic
None
Developmental
and
Reproductive
None
Carcinogenic
None
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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Animal
1. Oral (mg/kg-day)a
Short-term
5/0 Wistar rat, gavage, 7
and 28 days
7 day: 0, 25,
250, 607
28 day: 0, 25,
250, 527
Progressive periportal hepatocyte
necrosis, fibrosis, and hyperplasia;
increased relative liver weight
250
No fit
607 for
7-day study
and 527 for
the 28-day
study
Powell et al.
(1986)


5-10/0, albino Wistar
rat, diet, 25 days
0, 678,811
Decreased food consumption and
body-weight gains
678
Not run
811
Deichmann et al.
(1955a)


4 (sex not reported),
mongrel dog, diet,
28 days
0, 600, 900,
1371,2014
No effects observed
2014
Not run
N/D
Deichmann et al.
(1955b)

Subchronic
10/0 Wistar rat, diet,
8 weeks
0, 30, 151,755,
1132
Increased absolute and relative liver
weights; decreased body weight
N/D
Not run
30
Fulton et al.
(1980)


3 (sex not reported),
albino Wistar rat, diet,
90 days
0, 193,483,
772, 965, 1448
Increased mortality >483 mg/kg-
day; decreased food consumption
193
Not run
483 (FEL)
Deichmann et al.
(1955c)


5/5 F344 rat, diet,
7 weeks
Males: 0, 620,
1250, 2500,
5000
Females: 0, 700,
1411,2822,
5645
Decreased body weight; increased
hematopoiesis
High dose: 100% mortality
N/D
Not run
620
NCI (1979a)

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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Subchronic
5/5 B6C3F, mouse, diet,
7 weeks
Males: 0, 559,
1118, 2255,
4510,9019
Females: 0, 605,
1210, 2439,
4878, 9756
Decreased body weight
High dose: increased mortality and
slight centrilobular cytoplasmic
vacuolization of hepatocytes in
males
N/D
Not run
559
NCI (1979b)

Chronic
21/0 Fisher 344 rat, diet,
76 weeks
0, 8, 24, 79,
237, 474
Decreased body weight; increased
relative liver weight
79
Not run
237
Williams et al.
(1990)


27/0 Fisher 344 rat, diet,
110 weeks
0, 947
Decreased body weight; increased
mortality in both the control and
treated groups after 84 weeks
N/D
Not run
947
Williams et al.
(1990)


12/0, F344 rat, diet,
36 weeks
0, 700
25% mortality; decreased body
weight; increased relative liver and
kidney weights
Not
identified
Not run
700 (FEL)
Hirose et al.
(1993)


57/57 Wistar rat, diet,
104 weeks
Males: 0, 184,
736
Females: 0, 210,
842
Increased liver weight; decreased
spleen weights seen in females;
serum triglyceride and y-GTPO-
levels altered in treated males and
in total blood cholesterol in treated
females
High dose: increased mortality in
males after Week 96; decreased
body weights in males
N/D
Not run
184
Hirose et al.
(1981)


15/15 albino Wistar rat,
diet, 2 years
Males: 0, 147,
368, 589, 736
Females: 0, 168,
421,673, 842
Decreased body weight; increased
relative brain, lung, kidney and
liver weights in females; increased
relative brain, liver, kidney, and
testes weights in males
N/D
Not run
168
Deichmann et al.
(1955d)

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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Chronic
40/40 JCL:S-D rat, diet,
3-24 months
0, 2.5, 10, 160
Increased serum potassium and
serum cholesterol; decreased
transaminase activity
High dose: increased mortality at
24 months; increased liver weight;
altered liver and kidney
morphologies
N/D
Not run
N/D
Tokyo
Metropolitan
Research
Laboratory of
Public Health
(1992a); data
tables are barely
legible and,
therefore, the
qualitative
statements made
by the study
authors cannot be
verified
quantitatively
NPR

50/50 F344 rat, diet,
105 weeks
Males: 0, 237,
474
Females: 0, 275,
550
Decreased body weight; increased
focal alveolar histiocytosis in
females
275
157 for
increased
focal alveolar
histiocytosis
in female rats
550
NCI (1979c)


50/50 B6C3Fi mouse,
diet, 107 weeks
Males: 0, 515,
1029
Females: 0, 518,
1037
Decreased body weight; increased
incidence of hepatocytomegaly and
nonneoplastic lesions of the liver
(peliosis, hepatocellular
degeneration/necrosis, and
cytoplasmic vacuolation in males)
N/D
36 for
increased
incidence of
liver peliosis
in male mice
515
NCI (1979d)


50/50 B6C3Fi mouse,
diet, 104 weeks
Males: 0, 1640,
3480
Females: 0,
1750,4130
Decreased body weights; increased
number of foci of cellular
alterations in hepatocytes in males;
increased relative liver weights in
mice without tumors
N/D
Not run
N/D
Inai et al. (1988);
questionable dose
administration

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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Chronic
26-32/0, Syrian golden
hamster, diet, 16 weeks
0, 183
Mild hyperplasia in the forestomach
slightly increased; no severe or
papillomatous lesions; possible
interaction between chemical and
feed
N/D
Not run
N/D
Hirose et al.
(1986)


2, sex not reported,
mongrel dog, diet, 1 year
0, 170, 280,
470, 500, 640,
940
No effects
940
Not run
N/D
Deichmann et al.
(1955e)

Developmental
and
Reproductive
2/16 F0 Wistar rat, diet,
14 weeks
F1 Wistar rat (number
not reported), exposed
through lactation, 3 and
7 weeks
0, 500, 750,
1000 (F0)
F0 dams at >500 mg/kg-day:
increased liver weight; >750 mg/kg-
day: abnormal hepatocytes
(enlarged, vacuolized, proliferation
of ER), decreased body weight
Fl pups of dams >500 mg/kg-day:
reduced body weight
N/D
664 for
decreased
body weight
in F0 dams
Maternal and
Fetal: 500
McFarlane et al.
(1997a)


7/50 F0 Wistar rat, diet,
14 weeks
F1 Wistar rat (number
and sex not reported),
diet, postnatal exposure,
necropsied at 22 weeks
0, 25, 100, 500
(F0)
0, 25, 100, 250
(Fl)
F0 dams >500 mg/kg-day:
increased liver weight; abnormal
hepatocytes (enlarged, vacuolized,
proliferation of ER)
Fl pups >100 mg/kg-day: reduced
body weight; increased liver
weight; abnormal hepatocytes
Maternal:
100
Fl: 25
Not run
Maternal:
500
Fl: 100
McFarlane et al.
(1997b)


60/0 Wistar rat, diet, 22
months, interim
sacrifices at 1,6, 11, and
16 months; F1 rats were
generated from the
McFarlane et al. (1997b)
study
0, 25, 100, 250
Decreased body weight; increased
relative liver weight; enlarged and
eosinophilic centrilobular
hepatocytes; altered hepatic
nodules; thyroid hyperactivity
25
Not run
100
Price (1994) as
cited in OECD
SIDS (2002); this
study is a
continuation of
the McFarlane et
al. (1997b) study

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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Developmental
and
Reproductive
10/10 Crj:Cd-l mouse,
diet, dosed from
5-9 weeks before mating
through F2 weaning
F1 and F2 generations
were observed for
21 days for toxicological
effects
0, 29, 88, 263,
790
Fl pups: No effects
F2 pups: No effects
790
Not run
N/D
Tanaka et al.
(1993)


0/26-30 F0 JCL-ICR
mouse, gavage, GD 7-13
0/19-20 F0 JCL-ICR
mouse, gavage, GD 9
0, 70, 240, 800
0, 1200, 1800
F0 dams: increased spleen and
kidney weight at 800 mg/kg-day
F0 dam: increased mortality;
increased spleen and lung weights
in dams
N/D
Not run
N/D
Tokyo
Metropolitan
Research
Laboratory of
Public Health
(1992b); data
tables are
illegible, and,
therefore, the
qualitative
statements made
by the study
authors cannot be
verified
quantitatively
NPR

60/60 F0 Wistar rat,
diet, 13 weeks
100/100 F1 Wistar rat,
7 days/week, diet,
141-144 weeks
0, 25,100, 500
(F0)
0, 25,100, 250
(Fl)
Decreased maternal body weight
Fl animals: decreased body
weights
Maternal:
100
Fl:
Subchronic:
100
Chronic:
25
Not run
Maternal:
500
Fl:
Subchronic:
250
Chronic:
100
Olsen et al.
(1986)
PS
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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NO A EL'
BMDL/
BMCLa
LOAELab
Reference
(Comments)
Notes0
Carcinogenicity
21/0 Fisher 344 rat, diet,
76 weeks
0, 2, 6,21,64,
129
No tumors observed
Not
Identified
Not run
Not
identified
Williams et al.
(1990)


27/0 Fisher 344 rat, diet,
110 weeks
0, 257
No tumors observed; increased
mortality in both the control and
treated groups after 84 weeks
Not
Identified
Not run
Not
identified
Williams et al.
(1990)


57/57 Wistar rat, diet,
104 weeks
Males: 0, 52,
210
Females: 0, 54,
215
Increased pituitary gland adenomas
at the low-dose in females only
Not
Identified
Not run
Not
identified
Hirose et al.
(1981)


50/50 F344 rat, diet,
105 weeks
Males: 0, 64,
129
Females: 0, 66,
132
No tumors observed
Not
Identified
Not run
Not
identified
NCI (1979c)


50/50 B6C3Fi mouse,
diet, 107 weeks
0, 78, 156
Increased lung alveolar/bronchiolar
carcinomas or lung adenomas at the
low-dose level in females only
Not
Identified
Not run
Not
identified
NCI (1979d)


50/50 B6C3Fi mouse,
diet, 104 weeks
Males: 0, 249,
529
Females: 0, 262,
619
Increased incidence of
hepatocellular adenomas at the
high-dose level in males only
Not
identified
Not run
Not
identified
Inai et al. (1988);
questionable dose
administration


-60/0 Wistar rat, diet, 22
months, interim
sacrifices at 1,6, 11, or
16 months; F1 rats
generated from the
McFarlane et al. (1997b)
study
0,7.1,29,71
No tumors observed
Not
identified
Not run
Not
identified
Price (1994) as
cited in OECD
SIDS (2002); this
study is a
continuation of
the McFarlane et
al. (1997b) study

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Table 2. Summary of Potentially Relevant Data for Butylated Hydroxytoluene (CASRN 128-37-0)
Category
Number of
Male/Female, Strain
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL3
BMDL/
BMCL3
LOAEL3b
Reference
(Comments)
Notes0
Carcinogenicity
60/60 F0 Wistar rat,
diet, 13 weeks
100/100 F1 Wistar rat,
diet, 141-144 weeks
F1 Males: 0,
7.1, 28, 69
F1 Females: 0,
6.4,25,62
Dose-related increase in
hepatocellular tumors
(hepatocellular adenomas and
carcinomas; primarily in animals
exposed for 141 weeks or more)
Not
Identified
28 for
increased
total
hepatocellul
ar tumors
(adenomas
and
carcinomas)
in F1 male
rats
Not
identified
Olsen et al.
(1986)
PS
2. Inhalation (mg/m3)3
Subchronic
None
Chronic
None
Developmental
and
Reproductive
None
Carcinogenic
None
""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and to a human equivalent
dose (HED in mg/kg-d) for oral carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly)
exposure. Values from animal developmental studies are not adjusted to a continuous exposure. HED = (avg. mg test article avg. kg body weight number daily
dosed)1'4.
bNot reported by the study author, but determined from data.
°PS = principal study, indicated by bold text; NPR = not peer-reviewed.
N/D = Not Determinable.
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HUMAN STUDIES
Oral Exposures
The effects of oral exposure of humans to BHT have been evaluated in two case studies
of intentional oral self-administration of BHT (i.e., Grogan, 1986; Shlian and Goldstone, 1986),
one long-term epidemiologic study (i.e., Botterweck et al., 2000), as well as case studies
following dermal exposure (i.e., Dissanayake and Powell, 1989; Flyvholm and Menne, 1990).
No oral subchronic, reproductive, or developmental studies in humans were identified.
Short-term-duration Studies
Grogan (1986) described a case in which a 24-year-old female patient ingested 80 g of
BHT (suspended in safflower oil) on an empty stomach. The patient voluntarily consumed the
formula for BHT under the notion that it was a treatment for herpes. The patient experienced a
light-headed feeling 30-60 minutes after ingestion, followed by a headache and visual and
auditory hallucinations continuing for several hours. Within 1 day, she experienced slurred
speech, loss of balance, and complained that sounds seemed "far away." Examination revealed
dysarthria, wide-based gait, a positive Romberg test, slowed mentation without thought disorder,
and dysmetria of the left (nondominant) arm. Repeat examination 8 hours later showed no
abnormalities. A 6-month follow-up showed no long-term toxicity.
Shlian and Goldstone (1986) identified a trend of university students taking large doses
of BHT as a treatment for genital herpes simplex virus infections. The study authors described
one case in which a 22-year-old white female ingested 4 g of BHT on an empty stomach. Hours
after ingestion, the patient experienced severe epigastric cramping, weakness, nausea, and
vomiting, followed by dizziness, confusion, and a brief loss of consciousness. The woman was
taken to the emergency room and diagnosed with gastroenteritis before being released. The next
day, she was admitted to the hospital again complaining of vomiting, dizziness, epigastric
burning pain, and another brief loss of consciousness. The patient's blood pressure was 110/70,
with moderate orthostatic changes. She was afebrile, with a white-cell count of 7400, and was
within normal limits of liver-function tests, electrolyte measurements, electrocardiography, and
electroencephalography. The symptoms disappeared within a few days after she was given
hydration, prochlorperazine, and antacids.
Subchronic-duration Studies
No published studies investigating the effects of subchronic oral exposure to BHT in
humans have been identified for this review.
Chronic-duration Studies
Botterweck et al. (2000) performed a prospective case-cohort study of the association
between BHT and stomach cancer. Analyses were based on 192 stomach cancer cases and
2035 subcohort members (consisting of both men and women) after exclusion of prevalent
cancer cases at baseline and cases diagnosed in the first or second years of follow-up. The study
authors used cancer and pathology registries to determine incidence of cancer during 6.3 years of
follow-up time. Multivariate rate ratios of stomach cancer were computed for all variables, and
trends were analyzed by likelihood ratio tests. Mean intake of BHT was 351 |ig/day among
subcohort members. No statistically significant association between BHT intake and stomach
cancer risk was found, and the study authors noted a nonsignificant decrease in risk with
increased intake.
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Developmental and Reproductive Studies
Published studies investigating the developmental or reproductive toxicity of BHT via
oral exposure were not identified for this review.
Other Studies
No other oral studies of BHT exposure in humans are identified for this review.
Inhalation Exposures
No inhalation studies of BHT exposure in humans are identified for this review.
Other Exposures
Dissanayake and Powell (1989) published two peer-reviewed case studies describing
contact dermatitis in two leg ulcer patients treated with BHT-containing bandages. The cases
included a 77-year-old man with an 18-month history of stasis ulcers being treated using paste
bandages, and a 70-year-old male patient with bilateral venous stasis and intolerance to support
bandages. Patch testing revealed a positive reaction (i.e., eczema) to BHT. Both patients
improved when treatment with the BHT-containing bandages ceased.
Flyvholm and Menne (1990) conducted patch tests with BHT on 1336 consecutive
eczema patients (consisting of both men and women). Patients were tested from September 1987
to December 1989 and were all new referrals. Patch tests were left on the skin for a 2-day
period, and readings were performed after 2 days, 3 days, and 1 week. All of the patch tests with
BHT were negative, at all time points. Based on these results, the study authors concluded that
BHT does not cause allergic dermatitis.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to BHT have been evaluated in 3 subacute-
duration- (i.e., Powell et al., 1986; Deichmann et al., 1955a,b), 4 subchronic-duration-
(Fulton et al., 1980; Deichmann et al., 1955c; NCI, 1979a,b), 9 chronic-duration-
(Williams et al., 1990; Hirose et al., 1993, 1981, 1986; Deichmann et al., 1955d,e; Tokyo
Metropolitan Research Laboratory of Public Health, 1992a; NCI, 1979c,d; Olsen et al., 1986;
Inai et al., 1988), 4 developmental and reproductive toxicity (McFarlane et al., 1997a,b;
Tanaka et al., 1993; Tokyo Metropolitan Research Laboratory of Public Health, 1992b), and
5 carcinogenicity studies (Hirose et al., 1993; NCI, 1979c,d; Olsen et al., 1986; Inai et al., 1988).
Many additional studies on the effects of oral exposure of animals to BHT were identified, but,
because they do not instruct the POD, are excluded from this review. A list of these references is
available in an accompanying separate supplemental document.
Short-term Studies
In a published, peer-reviewed, subacute study, Powell et al. (1986) investigated the oral
toxicity of BHT (99.9% purity, in arachis oil vehicle) in male Wistar rats (200 g). It is unknown
if the study was conducted in compliance with Good Laboratory Practice (GLP). The study
authors administered 0, 25, 250, or 500 mg/kg-day BHT dissolved in arachis oil to groups of five
rats per dose via gavage once per day for 7 days. Rats in the 500-mg/kg group were initially
administered 750 mg/kg-day BHT for Days 1-3 and 500 mg/kg-day thereafter until study
termination for an average daily dose of 607 mg/kg-day [([750 mg/kg-day x 3 days] +
[500 mg/kg-day x 4 days]) ^ 7 days total]. In a second experiment, the study authors
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administered the same doses once per day for 28 days via gavage to groups of 10 rats (resulting
in an adjusted high dose of 527 mg/kg-day). The study authors measured animal body weights
periodically (frequency unspecified) throughout study duration. At study termination, the study
authors performed histological examinations on stained sections of the rats' livers and prepared
microsomal fractions for enzyme assay by homogenizing the liver tissue of each animal.
Additionally, in the second experiment, liver sections were stained for two enzymes of
cytochrome P-450. High-performance liquid chromatography (HPLC) was used to analyze BHT
concentrations in the liver and epididymal adipose tissue. Statistical analysis of all of the
endpoints measured was performed using the Student's Mest.
In the 7-day experiment, rats administered 250 mg/kg-day BHT had final body weights
slightly lower than those of the control group (Powell et al., 1986). Body weights decreased 7 g
per day on Days 1-3 when the high-dose group was fed 750 mg/kg-day BHT, but the weight was
gained back beginning on Day 4 when the daily dose was reduced to 500 mg/kg-day. Lower
body weights were observed in rats in the 607 mg/kg-day BHT dose group compared with the
concurrent control in the 28-day study. The study authors did not observe a dose-dependent
change in body weight in either experiment. A dose-related increase in relative liver weights
was reported at all doses with the greatest increase (166% above controls) seen in the
607-mg/kg-day BHT group in the 7-day experiment. BHT concentrations in epididymal adipose
tissue exhibited a dose-related trend in both the 28-day and the 7-day experiment (see
Table B. 1). Hepatic glucose-6-phosphatase activity and microsomal protein yield were
statistically significantly decreased in rats in the 607 mg/kg-day BHT dose group in the 7-day
experiment (see Table B.2). In the 28-day experiment, rats in the 250 and 527 mg/kg-day BHT
dose groups displayed statistically significantly decreased glucose-6-phosphatase activity and
increased microsomal protein yield. In addition, increased concentrations of hepatic cytochrome
bs were observed at the 250 mg/kg-day dose level in the 7-day experiment and at the 25- and
250 mg/kg-day dose levels (see Table B.2) in the 28-day experiment. Cytochrome P-450
concentrations were 288 and 218 nmol/whole liver (wet weight) in the 7-day experiment and 475
and 462 nmol/whole liver (wet weight) in the 28-day experiment, in the 250 and 500 mg/kg-day
dose groups, respectively. The study authors reported a dose-dependent increase in activities of
ethoxycoumarin O-deethylase and microsomal epoxide hydrolase, but not in ethoxyresorufin
O-deethylase. In both experiments, progressive periportal hepatocyte necrosis was observed
only at the 500 mg/kg-day dose level (607 mg/kg-day for 7-day study and 527 mg/kg-day for
28-day study), with 2/5 and 6/10 rats displaying lesions in the 7- and 28-day experiments,
respectively (see Table B.3). Morphological abnormalities in the periportal region were
observed at the 250 mg/kg-day (glycogen accumulation only) and 500 mg/kg-day
(607 mg/kg-day for 7-day study and 527 mg/kg-day for 28-day study) dose levels (fibrosis,
hepatocyte hypertrophy, hepatocyte hyperplasia, etc.) of both experiments. The study authors
further stated that hepatomegaly, as indicated by increased relative liver weight, was found at
autopsy in animals receiving BHT for 7 or 28 days. However, only qualitative statements and a
bar graph without quantitative data were presented for increased relative liver weight, and,
therefore, this effect cannot be used to derive a reference value.
Powell et al. (1986) concluded that animals dosed with BHT at 250 mg/kg-day or greater
for 28 days suffered progressive liver cell damage, as first indicated by decreased hepatic
glucose 6-phosphatase activity, considered as evidence of endoplasmic reticulum disruption.
Furthermore, the study authors suggested that their results may explain the increased
hepatocellular carcinomas following 250 mg/kg-day administration of BHT for 2.5 years
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described by Olsen et al. (1986), which the study authors stated may be caused by chronic liver
damage, evidenced by findings of hepatocellular necrosis. However, decreased hepatic glucose
6-phosphatase activity is not considered a critical effect. Therefore, based on increased
incidences of nonneoplastic lesions (i.e., necrosis, fibrosis, hepatocyte hypertrophy, and
hepatocyte hyperplasia) of the liver, a NOAEL of 250 mg/kg-day with a LOAEL of
607 mg/kg-day is identified from the 7-day study and a LOAEL of 527 mg/kg-day from the
28-day study.
Deichmann et al. (1955) conducted a series of exposure studies using varying exposure
duration, administration, and test species. The separate experiments in this peer-reviewed
publication are designated with postscripts a-e. While applicable toxicity information is
presented, many aspects of the methods are unreported, including animal husbandry details,
examination parameters, and statistical methods. There is no statement of certification; however,
this study was conducted prior to adoption of GLP. The results presented in this review are for
supporting purposes because the lack of methodological details precludes use of this study in
derivation of a provisional reference value.
Deichmann et al. (1955a) conducted a paired feeding experiment in male albino Wistar
rats for 25 days. The specific methods and endpoints measured were not reported beyond a
synthesis of the results. Five animals were given control feed or 0.8% BHT (purity not
reported), and 10 animals were given 1.0% BHT for 25 days. BHT concentrations in this
experiment were increased gradually with the animals receiving 0.4% (4000 ppm) BHT on
Days 1-4, 0.8% (8000 ppm) on Days 5-7 (or through Day 25 for the 0.8% group), and 1%
(10,000 ppm) for the duration of the study. The study did not report appropriate body-weight
data for use in the dose conversion although food consumption data were reported. However,
because food consumption and body weight are related parameters, values provided for male
Wistar rats by EPA (1994b) for body weight (0.217 kg) and food consumption (0.02 kg/day) are
used in the dosimetric calculation in the interest of consistency. The adjusted daily doses were
time weighted (i.e., [(4000 ppm x 4 -h 25 days) + (8000 ppm x 3-h 25 days) + (10,000 ppm x 18 ^
25 days)] x Food Consumption per Day x [l -h Body Weight] x [days dose ^ total days]).
Average daily food consumption was 12.3 g (84% of control) and 12.8 g (87% of control) for the
0.8%)- and 1.0%-dose groups, respectively. The corresponding adjusted daily doses are 678 and
811 mg/kg-day. Despite the decreased food intake in both dose groups compared to the control
group, mean total body-weight gain over the study period increased (120% of control) in the
0.8%-dose group, while it declined greatly (43% of control) in the 1.0%-dose group. Besides
body-weight gain and food consumption changes, no other BHT-related effects were reported.
Based on the diminished body-weight gain as compared to controls, a LOAELadj of
811 mg/kg-day is identified with a corresponding NOAELadj of 678 mg/kg-day.
In one subacute experiment among a series of experiments by Deichman et al. (1955b)
study, groups of four mongrel dogs (sex, age, and weight not reported) were administered doses
of0-, 1.4-, 2.1-, 3.2-, or 4.7-g/kg BHT in the diet 2 days per week for the first 2 weeks followed
by 3 days per week for the remaining 2 weeks in a 28-day study period. The corresponding daily
doses (adjusted for days dosed/total days) are 0, 600, 900, 1371, and 2014 mg/kg-day. All of the
animals exposed to BHT showed various degrees of diarrhea within 1-3 hours of exposure. This
effect was delayed over the course of the experiment, with diarrhea occurring 24 hours after
exposure by Week 4 of the study (data not reported). The study authors reported no other
clinical signs of toxicity. Gross pathology following sacrifice at the study termination showed
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hemorrhages, edema, and congestion in the lungs, which were attributed to the method (air
embolism) used for sacrifice (data not reported). No BHT-related pathologies were observed in
the gastrointestinal tract. Based on the study author's description of no chemical-related effects,
a NOAELadj of 2014 mg/kg-day is identified. This study will not support derivation of a
subchronic p-RfD due to a lack of methods and data reporting.
Subchronic-duration Studies
In a published, peer-reviewed study, Fulton et al. (1980) evaluated the effects of BHT
exposure on selected tissues in Wistar rats. Neat BHT (purity not specified) was administered to
10 male Wistar rats (weighing 36-63 g) at doses of 0, 0.02, 0.1, 0.5, or 0.75% of diet for
8 weeks. The study did not report appropriate body-weight data for use in the dose conversion
although food consumption data were reported. However, because food consumption and body
weight are related parameters, values provided for male Wistar weanling rats by EPA (1994b)
for body weight (0.053 kg) and food consumption (0.008 kg/day) are used in the dosimetric
calculation in the interest of consistency. The corresponding daily doses are 0, 30, 151, 755, or
1132 mg/kg-day. Food and water were available ad libitum. Food intake and body weights were
recorded daily during treatment but not reported. After 8 weeks, animals were sacrificed, and
liver weights were recorded. In addition, femoral bone marrow samples were obtained from
each animal. Erythrocyte counts were taken as an indicator of liver dysfunction. The proximal
ileum section of the small intestine was evaluated for villus height, crypt of Lieberkiihn depth,
and goblet cell count. Study authors performed statistical analysis using a partial correlation for
multivariant data. No information was provided regarding GLP compliance.
Table B.4 provides initial body weights, total food intake, body-weight gain, liver weight,
and relative liver-weight data reported by Fulton et al. (1980). Study authors reported
significantly higher food intake in the 0.02%- and 0.1%-dose groups and no statistical difference
in food consumed between other dose groups and controls. Total body-weight gain in the 0.5%-
and 0.75%-dose groups was significantly lower than that of controls, and body weight at
sacrifice decreased in a dose-related manner. Study authors stated there was no dose-related
trend in mean absolute liver weights but reported a statistically significant decrease in liver-to-
body-weight ratios as the level of BHT in the diet increased. However, this conclusion is not
supported by the data presented in the study report, which shows an increase with dose in the
liver-to-body weight ratio, as presented in Table B.4. Despite comments stating statistical
significance, statistical results associated with specific dose levels were not reported. An
independent statistical analysis could not be performed because only group means, without
standard deviations, were presented by the study authors.
Table B.5 presents the results of the ileal biopsy. Ileal biopsies revealed a nonsignificant
shortening and broadening of the villi with increasing dose and a statistically significant
(reported by the study authors) dose-related decrease in the depth of the crypts. Study authors
observed lower goblet cell counts in dose groups of 0.10% and higher but did not report
statistical analysis. Study authors observed no significant differences in the number of immature
erythrocytes between dose groups and controls upon examination of the femoral bone marrow.
The LOAELadj for Fulton et al. (1980) is identified to be 30 mg/kg-day in male rats based on
>10% increase in relative liver weight, considered to be biologically significant. Because the
LOAEL identified is the lowest dose administered, a NOAEL cannot be identified.
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In a 90-day albino Wistar rat study (Deichmann et al., 1955c), two different grades of
BHT (99.7 and 98.8%) were added to the diet for exposure concentrations of 0.2, 0.5, 0.8, and
1.0% BHT in 1.0% lard, resulting in eight exposure groups of three animals per group. Control
groups included feed alone as well as feed supplemented with 1.0% lard. Two additional groups
of rats were exposed to increasing concentrations of BHT (0.2% initially) at intervals of
3-4 days until a BHT concentration of 1.5% was reached. The sex, age, and weights of the rats
used were not reported. Appropriate body-weight data and food consumption data for dose
conversion were not provided in the study. Therefore, average values provided for Wistar rats
by EPA (1994b) for body weight (0.1865 kg for both sexes) and food consumption (0.018 kg/day
for both sexes) are used in the dosimetric calculation. It is also assumed that because the details
of the ramping method of dosing are not provided, the doses were consistent over the exposure
duration. The corresponding adjusted daily doses are 193, 483, 772, 965, and 1448 mg/kg-day,
respectively. The specific endpoints examined are not specified, along with many
methodological details such as the age and weight of animals at study initiation. No differences
in animals treated with the two BHT grades were observed. No effects were reported in the
animals exposed to 193 mg/kg-day. Increased mortality was observed at the 483 mg/kg-day and
greater doses (1/6, 2/6, 1/6, at the 483, 772, and 965 mg/kg-day doses, respectively, at 12 days, 4
and 5 weeks, and 13 weeks, respectively). Animals in the 1448-mg/kg-day group refused to eat,
and mortality in this group was 4/6 animals with one animal found dead at 4, 4, 9, and 11 weeks,
respectively. For this study, a NOAEL of 193 mg/kg-day is identified, but no LOAEL can be
determined because the next highest dose of 483 mg/kg-day is an FEL.
NCI (1979a) conducted a rat subchronic-duration study, which is summarized here, as
well as a mouse subchronic-duration study, which is summarized separately. Researchers
administered neat BHT (purity 99.9%) by diet to 5 F334 rats per sex per dose at levels of 0,
6200, 12,500, 25,000, or 50,000 ppm for 7 weeks and then observed animals for 1 week.
Appropriate body-weight data and food consumption data for dose conversion were not provided
in the study. Therefore, average values provided for F344 rats by EPA (1994b) for body weight
(0.18 kg for males and 0.124 kg for females) and food consumption (0.018 kg/day for males and
0.014 kg/day for females) are used in the dosimetric calculations. The corresponding adjusted
daily doses are 0, 620, 1250, 2500, and 5000 mg/kg-day for males and 0, 700, 1411, 2822, and
5645 mg/kg-day for females. Feed and water were supplied continuously ad libitum for the
duration of the study. The study authors weighed each animal prior to the study period and then
twice weekly until study termination; however, body-weight data were not reported in the study.
All of the animals were sacrificed using CO2 and necropsied at the conclusion of the study. The
study authors conducted histopathological examinations (specific endpoints unreported) of each
animal following necropsy. The study authors used a 10% depression in body weight as a major
criterion for the estimation of maximum tolerated doses (MTDs). Least squares regression of
mean body weight per number of days was applied to estimate the final mean body weights of
each group. Study authors plotted probits of the percentage weights of each dose group at
Day 49 relative to those of the corresponding control groups against the logarithms of the doses.
Study authors then used fitted least squares regression to estimate the doses that induced 10%
reduction in body weight.
NCI (1979a) observed decreased survival rates with increasing dose and 100% mortality
rate for both males and females at the highest dose level (see Table B.6). Decreased mean body
weights also were observed with increasing dose, and this trend was more pronounced in males
than females. The 10% depression threshold in mean body weight was recorded at
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620 mg/kg-day in males and 1411 mg/kg-day in females. Study authors also reported slight
increased hematopoiesis in rats treated with 1250 mg/kg-day in males and 1411 mg/kg-day in
females (date not shown). Based on a 10% weight loss in male rats, a LOAELadj of
620 mg/kg-day is identified. This LOAEL corresponds to the dose at which males lost at least
10% mean body weight. Because the dose at which the LOAEL is reported was the lowest dose
administered, a NOAEL cannot be identified.
Parallel to the subchronic-duration study in rats, NCI (1979b) published results from a
subchronic-duration study in which they administered groups of five B6C3Fi mice per sex per
dose dietary concentrations of 0, 3100, 6200, 12,500, 25,000, or 50,000 ppm BHT for 7 weeks,
followed by 1 week of observation. Appropriate body-weight data and food consumption data
for dose conversion were not provided in the study. Therefore, average values provided for
B6C3Fi mice by EPA (1994b) for body weight (0.0316 kg for males and 0.0246 kg for females)
and food consumption (0.0057 kg/day for males and 0.0048 kg/day for females) are used in the
dosimetric calculation. The corresponding adjusted daily doses are 0, 559, 1118, 2255, 4510,
and 9019 mg/kg-day for males and 0, 605, 1210, 2439, 4878, and 9756 mg/kg-day for females.
The study was performed using the same methods of exposure, examination parameters, and
statistical analysis as the subchronic-duration study in rats described previously (NCI, 1979a).
NCI (1979b) reported mortality in the males (1/5) and females (4/5) in the high-dose
group (see Table B.7). Study authors observed a decreasing trend in mean body weight with
increasing dose in males. A similar trend was also observed in females, except in the highest
dose group, in which the remaining single surviving female had a mean body weight of 97% of
control. Males and females in the lowest dose group experienced a threshold decrease in mean
body weight. In addition to body-weight changes, the study authors also reported observing
findings of centrilobular cytoplasmic vacuolation in the livers of male animals in the highest
dose group (quantitative data not reported). Based on a 10% loss in body weight in male mice, a
LOAELadj of 559 mg/kg-day is identified. Because this was the lowest dose administered, a
NOAEL cannot be identified.
Chronic-duration Studies
In a peer-reviewed publication, Williams et al. (1990) investigated the oral toxicity of
BHT (99% purity determined by thin layer chromatography [TLC]) in male F344 rats in two
studies. The study authors administered a basal diet (NIH-07) containing 100, 300, 1000, 3000,
or 6000 ppm BHT to groups of 21 six-week-old male (100 g) rats for 76 weeks in one study, and
a single 12,000 ppm BHT dose to groups of 27 eleven-week-old male (200 g) rats for 110 weeks
in the second study. A separate group of 36 six-week-old (100 g) and 27 el even-week-old male
(200 g) rats served as control groups, respectively. Appropriate body-weight data and food
consumption data for dose conversion were not provided in the studies. Therefore, average
values provided for male Fisher 344 rats by EPA (1994b) for body weight (0.38 kg) and food
consumption (0.03 kg/day) are used in the dosimetric calculation. The adjusted daily doses are
8, 24, 79, 237, and 474 mg/kg-day for the 76-week study and 0 and 947 mg/kg-day for the
110-week study. Four rats from each group treated for 76 weeks were sacrificed 12, 36, 48, and
76 weeks after study commencement for hepatocellular foci analysis. The study authors
recorded body weights every 4 weeks. Complete autopsies on all animals were performed at
study termination (i.e., 76 and 110 weeks). The study authors recorded liver weights, took slices
from each lobe for staining, and tested for iron to identify iron-deficient lesions.
Histopathological examinations were performed on the liver and other unspecified organs.
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Morphological analysis of altered cellular foci in the liver was performed by microscopic
quantization of the number of foci per cm2, adjusted, and presented to be relative to the total area
of the liver section. Statistical analysis was performed using the Student's Mest to analyze
differences between groups and Fisher's exact probability test to analyze neoplasm incidence.
No information is presented regarding GLP compliance.
Williams et al. (1990) reported no mortalities in rats administered BHT for 76 weeks.
Rats in the control and treatment groups exposed for 110 weeks displayed increased mortality
beginning at Week 84, with 11/27 and 9/27 mortalities, respectively, noted at study termination.
Body-weight gain was reduced significantly in rats administered 3000- (89% of control) and
6000-ppm BHT (89% of control) for 76 weeks and in rats administered 12,000-ppm BHT (89%
of control) for 110 weeks, relative to controls (see Table B.8). Increased absolute liver weights
were observed in rats in the 76-week 6000-ppm dose group (32% over control), relative to
controls. Relative liver weight (per 100 g body weight) was biologically significantly increased
(11% over control) in the 3000-ppm dose group and statistically and biologically significant
(50%) over control) in the 6000-ppm dose group. The size of hepatocellular foci increased
slightly—but not significantly—in the 6000-ppm dose group (76 weeks) and decreased
slightly—but not significantly—in rats in the 12,000-ppm group (110 weeks) (see Table B.9).
The study authors observed mild hyperplasia in the stomachs of all examined groups, including
control (controls, 6000 ppm, controls, and 12,000 ppm) (see Table B.10). Incidence was not
dose related, and no other gastric lesions were identified in any of the groups.
Neoplasm incidence was measured by Williams et al. (1990) and was not significant in
either study and the study authors concluded that BHT alone is not a carcinogen. For the 76-
week study, the biologically significant (>10%) increase in relative liver weight in animals
exposed to 3000- and 6000-ppm BHT supports a LOAELadj of 237 mg/kg-day and a
corresponding NOAELadj of 79 mg/kg-day. For the 110-week study, a LOAELadj of 947
mg/kg-day is identified based on decreased body weight (>10%). Because this is the only dose
tested, identification of a NOAEL is precluded.
Hirose et al. (1993) conducted a published, peer-reviewed 36- to 40-week study in which
they exposed groups of F344 male rats (5 weeks of age, weight unreported), obtained from
Charles River Japan, Inc. (Kanagawa, Japan), to a series of carcinogens, followed by treatment
with antioxidants including BHT. The study authors also exposed rats to BHT (>98% purity) or
other potential antioxidants individually. Hirose et al. (1993) exposed a group of 12 animals to
0.7%- (7000-ppm) BHT in powdered basal diet for 36 weeks, whereas 11 control animals
received only basal diet. Appropriate body-weight data and food consumption data for dose
conversion were not provided in the study. Therefore, average values provided for F344 male
rats by EPA (1994b) for body weight (0.18 kg) and food consumption (0.018 kg/day) are used in
the dosimetric calculation. The adjusted daily doses are 0 and 700 mg/kg-day. Rats were housed
five to a cage in plastic cages at 24 ± 2°C on 12-hour-light/dark cycle. Their basal diet consisted
of Oriental MF (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum. The animals were
weighed every 2-4 weeks during treatment with BHT. Surviving animals with tumors in any of
the experimental groups were included for analysis. Animals were sacrificed and autopsied after
36 weeks of treatment. At study termination, liver and kidneys were weighed, and portions were
removed for immunohistochemical staining for glutathione ^'-transferase placental form
(GST-P). The number of lung lesions and the number and area of GST-P-positive foci were
measured. After the data collection, Student's Mests and Fisher's exact probability tests were
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used in the statistical analysis of the data. No information on GLP compliance was provided by
the study authors.
Hirose et al. (1993) reported statistically significantly decreased body weights (87% of
control) in rats treated with BHT (n= 12) compared to those of the control groups (n = 11) (see
Table B. 11). Relative liver weight was significantly greater in rats treated with BHT (176%).
The relative kidney weight also was increased significantly (128%) (see Table B.l 1). However,
3 of 12 rats treated only with BHT died of abdominal hemorrhage. No other treatment-related
effects of BHT were reported by the study authors, and no further data were provided.
As noted above, Hirose et al. (1993) observed that in the only dose of BHT administered
(700 mg/kg-day or 0.7% in powdered food), 3 of the 12 rats died of hemorrhage. Due to this
frank effect, an FEL of 700 mg/kg-day is identified. Because this is the only dose tested,
identification of a NOAEL is precluded.
Hirose et al. (1981) published a 2-year, peer-reviewed, chronic-duration study
investigating the toxicity of BHT. The study authors administered a basal diet containing 0%,
0.25% (2500 ppm), and 1% (10,000 ppm) BHT (purity not reported) in pellet form to 36, 57, and
57 seven-week-old male and female Wistar rats (100-200 g at study initiation), respectively.
Appropriate body-weight data and food consumption data for dose conversion were not provided
in the study. Therefore, average values provided for Wistar rats by EPA (1994b) for body
weight (0.462 kg for males and 0.297 kg for females) and food consumption (0.034 kg/day for
males and 0.025 kg/day for females) have been used in the dosimetric calculation. Adjusted
daily doses are 0, 184, and 736 mg/kg-day for males and 0, 210, and 842 mg/kg-day for females.
The study authors recorded body weights weekly for the duration of the study and measured food
consumption at unspecified regular intervals. After the treatment period, study authors sacrificed
animals and collected blood samples for measurement of clinical chemistry parameters including
red and white blood cell counts, hemoglobin, hematocrit, glutamic-oxaloacetic transaminase
(GOT), glutamic-pyruvic transaminase (GPT), alkaline phosphatatase, cholinesterase, y-glutamyl
transpeptidase (y-GTP), total protein, albumin/globulin, thymol turbidity, total cholesterol,
triglyceride, P-lipoprotein, blood urea nitrogen, creatinine, uric acid, total bilirubin, sodium,
potassium, chloride, and inorganic phosphate. They also measured organ weights for the liver,
spleen, and kidneys and performed histological examinations of the liver, pancreas, mammary
gland, uterus, pituitary gland, adrenal gland, lung, thyroid, and kidneys. Study authors did not
provide any information on GLP compliance. Statistical analysis did not include rats surviving
less than 69 weeks. Study authors performed statistical analysis using the chi-square test when
comparing groups and the Student's Mest to analyze variance and differences between means.
Hirose et al. (1981) reported increased mortality in high-dose males from Week 96 until
the end of the study (approximately 60% when compared to 35% of controls, estimated using
graph-digitizing software). Mortality prior to Week 96 was 28% (10/36), 25% (14/57), and 33%
(19/57) in males and 11% (4/36), 19% (11/57), and 10.5% (6/57) in females in the 0-, 0.25-, and
1%-dose groups, respectively. Dietary administration of BHT significantly decreased body
weight in male rats with recovery occurring by Week 36 in the low-dose group and by Week 60
in the high-dose group. Females administered 0.25%-BHT experienced a significant decrease in
body-weight gain at Weeks 12 and 48, and the females administered 1%-BHT also experienced a
decrease throughout the study. Most animals recovered over the course of the study period, as
reported by the study authors, although the body-weight data, presented graphically by the study
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authors, cannot be confirmed by graph-digitizing software used for this review. Males and
females in all BHT dose groups displayed increased absolute (data not shown) and relative liver
weights at study termination (see Table B. 12). Average relative liver weights were 2.5, 4.1 (64%
over control), and 3.7 g/100 g (48% over control) body weight in males and 2.8, 2.9 (4% over
control), and 3.5 g/100 g (25% over control) body weight in females in the 0-, 0.25-, and
1%-dose groups, respectively. Male rats did not display decreased spleen weights, but average
relative spleen weights were decreased (62 and 68% of controls) per 100 g body weight in
females in the 0.25- and 1%-dose groups compared to controls. Individual animal spleen
weights and the variance of the mean or absolute spleen-weight data were not reported. No other
organ-weight changes were reported. Treatment-related effects seen during hematological and
serum biochemistry evaluations are provided in Table B.13. At the end of the study, females in
both treated groups had increased red blood cell (RBC) counts. White blood cell (WBC) counts
were decreased at the low dose in males and in females at the low- and high-dose levels (data not
reported by study authors). Changes in RBC and WBC counts were not dose dependent. In the
0.25- and 1%- groups, serum triglyceride in males was significantly lower (140 and 137 mg/dl,
respectively, compared to 180 mg/dl in the control group), y-GTP in males was significantly
higher (3.8 and 4.4 mU/dl, respectively, compared to 2.8 mU/dl in the control group), and total
cholesterol in females was significantly higher (99.2 and 112 mg/dl, respectively, compared to
73.4 mg/dl in the control group). Total cholesterol in males was significantly higher only in the
0.25%-group (94.8 mg/dl compared to 83.1 mg/dl in the control group). No other changes in
hematology or serum biochemistry were reported. While a significant increase in pituitary
adenomas (13%) was reported in the low-dose females, the effect was reportedly not related to
chemical exposure, as the incidence was not as high in the high-dose females (11.8%). No
treatment-related nonneoplastic or neoplastic lesions were observed in BHT-treated animals
although nonsignificant increases were reported in the incidence of total tumors, which were
higher in the 0.25%-dose group than the 1%-dose group, and, therefore, reported not to be dose
related. In males, there was a small increase in tumors of the pancreas of treated animals
(carcinomas: 2.6% in the 1.0%-dose group as compared with 0% in the 0.25%- and control
groups; islet-cell adenoma: 2.3% in the 0.25%-dose group, and 5.3% in the 1.0%-dose group, as
compared to 0% in the control group). Female animals showed small increases in the incidence
in tumors of the liver (6.5% in the 0.25%-dose group and 5.9% in the 1%-dose group, as
compared to 0% in the control group) (see Table B.14).
Statistically significant changes were observed in levels of triglycerides in male rats,
y-GTP in male rats, and total cholesterol in both sexes. These enzyme effects are supported by
increased relative liver weights, indicating liver injury. Changes in the RBC and WBC levels,
while significant, are not supported by examination of the hematopoietic system for
morphological abnormalities by the study authors. Total tumor incidence was reported by the
study authors as not related to BHT treatment because dose-related increases in tumors were not
observed. Due to the increased mortality representing a frank effect in the high-dose treated
males (60%) as compared to controls (35%), the endpoints measured at this level cannot be
considered. A LOAELadj of 184 mg/kg-day is identified based on a 64% increase in relative
liver weight in male rats, considered to be biologically significant; a NOAEL cannot be
identified because liver effects were seen at the lowest administered dose.
Two chronic-duration studies, peer-reviewed and published by Deichmann et al.
(1955d,e), were performed according to the same methods as previously described for the
subacute and subchronic studies. These studies were performed prior to adoption of GLP. In a
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2-year chronic toxicity study (Deichmann et al., 1955d), study authors administered groups of
15 male and 15 female albino Wistar rats 0.2% (2000 ppm), 0.5% (5000 ppm), 0.8%
(8000 ppm), and 1.0% (10,000 ppm) BHT (purity not reported) in 1% lard in diet. The study
authors included a control group of rats fed 1% lard and a group exposed to 0.5% (5000 ppm)
BHT dissolved in lard and heated at 150° C for 30 minutes. This experiment was conducted to
assess the impact of heating on the stability and toxicity of BHT. Appropriate body-weight data
and food consumption data for dose conversion were not provided in the study. Therefore,
average values provided for Wistar rats by EPA (1994b) for body weight (0.462 kg for males and
0.297 kg for females) and food consumption (0.034 kg/day for males and 0.025 kg/day for
females) are used in the dosimetric calculation. The adjusted daily doses are 147, 368, 589, and
736 mg/kg-day for males and 168, 421, 673, and 842 mg/kg-day for females. Body weights
were measured once per week until stable (time point unspecified) at which point body weights
were determined once per month. Measurements of blood counts and hemoglobin were taken "at
intervals" in randomly selected animals although the study authors reportedly selected animals
that appeared less healthy (Deichmann et al., 1955d). Study authors conducted gross
pathological examination on all animals surviving until study termination including recording
weights of major organs and preparing tissue for micropathological analysis. Microscopic
analysis of organs (brain, lung, heart, spleen, liver, kidneys, and enteric tract) involved paraffin
embedding of tissue, followed by staining with hematoxylin, eosin, and/or Sudan III.
Deichmann et al. (1955d) reported mortalities in various groups (data were not reported
by study authors), but concluded that they were not dose-related. There was no noteworthy
effect on the number of erythrocytes and leucocytes or the hemoglobin concentration (see
Tables B.15 and B.16). There were also biologically significant increases in relative liver and
kidney weights at some doses in both male and female rats. Specifically, there was an 11%
increase in relative kidney weight in female rats at a dose of 168 mg/kg-day, see Table B.16.
Pathological examinations indicated no dose-related effects. Both males and females suffered
from pneumonia, but incidence was higher in the low-dose groups and controls compared to the
high-dose group. Similarly, neoplasms were identified, however incidence in the control group
(4/6 females, 2/6 males), and a lack of incidence in the high-dose group, indicated that
neoplasms were not related to dosing. There were no abnormalities identified during
pathological examinations of animals in the 1%-dose group. A LOAELadj of 168 mg/kg-day
(0.2-dose group in females) is identified for causing a biologically significant increase in relative
kidney weight. Because this was the lowest dose administered, a NOAEL cannot be identified.
In an unpublished (not peer-reviewed) chronic toxicity study to examine changes in
growth, mortality, lifespan, and appearance of tumors (Tokyo Metropolitan Research Laboratory
of Public Health, 1992a), male and female JCL:S-D rats were fed BHT (purity not reported) in
the diet at doses of 0, 2.5, 10, or 160 mg/kg-day (40/sex/dose group) for 3, 6, 12, or 24 months or
throughout the lifespan (total number of weeks not specified). BHT was added to powder feed
CE-2 of Nihon Clea Co., Ltd. The animals were housed individually in cages suspended on
belt-type racks that were equipped with an automatic water supply system. Diet and water were
given ad libitum. Environmental conditions were described by a temperature of 25 ± 1°C,
55 ± 5% humidity, and a 13-hour light/11-hour dark cycle. No information is provided regarding
GLP compliance.
The Tokyo Metropolitan Research Laboratory of Public Health (1992a) observed
behavior daily; body weight and food intake were measured in the lifespan group weekly for the
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first 3 months, every 2 weeks from 4-8 months of feeding, then every 4 weeks until 24 months
of feeding. At the end of the 3-, 6-, and 12-month feedings, animals were sacrificed from all
three groups, and liver, kidney, heart, spleen, thyroid, and caecum weights were recorded.
Additionally, study authors performed hematological examination, serum biochemical
examination, urinalysis, and histological examination of tissues. After necropsy of animals in
the 24-month feeding group, study authors recorded weights of heart, liver, kidney, spleen,
pituitary, thyroid, adrenal, testis, prostate, ovary, and uterus. Study authors also recorded
mortality, and completed hematological examination, serum-biochemical examination,
urinalysis, and histological examination of tissues and neoplasms. For the lifespan group,
detailed methods of observation were not provided in the text of the report. However, review of
the study tables indicates that the same observations and measurements were recorded as those
for the 24-month feeding group. Animals in moribund condition were sacrificed and some
(unspecified) examinations were performed. Rats found dead were autopsied, and histological
examinations of organs, tissues, and neoplasms were conducted. Statistical analysis of results
was performed using the Student's Mest and the rank sum test.
The Tokyo Metropolitan Research Laboratory of Public Health (1992a) reported no
noticeable differences in behavior between the control group and each of the treated groups. For
the lifespan feeding group, body weights and rates of body weight change were presented in a
report table, but most values in that table were illegible. Food intake rates for the lifespan
feeding group were presented in report tables, but values in those tables were generally illegible.
However, study authors reported that there were no noticeable differences in mean daily food
intake in each treated group compared to the control for animals in the lifespan feeding group.
Pages of the Tokyo Metropolitan Research Laboratory of Public Health (1992a) report
containing tables and figures with mortality data for the 24-month and other durations appear to
be missing. However, the study authors indicated that there were no treatment-related increases
or decreases in mortality for the 3-, 6-, or 12-month feeding groups. Males and females in the
24-month feeding group experienced increased mortality compared to controls—mortality in the
160-mg/kg-day dose group was noted to be statistically significantly higher compared to the
control (using the Fisher direct probability calculation). Mortality in the lifespan feeding group
was not significantly different than controls.
The Tokyo Metropolitan Research Laboratory of Public Health (1992a) described
findings of the urinalyses, hematological examinations, and macroscopic examinations during
necropsy in the report; however, tables with results are missing from the study report. The study
authors stated that there were no marked differences between the control and each BHT-treated
group in these parameters. Study authors present findings of the serum-biochemical examination
in the text of the study report; however, tables with results are missing from the report.
Statistically significant results were reported as follows for specific tests.
•	Serum K+ was significantly elevated compared to controls in the 3-month feeding
group for males at 2.5, 10, and 160 mg/kg-day and females at 160 mg/kg-day. It was
suggested by the study authors that significantly increased K+ in both sexes at
160 mg/kg-day indicates the effect of BHT at an early stage of feeding.
•	For serum cholesterol, slightly higher values were generally observed in treated rats
until the 12-month feeding. Cholesterol was significantly elevated in the 12-month
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feeding group females at 2.5 mg/kg-day, but was significantly lowered in the
24-month females at 2.5 mg/kg-day.
• Transaminase activities (i.e., GOT and GPT) were significantly lower in the 6-month
feeding group males at 160 mg/kg-day compared to controls.
The 12-month feeding group males at 160 mg/kg-day showed higher values in all
biochemical examinations compared to mean values; however, study authors suggested that this
was due to abnormally high values in one animal (Tokyo Metropolitan Research Laboratory of
Public Health, 1992a). The study authors also observed abnormal values for some examinations
in the 24-month feeding group, but none were statistically significant. Results of organ weight
measurements are described in the study report; however, tables with results are missing from
the report. In the 160-mg/kg-day dose group, the study authors observed a trend for increased
liver weight in each feeding group related to BHT intake. Histopathological findings in all
feeding groups were presented in report tables; however, values in those tables were illegible. In
the text, the study authors noted histological changes were believed to be related to BHT intake.
In the 160-mg/kg-day dose group, there was slight swelling of hepatic cells (after 3 months) and
aggregation of basophilic substances at the hepatic cytoplasmic peripheral region (after
6 months) but these findings were not observed in the 24-month and lifespan feeding groups.
There was vacuolized degeneration of renal tubule epithelium, but the dose levels at which this
was seen were not specified.
The Tokyo Metropolitan Research Laboratory of Public Health (1992a) did not observe
tumors in the 3-, 6-, or 12-month feeding groups. Tumors that occurred relatively frequently in
the 24-month and lifespan groups included: mammary gland in females, pituitary in males and
females, and adrenal gland in males; all tumors were observed in each BHT-treated group as well
as the control group. Other malignant tumors occurred sporadically (one or two cases) in other
tissues, including the abdominal muscle, kidney, skin and subcutaneous tissue, thoracic cavity,
abdominal cavity, pancreas, intestinal wall, and uterus.
At the highest dose (160 mg/kg-day BHT), the Tokyo Metropolitan Research Laboratory
of Public Health (1992a) noted a tendency toward increased liver weight, serum cholesterol, and
serum K+. Histological changes in the liver and kidney were also observed at this dose.
However, there were no BHT-related changes in the quantity of food intake, body-weight gain,
mortality, and mean lifespan. The study authors also believed that BHT did not cause induction
of malignant tumors and indicated that their occurrence in treated rats happened spontaneously.
The study authors concluded that ingestion of BHT in feed (at 2.5, 10, or 160 mg/kg-day) did not
cause harmful effects on the study animals. As mentioned several times in the summary of this
study, the data tables are barely legible and therefore the qualitative statements made by the
study authors cannot be verified quantitatively, making this study unusable for derivation of a
subchronic p-RfD.
In a published, peer-reviewed, chronic-duration oral study, NCI (1979c,d) examined
toxicity and carcinogenicity of BHT on F344 rats and B6C3Fi mice. The chronic rat portion of
the study (NCI, 1979c) is summarized here, and the chronic mouse portion (NCI, 1979d) is
summarized separately. Neat BHT (99.9% purity) was administered by diet to 50 six-week-old
animals per sex per dose at doses of 0, 3000, or 6000 ppm for 105 weeks. Appropriate
body-weight data and food consumption data for dose conversion were not provided in the study.
Therefore, average values provided for F344 rats by EPA (1994b) for body weight (0.38 kg for
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males and 0.229 kg for females) and food consumption (0.03 kg/day for males and 0.021 kg/day
for females) are used in the dosimetric calculation. Corresponding adjusted daily doses are 0,
237, or 474 mg/kg-day for males and 0, 275, or 550 mg/kg-day for females. Study authors did
not report whether or not they adhered to GLP standards. BHT was mixed with Wayne®
Sterilizable Lab Meal and made available ad libitum. Study authors examined animals twice
daily for sick, moribund, and tumor-bearing animals and performed clinical examinations and
palpations for masses each month. Animals were weighed at least once per month. The study
authors made peripheral blood smears for each animal "whenever possible." Upon termination
of the study, surviving animals were sacrificed using CO2 and necropsied. All animals found
dead were necropsied as well, except in cases of advanced autolysis or cannibalism.
Study authors of the NCI (1979c) study used the Carcinogenesis Bioassay Data System to
record and process data, using data elements recommended by the International Union Against
Cancer. Microscopic pathological parameters included skin, lungs and bronchi, trachea, bone
marrow (femur), spleen, lymph nodes (mesenteric and submandibular), thymus, heart, salivary
glands (parotid, sublingual, and submaxillary), liver, pancreas, esophagus, stomach (glandular
and non glandular), small and large intestine, kidney, urinary bladder, pituitary gland, adrenal
gland, thyroid, parathyroid, testes, prostate, uterus, ovaries, brain (cerebrum and cerebellum),
and all tissue masses. Study authors based all statistical tests on animals that survived at least
52 weeks or until the first tumor at a particular site was discovered. The study authors used the
product-limit procedure of Kaplan and Meier to estimate probabilities of survival. They
performed an analysis of dose-related effects on survival between two groups by the method of
Cox and tested for trend using Tarone's extension of the Cox method. Study authors used the
Fisher exact test to compare tumor incidence between dose groups and controls and applied the
Bonferroni inequality correction when testing multiple groups. The study authors tested for
linear trend in proportions using the Cochran-Armitage test. They used life-table methods to
analyze the incidence of tumors, comparing survival curves using the method of Cox and
Tarone's extension. The study authors computed 95% confidence intervals for the relative risk
of each dose group compared to the control from the exact interval on the odds ratio.
Study authors noted that treatment in the NCI (1979c) study resulted in dose-related
decreases of mean body weight in both males and females (no data provided). The Tarone test
for dose-related mortality did not provide significant results for males or females although
mortality rates at both dose levels were lower than controls for both sexes (see Table B.17).
Histopathological examination revealed an apparent dose-related incidence of focal alveolar
histiocytosis in the lungs of males and females (see Table B. 18) although the study authors
reported no statistical analysis. Independent statistical analysis performed for this review
(X test) indicated a significant increase in focal alveolar histiocytosis in female rats at the
high-dose level. Other lesions commonly seen in aged F344 rats were observed but were not
dose-related. The Cochran-Armitage test for positive dose-related incidence of tumors and the
Fisher exact test comparing incidence of tumors in dose groups to controls were not significant
for all examination parameters for each sex. However, the study authors observed significant
results in the negative direction for incidence of adenomas of the pituitary gland in high-dose
females (see Table B. 19). No positive dose-related trends were apparent in primary tumors,
benign tumors, or malignant tumors (see Table B.20). Study authors noted that the confidence
interval for each measured tumor incidence, except that for incidence of adenomas in the
pituitary gland of high-dose females, displayed an upper limit greater than one, indicating the
possibility of tumor induction by BHT that could not be detected in this test.
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Given the histopathological and statistical findings, NCI (1979c) concluded that BHT did
not induce neoplastic or nonneoplastic lesions and, thus, it is not carcinogenic in the F344 rat at
the doses administered in this study. Based on the increased incidence of alveolar focal
histiocytosis in female rats exposed to BHT, a LOAELadj of 550 mg/kg-day is identified, with a
corresponding NOAELadj of 275 mg/kg-day. The data for increased incidence of alveolar focal
histiocytosis in female rats are further evaluated with the BMDS modeling program for
determination of a POD for the chronic p-RfD.
Parallel to the NCI (1979c) study involving rats, NCI (1979d) published a peer-reviewed
chronic-duration study in which study authors administered BHT (99.9% purity) in the diet at
concentrations of 0, 3000, or 6000 ppm to 50 six-week-old B6C3Fi mice per sex per dose for
107 weeks. Appropriate body-weight data and food consumption data for dose conversion were
not provided in the study. Therefore, average values provided for B6C3Fi mice by EPA (1994b)
for body weight (0.0373 kg for males and 0.0353 kg for females) and food consumption
(0.0064 kg/day for males and 0.0061 kg/day for females) are used in the dosimetric calculation.
Corresponding daily doses are 0, 515, or 1029 mg/kg-day for male mice and 0, 518, or
1037 mg/kg-day for female mice. Study authors applied administration methods, examination
parameters, and statistical analyses as described in the 105-week NCI (1979c) study on rats. No
information was provided regarding GLP compliance.
Treatment resulted in dose-related reduction of mean body weights of males and females
(data not shown) (NCI, 1979d). In females, the Tarone test for dose-related mortality rate was
not significant; however, the test was significant for males in the negative direction (i.e., treated
lived longer then controls). Table B.29 displays the data for survival of male and female mice.
Histopathological examination revealed a high incidence of multiple proliferative lesions of the
liver and lungs in both sexes. Lesions also occurred in the lacrimal gland but were only
examined when grossly apparent and, thus, cannot be evaluated in relation to BHT treatment (see
Table B.30). Statistical tests for dose-related increased incidence of the liver adenoma and
carcinoma were only significant for hepatocellular carcinomas at the highest dose in males.
Dose-related and statistically significant increased incidences of hepatocytomegaly and other
nonneoplastic liver lesions including peliosis, hepatocellular degeneration/necrosis, and
cytoplasmic vacuolation, were observed in males but not in females. Incidence of
alveolar/bronchiolar carcinomas or adenomas in low-dose females was significantly higher than
the control, but the Cochran-Artmitage test of trend was not significant because a lower
incidence was seen with the high dose. Study authors found a significant dose-related trend in
incidence of adenomas of the lacrimal gland in males, but results of the Fisher exact test were not
significant. Cochran-Armitage and Fisher exact tests for all other examination parameters were
not significant. NCI (1979d) reported that no dose-related tumors were observed following
treatment with BHT (see Table B.31).
NCI (1979d) concluded that BHT was not carcinogenic in B6C3Fi mice of either sex.
However, there were dose-related increases in nonneoplastic lesions in livers of male mice.
Based on these observations, a LOAELadj of 515 mg/kg-day is identified. Effects were
observed at the lowest dose administered, precluding identification of a NOAEL. The data for
these effects (i.e., hepatocytomegaly, liver peliosis, cytoplasmic vacuolation, and hepatocellular
degeneration/necrosis) are further evaluated with the BMDS modeling program for
determination of a POD for the chronic p-RfD.
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Inai et al. (1988) conducted a published, peer-reviewed study of the chronic effects of
BHT in mice. B6C3Fi mice were obtained from Charles River Japan at 4 weeks of age and
observed for another 4 weeks prior to testing. Mice were housed in plastic cages, 5 males or
10 females per cage, in the same air-conditioned room. Male and female mice, 50/sex/group,
were administered 0, 1, or 2% BHT (96% purity) in diet for 104 weeks, followed by a 16-week
recovery period. Average daily doses reported by the study authors were 0, 1640, and
3480 mg/kg-day for males and 0, 1750, and 4130 mg/kg-day for females. However, the study
authors note that incorporation of BHT into the food pellets was approximately 50% of the
original content. Further data on this is not provided in the report, making assessment of the
actual doses administered to each animal problematic. All animals were given feed and water ad
libitum. The amount of food consumed and body weight were measured biweekly for the first
12 weeks and once every 4 weeks for the rest of the treatment period. At the end of the study, all
of the surviving animals were sacrificed and necropsied. Animals that died prematurely also
were necropsied. Tissues that were evaluated microscopically included the liver, lung,
hematopoietic system, spleen, integumentary system, uterus, ovary, breast, pancreas, esophagus,
forestomach, intestine, brain, pituitary gland, parathyroid gland, heart, and eyelid. Data on
clinical chemistry were not reported. Organ weight is only provided for the liver. No
information on GLP compliance is provided by the study authors.
Inai et al. (1988) reported a dose-dependent decrease in survival throughout the study in
males but not in females. Dose-dependent decreases in average body weights were reported in
the males and females (quantitative data not reported and graphical data could not be analyzed
by the graph digitizer and thus data were not shown). Study authors reported increased absolute
and relative liver weights in mice that did not develop liver tumors (see Table B.32). No
changes in absolute or relative organ weights were seen in mice with hepatocellular tumors or in
total mice groups. Hepatocellular lesions for male mice are shown in Table B.33.
Dose-dependent increases in the number of foci of cellular alterations in hepatocytes and
hepatocellular adenomas were seen in males but not females. The study authors also stated that
nuclear pleomorphisms of hepatocytes in nontumorous areas were present but there were no
hepatocellular necrosis, bile duct hyperplasia, or peliosis (data not reported). However, due to
concerns regarding the accuracy of the reported doses, this study can only serve as supporting
information, and a dose-response assessment is not appropriate. For these reasons, neither a
NOAEL nor LOAEL can be derived from this study.
Hirose et al. (1986) published a peer-reviewed 16-week chronic study investigating the
toxicity of BHT. The study authors administered a basal diet (5.1% fatty acid, 24.5% oleic acid,
48.5%) linoleic acid, and 14.1%> palmitic acid) containing 0 or 1% BHT (>98% purity) in
powdered form to 26-32 (specific number not reported) 7-week-old male Syrian golden
hamsters (weighing 85-115 g at study initiation) for 16 weeks. Adjusted daily doses were 0 and
183 mg/kg-day for males. The study authors recorded body weight weekly for the first 4 weeks
and then monthly for the remainder of the study. The study authors did not provide data on food
consumption throughout the study. In each treatment group, three hamsters were sacrificed at 1,
2, 3, and 4 weeks, and all of the remaining animals were sacrificed at 16 weeks for histological
and autoradiographic examination. The histological examination included a complete
postmortem examination and measurement of liver and kidney weight. No information on GLP
compliance was provided by the study authors. No information on the statistical analysis
performed by the study authors was presented although significant findings were listed in the
results section. No raw data were provided by the study authors.
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Hirose et al. (1986) noted that dietary administration of 1% BHT did not significantly
affect body weight compared to control. Absolute liver weight was higher (although not
significantly) from Day 3 to the conclusion of the study compared to the control. BHT
administration did not significantly increase forestomach lesions or the labeling index (indicative
of cell proliferation) in the forestomach in hamsters after BHT exposure.
Hirose et al. (1986) also administered 2-fert-BHA, 3-/C/7-BHA, and crude BHA in
parallel to the BHT experiment using the same protocol. The study authors found increased
incidence of labeled cells in the forestomach epithelium for animals exposed to BHA. The study
authors discussed the possibility that the fatty acids in the basal diet may have become oxidized,
allowing the test substance to attack the forestomach cells. They further suggested that BHT did
not release enough free radicals to do significant damage as compared to BHA. Despite not
identifying adverse effects of BHT administration, their finding presents an interesting
explanation of interaction between the exposure compound and the components of the diet.
Hirose et al. (1986) did not identify a NOAEL or a LOAEL in this study. The possibility of
chemical interactions between the feed and the chemicals administered, together with the lack of
raw data, preclude identification of a NOAEL or a LOAEL.
Deichmann et al. (1955e) exposed mongrel two dogs for 1 year via diet to average doses
of BHT of 170, 280, 500, 470, 640, or 940 mg/kg-day (purity unreported) in ground beef. Dogs
showed no signs of toxicity and unlike in the subacute study in dogs did not experience diarrhea.
Histopathological analysis of several tissues (brain, pituitary, thyroid, thymus, heart, aorta,
trachea, lungs, spleen, stomach, intestine, pancreas, liver, mesenteric lymph nodes, adrenal
glands, kidneys, urinary bladder, uterus, ovaries, testes, epididymis, and prostate) did not show
any pathologies related to BHT administration. The results presented in this review are for
supporting purposes due to the lack of methodological details. Based on the lack of effects
reported in the study a NOAEL of 940 mg/kg-day is identified. A LOAEL cannot be identified
because no effects were reported at any of the administered doses.
Developmental and Reproductive Studies
McFarlane et al. (1997) published the results of a peer-reviewed range-finding study
(McFarlane et al., 1997a) as well as a more extensive two-generation exposure study
(McFarlane et al., 1997b). These studies were performed according to GLP compliance. The
range-finding study exposed 2 male (200 g) and 16 female (60 g) FO-generation Wistar albino
rats (Bantin and Kingman, Hull, U.K.) per dose group to doses of 0-, 500-, 750-, or
1000-mg/kg-day BHT (99.9 % purity) by diet and allowed them to cohabitate for 8 weeks.
Except when paired together during mating, the males were housed individually in polycarbonate
cages. Females were housed in groups of seven or eight, until pregnancy was confirmed, at
which point they were housed singly. Standard breeding diet (CRM, Labsure, Manea,
Cambridge, U.K.) and tap water were provided ad libitum. Environmental conditions were
maintained at a temperature of 20 ± 3°C, relative humidity of 30-70%, and a 12-hour
light/12-hour dark cycle. Dosing at the same levels continued through pregnancy and lactation.
Between Postnatal Days (PNDs) 6 and 10, litters were reduced or increased as needed to eight
pups (Fl), leaving a "maximum number of males," although the exact number was unspecified
(McFarlane et al., 1997a). Dams and all but 14 pups per dose group were sacrificed at PND 21.
The remaining pups (Fl) were fed a control diet for the following 4 weeks.
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Over the course of the study, all animals (F0 and Fl) were weighed weekly and examined
daily; Concentrations of BHT were adjusted in the feed biweekly, to maintain constant exposure,
with the exception of pregnancy and lactation, when no dosing adjustments were made
(McFarlane et al., 19997a). The liver, kidneys, adrenal, thyroid, spleen, pancreas, and lungs
were taken from the sacrificed dams for examination. Study authors conducted autopsies on one
pup per litter, and liver, kidneys and adrenals were removed for gross and microscopic
examination. Study authors performed statistical analysis using the Student's /-test. Although
the results of the histological exams are discussed in the study, no quantitative data is presented.
Treatment with BHT did not affect the body weights of F0 animals in any dose group
prior to pregnancy (see Table B.34) (McFarlane et al., 1997a). Dams exposed to 750 and
1000 mg/kg-day gained less weight at the end of their pregnancies (89 and 80% of control,
respectively) and did not show increased food consumption during lactation (which caused
decreased body weights), in comparison to controls (data not provided). Dams in the
500-mg/kg-day dose group also lost body weight during lactation (96% of control) although this
change did not reach the level of statistical significance. Mating success was not affected in any
dose group. Examinations of the liver showed increased relative liver weights in animals in each
dose group (151%, 162%, and 166% at 500, 750, and 1000 mg/kg-day, respectively).
Histopathological analysis showed no effects in the liver of dams sacrificed at gestational days
(GD) 19 or 20 (data not reported; methods describing which animals or how many were
sacrificed at this time point were not reported). Although the quantitative data were not
provided, study authors reported that dams exposed to 750 and 1000 mg/kg-day had hypertrophy
of the centrilobular hepatocytes after lactation. Additionally, cytoplasmic vacuolization was
noted in the enlarged cells as well as loss of glycogen. Dilation of the sinusoids in the
centrilobular zone, bile duct proliferation (without evidence of inflammation or fibrosis), and
proliferation of the endoplasmic reticulum (ER) of hepatocytes in liver sections were also
reported. No other changes related to BHT exposure were observed in other tissues.
Table B.35 summarizes effects from exposure to BHT on the Fl pups, as reported by
McFarlane et al. (1997a). No difference was observed in the number of pups born per litter to
exposed dams compared to the control group. However, treated dams in the 500, 750, and
1000-mg/kg-day dose groups had reduced litter weights as compared to controls (96, 88, and
83%), respectively). Pup body weight at birth was only statistically significantly decreased in the
750-mg/kg-day group (91%>, as compared to controls). The study authors observed normal
postnatal development up to 1 week following birth but noted retarded development in the
treated pups by the second week. Growth of pups in the 750-mg/kg-day and higher dose group
before litter reduction was severely stunted (48 and 41%, as compared to controls, respectively).
Reduction of the litters did not impact body weight reductions. Lethargy and poor fur also were
observed in these animals. The weights of the pups in the 500-mg/kg-day dose group were
decreased (66%, as compared to control). Absolute liver weights in pups in all dose groups were
decreased, but when liver weights were compared to body weights, the relative liver weights
were unchanged compared to controls. Notably, pups from smaller litters, where the study
authors could not foster pups to make a litter of eight, were less affected.
McFarlane et al. (1997a) reported that pups maintained on a regular diet after weaning
appeared healthy. Some (number not reported) of the 1000 mg/kg-day exposed pups suffered
from weakness and were euthanized. Animals from this dose group that survived 1 week were
healthy by 4 weeks past weaning. Weaned animals showed no gross abnormalities, no change in
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relative liver weight, and no abnormal results after histopathological examination of the liver.
Despite a lack of quantitative data, the results of histological analysis showed a lack of glycogen
in animals exposed to 500 mg/kg-day or more. According to the study authors, the zona
fasciculata had few lipid droplets as compared to control animals. No differences in the thyroids
of exposed pups could be found as compared to controls. Based on the increased relative liver
weights in the exposed dams, a maternal LOAEL of 500 mg/kg-day is identified. This effect is
noted at the lowest dose administered in the study precluding identification of a maternal
NOAEL. Based on the reduced body weights of F1 pups, LOAEL of 500 mg/kg-day is
identified. This effect is noted at the lowest dose administered in the study precluding
identification of a fetal NOAEL. The data for decreased body weight, increased relative liver
weight in maternal dams, and decreased body weight of pups before reduction are further
evaluated with the BMDS modeling program for determination of a POD for the chronic p-RfD.
The more extensive, two-generation exposure study (McFarlane et al., 1997b) was
conducted using the same methods as described above for the range-finding study (no GLP
information provided), with the exception that 7 male and 50 female rats per dose group were
exposed to 0, 25, 100, or 500 mg/kg-day BHT. Animals were evaluated as described for the
range-finding study with the following changes. Animals were allowed to cohabitate for
3 weeks after 5 weeks of exposure to BHT. Five dams from each dose group were sacrificed on
GD 19 or 20 and analyzed as previously described. GD 19 or 20 fetuses were removed,
sacrificed, weighed, and examined for abnormalities. Five of the fetuses were fixed for
histological analysis, and five fetuses' livers were removed for ultrastructural examination or
were pooled and homogenized for biochemical analysis. Five dams from each dose group were
sacrificed at weaning (PND 21) as were their litters and all F0 males. The livers were removed
from these dams for histological analysis. Liver, kidneys, adrenal glands, and thyroid were
removed from at least four of the pups per dam for histological analysis. Additionally, livers
from five control and 500 mg/kg-day treated pregnant and nonpregnant females were removed
for microscopic and biochemical analysis. Sixty pups per dose group were sacrificed at 4 and
22 weeks after weaning from all dose groups. Biochemical analysis included: glucose-
6-phosphatase activity, cytosolic glutathione-»Y-transferase, total glutathione, 7-ethoxyresorufin
O-deethylase, benzphetamine A'-demethylase, pentoxyresorufin O-depentylase, epoxide
hydrolase, total microsomal cytochrome p-450 and homogenate, and cytosolic and microsomal
protein levels.
Similar to the range-finding study (McFarlane et al., 1997a), no significant differences in
mating success, weight gain, or food consumption were seen prior to lactation in F0 rats exposed
to 25, 100, or 500 mg/kg-day BHT (McFarlane et al., 1997b). Table B.36 shows that relative
liver weight was increased in the 500 mg/kg-day dams sacrificed on GD 19 or 20 (106% as
compared to control) but did not reach the level of significance. The relative liver weights of
lactating and the time-control nonlactating dams were increased 135% for both groups compared
to their respective control (see Table B.37). Lactating females (PND 21) were reported to have
the same hypertrophy of the liver that was observed in the range-finding study (data not
reported) (McFarlane et al., 1997a). Although no quantitative data were presented, livers of
dams after lactation showed dose-dependent centrilobular cell enlargement at the 100- and
500-mg/kg-day but not at 25 mg/kg-day. According to the study authors, no other organs were
affected by treatment, except the thyroid, where hyperactivity in the 100- and 500-mg/kg-day
dose groups was observed. Microscopic examination of livers from dams exposed to
500 mg/kg-day showed a dose-dependent proliferation of smooth ER. Dams sacrificed on
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PND 21 had a dose-related reduction in fat surrounding the body wall, kidneys, and adrenal
glands.
Biochemical analysis conducted by McFarlane et al. (1997b), summarized in Table B.37,
showed that glucose-6-phosphatase levels were lower in lactating and nonlactating rats exposed
to 500 mg/kg-day than those in controls (76% and 47%, respectively). Total glutathione in
500 mg/kg-day lactating rats was significantly decreased as compared to lactating control (39%),
but increased in nonlactating 500 mg/kg-day rats as compared to control (110%). Nonlactating
and lactating rats exposed to 500 mg/kg-day BHT had increased total cytochrome p-450 levels
(165%) and 169%), respectively) and glutathione-»Y-transferase levels (215% and 294%,
respectively), with the greatest increases seen in the lactating rats. Nonlactating and lactating
rats exposed to 500 mg/kg-day had significantly up-regulated pentoxyresorufin O-deethylase
(6462%) and 8449%), respectively) and slightly down-regulated (not statistically significant)
ethoxyresorufin O-deethylase (96%> and 83%, respectively).
McFarlane et al. (1997b) reported no effects on litter number of dams sacrificed at GD 19
or 20 (see Table B.38). Body weight of fetuses also were statistically and biologically decreased
in the second highest dose group. In the highest dose group, decreased fetal body weight was not
statistically changed but was still biologically significantly (>5% change considered to be
biologically significant in non-adult pups) decreased. Relative liver weight was statistically and
biologically significantly increased at the highest dose tested. Histopathological and biochemical
examination of livers from treated fetuses showed no changes compared to controls.
Biochemical analysis of these fetuses did not show changes in liver enzyme levels (glutathione
^-transferase, total glutathione, 7-ethoxyresorufin O-deethylase, benzphetamine iV-demethylase,
and epoxide hydrolase) compared to controls, with the exception of a decrease in glucose-
6-phosphatase (58% of control) at the 100 mg/kg-day dose-level. Pups born to dams receiving
500 mg/kg-day of BHT during gestation and lactation and then fed diets providing
250 mg/kg-day after weaning, examined at weaning (PND 21) had an increased relative liver
weight (125%>) compared to controls (see Table B.39). Biochemical analysis of these treated
pups (PND 21) revealed increased levels of glutathione ^-transferase, 7-ethoxyresorufin
O-deethylase, benzphetamine A'-demethylase, and epoxide hydrolase in the pups exposed to
250 mg/kg-day, as well as increased levels of 7-ethoxyresorufin O-deethylase, benzphetamine
iV-dem ethyl ase, and epoxide hydrolase in the 100-mg/kg-day group. No change was seen in the
total glutathione levels, but dose-related, statistically nonsignificant decreases in glucose-
6-phosphatase were seen in the 100- and 250-mg/kg-day dose groups (82% and 77%,
respectively) (see Table B.39).
McFarlane et al. (1997b) noted an increase in relative liver weights in pups born to
500 mg/kg-day dams, fed 250 mg/kg-day after weaning, and sacrificed at 4 and 22 weeks after
weaning (111% and 107%, respectively), and body weights decreased (93% and 87%) (see
Tables B.40 and B.41). No liver histopathology findings were reported, aside from a slight
dilation of the sinusoids in exposed animals. No changes were observed in other organs of the
100- and 250-mg/kg-day dose groups except for mild hyperactivity of the thyroid and
hypertrophy of the zona fasciculata cells of the adrenal glands. Proliferation of the smooth ER
was reported in the livers of pups exposed to 250 mg/kg-day, which the study authors state is
supported by observed centrilobular eosinophilia and up-regulation of cytochrome p-450 (124%,
only at 22 weeks). Dilation of the sinusoids and loss of glycogen also were observed. Other
observations included large vacuoles in the hepatocytes and osmiophilic material in the lumen of
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the bile canaliculi. Biochemical analysis of treated pups revealed no significant changes in the
levels of benzphetamine /V-dem ethyl ase at 4 and 22 weeks postweaning (see Tables B.40 and
B.41). Ethoxyresorufin O-deethylase was up-regulated at 4 weeks postweaning in all dose
groups (25 mg/kg-day: 146%, 100 mg/kg-day: 138%, 250 mg/kg-day: 147%), but at 22 weeks
after weaning the increase was statistically significant only in the animals exposed to
100 mg/kg-day (140%). Glutathione ^'-transferase and epoxide hydrolase were significantly
increased in the 250-mg/kg-day dose group at 4 weeks (158%) and 22 weeks (146%) after
weaning and in the 100-mg/kg-day group at 4 weeks (143%) postweaning. Total glutathione
was decreased in the 250 mg/kg-day-treated pups at 4 weeks (73%) and 22 weeks (63%) after
weaning while both the 100 and 250 mg/kg-day offspring animals had glucose 6-phosphatase
reduced to 72-80% of the control values during both study periods (see Tables B.40 and B.41).
The study authors conclude that for both the range-finding (McFarlane et al., 1997a) and
the main study (McFarlane et al., 1997b) administration of BHT had no systemic effect on
treated animals. There is no evidence of exposure influencing mating success or fetal
development. Dams given doses of 500 mg/kg-day and greater had pups that did not gain as
much weight between nursing and weaning of pups, as compared to controls, and this trend
continued after being given the control feed. The study authors suggested that this weight
retardation is due to malnutrition, rather than as a result of BHT exposure because pups in
reduced litter sizes in these exposure groups had a less severe stunting of their weight (data not
reported). Pups exposed to 100 and 250 mg/kg-day reportedly had mild hyperactive thyroid and
hypertrophy of the zona fasciculata cells in the adrenal glands. Pups of the dams given doses of
500 mg/kg-day and greater had upregulated liver xenobiotic-metabolizing enzymes, increased
relative liver weights after weaning, and hepatocyte abnormalities (proliferation of the smooth
ER and eosinophilia). Liver changes, including vacuolization and proliferation of the ER were
also clearly seen in the lactating dams treated with 500 mg/kg-day or more, which was supported
by the observed decrease in glucose-6-phosphatase (a possible indicator of liver damage). The
study authors reported that BHT in combination with lactation exacerbated the stress on the liver,
reducing the nutrient content available to the pups. Lactation also reportedly increased food
consumption and thus the exposure levels of the dams.
Based on the liver effects observed in dams, as well as females exposed during mating,
gestation, and lactation (estimated 14 weeks), a maternal LOAEL of 500 mg/kg-day is identified
along with a NOAEL of 100 mg/kg-day. Based on a biological (>5% change considered to be
adverse for fetal effects) and statistical significant decrease in body weight observed in the F1
generation, a LOAEL of 100 mg/kg-day is identified from this study, with a corresponding
NOAEL of 25 mg/kg-day.
Price (1994) conducted a study using F1 Wistar rats generated from the study by
McFarlane et al. (1997). The original report was not obtainable; limited information was cited in
the OECD SIDS (2002) report. This study was performed according to GLP guidelines. Male
F1 rats (approximately 60/dose group) were administered BHT (99.9 % purity) in the diet for 22
months postweaning with interim sacrifices at 1, 6, 11, and 16 months. As discussed in the
OECD SIDS (2002) report, doses are 0, 25, 100, or 250 mg/kg-day. The study authors reported
decreased body weight in the mid- and high-dose groups throughout the 22-month treatment
duration. The following liver effects were observed: increased relative liver weight and altered
hepatic nodules at 16 months, enlarged and eosinophilic centrilobular hepatocytes at 6 months,
and periportal induction of gamma-glutamyl transferase at 11 months. Immunocytochemistry
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was performed for only the high-dose group and revealed increased altered hepatic foci
cytochrome P450 IB at 16 months and total cytochrome P450 at 11, 16, and 22 months.
Microscopic examination of the thyroid revealed hyperactivity in the mid- and high-dose groups at
11 months. Chronic progressive nephropathy was observed in all rats including controls at 11
months. No effects on the adrenals were observed. The study authors reported that no tumors were
observed. The OECD SIDS (2002) report identified a NOAEL of 25 mg/kg-day based on liver,
kidney, thyroid and adrenal effects.
Tanaka et al. (1993) published a peer-reviewed, three-generation dietary exposure study
in which they exposed male and female Crj :CD-1 mice (weights not reported) to BHT (purity not
stated). Mating occurred at 9 weeks of age, and rearing was consistent across all three
generations. One hundred male and female four-week-old Crj :CD-1 mice were acquired from
Charles River Japan Inc., Kanagawa, Japan. Mice were housed individually in polycarbonate
solid-floored cages with wood flakes at 24 ± 1°C and 55 ± 5% humidity. Of these, 10 male and
10 female mice and their subsequent offspring from mated nonsiblings served as controls. Ten
male and ten females at 5 weeks of age were fed diets containing 0.015% (150 ppm), 0.045%
(450 ppm), 0.135%) (1350 ppm), or 0.405% (4050 ppm) BHT (purity not reported). Appropriate
body-weight data and food consumption data for dose conversion were not provided in the study.
Therefore, average values provided for different strains of mice by EPA (1994b) for body weight
(0.024725 kg for both sexes) and food consumption (0.004825 kg/day for both sexes) are used in
the dosimetric calculation. Corresponding adjusted daily doses are 29, 88, 263, and
790 mg/kg-day for both males and females. At 9 weeks of age, F0 females and F0 males in the
same dose group were allowed to cohabitate for 5 days. F1 pups were weaned at 4 weeks of age
and mated at 9 weeks of age, while avoiding sibling matings to produce an F2 generation. This
study's conformance with GLP guidelines could not be determined.
After birth, on PND 0, Tanaka et al. (1993) recorded litter size, litter weight, and sex ratio
(male/female) of the F1 generation. No characteristics of the dams at birth or during gestation
were reported. F1 pups were evaluated for functional and behavioral endpoints including:
surface righting and negative geotaxis on PNDs 4 and 7, cliff avoidance on PND 7, swimming
behavior (direction, head angle, and limb movement) on PNDs 4 and 14, and olfactory
orientation on PND 14. Field activity was assessed at 3 weeks of age (PND 21) for 3 minutes by
observing ambulation, rearing, 180° turn, defecation, urination, and preening. Litter size, litter
weight, and body weight were analyzed statistically using Bonferroni's multiple comparison test
following an ANOVA or Kruskal-Wallis test. A chi-square test was used to analyze the sex
ratio. Neurobehavioral parameters were analyzed with the Mann-Whitney I /-test. All endpoints
were consistently measured between different generations.
Tanaka et al. (1993) reported no dose-related effects of BHT on number of litters, litter
size, litter weight at birth, or sex ratio in the F1 or F2 generations (see Table B.42). The
low-dose group's body weights increased statistically significantly compared to control in both
generations, whereas, in the first generation, pup birth weights decreased in the two highest-dose
groups (quantitative data not reported and thus data not shown here). This effect was not
observed in the F2 generation. Neurobehavioral effects in F1 and F2 pups were also inconsistent
across doses (see Tables B.43 and Table B.44). All exposed F2 males turned 180° significantly
less than controls (50%>, 58%>, 52%>, and 45%>, in the 29, 88, 263, and 790-mg/kg-day groups,
respectively) (see Table B.44). Negative geotaxis in the highest-dose group of F1 males on
PND 4 was significantly greater than controls. All other neurobehavioral effects that were
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significantly different did not increase with increasing dose. Among these, only F2 male pups at
PND 4 had any other neurobehavioral effects present at higher doses. The most significant
neurobehavioral effects therefore were observed in F2 males (see Tables B.43 and Table B.44).
The dose levels of BHT resulted in no dose-related effects on neurobehavioral or
reproductive endpoints, except for a statistically significant decrease in the 180° turn test in F2
males that was not observed in the F2 females. Based on this effect alone the results are not
significantly adverse to warrant assigning a LOAEL to this compound. A NOAELadj of
790 mg/kg-day is identified.
The Tokyo Metropolitan Research Laboratory of Public Health (1992b) sponsored an
unpublished teratogenicity study in JCL-ICR mice with BHT (purity not reported) in olive oil
that was not peer-reviewed. Two types of testing were conducted: a repeat-dose and a
single-dose administration. The study authors did not state whether the study protocol adhered
to GLP standards. In the repeated-dose test, BHT was administered by gavage once per day for
7 days to pregnant mice (n = 26-30) on GDs 7-13 at dose levels of 70, 240, or 800 mg/kg-day,
including both negative controls in which no substance was administered and vehicle controls
which were administered olive oil. In the single-dose test, BHT was administered by gavage to
pregnant female mice (n = 19-20) on GD 9 at dose levels of 1200 or 1800 mg/kg-day, including
a negative control group. Study authors measured body weights and observed general health of
the animals daily. At GD 18, dams were sacrificed, and investigators counted the number of
implantation sites, corpus luteum absorbed embryos, and dead and live fetuses. Also at
necropsy, study authors measured organ weights and recorded gross observations of toxicity in
the dams. In the surviving fetuses, investigators measured body weights and major organ
weights, calculated the sex ratio, and examined embryos for external malformations. Study
authors also randomly selected five dams (dose group not specified) to examine the internal and
skeletal abnormalities of the live fetuses.
In the 7-day repeat-dose test, the Tokyo Metropolitan Research Laboratory of Public
Health (1992b) did not observe any significant changes in body weight or behavior in dams
treated at any dose level of BHT versus the vehicle or negative controls. The mean spleen and
kidney weights of dams in the high-dose (800-mg/kg-day) group were significantly different
from control (no further data provided; data table in study report is illegible). There were no
significant differences in gestation rate, numbers of corpus luteum and implantations, dead and
live fetuses, and sex ratio between any of the treatment groups versus controls. Although body
weights were significantly lower in male fetuses of dams treated with BHT, no dose-response
trend was observed, and study authors did not consider this finding to be biologically significant.
Investigators also reported significantly lower incidence of accessory sternebrae (1.4%) and a
significantly higher incidence (11.1%) of cervical ribs in the high-dose (800-mg/kg-day) group
versus controls, but these differences were not considered treatment related when compared to
background incidences. Study authors did not consider the observations of cleft palate, eyelid
opening, and polydactyl in the BHT-treated fetuses to be treatment-related due to similar
incidences observed in the control groups. No internal abnormalities or differences in
ossification rates were observed in any of the fetuses in the BHT-treated groups.
In the single-dose test, dams in the 1200- and 1800-mg/kg-day groups experienced 10%
(2/20) and 25% (5/20) mortality, respectively (Tokyo Metropolitan Research Laboratory of
Public Health, 1992b). The mean spleen weight of the dams in the 1200-mg/kg-day dose group
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and the mean spleen and lung weights of the dams in the 1800-mg/kg-day dose group were
significantly higher than the negative control group (no further data provided; data table in study
report is illegible). One dam in the 1200-mg/kg-day dose group had no surviving fetuses; all
died at an early stage. Though the 1800-mg/kg-day dose group did not have a similar occurrence
and the incidence of dead fetuses in this group was similar to controls, the study authors did not
consider the effect in the 1200-mg/kg-day dose group to be biologically significant. There were
no significant differences in gestation rate, numbers of corpus luteum and implantations, dead
and live fetuses, and sex ratio between any of the treatment groups compared to controls.
Investigators observed no significant differences in external, internal, or skeletal abnormalities in
the live embryos of BHT-treated dams versus controls. Study authors did not report a NOAEL
or LOAEL for either experiment. As mentioned previously, the data tables are largely illegible
making these studies unusable for derivation of a subchronic p-RfD.
Olsen et al. (1986) is selected as the principal study for deriving the subchronic and
chronic p-RfDs and the p-OSF. Olsen et al. (1986) published a peer-reviewed developmental,
reproductive, and carcinogenicity study in rats exposed in utero to BHT. The study authors
reported both subchronic and chronic noncancer effects as well as chronic cancer effects.
Groups of male and female specific-pathogen-free (SPF) 7-week-old F0 Wistar rats (body
weights unreported) were treated with 0, 25, 100, or 500 mg/kg-day BHT (>99.5% purity) in a
semisynthetic diet (n = 60, 40, 40, and 60, respectively, for each dose group) confirmed to be
free of aflatoxin and nitrosamines. After 13 weeks of exposure, 40, 29, 30, and 44 litters from
each dose group were used to populate exposure groups (0, 25, 100, and 250 mg/kg-day) with
100, 80, 80, or 100 male and female rats, respectively. F0 animals were excluded from the study
after mating for the males and lactation for the females. Because F0 female rats treated with
500 mg/kg-day BHT exhibited an adverse effect in the liver, the F1-generation rats were
administered a high dose of 250 mg/kg-day instead of 500 mg/kg-day. After weaning, F1
animals were exposed to BHT in a semisynthetic diet until 141-144 weeks of age. No
information was provided regarding GLP compliance.
Olsen et al. (1986) recorded the rats' body weights on a weekly basis until the animals
were 31 weeks of age; afterward, they recorded body weights once every 2 weeks. Food
consumption was recorded every week. Hematocrit and hemoglobin levels were determined in
whole blood and in red and white blood cells using blood collected from 20 F1 male and female
rats after 9, 19, 43, and 108 weeks of BHT treatment. Serum was collected and analyzed for
glucose, free and total cholesterol, triglycerides, blood urea nitrogen (BUN), and phospholipid
levels. All F1 rats were inspected on a regular basis (time frame not specified) for the presence
of tumors. At study termination, all surviving animals (141-144 weeks of age) were sacrificed.
A gross necropsy was performed on animals that were sacrificed at study termination and
animals that died during the course of the experiment. Liver, kidneys, lungs, brain, heart, spleen,
pituitary gland, thymus, pancreas, thyroid, adrenal glands, testes, ovaries, uterus, seminal glands,
mesenteric and axillary lymph nodes, salivary glands, urinary bladder, gastrointestinal tract,
spinal cord, skeletal muscle, peripheral nerve, bone, skin, mammary gland, eyes, and the
Harderian gland were preserved for further examination. Animals that developed the first tumors
beyond 43 weeks were included in the total effective number of animals. Biochemical,
hematological, and other biological data were statistically analyzed using the Student's t-test,
while the Armitage-Cochran test for linear trend was used to determine preweaning mortality in
various litters. Mortality and tumor incidence were analyzed by the Peto method (a comparison
of observed and expected values).
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Olsen et al. (1986) reported that F0 rats treated with 25, 100, or 500 mg/kg-day BHT did
not exhibit any differences in food consumptions rates when compared to the concurrent control
group. The study authors reported a statistically significant decrease in body weight compared to
the controls in male and female F0 rats treated with 500 mg/kg-day BHT (data not reported).
This decrease was noted beginning in Week 6 of the treatment, and it persisted throughout their
lifetimes. The study authors reported that the number of litters with 10 or more pups decreased
in a dose-dependent manner, with dams treated with 500 mg/kg-day BHT exhibiting a
statistically significant decrease in the number of litters, as indicated by the Cochran-Armitage
statistical analysis. Pup viability was not affected as a result of exposure to BHT. Average F1
pup birth weights in the 100- and 500-mg/kg-day dose groups were slightly lower (97%)
compared with the concurrent control groups (see Table B.21). At weaning, F1 pups exposed to
BHT exhibited significant and dose-dependent depression in body-weight gain compared with
the corresponding control group (95% at 25 mg/kg-day, 94% at 100, and 60% at
500 mg/kg-day).
F1 rats did not exhibit any change in food consumption rates as a result of exposure to
BHT (Olsen et al., 1986). A dose-dependent and significant depression in mean body weight
was noted in animals treated with BHT compared to the concurrent control group (see
Table B.22). For example, at 138 weeks, male body weights were 92, 89, and 85% and female
body weights were 100, 97, and 90%, for the 25, 100, and 250 mg/kg-day doses, respectively.
The body weight changes at 100 and 250 mg/kg-day in males and at 250 mg/kg-day in females
are considered to be biologically significant based on a BMR of 10% for changes in adult
animals. Mortality was reported to be higher in F1 control groups compared with the
BHT-treated groups (see Table B.23). At 104 weeks, 72% of males and 86% of female F1 rats
treated with 250 mg/kg-day BHT survived compared to 70% of males and 69% of females in the
control group. At study termination, 44 and 39% of male and female F1 rats survived in the
250-mg/kg-day dose group compared to 16 and 17% in the male and female control groups,
respectively (see Table B.23). The study authors reported that there was a significant difference
in longevity in both sexes. The higher mortality of control F1 male rats compared with
BHT-treated male F1 rats was mainly attributed to inflammation of the bladder that was often
associated with kidney stones. In female F1 rats, higher mortality of controls compared with the
BHT-treated groups was primarily due to the occurrence of nephropathy and tumors in the
pituitary gland. Exposure to BHT had no effect on clinical appearance or animal behavior.
However, a slight reddish discoloration of the urine was noted in male F1 rats exposed to
250 mg/kg-day BHT. Hematological analysis indicated no treatment-related changes in the F1
rats. Serum chemistry analysis exhibited elevated cholesterol and phospholipid levels during the
first year of exposure in female F1 rats treated with 250 mg/kg-day BHT compared with the
concurrent control group (see Table B.24). Triglyceride levels were statistically significantly
lower in both male and female F1 rats in the BHT-treated groups compared with the
corresponding controls during Weeks 19, 43, and 108 (see Table B.24).
Olsen et al. (1986) reported that incidences of hepatocellular adenomas and carcinomas
in F1 males and hepatocellular adenomas in F1 females were elevated in BHT-treated animals
compared to the corresponding control groups (see Table B.25). In the 0, 25, 100, and
250-mg/kg-day dose groups, the percent incidences of hepatocellular adenomas were 1, 1,6, and
18%) in male rats and 2, 4, 8, and 12% in female rats, respectively. The respective percent
incidences of hepatocellular carcinoma were 1,0, 1, and 8% in males and 0, 0, 0, and 2% in
females. The first carcinoma in BHT-treated F1 rats was observed during Week 132 in one male
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treated with 250 mg/kg-day BHT. The rest of the carcinomas were observed at study
termination. Only one control F1 male exhibited carcinoma at 117 weeks of age. The first
adenoma was observed in one male treated with 250 mg/kg-day BHT after 115 weeks, but most
adenomas were reported in both sexes at study termination (Weeks 141-144; Table B.26). There
was no intralitter correlation among rats diagnosed with hepatocellular tumors. Gross
examination of hepatocellular adenomas indicated that the adenomas were 4-30 mm in diameter,
whereas the carcinomas were 15 mm or more in diameter. Neoplasms were not preferentially
located in the liver. Occasional ascites in connection with large carcinomas also were observed.
Although basophilic adenomas were observed on occasion, eosinophilic adenomas were
observed more frequently. Hepatocellular carcinomas were comprised of basophilic hepatocytes
that formed a trabecular pattern, and, in some carcinomas, a projection of irregular cords without
endothelial lining was seen in dilated sinusoids. The study authors reported that metastases of
the carcinomas were not observed. The incidence of hepatocellular adenomas was high in both
the male and female F1 rats in the 250-mg/kg-day dose group, but a higher number of
hepatocelluar carcinomas was observed in the F1 males compared with the F1 females (see
Table B.25), indicating that the males were more susceptible to BHT than females.
Besides hepatocellular adenomas and carcinomas, the following tumors were observed by
Olsen et al. (1986) in either males or females or in both sexes: thyroid C-cell adenoma (females),
thyroid C-cell carcinoma (males), islet-cell adenoma in both males and females, exocrine
adenomas of the pancreas and haemangioma and reticulum-cell sarcoma of the
reticuloendothelial system in males, theca granulosa-cell adenoma in females, adenoma and
adenocarcinoma of the uterus and thymoma in females, and ductular adenoma of the mammary
gland in females. However, the incidence of these tumors was not statistically significantly
higher when compared to the corresponding control group. The low incidence of theca
granulosa-cell adenomas in the F1 females was significantly higher in the trend analysis but only
in the 250-mg/kg-day dose group. The study authors reported that overall the number of F1 male
and female rats exhibiting malignant tumors or multiple tumors, excluding hepatocellular
tumors, was slightly—but not significantly—higher in the 250-mg/kg-day dose group compared
with the concurrent control group (see Table B.27).
The study authors also reported the occurrence of nonneoplastic lesions in the liver
including a dose-dependent increase in the incidence of bile duct proliferation and cysts in F1
males, and focal cellular enlargement in F1 females (see Table B.28). Heart nephropathy and
fibrosis also were noted to occur less frequently in rats treated with BHT than in corresponding
controls. Other nonneoplastic lesions that were observed occurred on an incidental basis and
according to study authors are not related to BHT exposure.
After utilizing tests of heterogeneity using the trend analysis, Olsen et al. (1986)
concluded that the number of hepatocellular adenomas and carcinomas was increased
significantly in F1 male rats, but, in F1 females, only the hepatocellular adenomas were
increased significantly (see Table B.25). A dose-related increase in total hepatocellular tumors
(sum of adenomas and carcinomas) was determined in both sexes and was statistically significant
at the highest dose tested. Hepatocellular tumors were detected when F1 rats were at least
2 years of age (see Table B.26). Tumors observed in other organs had a low incidence and were
not statistically significantly different from the corresponding control group. Maternal
LOAELadj of 500 mg/kg-day, and a corresponding NOAELadj of 100 mg/kg-days are identified
based on reported decreases in weight in the dams. Because study authors reported both
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subchronic and chronic effects for F1 adult rats, a NOAEL and LOAEL can be identified for
both study durations. For subchronic effects, a LOAELadj of 250 mg/kg-day and a
corresponding NOAELadj of 100 mg/kg-day are identified based on decreased body weight. For
chronic effects, a LOAELadj of 100 mg/kg-day and a corresponding NOAELadj of
25 mg/kg-day are identified based on chronic body-weight depression.
Other Studies
No other quantitative data were located regarding the toxicity of BHT to animals
following other exposures.
Inhalation Exposures
No quantitative data were located regarding the toxicity of BHT to animals following
subchronic or chronic inhalation exposure.
Other Exposures
No quantitative data were located regarding the toxicity of BHT to animals following
other exposures.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Table 3 summarizes studies examining the genotoxicity and neurotoxicity of BHT, as
well as its effects on the transcriptome, metabolism, and toxicokinetics. Toxicokinetic and
transcriptome analysis are discussed in greater detail due their importance in supporting the
provisional toxicity values. Details of the neurotoxicity study are also presented because of the
toxicological significance of neurological effects. A summary of the genotoxicity results is
provided because BHT has been shown to be negative for genotoxic activity, and thus these
results do not affect the derivation of the provisional toxicity values, or any associated mode of
action.
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Neurotoxicity
(unreported number and sex) F1 Swiss
Webster mouse, gestation, lactation and
0.5% (5000 ppm) diet exposure
GD1-6 weeks.
Exposure decreased sleeping and
increased fighting as compared to
control. Exposed animals did not
exhibit learned behaviors in the
automated avoidance conditioning
climbing screening. Males showed
significant increases in isolation-
induced aggression when compared to
control.
Due to exposure pre- and postnatally to
BHT, it was not possible to determine
which stage caused the adverse effects.
The study authors believed that the change
in aggressive behavior was developmental
due to the results of the acute testing. The
study authors further noted that BHT may
be largely lipophilic in nature and
therefore interferes with the synaptic
vesicle membrane.
Stokes and
Scudder(1974)
Transciptome
analysis
Male Sprague-Dawley rat, diet (0, 28, 88,
167, 321, or 1159 mg/kg-day), 28 days.
Hepatic mRNA levels of the phase 1
cytochrome P450 isozymes,
CYP2B1/2, increased. BHT also
increased the mRNA levels of GST 1
among the phase 2 metabolizing
enzymes in a dose-dependent manner.
Indicates potential role of liver enzyme
induction in the metabolism and liver
effects seen after exposure.
Stierum et al.
(2008)
Toxicokinetic
Male and female DDY/Slc mice and male
Sprague-Dawley rat given single doses
labeled with 14C ([14C])BHT of 20 or 500
mg/kg by stomach intubation.
Concentrations were highest in the
stomach, intestines, gall bladder, and
urinary bladder. Excretion over 7 days
was 41-65%, in the urine at 26-50%,
and in expired air at 6-9%. Forty-three
metabolites were identified in the urine
and feces of rats and mice. All traces
of BHT or metabolites were completely
excreted by 7 days.
BHT is highly metabolized, and is
absorbed primarily into part of the
digestive and urinary system.
Matsuo et al.
(1984)
Toxicokinetic
White male Wistar rats were given doses
of [14C]-labeled BHT (or its related
compounds) via intravenous (i.v.) or
intraperitoneal (i.p.) injection.
Intravenous: 46% of administered BHT
was excreted in the bile in the first hour
and 94% was excreted in the bile 6
hours after dosing.
Intraperitoneal: 32% excreted in the
bile 2 hours following dosing, and 52%
excreted 6 hours after dosing.
BHT and related metabolites are rapidly
metabolized and excreted in bile. The
differences between the i.v. and i.p.
administrations suggest differences in the
kinetics, potentially in the peritoneum,
which may influence the metabolism of
BHT.
Holder et al.
(1970)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Salmonella strains TA98, TA100, and
TA1537 were exposed to 0.05, 0.5, 5, 50,
or 500 ng/plate BHT in an Ames assay.
Colonies were evaluated for mutagenic
effects with and without metabolic
activation by S9 liver fraction. Positive
responses were characterized by a 50%
increase in the number of revertant
colonies over the spontaneous frequency.
Study authors reported negative results
for all strains at all dose levels with and
without S9 activation.
These results suggest that BHT is not
mutagenic in Salmonella strains TA98,
TA100, or TA1537.
Bruce and Heddle
(1979)
Genotoxicity
Male Drosophila melanogaster were
injected with 0.02^1 of 0.001% or
0.0001% BHT and mated. Female
offspring were evaluated for sex-linked
recessive lethal mutations and males for
chromosome translocations. Some males
were also exposed to 2.4 krads gamma
rays and evaluated for radiosensitizing
effects.
Study authors found no mutagenic
effects from BHT exposure alone, but
reported extreme radiosensitizing
effects of gamma ray-induced sex-
linked recessive lethal mutations and
chromosome translocations.
Study authors concluded that BHT alone
does not cause sex-linked recessive lethals
or chromosome translocations in mature
sperm of Drosophila melanogaster, but
that BHT is an effective radiosensitizer of
these mutations.
Kamra (1973)
Genotoxicity
Male Drosophila melanogaster were
either injected with 0,4|il of a 0.05%
solution BHT in saline ethanol or fed a
0.2% solution BHT and mated. Offspring
were evaluated for II-III translocations and
sex-linked recessive lethals. Offspring of
males that were also treated with 2krads of
X-rays were also evaluated for dominant
lethals.
Study authors found that BHT
treatment alone caused no increased
frequency of translocations or sex-
linked recessive lethals. Treatment
with BHT as well as irradiation did not
increase the frequency of II-III
translocations, or dominant lethals,
however, study authors reported a
decrease in sex-linked recessive lethals.
These results suggest that BHT does not
increase radiation-induced genetic damage
in Drosophila melanogaster.
Barnett and
Munoz (1980)
Genotoxicity
Salmonella strains TA98, TA100,
TA1537, and TA1538 were exposed to
BHT in concentrations below
10 |imo 1/plate (according to a toxicity
determination assay) as well as known
mutagens in an Ames assay to determine
the inhibitory effects of BHT on
mutagenic effects of these chemicals.
Study authors reported that BHT
reduced inversions induced by every
mutagen requiring S9 activation, but
did not reduce inversions induced by
direct-acting chemicals.
Study authors concluded that in nontoxic
concentrations, BHT inhibits an enzymatic
activation mechanism of promutagens,
reducing inversions in salmonella strains
TA98, TA100, TA1537, and TA1538.
McKee and
Tometsko (1979)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Chinese hamster V79 cells were exposed
to BHT along with benzo(a)pyrene and
treated with mouse liver S9 fraction to
evaluate the inhibitory effects of BHT on
benzo(a)pyrene-induced mutations.
Study authors reported fewer mutations
in cultures exposed to both BHT and
benzo(a)pyrene than in cultures
exposed to benzo(a)pyrene alone. At a
ratio of 1:1 benzo(a)pyrene to BHT,
there was no significant difference
between mutant frequencies and those
of spontaneous background level.
These results suggest that BHT has an
inhibitory effect onbenzo(a)pyrene-
induced mutations.
Paschin and
Bahitova (1984)
Genotoxicity
Male Drosophila melanogaster were
injected with 0.001% BHT and exposed to
0, 1.2, 2.4, or 3.6 krads gamma-rays and
mated. Generation F1 was evaluated for
XB chromosome loss and sex ratio and the
F2 generation for sex-linked recessive
lethal mutations.
Study authors reported increased
frequency of XB chromosome loss in
generation F1 and of sex-linked
recessive lethals in F2, however, these
effects were not significant. Study
authors attributed this increase in sex-
linked recessive lethals to
radiosensitization effects of BHT.
These results suggest that BHT enhances
the mutagenic effects of gamma-rays in
Drosophila melanogaster.
Prasad and Kamra
(1974)
Genotoxicity
Blood leukocyte cultures drawn from a
male human were exposed to
7,12-dimethylbenzanthracene along with 0
or 0.21 |imol BHT and evaluated for
chromosomal aberrations.
Study authors reported a 63.8%
reduction in the number of
chromosome breaks in cultures
exposed to dimethylbenzanthracene as
well as BHT.
These results suggest that BHT has an
inhibitory effect on 7,12-
dimethylbenz(a)anthracene- induced
chromosomal breakage.
Shamberger et al.
(1973)
Genotoxicity
Salmonella strains TA98 and TA100 were
exposed to 0-50 ng/plate BHT and
evaluated for mutagenic effects with and
without Aroclor 1254-induced rat liver S9
fraction. Strains also were exposed to
BHT in conjunction with aflatoxinBi and
evaluated for effects on mutagenicity.
Study authors reported no mutagenic
effects of BHT alone in either strain
with or without metabolic activation,
but reported toxic effects at doses of
20 ng/plate and higher. A two-fold
increase in revertant colonies was
reported in both strains with the
addition of 5-20 |ig/plate BHT to
aflatoxin B,.
These results suggest that the presence of
BHT enhances the mutagenic effects of
aflatoxinBi.
Shelef and Chin
(1980)
Genotoxicity
Male Sprague-Dawley rats were fed a diet
of 0.4% BHT for 10 weeks and mated.
Females were sacrificed and evaluated for
presence of dominant lethals by the
number of live and dead implants.
Study authors reported weak but
statistically significant increases in the
frequency of dominant lethal
mutations.
These results suggest that BHT causes
dominant lethal mutations in Sprague-
Dawley rats.
Sheuetal. (1988)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Male hybrid (101 x C3H) mice were fed a
diet of 1% BHT for 8 weeks and mated
with hybrid (SEC x C57BL and C3H x
C57BL) female mice. Females were
evaluated for presence of dominant lethals
and offspring were evaluated for presence
of translocations.
Study authors reported no evidence of a
dominant lethal effects or induction of
heritable translocations.
These results suggest no mutagenicity of
BHT in hybrid mice.
Sheuetal. (1988)
Genotoxicity
Salmonella typhimurium strain TA98 was
exposed to 0, 10, 50, 100, 500, 1000, or
5000 ng/plate BHT with and without
promutagens benzo(a)pyrene and 2-
aminoanthracene. Colonies were
evaluated for mutagenic effects with
Aroclor-induced rat liver S9 fraction.
Study authors found no mutagenic
effects of BHT with metabolic
activation. No effects of the presence
of BHT on benzo(a)pyrene were
reported, however, a significant
increase in the number of revertant
colonies was reported in cultures
treated with 2-aminoanthracene and
10 ng BHT and higher.
These results suggest that BHT has an
enhancing effect on the mutagenicity of 2-
aminoanthracene in salmonella strain
TA98.
Dertinger et al.
(1993)
Genotoxicity
Male ICR/Ha Swiss mice were injected
(i.p.) with 250, 500, 1000, or 2000 mg/kg-
body weight BHT and subsequently mated
with females. Females were sacrificed and
evaluated for dominant lethal mutations by
numbers of live implants and early and
late fetal deaths.
Study authors reported no significant
evidence of dominant lethal mutations
at any dose level.
These results suggest that BHT in not
mutagenic to ICR/Ha Swiss mice.
Epstein et al.
(1972)
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Table 3. Other Studies
Test
Materials & Methods
Results
Conclusions
References
Genotoxicity
Male Drosophila melanogaster were
exposed to 0.5% BHT and X-irradiated or
treated with diepoxybutane or
diethylnitrosamine to determine effects of
BHT on frequency of sex-linked recessive
lethal mutations, autosomal translocations,
sex chromosome losses, or dominant
lethals.
Study authors reported a lower
frequency of sex-linked recessive
lethals and translocations in Drosophila
spermatids treated with X-rays and
BHT. No significant effects of BHT on
the induction of dominant lethals, sex-
linked recessive lethals, or sex
chromosome losses in mature
spermatozoa treated with X-rays were
reported. Spermatids treated with BHT
and diepoxybutane or
diethylnitrosamine demonstrated
significantly fewer sex-linked recessive
lethal mutations.
These results suggest inhibitory effects of
BHT on mutagenic effects in spermatids
of Drosophila melanogaster.
Sankaranarayanan
(1983)
Genotoxicity
B. subtilis strains H-17 (rec+) and M-45
(rec ) were exposed to an unknown
concentration of BHT in a rec assay.
Differences in inhibition length were
recorded and used to identify mutagenic
effects.
Study authors reported no differences
in inhibition length.
These results suggest no mutagenic effects
of BHT on B. subtilis.
Kinae et al. (1981)
Genotoxicity
Salmonella strains TA98, TA100, and
TA1537 were exposed to 10 |ig/plate BHT
in an Ames reversion assay. Cultures
were evaluated for mutagenic effects with
and without metabolic activation by S9
Wistar rat liver fraction.
Study authors reported negative results
for all strains with and without
metabolic activation.
These results suggest no mutagenic effects
of BHT on Salmonella strains TA98,
TA100, or TA1537.
Kinae et al. (1981)
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Stokes and Scudder (1974) published a peer-reviewed, behavioral development dietary
exposure study in which they exposed multigenerations of male and female Swiss Webster mice
(age and weight not reported) to BHT (purity not stated). Pups from mated pairs were culled to
eight pups per pair and weaned at 21 days of age, and then performed several behavioral tests.
Mice were housed eight per cage at a constant temperature (21° C), with a 16-hour light/8-hour
dark cycle. Water and food were available ad libitum. Mated pairs and pups were fed Purina
Rat Chow and Pet Milk. Groups of 12 mice were randomly selected for a control group and an
exposure group. The exposed parents received 0.5% (5000 ppm) BHT by diet weight. Pups
were weaned at 21 days of age and exposed to 0.5% (5000 ppm) BHT by diet. Water and food
were provided ad libitum. This study's conformance with GLP guidelines could not be
determined. The dosing regimen is not explicit in the methods of the study in terms of the fetus'
exposure time.
At 6 weeks of age, mice were selected randomly for behavioral testing (Stokes and
Scudder, 1974). Social behavior was tested by placing naive pairs of male and female mice into
six small chambers, and interaction was recorded for 80 minutes. Study authors noted all
instances of contractual behavior, digging, stereotypic behavior, freezing, ingestion, carrying,
being groomed, grooming self, grooming others, sleeping, exploration, and aggression. Second,
at 7 weeks of age, to test learning, mice were placed in an automated avoidance conditioning
climbing screen where they were shocked every 5 seconds if they did not climb to a higher
platform. Third, 10 males from each test group were kept isolated for 3 weeks and then tested
for isolation-induced aggression. In conjunction with the third experiment, one group of
10 males, previously untreated, was fed the 0.5% (5000 ppm) BHT diet for the last 7 days of the
3-week	study to evaluate acute effects on aggression. Study authors evaluated statistical
significance for all tests, except for aggression results, using Student's /-test. The Wilcoxon
two-sample rank test was used to determine statistical significance of the isolation-induced
aggression testing.
Stokes and Scudder (1974) found that 0.5% BHT administered by diet significantly
decreased sleeping and increased fighting compared to controls. Additionally, animals exposed
to BHT did not exhibit learned behaviors in the automated avoidance conditioning climbing
screening. Last, males showed significant increases in isolation-induced aggression when
compared to controls. Table B.45 provides the mean and standard deviation of measured
instances of behavior in the control and 0.5% exposure group. Study authors stated that due to
animals being exposed pre- and postnatally to BHT it was not possible to determine which stage
caused the adverse effects. The study authors believed that the change in aggression behavior
was developmental because these effects were not seen in mice that were not treated in utero, but
were administered 0.5% (5000 ppm) via diet for 7 days. The study authors further estimated that
BHT may be largely lipophilic in nature and therefore interferes with the noted synaptic vesicle
membrane.
In a study conducted by Holder et al. (1970), the biliary metabolism of BHT in the rat
was examined and compared to the biliary metabolism of other BHT compounds—3,5-di-t-
butyl-4-hydroxybenzyl alcohol (BHT-CH20H); 3,5-di-t-butyl-4-hydroxybenzaldehyde
(BHT-CHO); 3,5-di-t-butyl-4-hydroxybenzoiac acid (BHT-COOH); and l,2-bis(3,5-di-t-butyl-
4-hydroxyphenyl)ethane	(B-B). White male Wistar rats (total number of animals unclear) were
given 100-fj.g doses of [14C]-labeled BHT (or other amounts of its related compounds) in
aqueous ethanol via intravenous (i.v.) or intraperitoneal (i.p.) injection. Animals were housed in
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metabolism cages separated by glass (environmental conditions not specified) where urine and
feces samples were collected. Bile was collected through a biliary cannula for periods of
6-8 hours. The study was conducted over a 5-day period.
Mean hourly biliary excretion values for radioactive BHT and its biliary metabolites
(BHT-CH20H, BHT-CHO, BHT-COOH, and B-B) were reported for 6 hours after a single
intravenous dosing (six animals per compound, except for BHT-CHO, which included four
animals) (Holder et al., 1970). Excretion values were recorded every 2 hours for an 8-hour
period for the same compounds (except B-B) after single i.p. dosing (six animals for BHT and
two animals for the other compounds). The results showed that these BHT compounds (with the
exception of B-B) are rapidly absorbed, metabolized, and excreted in the bile.
Five days after single intraperitoneal dosing, hourly biliary excretion was measured over
a 6-hour period for BHT-CH20H, BHT-CHO, and BHT-COOH. After that 6-hour period
(i.e., 126 hours after single i.p. dosing), the total recoveries (in % dose) for urine, feces, and bile
of radioactive metabolites of BHT, BHT-CH20H, BHT-CHO, and BHT-COOH were
determined (numbers of animals varied) (Holder et al., 1970). There were individual differences
among compounds in the ratio of urinary to fecal excretion of 14C. However, there were no
significant differences in the total radioactivity excreted over 126 hours after dosing. In general,
approximately 70% of the dosed radioactivity for each BHT compound was recovered.
Potential differences in metabolism of i.v. administered BHT and i.p. administered BHT
were suggested given differences in rates of excretion (Holder et al., 1970). Approximately 46%
of i.v. administered BHT was excreted in the bile in the first hour, with 94% excreted 6 hours
after dosing; approximately 32% of the administered i.p. dose was excreted in the bile 2 hours
following dosing, with 52% excreted 6 hours after dosing. Furthermore, excretion data
demonstrated that BHT-COOH and its ester glucuronide are the primary compounds in
enterohepatic circulation.
Holder et al. (1970) identified the metabolites of BHT and its derivatives in urine, feces,
and bile. In all biological extracts, BHT-COOH and/or ester glucuronide were the major
metabolites present.
Stierum et al. (2008) examined the mechanism of BHT-induced liver changes in an in
vivo assay using transcriptomic analysis. Groups of 7-week-old male Sprague-Dawley rats were
treated with 0, 25, 75, 150, 300, or 1000 mg/kg-day BHT (purity 99.9%) via diet for 28 days
(n = 10 for the control group and n = 6 for BHT-treated groups). The study authors reported that
the actual dietary intakes based on animal body weight and food intakes in rats were 0, 28, 88,
167, 321, and 1159 mg/kg-day BHT. At study termination, livers of animals were dissected, and
samples were obtained for cDNA microarray analysis. In addition, samples from the left lobe
also were obtained for RT-PCR analysis. Sample selection for transcriptome analysis was made
after histopathological and clinical chemistry analysis. For BHT, microarray analysis was
performed using tissues obtained from the 150-, 300-, and 1000-mg/kg-day dose groups because
no histological or clinical chemistry changes were observed at these doses. In addition to cDNA
analysis, the study authors also determined CYP1A2 and CYP2B1/2 and glutathione
^'-transferase (GST) activity.
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cDNA analysis indicated that the expression of 10 genes was impacted after treatment
with BHT (Stierum et al., 2008). Hepatic mRNA levels of the phase 1 cytochrome P450
isozymes, CYP2B1/2, CYP3A9, and CYP2C6 were increased, with CYP2B1/2 levels exhibiting
a clear dose-response trend. BHT also increased the mRNA levels of GST 1 among the phase 2
metabolizing enzymes in a dose-dependent manner. In addition to these phase 1 and 2 enzymes,
mRNA levels of carboxylesterase 10 precursor, interleukin 15, hematopoietic cell tyrosine
kinase, zinc finger protein 179, tryptophan-2-3-dioxygenase, and tropomyosin isoform 6 also
were increased after BHT treatment. RT-PCR analysis indicated that there was a dose-dependent
and statistically significant (p < 0.001) increase in mRNA levels of both CYP2B1 and
CYP2B1/2 that correlated well with the cDNA microarray data. To confirm the cDNA
microarray increase in GST \i2 levels after BHT treatment, the study authors conducted an assay
for GST activity toward l,2-dichloro-4-nitrobenzene (DCNB) as an indicator substrate. A
dose-dependent and statistically significant (p < 0.001 at the three highest doses) increase was
noted in GST \i2 activity toward DCNB compared to the concurrent control group. Based on
these results, the study authors concluded that gene expression analysis provides new insights
regarding the dose-dependent mode of action of BHT after in vivo administration in male rats.
Induction of both phase 1 and 2 metabolizing enzymes may provide an understanding regarding
the toxicity of BHT after oral administration.
In a published, peer-reviewed, comparative metabolism study (Matsuo et al., 1984),
5-week-old male and female DDY/Slc mice (4/sex/dose group) and 5-week-old male
Sprague-Dawley rats (4/dose group) were given a single oral dose of BHT labeled at the
/;-methyl group with 14C ([14C]BHT; radiochemical purity >99%) of 20 or 500 mg/kg in a 5-mL
corn oil suspension by stomach intubation. No information regarding GLP compliance was
provided. The study authors also indicated that other dosing groups were examined. A group of
male mice (number of animals unclear) was given daily oral doses of 20 mg [14C]BHT/kg for
10 consecutive days for the purpose of tissue residue examination, and another group of 50 male
mice was given a single oral dose of 500 mg [14C]BHT/kg for the purpose of metabolite
characterization.
For the duration of the experiment, animals were housed individually in metabolism
cages in an air-conditioned room (25 ± 2°C) and supplied a diet of CE-2 of Clea Japan, Inc. and
water, which were given ad libitum (Matsuo et al., 1984). Urine, feces, and expired air samples
were collected. Seven days after treatment, the mice were sacrificed, and the following tissues
were obtained: blood, heart, kidney, liver, spleen, pancreas, lung, brain, adrenal gland, muscle,
sciatic nerve, spinal cord, salivary gland, fat, stomach, intestine, caecum, hair, skin, bone, testis,
uterus, and ovary.
In mice given the single oral doses, absorption measured at 3 and 16 hours after treatment
was highest in the stomach, intestines, gall bladder, and urinary bladder, and was present to a
lesser extent in the liver, kidney, spleen, and salivary gland (Matsuo et al., 1984). At 24 hours
minimal concentrations were found in the gall bladder, urinary bladder, liver, kidney, spleen, and
digestive organs. By 168 hours, no concentrations were detected. Over the 7 days after
treatment, 41-65% of [14C]BHT was excreted in the feces, 26-50% in the urine, and 6-9% in
expired air (total recovery 96-98%). For the 20 mg [14C]BHT/kg single-dose group, 14C-residue
levels in tissues were low (<1 ppm). For the 500 mg [14C]BHT/kg single-dose group,
14C-residue levels in tissues were higher (up to 11 ppm). For mice given 20 mg [14C]BHT/kg for
10 consecutive days, the study authors reported that tissue residues of 14C increased over time
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and appeared to move toward a steady state after 10 days. One to two days after treatment
ended, a slight increment in tissue residues was found, after which tissue residues began to
rapidly decrease. In male rats given a single oral dose of 20 or 500 mg, excretion of [14C]BHT
was 64-70% in feces and 16-19% in urine with the total recovery being 83-86%) at 3 days
following administration. Tissue residue analyses were not performed in rats.
More than 43 metabolites were identified in the urine and feces of both species
(Matsuo et al., 1984). For mice and rats, the oxidation of the />m ethyl group was the major
metabolic reaction of [14C]BHT. Oxidation of the tert-butyl groups was also a major metabolic
reaction for mice, but this was only a minor reaction in rats.
Generally tests to assess the mutagenicity of BHT are negative. Ames assays testing the
genotoxicity of BHT found no evidence of genotoxic effects in various strains of S. typhimurium
(Bruce and Heddle, 1979; Shelef and Chin, 1980; Dertinger et al., 1993; Kinae et al., 1981) or
B. subtilis (Kinae et al., 1981). McKee and Tometsko (1979) concluded that in nontoxic
concentrations, BHT reduces chromosomal inversions by chemicals requiring metabolic
activation. Examination of frequencies of sex-linked recessive lethal mutations in Drosophila
melanogaster exposed to BHT in conjunction with radiation revealed radiosensitizing properties
of BHT, but did not suggest BHT-induced genetic damage (Kamra, 1973; Barnett and Munoz,
1980; Prasad and Kamra, 1974; Sankaranarayanan, 1983). Shamberger et al. (1973) and Paschin
and Bahitova (1984) reported evidence of inhibitory effects of BHT on chromosomal breakage
induced by exposure to dimethylbenzanthracene in human blood leukocytes and benzo(a)pyrene
in hamster V79 cells, respectively. Epstein et al. (1972) found no evidence of mutagenic effects
of BHT in mice. Sheu et al. (1988) also reported no mutagenic effects of BHT in mice, but
observed an increase in dominant lethal mutations in rats exposed to BHT.
Based on the results of studies presented in Table 3, a mode of action for BHT cannot be
determined.
DERIVATION OF PROVISIONAL VALUES
DERIVATION OF ORAL REFERENCE CONCENTRATIONS
Table 4 presents a summary of noncancer reference values. Table 5 presents a summary
of cancer values. The cancer and inhalation toxicity values are converted to HEC/HED units,
and the conversion process is presented as footnotes. IRIS data are indicated in the table if
available.
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
The study by Olsen et al. (1986) is selected as the principal study for the derivation
of the subchronic p-RfD. The study is published and peer-reviewed. Details are provided in
the "Review of Potentially Relevant Data" section. The critical effect selected is decreased body
weight observed at 9 weeks of treatment in F1 Wistar rats directly exposed to BHT via diet for
141-144 weeks (see Table B.22). This endpoint is supported by decreased body weight reported
in several other studies of subchronic duration (Fulton et al., 1980, Powell et al., 1986;
Hirose et al., 1993; NCI, 1979a,b; McFarlane et al., 1997a,b).
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Table 4. Summary of Noncancer Reference Values for Butylated Hydroxytoluene (CASRN 128-37-0)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Valuea
POD Method
PODb
UFC
Principal Study
Subchronic p-RfD
(mg/kg-day)
Rat/M
Decreased F1 body weight at
9 weeks
1 x 10°
NOAEL
100
100
Olsen et al. (1986)
Chronic p-RfD
(mg/kg-day)
Rat/M
Decreased F1 adult weight at
138 weeks
3 x 10"1
NOAEL
25
100
Olsen et al. (1986)
Subchronic p-RfC
(mg/m3)
None
Chronic p-RfC
(mg/m3)
None
ap-reference values are presented in mg/kg-day.
bDosimetry: POD values are converted from a discontinuous to a continuous (daily) exposure in mg/kg-day.
Table 5. Summary of Cancer ReferenceValues for Butylated Hydroxytoluene (CASRN 128-37-0)
Toxicity Type
Species/Sex
Tumor Type
Cancer Valuea
Principal Study
p-OSF
Rat/M
Total hepatocellular tumors
3.6 x 10"3(mg/kg-day)"1
Olsen et al. (1986)
p-IUR
No information available
""Dosimetry: POD values are converted from a discontinuous to a continuous (daily) exposure in mg/kg-dayand further converted to an HED.
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Based on the available database (see Table 2), it appears that the liver is a target organ of
BHT toxicity. Although there is support for a role of BHT in causing liver injury, liver effects
may not be the most sensitive toxicological endpoint due to BHT exposure. The most sensitive
subchronic liver endpoint appears to be increased relative liver weight in male Wistar rats with a
LOAEL of 30 mg/kg-day from the Fulton et al. (1980) study. However, this LOAEL is not
consistent with other values observed for increased relative liver weight from other experimental
studies. In a chronic-duration study by Williams et al. (1990), a NOAEL of 79 mg/kg-day with a
corresponding LOAEL of 237 mg/kg-day are identified for increased relative liver weight in
male F344 rats. Second, a NOAEL of 147 mg/kg-day and a LOAEL of 368 mg/kg-day are
identified for increased relative liver weight in male Wistar rats from the chronic-duration study
by Deichmann et al. (1955d). The LOAELs from the Williams et al. (1990) and the
Deichmann et al. (1955d) studies are nearly 7-fold higher than that identified for increased
relative liver weight in male Wistar rats from the Fulton et al. (1980) study, suggesting that the
LOAEL of 30 mg/kg-day may not be reliable because of its inconsistency with other values
identified for the same endpoint. The next most sensitive subchronic value, a NOAEL of
250 mg/kg-day for increased incidences of nonneoplastic hepatic lesions (i.e., necrosis, fibrosis,
hepatocyte hypertrophy, and hepatocyte hyperplasia) identified from the 28 day study by
Powell et al. (1986), could be a potential POD for derivation of the subchronic p-RfD.
Another common toxicological effect of BHT is decreased body weight (see Table 2),
which may be a more sensitive endpoint of BHT exposure than liver. From the developmental
and reproductive study by McFarlane et al. (1997b), a LOAEL of 100 mg/kg-day is determined
from this study by causing a 20% decrease in fetal body weight with a corresponding NOAEL of
25 mg/kg-day (see Table B.38). However, the effect of BHT on decreased fetal body weight is
not consistent with other studies. Olsen et al. (1986) observed no BHT-related biologically
significant effects on fetal body weight (see Table B.21). These data suggest that the decreased
fetal body weight observed in the McFarlane et al. (1997b) study may not be a toxicological
effect of BHT exposure and thus should not be used for derivation of a reference value.
Although Olsen et al. (1986) observed no changes in fetal body weight, decreased body
weight was noted throughout the 141-144 weeks in which F1 rats were exposed to BHT via diet
(see Table B.22). Because body-weight data were recorded throughout the study, subchronic
effects of BHT can be delineated even though the study was performed with the purpose of
determining chronic effects. At 9 weeks of exposure (i.e., subchronic-duration), there was a
biologically (>10% change) and statistically significant decrease in body weight in male and
female rats at the highest dose tested (i.e., 250 mg/kg-day). These data from Olsen et al. (1986)
are not amenable to BMD modeling because no data variability is provided, which is necessary
for BMDS. A NOAEL of 100 mg/kg-day is identified from this study for an 11% decrease in
body weight in male rats with a corresponding LOAEL of 250 mg/kg-day. The selection of this
NOAEL of 100 mg/kg-day as the POD would protect against BHT-decreased body weight but
also the liver effects observed in the 28 day study by Powell et al. (1986). Therefore, the critical
effect selected is decreased body weight at 9 weeks in F1 male Wistar rats (Olsen et al., 1986).
The NOAEL of 100 mg/kg-day based on this effect is chosen as the POD to derive a subchronic
p-RfD.
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Adjusted points for daily exposure:
The following dosimetric adjustments were made for each dose in the principal study for
dietary administration. Dosimetric adjustment for 100 mg/kg-day is presented below.
(DOSEadj) = DOSEoisenetai. 1986 x [conversion to daily dose]
= 100 mg/kg-day x (days of week dosed ^ 7)
= 100 mg/kg-day x (7 -h 7)
= 100 mg/kg-day
A subchronic p-RfD for BHT, based on a NOAEL of 100 mg/kg-day in male rats
(Olsen et al., 1986), is developed as follows:
Subchronic p-RfD = NOAEL -h UFc
= 100 mg/kg-day -M00
= 1 x 10 mg/kg-day
Tables 6 and 7, respectively, summarize the uncertainty factors and the confidence descriptor for
the subchronic p-RfD for BHT.
Table 6. Uncertainty Factors for Subchronic p-RfD of Butylated Hydroxytoluene
UF
Value
Justification
ufa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic
and toxicodynamic differences between rats and humans. There are no data to determine
whether humans are more or less sensitive than rats to subchronic oral exposure to BHT.
ufd
1
A UFd of 1 is selected because the database includes one acceptable three-generation
reproduction study in mice (Tanaka et al., 1993) and two developmental studies in rats and
mice (McFarlane et al., 1997).
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response in humans.
ufl
1
A UFl of 1 is applied for using a POD based on a NOAEL.
UFS
1
A UFS of 1 is applied because a subchronic study was utilized as the principal study.
UFC
100

a01sen et al. (1986).
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Table 7. Confidence Descriptor for Subchronic p-RfD for Butylated Hydroxytoluene
Confidence Categories
Designation"
Discussion
Confidence in the study
H
Confidence in the key study is high. Olsen et al. (1986)
is a toxicity study in rats that was performed to
investigate developmental, reproductive, and
carcinogenic effects of BHT administration on Wistar
rats. Multiple studies in the literature support decreased
body weight as an index of BHT toxicity.
Confidence in the database
H
Confidence in the database is high. The database
includes subchronic and chronic toxicity studies in four
species (rat, mouse, hamster, and dog), developmental
toxicity studies in two species (rat and mouse), and a
two-generation reproduction study (mouse).
Confidence in the subchronic p-RfDb
H
The overall confidence in the subchronic p-RfD is high.
aL = low; M = medium; H = high.
bThe overall confidence cannot be greater than the lowest entry in the table.
The confidence of the subchronic p-RfD for BHT is high, as explained in Table 7.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
To fully evaluate noncancer effects due to chronic exposure of BHT, BMD modeling was
performed on appropriate data from chronic studies listed in Table 2. The studies for each effect
are presented in Table 8 with their corresponding benchmark responses (BMRs), BMDs, and
BMDLs.
Whenever possible, dose-response models were fit to the data from the aforementioned
studies to estimate potential PODs from which to derive the RfD. All of the common models
(i.e., Gamma, Multistage, Logistic, Probit, Weibull, and Quantal-Linear models for dichotomous
data; Linear, Polynomial, Power, and Hill models for continuous data) available in the EPA's
Benchmark Dose Software (BMDS, version 2.1.2) were fit to the data, and results are
summarized in Table 8 below. A detailed summary of BMD methodology and modeling results
is provided in Appendix C. These results and their associated studies were reviewed to establish
a principal study and a POD.
The study by Olsen et al. (1986) is selected as the principal study for the derivation
of the chronic p-RfD. The study is published and peer-reviewed. Details are provided in the
"Review of Potentially Relevant Data" section. The critical effect selected is decreased body
weight at 138 weeks in F1 Wistar rats exposed to BHT via diet for 141-144 weeks (see
Table B.22). This study provides the most sensitive toxicological endpoint.
BMD modeling was performed as described in Appendix C, to fully evaluate noncancer
effects due to chronic exposure of BHT. After reviewing the modeling results, it was determined
that liver peliosis in male mice from the NCI (1979d) study, was the most sensitive liver effect
due to chronic exposure of BHT. For the endpoint of increased incidence of liver peliosis, the
Log-logistic model was considered the best fit and produced a BMDio and BMDLio of 73 and
14 mg/kg-day (see Table 8). However, the BMDLio of 14 mg/kg-day is 37-fold lower than
lowest dose (i.e., 515 mg/kg-day) tested for this effect, suggesting that the BMD modeling of
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liver peliosis is not reliable and the resulting BMDLio is not suitable as a potential POD. A
similar situation was determined for the liver necrosis data with a BMDLio of 16 mg/kg-day.
The next most sensitive and reliable, possible POD for liver effects from the NCI (1979d) study
is a BMDLio of 100 mg/kg-day for increased incidence of hepatic cytoplasmic vacuolation in
male rats.
In the study by Olsen et al. (1986), biologically and statistically decreases in body weight
were observed in F1 Wistar rats, throughout the duration of the experiment. At the end of
138 weeks, a NOAEL of 25 mg/kg-day is identified with a corresponding LOAEL of
100 mg/kg-day for an 11% decrease in body weight of male rats. Therefore, the critical effect
selected is decreased body weight at 138 weeks in F1 male Wistar rats (Olsen et al., 1986). The
NOAEL of 25 mg/kg-day based on this effect is chosen as the POD to derive a chronic p-RfD.
The selection of the NOAEL of 25 mg/kg-day based on chronic body-weight depression as the
POD will not only protect against this effect but also the liver effects of BHT observed in the
NCI (1979d) study.
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Table 8. BMDLs for Multiple Noncancer Effects Following Chronic Oral Exposure to BHT

Dose (mg/kg-day)
Comments
BMRa
BMD
BMDL
NCI (1979c)
Focal alveolar histiocytosis
10%
199 (F)
157 (F)
Increased incidence of focal alveolar histiocytosis in female F344 rats. M and F notations in this
case refer to the sex of rats.
NCI (1979d)
Hepatocytomegaly
10%
355 (M)
284 (M)
Increased incidence of hepatocytomegaly in B6C3F, mice. M and F notations in this case refer to
the sex of mice.
NCI (1979d)
Liver peliosis
10%
73 (M)
14 (M)
Increased incidence of liver peliosis in B6C3F, mice. M and F notations in this case refer to the sex
of mice. BMD results are not reliable.
NCI (1979d)
Liver necrosis
10%
142 (M)
16 (M)
Increased incidence of liver necrosis in B6C3F, mice. M and F notations in this case refer to the
sex of mice. BMD results are not reliable.
NCI (1979d)
Hepatic cytoplasmic
vacuolation
10%
180 (M)
100 (M)
Increased incidence of hepatic cytoplasmic vacuolation in B6C3F, mice. M and F notations in this
case refer to the sex of mice.
McFarlane et al. (1997a)
Body weight of F0 dams
10%
730
664
Decreased body weight in F0 Wistar rat dams.
McFarlane et al. (1997a)
Relative liver weight
10%
No fit
No fit
Increased relative liver weight in Wistar rat dams.
McFarlane et al. (1997a)
Body weight of pups
10%
No fit
No fit
Decreased body weight in Wistar pups.
aBMR = benchmark response.
BMD input data for these liver data are presented in Tables B. 18, B.30, B.34, and B.35. The curves and BMD output text are
provided in Appendix C.
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Adjusted points for daily exposure:
Daily doses in mg/kg food were provided by Olsen et al. (1986). The following
dosimetric adjustments were made for each dose in the principal study for dietary administration.
Dosimetric adjustment for 25 mg/kg-day is presented below.
(DOSEadj) = DOSEoisenetai. 1986 x [conversion to daily dose]
= 25 mg/kg-day x (days of week dosed ^ 7)
= 25 mg/kg-day x (7 -h 7)
= 25 mg/kg-day
The chronic p-RfD for BHT, based on the NOAEL of 25 mg/kg-day in male mice
(Olsen et al, 1986), is derived as follows:
Chronic p-RfD = NOAEL - UFC
= 25 mg/kg-day -MOO
= 3 x 10"1 mg/kg-day
Tables 9 and 10, respectively, summarize the uncertainty factors and the confidence
descriptor for the chronic p-RfD for BHT.
Table 9. Uncertainty Factors for Chronic p-RfD
of Butylated Hydroxytoluene (Olsen et al., 1986)
UF
Value
Justification
ufa
10
A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic
and toxicodynamic differences between mice and humans. There are no data to determine
whether humans are more or less sensitive than mice to chronic oral exposure to BHT.
ufd
1
A UFd of 1 is selected because the database includes one acceptable three-generation
reproduction study in mice (Tanaka et al., 1993) and two developmental studies in rats and
mice (McFarlane et al., 1997).
UFh
10
A UFh of 10 is applied for intraspecies differences to account for potentially susceptible
individuals in the absence of information on the variability of response in humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
1
A UFS of 1 is applied because a chronic study was utilized as the principal study.
UFC
100

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The confidence of the chronic p-RfD for BHT is high, as explained in Table 10.
Table 10. Confidence Descriptor for Chronic p-RfD for Butylated Hydroxytoluene
Confidence Categories
Designation"
Discussion
Confidence in the study
H
Confidence in the key study is high. Olsen et al. (1986)
is a toxicity study in rats that was performed to
investigate developmental, reproductive, and
carcinogenic effects of BHT administration on Wistar
rats. Multiple studies in the literature support decreased
body weight as an index of BHT toxicity.
Confidence in the database
H
Confidence in the database is high. The database
includes subchronic and chronic toxicity studies in four
species (rat, mouse, hamster, and dog), developmental
toxicity studies in two species (rat and mouse), and a
two-generation reproduction study (mouse).
Confidence in the chronic p-RfDb
H
The overall confidence in the chronic p-RfD is high.
aL = low; M = medium; H = high.
bThe overall confidence cannot be greater than the lowest entry in the table.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No human or animal studies examining the toxicity of BHT following inhalation
exposure have been identified. Therefore derivation of a subchronic p-RfC or chronic p-RfC is
precluded.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Cancer Weight-of-Evidence Descriptor
Table 11 identifies the cancer weight-of-evidence descriptor for BHT.
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Table 11. Cancer Weight-of-Evidence Descriptor for Butylated Hydroxytoluene"
Possible WOE Descriptor
Designation1"
Route of Entry (Oral,
Inhalation, or Both)
Comments
"Carcinogenic to Humans "
N/A
N/A
No human cancer studies are available.
"Likely to be Carcinogenic
to Humans "
N/A
N/A
The evidence from animal studies is mixed
with both positive and negative results
regarding the carcinogenic potential of BHT.
Therefore, BHT cannot be considered
"Likely to be carcinogenic to humansT
"Suggestive Evidence of
Carcinogenic Potential"
Selected
Oral
Liver tumors related to BHT treatment
have been reported in both male and
female rats (Olsen et al., 1986) as well as
in male mice (Inai et al., 1988). However,
there are also four animal studies that
reported negative tumor findings in
rodents treated with BHT (Williams et al.
1990; NCI, 1979c; Price, 1994). Therefore
based on the mixed results from the
available cancer studies, BHT is
considered to have "Suggestive evidence of
carcinogenic potential."
"Inadequate Information to
Assess Carcinogenic
Potential"
N/A
N/A
Adequate information is available to assess
carcinogenic potential.
"Not likely to be
Carcinogenic to Humans"
N/A
N/A
No evidence of noncarcinogenicity is
available.
aBold text indicates choice of cancer weight-of-evidence descriptor.
bN/A = not applicable.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
As noted in Table 11, EPA concluded that there is suggestive evidence of carcinogenic
potential for BHT. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) state:
"When there is suggestive evidence, the Agency generally would not attempt a dose-response
assessment, as the nature of the data generally would not support one; however, when the
evidence includes a well-conducted study, quantitative analyses may be useful for some
purposes, for example, providing a sense of the magnitude and uncertainty of potential risks,
ranking potential hazards, or setting research priorities. In each case, the rationale for the
quantitative analysis is explained, considering the uncertainty in the data and the suggestive
nature of the weight of evidence. These analyses generally would not be considered Agency
consensus estimates."
In the case of BHT, although there are no epidemiologic studies that have evaluated the
carcinogenicity in humans, the carcinogenicity of BHT has been evaluated in several studies in
both rats and mice. As described in Table 11, these studies indicate that there are mixed results
regarding the carcinogenic potential of BHT. However, the study by Olsen et al. (1986) is a
well-conducted study showing evidence of increased incidence of tumors in both sexes of one
species at multiple exposure levels, and the data from this study are adequate to support a
quantitative cancer dose-response assessment. Considering these data and uncertainty associated
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with the suggestive nature of the tumorigenic response, it was concluded that quantitative
analyses may be useful for providing a sense of the magnitude of potential carcinogenic risk.
Based on the weight of evidence, a dose-response assessment of the carcinogenicity of BHT is
deemed appropriate.
The rat study by Olsen et al. (1986) is selected as the principal study for derivation of the
p-OSF. The critical endpoint is increased incidence of total hepatocellular tumors (sum of
adenomas and carcinomas) in F1 Wistar rats. This study is well conducted, peer-reviewed, and
data from this study can support a quantitative cancer dose-response assessment. It is, however,
unclear if this study was performed according to GLP standards. Details are provided in the
"Selection of Potentially Relevant Studies" section.
Olsen et al. (1986) conducted a two-generation feeding study that yielded evidence of
BHT-induced cancer. Dose-related increases of hepatocellular adenomas, carcinomas, and total
hepatocellular tumors were reported in male and female (only increases in adenomas and total
tumors) rats that had been exposed in utero, during lactation, and through 141-144 weeks of life.
The data for increased incidence of hepatocellular carcinomas in male rats is not amenable to
BMD modeling because there are no data at the low response range which is necessary for BMD
modeling. Therefore, only the dose-response data for hepatocellular adenomas and total
hepatocellular tumors (sum of adenomas and carcinomas) in male and female rats can be used to
derive a p-OSF for BHT (see Tables 13 and B.25). The curves and BMD output text are
provided in Appendix D. Table 14 presents a summary of the results for the BMD modeling.
The incidence of total hepatocellular tumors in male rats was considered the most sensitive
tumor response because the modeled data produced a slightly lower BMDio and BMDLio of 41
and 28 mg/kg-day, respectively, compared to other tumor data (i.e., total tumors and adenomas)
from male and female rats. The selection of total hepatocellular tumors as the critical endpoint is
supported by the results of Inai et al. (1988), who reported increased liver tumors in male mice,
as well as other pathologies noted in the liver (liver enlargement, increased xenobiotic liver
enzymes, vacuolation of hepatocytes, liver necrosis, and peliosis), which may indicate a
nongenotoxic mode of action for tumor formation (OECD SIDS, 2002). However, there are
currently insufficient data to support a carcinogenic mode of action for BHT.
Table 12 summarizes the relevant oral carcinogenicity studies for BHT.
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Table 12. Summary of Positive Oral Cancer Studies For Butylated Hydroxytoluene
References
# M/F,
Species
Exposure
(mg/kg-day)
Frequency/
Duration
NOAEL,,,;,/1
(mg/kg-day)
LOAEL,,,.,)
(mg/kg-day)
Critical Endpoint
Olsen et al.
(1986)
60/60 F1
Wistar
rat
F1 Males: 0,
7.1,28, 69
F1 Females:
0, 6.4, 25, 62
7 d/wk for
141-144 weeks
in diet
N/A
N/A
Increased incidence of
hepatocellular tumors
(hepatocellular
adenomas and
carcinomas in males
and adenomas in
females)
Inai et al.
(1988)
50/50
B6C3FJ
mouse
Males: 0,
249, 529
Females: 0,
262, 619
7 d/wk for 104
weeks in diet
N/A
N/A
Increased incidence of
liver adenomas in
males
""Dosimetry: NOAEL and LOAEL values are converted to human equivalent dose (HED in mg/kg-day). All exposure
values are converted from a discontinuous to a continuous (weekly) exposure. Values for oral (cancer only) are
further converted to an HED using the following equation: HED = Dose x (Body Weight Animal ^ Body Weight
Human)1'4.
N/A= not applicable.
Adjusted points for daily exposure:
Daily doses in mg/kg-day were provided by Olsen et al. (1986). The following
dosimetric adjustments were made for dietary treatment in adjusting doses for oral cancer
analysis (see example calculation below):
DOSE hed
Body-weight adjustment
BWh
BW
A
Body-weight adjustment
DOSEhed
Dose x (Body Weight Animal + Body Weight
Human)
0.25
0.25
= (BWa - BWh)'
= 70 kg (human reference body weight
(U.S. EPA, 1997)
= 0.468 kg (time-weighted average body weight for
male Wistar rats calculated using body-weight data
from Olsen et al., 1986)
= (0.468 - 70)0'25 = 0.285
= (Dose)n x 0.285
= 25 mg/kg-day x 0.285
= 7.1 mg/kg-day
Table 13 presents the BMD input for incidence of total hepatocellular tumors (sum of
adenomas and carcinomas) in male and female rats exposed to BHT for 141-144 weeks.
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Table 13. Dose-Response Data for Butylated Hydroxytoluene-Induced
Total Hepatocellular Tumors (Adenomas and Carcinomas) in Rats
Exposed via Diet for 141-144 Weeks"
Dose (mg/kg-day)
DoseHED (mg/kg-day)b
Incidence
Male Rats
0
0
2/100
25
7.1
1/80
100
28
6/80
250
69
26/99
Female rats
0
0
2/100
25
6.4
3/79
100
25
6/80
250
62
14/99
a01sen et al. (1986).
bDoses converted to human equivalency doses using:
HED = Dose x (Body Weight Animal ^ Body Weight Human)14.
Table 14. Goodness-of-Fit Statistics, BMDiohed, and BMDLiohed Values for a
Dichotomous Model for Hepatocellular Tumors in F1 Wistar Rats Treated
with BHT in the Diet for 141-144 weeksa'b
Model
Goodness-of-Fit
p-Valuec
AIC for
Fitted Model
BMDiohed
(mg/kg-day)
BMDLiohed
(mg/kg-day)
Conclusions
Multistage -Cancer
Male
(adenomas)
0.667
159.432
42
30

Multistage -Cancer
Female
(adenomas)
0.906
165.063
58
36

Multistage-Cancer
Male
(total tumors)
0.496
193.48
41
28
Lowest BMDL
Multistage -Cancer
Female
(total tumors)
0.979
172.471
49
32

a01sen et al. (1986).
bBold text indicates BMD model selected to derive the p-OSF.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose.
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p-OSF = 0.1
= 0.1
= 3.6
- BMDL10
28 mg/kg-day
x 10 3 (mg/kg-day)"1
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of BHT following inhalation
exposure have been identified. Therefore, derivation of a p-IUR is precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
No screening values are presented.
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APPENDIX B. DATA TABLES
Table B.l. Concentration of BHT in Tissues of Male Wistar Rats Administered
Butylated Hydroxytoluene by Gavage for 7 or 28 Days


Exposure Group
Parameter
0 mg/kg-day
25 mg/kg-day
250 mg/kg-day
500 mg/kg-day3
7-day study
Liver (mg/kg wet weight)
<1
<1
<1
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Table B.2. Liver Biochemistry in Male Wistar Rats Administered
Butylated Hydroxytoluene by Gavage for 7 or 28 Days
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
250 mg/kg-day
500 mg/kg-daya
7-day study
Hepatic microsomal protein yield
(mg/g wet weight liver)b
20.0 ±2.31
17.7 ±2.88 (89)
19.0 ± 1.81 (95)
15.3 ±2.63 (77)°
Hepatic glucose 6-phosphatase
activity (|imol/min/mg whole
homogenate protein)b
0.05 ± 0.007
0.06 ± 0.003
(120)
0.05 ± 0.003
(100)
0.03 ± 0.009
(60)°
Hepatic cytochrome b5 concentration
(nmol/mg microsomal protein)b
0.52 ±0.09
0.61 ±0.06
(117)
0.65 ±0.06
(125)°
0.59 ±0.10
(113)
28-day study
Hepatic microsomal protein yield
(mg/g wet weight liver)b
24.0 ±3.16
23.7 ± 3.44 (99)
28.2 ±3.24
(118)°
29.5 ± 1.50
(123)°
Hepatic glucose 6-phosphatase
activity (|imol/min/mg whole
homogenate protein)b
0.04 ± 0.002
0.04 ± 0.003
(100)
0.03 ± 0.003
(75)°
0.03 ±0.002
(75)°
Hepatic cytochrome b5 concentration
(nmol/mg microsomal protein)b
0.55 ±0.04
0.62 ±0.08
(113)°
0.66 ± 0.06
(120)°
0.57 ±0.05
(104)
aThe 500-mg/kg-day dose group was treated with 750 mg/kg-day for the first three days of the exposure period,
resulting in an average daily dose of 607 mg/kg-day for the 7-day study and 527 mg/kg-day for the 28-day study.
The equation used for calculating the daily average is:
DoseADD = [(daily dose (mg/kg-day) x days dosed) + (daily dose (mg/kg-day) x days dosed)] total days.
bMean ± SD, (corresponding percentage of control); calculated for this review.
Significantly different from control (p < 0.05) by the Student's t-test conducted by the study authors.
Source: Powell et al. (1986).
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Table B.3. Hepatic Lesions in Male Wistar Rats Administered

Butylated Hydroxytoluene by Gavage for 7 or 28 Days



Exposure Group
Parameter

25 mg/kg-day
250 mg/kg-day
500 mg/kg-daya
7-day study
Periportal regionb
Hepatocyte necrosisc
0/5 (0)
0/5 (0)
2/5 (40)

Fibrosis0
0/5 (0)
0/5 (0)
3/5 (60)

Hepatocyte hypertrophy0
0/5 (0)
0/5 (0)
3/5 (60)

Hepatocyte hyperplasia0
0/5 (0)
0/5 (0)
4/5 (80)

Glycogen accumulation0
0/5 (0)
4/5 (80)
4/5 (80)
28-day study
Periportal regionb
Hepatocyte necrosis0
0/10 (0)
0/10 (0)
6 /10 (60)

Fibrosis0
0/10 (0)
0/10 (0)
5/10 (50)

Bile-duct cell proliferation0
0/10 (0)
0/10 (0)
4/10 (40)

Hepatocyte hypertrophy0
0/10 (0)
0/10 (0)
2/10 (20)

Hepatocyte hyperplasia0
0/10 (0)
0/10 (0)
3/10 (30)

Pigment-laden macrophages0
0/10 (0)
0/10 (0)
3/10 (30)

Glycogen depletion0
0/10 (0)
0/10 (0)
7/10 (70)

Glycogen accumulation0
0/10 (0)
8/10 (80)
0/10 (0)
Mid-zonal glycogen accumulation0
0/10 (0)
0/10 (0)
5/10 (50)
aThe 500-mg/kg-day dose group was treated with 750 mg/kg-day for the first three days of the exposure period,
resulting in an average daily dose of 607 mg/kg-day for the 7-day study and 527 mg/kg-day for the 28-day study.
The equation used for calculating the daily average is:
DoseADD = [(daily dose (mg/kg-day) x days dosed) + (daily dose (mg/kg-day) x days dosed)] total days.
bNo tests for significance were performed due to lack of control data.
°Number of animals with lesions/number examined per dose group, () -corresponding percentages; calculated for
this review.
Source: Powell et al. (1986).
Table B.4. Mean Weight Gain and Relative Liver Weight of Male Wistar Rats
Orally Exposed to Butylated Hydroxytoluene for 8 Weeks
Parameter
Exposure Group (Daily Dose, mg/kg-day)
0
0.02% (30)
0.10% (151)
0.50% (755)
0.75% (1132)
Initial body weight (g)a
37.0
39.2
43.4
48.1
51.6
Total food intake (g)a
472
509b
553b
435
461
Mean body-weight gain (g)a
159.6
164.4 (103)
152.6 (96)
77.8 (49)b
51.8 (32)b
Mean liver weight (g)a
6.3
7.7
8.2
6.7
5.4
Liver to body-weight ratio3
0.03
0.04 (|33)
0.04 (|33)
0.06 (|100)
0.05 (|67)
aMean, (percent difference from control); calculated for this review.
bSignificantly different from control (p < 0.05) by a partial correlation for multivariant data test conducted by the
study authors.
Source: Fulton etal. (1980).
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Table B.5. Ileal Biopsy Data of Male Wistar Rats Orally Exposed to
Butylated Hydroxytoluene for 8 Weeks
Parameter
Exposure Group (Daily Dose, mg/kg-day)
0
0.02% (30)
0.10% (151)
0.50% (755)
0.75% (1132)
Villus:
Height
230.6
221.9 (96)
179.2 (78)
147.8 (64)
137.3 (60)
Goblet count3
22.3
23.0 (103)
14.2 (64)
13.0(58)
13.3 (60)
Crypt:
Depth
59.8
47.2 (79)
35.8 (60)
40.9 (68)
40.0 (67)
Goblet count3
7.2
7.6 (106)
2.8 (39)
2.1 (29)
2.7 (38)
aMean, (percentage of control); calculated fortius review.
Source: Fulton et al. (1980)
Notes: Quantitative statistics were not provided for this data from the study and cannot be performed independently
due to lack of information.
Table B.6. Survival and Weight Loss of Male and Female Fischer F344 Rats Following
Neat Administration of Butylated Hydroxytoluene for 7 Weeks


Exposure Group (Adjusted Daily Dose, mg/kg-day)3
Parameter
0 ppm
6200 ppm
(620)
12,500 ppm
(1250)
25,000 ppm
(2500)
50,000 ppm
(5000)
Male Rats
Sample Size
5
5
5
5
5
Survival
5
5
4
5
0
Mean Weight (% of
control)b
100
88
74
38
-

Exposure Group (Adjusted Daily Dose, mg/kg-day)3
Parameter
0 ppm
6200 ppm
(700)
12,500 ppm
(1411)
25,000 ppm
(2822)
50,000 ppm
(5645)
Female Rats
Sample Size
5
5
5
5
5
Survival
5
5
5
5
0
Mean Weight (% of
control)b
100
93
84
44
-
aDoses were converted to adjusted daily doses using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bAt Week 7 as percentage of control.
Source: NCI (1979a).
Notes: Quantitative statistics were not provided for this data from the study and cannot be performed independently
due to lack of information.
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Table B.7. Survival and Weight Loss of Male and Female B6C3Fi Mice After Neat
Administration of Butylated Hydroxytoluene for 7 Weeks
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0 ppm
3100 ppm
(559)
6200 ppm
(1118)
12,500 ppm
(2255)
25,000 ppm
(4509)
50,000 ppm
(9019)
Male Mice
Sample Size
5
5
5
5
5
5
Survival
5
5
5
5
5
4
Mean Weight (% of
control)b
100
89
94
78
79
73
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0 ppm
3100 ppm
(605)
6200 ppm
(1210)
12,500 ppm
(2439)
25,000 ppm
(4878)
50,000 ppm
(9756)
Female Mice
Sample Size
5
5
5
5
5
5
Survival
5
5
5
5
4
1
Mean Weight (% of
control)b
100
88
83
82
74
97
aDoses were converted to adjusted daily doses using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bAt Week 7 as percentage of control.
Source: NCI (1979b).
Notes: Quantitative statistics were not provided for this data from the study and cannot be performed independently
due to lack of information.
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Table B.8. Body and Liver Weights of Fisher 344 Rats Administered Butylated
Hydroxytoluene by Diet for 76 and 110 Weeks
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0 ppm
(0)
100 ppm
(8)
300 ppm
(24)
1000 ppm
(79)
3000 ppm
(237)
6000 ppm
(474)
76-week study
Body weight (g)b
409 ± 35
424 ± 42
(104)
409 ± 34
(100)
411 ±20
(100)
362 ± 35°
(89)
367 ± 49d
(90)
Liver weight (g)b
14.5 ±2.1
14.2 ± 1.3
(98)
15.0 ± 1.0
(103)
15.2 ± 1.3
(105)
14. 7 ±3.4
(101)
19.1 ± 3.3e
(132)
Relative liver weight
(g/100 g body weight)b
3.6 ±0.5
3.4 ±0.4
(94)
3.7 ±0.3
(103)
3.7 ±0.4
(103)
4.0 ±0.6
(HI)
5.4 ± 1.4'
(150)
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0 ppm (0)
12000 (947)
110-week study
Body weight (g)b
425 ± 35
379 ± 28s (89)
Liver weight (g)b
18.9 ±3.8
15.6 ±2.4' (83)
Relative liver weight
(g/100 g body weight)b
4.4 ±0.8
4.0 ±0.5 (91)
aDoses were converted from then ppm intake in food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bMean ± SD, (percentage of control); calculated for this review.
Significantly different from control (p < 0.02) by Student's t-test performed by the study authors.
Significantly different from control (p < 0.05) by Student's /-test performed by the study authors.
"Significantly different from control (p < 0.002) by Student's /-test performed by the study authors.
Significantly different from control (p < 0.01) by Student's t-test performed by the study authors.
8Significantly different from control (p < 0.001) by Student's /-test performed by the study authors.
Source: Williams et al. (1990).
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Table B.9. Liver Lesions in Fisher 344 Rats Administered Butylated
Hydroxytoluene by Diet for 76 and 110 Weeks
Parameter
Exposure Group (Human Equivalency Dose, mg/kg-day)a'b
0 ppm
(0)
100 ppm
(2)
300 ppm
(6)
1000
ppm (21)
3000 ppm
(64)
6000 ppm
(129)
76-week study
Hepatocellular Altered Foci
Incidence (%)
1/4 (25)
1/4 (25)
1/4 (25)
1/4 (25)
1/4 (25)
1/4 (25)
Profiles0
(No./cm2)
0.1 ±0.6
0.3 ±0.6
0.3 ±0.5
0.3 ±0.6
0.4 ±0.6
0.7 ± 1.2
Area0 (mm2)
0.3 ±0.1
0.3 ±0.2
0.4 ±0.3
0.4 ±0.1
0.4 ±0.1
0.7 ±0.5
Area (%)
0
0
0.03 ± 0.04
0
0.03 ±0.04
0.03 ±0.04
Neoplasms
No. of adenomas
3
1
2
2
1
2
No. of carcinomas
0
0
0
0
0
0
Multiplicity0
0.5 ± 1.4
0.1 ±0.3
0.3 ±0.5
0.3 ±0.5
0.1 ±0.5
0.3 ±0.5
Incidence (%)
3/7 (17)
1/7 (14)
2/7 (29)
2/7 (29)
1/7 (14)
2/6 (33)
Parameter
Exposure Group (Human Equivalency Dose, mg/kg-day)a'b
0 ppm (0)
12000 (257)
110-week study
Hepatocellular Altered Foci
Incidence (%)
16/25 (64)
9/23 (39)
Profiles0
(No./cm2)
0.9 ±0.9
0.5 ±0.8
Area0 (mm2)
0.2 ±0.2
0.1±0.1
Area0 (%)
0.3 ±0.3
0.1 ±0.1
Neoplasms
No. of adenomas
9
3
No. of carcinomas
0
0
Multiplicity0
1.0 ± 1.6
0.1 ± 0.3d
Incidence (%)
9/25 (36)
3/23 (13)
aDoses were converted from then ppm intake in food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses were converted from adjusted daily doses to human equivalency doses using the following
formula:
Dose hed = DoseADi x (Body Weight Animal ^ Body Weight Human)A(0.25).
°Mean± SD.
Significantly different from control (p < 0.02) using Fisher's exact probability test, as reported by the
study authors.
Source: Williams et al. (1990).
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Table B.10. Gastric Lesions in Fisher 344 Rats Administered Butylated
Hydroxytoluene by Diet for 76 and 110 Weeks


Exposure Group (Human Equivalency Dose, mg/kg-day)a'b
Parameter
0 ppm (0)°
6000 ppm (129)°
0 ppm (0)d
12000 ppm
(257)
Squamous stomach hyperplasia
Milde
2/5 (40)
4/10 (40)
12/25 (48)
14/23 (61)
Moderate
0/5 (0)
0/10 (0)
0/25 (0)
0/23 (0)
Severe
0/5 (0)
0/10 (0)
0/25 (0)
0/23 (0)
Squamous stomach neoplasms
Squamous cell papilloma
0/5 (0)
0/10 (0)
0/10 (0)
0/10 (0)
Gladular stomach dysplasia
Mild
0/5 (0)
0/10 (0)
0/10 (0)
0/23 (0)
Moderate
0/5 (0)
0/10 (0)
0/10 (0)
0/23 (0)
Severe
0/5 (0)
0/10 (0)
0/10 (0)
0/23 (0)
aDoses were converted from then ppm intake in food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses were converted from adjusted daily doses to human equivalency doses using the following
formula:
Dose hed = DoseADi x (Body Weight Animal ^ Body Weight Human)A(0.25).
°Study period was 76 weeks.
dStudy period was 110 weeks.
"Incidence/total number of animals, () -corresponding percentage; calculated for this review.
Source: Williams et al. (1990)
Table B.ll. Final Body Weight, Organ Weight, and Food Consumption in
F344 Male Rats Administered Butylated Hydroxytoluene by Diet for 36 Weeks
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.7% (700)
Sample size
11
12
Body weight (g)b
402 ±21
348 ± 16° (87)
Relative liver weight (g/100 g bw)b
2.69 ±0.16
4.73 ±0.22c (176)
Relative kidney weight (g/100 g bw)b
0.53 ±0.04
0.68 ±0.03c (128)
Food consumption (g/day/rat)b
15.8
15.0 (95)
aDoses are converted from % of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), and then ppm intake in
food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bMean ± SD, (percentage of control); calculated for this review.
"Significantly different from controls (p < 0.001) by Students /-test performed by the study authors.
Source: Hirose et al. (1993).
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Table B.12. Average Relative Organ Weights in the Wistar Rat Exposed to
Butylated Hydroxytoluene by Diet for 104 Weeks
Parameter (g/100 g bw)
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.25% (184)
1% (736)
Male Rats
Relative Liver Weightb
2.5
4.1 (164)
3.7(148)
Parameter (g/100 g bw)
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.25% (210)
1% (842)
Female Rats
Relative Liver Weightb
2.8
2.9 (104)
3.5 (125)
Relative Spleen Weightb
0.34
0.21(62)
0.23 (68)
aDoses were converted from % food to ppm by dividing by 10,000 (1% = 10,000 ppm) and then converted
to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bMean, (corresponding percentage of control); calculated for this review.
Source: Hirose et al. (1981).
Notes: Quantitative statistics were not provided for this data from the study and cannot be performed
independently due to lack of information.
Table B.13. Selected Hematology and Clinical Chemistry Parameters in the Wistar
Rat Exposed to Butylated Hydroxytoluene by Diet for 104 Weeks
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.25% (184)
1% (736)
Male Rats
Serum Triglycerides (mg/dl)
180
140b
137b
y-GTP (mU/dl)
2.8
3.8C
4.4C
Total Cholesterol (mg/dl)
83.1
94.8d
NR
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.25% (210)
1% (842)
Female Rats
Red Blood Cells (xl04/mm3)
585
612d
606d
Total Cholesterol (mg/dl)
73.4
99.2C
112°
aDoses were converted from % food to ppm by dividing by 10,000 (1% = 10,000 ppm) and then converted to
adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bSignificantly different from control (p < 0.01) using the Student /-test, as reported by the study authors.
"Significantly different from control (p < 0.001) using the Student /-test, as reported by the study authors.
dSignificantly different from control (p < 0.05) using the Student /-test, as reported by the study authors.
Source: Hirose et al. (1981).
Notes: NR= Not Reported.
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Table B.14. Tumor Incidence in Wistar Rats Administered Butylated

Hydroxytoluene by Diet for 104 Weeks



Exposure Group (Human Equivalency Dose, mg/kg-day)a'b

Males
Females

0%


0%
0.25%
1%
Parameter
(0)
0.25% (52)
1% (210)
(0)
(54)
(215)
Sample size
26
43
38
32
46
51
Liver
Hyperplastic nodule0
2 (7.7)
2 (4.7)
1 (2.6)
0
3 (6.5)
3 (5.9)
Pancreas
Carcinoma0
0
0
1 (2.6)
0
1 (2.2)
4 (7.8)
Islet-cell adenoma0
0
1 (2.3)
2 (5.3)
0
0
0
Mammary gland
Fibro-adenoma0
NA
NA
NA
6(18.8)
8 (17.4)
8 (15.7)
Adenoma0
NA
NA
NA
1 (3.4)
1 (2.2)
1 (2.0)
Uterus
Leiomyoma0
NA
NA
NA
1 (3.4)
1 (2.2)
0
Carcinoma0
NA
NA
NA
1(3.1)
2 (4.3)
1 (2.0)
Pituitary gland
Adenoma0
2 (7.7)
3 (7.0)
1 (2.6)
0
6 (13.0)d
3 (11.8)
Carcinoma0
0
2 (4.7)
5 (13.2)
3 (9.4)
3 (6.5)
7 (13.7)
Adrenal gland
Adenoma0
1 (3.8)
3 (7.0)
0
0
2 (4.3)
1 (2.0)
Carcinoma0
0
0
0
0
0
1 (2.0)
Other00
2 (7.7)
2 (4.7)
4(10.5)
2 (6.3)
4 (8.7)
3 (11.8)
Total0
6 (23.1)
13 (30.2)
10 (26.3)
11 (34.4)
25 (54.3)
25 (49.0)
aDoses were converted from % food to ppm by dividing by 10,000 (1% = 10,000 ppm) and then converted
to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses were converted from adjusted daily doses to human equivalency doses using the following
formula:
Dose hed = DoseADi x (Body Weight Animal ^ Body Weight Human)A(.25).
incidence, (corresponding percentage); reported by the study authors; Animals that survived more than
69 weeks were included.
Significantly different from control (p < 0.05) using the chi-square test, as reported by the study authors.
"Other tumors were a malignant lymphoma of the lung and an osteosarcoma in the limb of one male
control; a subcutaneous fibroma and a thyroid adenoma in male rats given 0.25% BHT diet; a
subcutaneous fibroma, a subcutaneous lipoma, a chronic myelogenous leukemia and a thyroid adenoma in
male rats fed 1% BHT diet; and osteosarcoma in the limb and a thyroid adenoma in a female control; a
kidney liposarcoma, a subcutaneous rhabdomyosarcoma, a uterine lipoma and a subcutaneous squamous-
cell carcinoma in female rats fed 0.25% BHT diet; a rhabdomyosarcoma in the retroperitoneum, an
osteosarcoma in a limb and a subcutaneous fibroma in female rats fed 1% BHT diet.
Source: Hirose et al. (1981).
Notes: NA = Not Applicable.
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Table B.15. Selected Hematology, Body and Organ Weights in Albino Wistar
Male Rats Exposed to Butylated Hydroxytoluene via Diet for 24 Months
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.2% (147)
0.5% (368)
0.5%
(589)b
0.8% (589)
1.0% (736)
Body-weight gain (g)°
367
362
(99)
352
(96)
389
(106)
324
(88)
241
(66)
Brain (% bw)°
0.476
0.481 (101)
0.483 (102)
0.478 (100)
0.537 (113)
0.663 (139)
Heart (% bw)°
0.397
0.387 (98)
0.388 (98)
0.330 (83)
0.361 (91)
0.366 (92)
Lung (% bw)°
1.12
1.34
(89)
1.00
(89)
1.04
(93)
0.781 (68)
0.871 (78)
Kidney (% bw)°
0.830
0.884 (107)
0.908 (113)
0.804 (97)
0.893 (108)
0.897 (108)
Spleen (% bw)°
0.402
0.278 (69)
0.376 (94)
0.303 (75)
0.291 (72)
0.316(79)
Liver (% bw)°
3.85
4.04 (105)
4.55 (118)
4.51 (117)
4.87 (127)
5.85 (152)
Testes (%bw)°
0.774
0.804 (104)
0.758 (98)
0.800 (103)
0.862(111)
1.01 (131)
Erythrocytes
(millions/mm3)d
8.9 [7.3 to
10.4]
9.2 [7.0 to
12.1]
9.8 [6.7 to
13.7]
9.0 [6.8 to
12.6]
9.4 [6.9 to
12.0]
NR
Leucocytes
(thousands/mm3)d
15.7 [5.5 to
29.7]
16.4 [7.6 to
32.5]
12.3 [5.6 to
19.7]
15.8 [9.4 to
23.9]
16.4 [8.2 to
32.0]
NR
Hemoglobin
(g/100 mL)d
14.8 [13.5
to 16.4]
14.6 [13.6
to 16.2]
14.5 [13.6
to 15.5]
14.5 [13.2
to 15.7]
14.5 [12.7
to 16.0]
NR
aDoses were converted from % food to ppm by dividing by 10,000 (1% = 10,000 ppm) and then converted to
adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
b0.5% BHT dissolved in lard, heated for 30 minutes at 150 °C and administered in feed.
°Mean, (corresponding percentage of control); calculated for this review.
dMean [90% values].
Source: Deichmann et al. (1955d).
Notes: Quantitative statistics not provided for this data from the study and cannot be performed independently
due to lack of information. NR = Not Reported.
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Table B.16. Selected Hematology, Body and Organ Weights in the Albino Wistar Female
Rat Exposed to Butylated Hydroxytoluene via Diet for 24 Months
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
0.2% (168)
0.5% (421)
0.5%
(421)b
0.8% (673)
1.0% (842)
Body-weight gain (g)°
237
224 (95)
241 (102)
243 (103)
228 (96)
172 (73)
Brain (% bw)°
0.616
0.681 (111)
0.630 (102)
0.606 (98)
0.608 (99)
0.765 (124)
Heart (% bw)°
0.480
0.531 (111)
0.475 (99)
0.469 (98)
0.468 (98)
0.551 (115)
Lung (% bw)°
0.996
0.741 (74)
0.768 (77)
1.04 (104)
1.02 (102)
1.20(121)
Kidney (% bw)°
0.910
1.01 (111)
0.854 (94)
0.920(101)
0.778 (85)
0.897 (99)
Spleen (% bw)°
0.290
0.290 (100)
0.257 (89)
0.308 (106)
0.249 (86)
0.239 (82)
Liver (% bw)°
4.57
4.25 (93)
4.10(90)
5.20(114)
5.19(114)
5.32(116)
Erythrocytes
(millions/mm3)d
8.2 [6.6 to
9.2]
8.2 [6.8 to
9.9]
8.7 [7.2 to
10.5]
8.1 [6.9 to
9.1]
8.9 [7.6 to
10.8]
9.6 [7.9 to
11.3]
Leucocytes
(thousands/mm3)d
13.4 [7.7 to
22.6]
9.6 [5.4 to
18.8]
10.1 [7.0 to
15.8]
10.8 [5.3 to
15.1]
10.4 [6.2 to
17.5]
9.9 [6.0 to
16.0]
Hemoglobin (g/100 mL)d
14.5 [12.2
to 16.8]
14.1 [13.0
to 15.1]
14.4 [12.8
to 15.3]
15.0 [13.4
to 16.1]
14.6 [12.3
to 16.8]
14.3 [13.0
to 15.5]
°Mean, (corresponding percentage of control); calculated for this review.
dMean [90% values].
b0.5% BHT dissolved in lard, heated for 30 minutes at 150 °C and administered in feed.
aDoses were converted from % food to ppm by dividing by 10,000 (1% = 10,000 ppm) and then converted to
adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
Source: Deichmann et al. (1955d).
Notes: Quantitative statistics not provided for this data from the study and cannot be performed independently due
to lack of information.
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Table B.17. Survival of Male and Female F344 Rats after Oral Exposure to Butylated

Hydroxytoluene for 105 Weeks


Exposure Group (Adjusted Daily Dose, mg/kg-day)a
Parameter
0 ppm (0)
3000 ppm (237)
6000 ppm (474)
Male Rats
Sample size
20
50
50
Survival13
13 (65)
39 (78)
36 (72)

Exposure Group (Adjusted Daily Dose, mg/kg-day)a
Parameter
0 ppm (0)
3000 ppm (275)
6000 ppm (550)
Female Rats
Sample size
20
50
50
Survival13
13 (65)
37 (74)
39 (78)
aDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bNumber of animals surviving to the end of the study period, (corresponding percentage).
Source: NCI (1979c).
Table B.18. Incidence of Nonneoplastic Focal Alveolar Histiocytosis in Male and
Female Rats after Oral Exposure to Butylated Hydroxytoluene for 105 Weeks
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0 ppm (0)
3000 ppm (237)
6000 ppm (474)
Male Rats
Sample size
20
49
49
Focal alveolar histiocytosis'3
1(5)
4(8)
7(14)
Parameter
Exposure Group (Adjusted Daily Dose, mg/kg-day)b
0 ppm (0)
3000 ppm (275)
6000 ppm (550)
Female Rats
Sample size
18
48
49
Focal alveolar histiocytosis'3
2(11)
12 (25)
21 (43)°
aDoses converted to adjusted daily dose using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days)
bNumber of animals with observation, (corresponding percentage)
"Statistically significant (p < 0.05) from independent 72 test performed for this analysis
Source: NCI (1979c).
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Table B.19. Selected Incidence of Neoplasms in Male and Female F344 Rats
After Oral Exposure to Butylated Hydroxytoluene for 105 Weeks
Parameter
Exposure Group
(Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (64)
6000 ppm (129)
Male Rats
Lung
Squamous cell carcinoma, metastac
0/20 (0)
1/49 (2)
0/49 (0)
Alveolar/bronchiolar adenomac
0/20 (0)
0/49 (0)
2/49 (4)
Alveolar/bronchiolar carcinoma0
1/20 (5)
1/49 (2)
1/49 (2)
Liver
Bile duct carcinoma0
0/20 (0)
0/48 (0)
1/48 (2)
Neoplastic nodulec
0/20 (0)
1/48 (2)
1/48 (2)
Hepatocellular carcinoma0
0/20 (0)
1/48 (2)
1/48 (2)
Small intestine
Lipoma0
0/18 (0)
0/48 (0)
1/48 (2)
Kidney
Nephroblastoma0
0/20 (0)
1/49 (2)
0/48 (0)
Urinary bladder
Transitional-cell carcinoma0
0/20 (0)
1/47 (2)
0/46 (0)
Testis
Interstitial-cell tumor40
15/20 (75)
42/49 (86)
32/49 (65)
Parameter
Exposure Group
(Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (66)
6000 ppm (132)
Female Rats
Lung
Alveolar/bronchiolar adenoma0
1/18 (6)
2/48 (4)
0/49 (0)
Alveolar/bronchiolar carcinoma0
0/18 (0)
1/48 (2)
1/49 (2)
Pituitary gland
Adenoma, NOSdo
8/18 (44)
9/48 (19)
5/49 (10)
Uterus
Carcinoma, NOS°
0/17 (0)
0/49 (0)
1/49 (2)
Endometrial stromal polyp0
2/17 (12)
8/49 (16)
6/49 (12)
aDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses converted to human equivalency doses using: HED = NOAELadj x (Body Weight Animal ^ Body Weight
Human)A(.25).
°Number of animals with observation/ total number examined () -corresponding percentage; reported by study
authors.
Significant negative dose-related trend by Cochran-Armitage test performed by authors.
Source: NCI (1979c).
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Table B.20. Selected Tumor Incidence in Male and Female F344 Rats after
Oral Exposure to Butylated Hydroxytoluene for 105 weeks
Parameter
Exposure Group
(Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (64)
6000 ppm (129)
Male Rats
Animals in study
20
50
50
Animals examined histopathologically
20
49
49
Animals with primary tumors0
19 (95)
46 (94)
44(90)
Animals with benign tumors0
18 (90)
45 (92)
41 (84)
Animals with malignant tumors0
9(45)
19 (39)
20 (41)
Parameter
Exposure Group
(Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (66)
6000 ppm (132)
Female Rats
Animals in study
20
50
50
Animals examined histopathologically
18
49
50
Animals with primary tumors0
12 (67)
36 (73)
26 (52)
Animals with benign tumors0
11(61)
27 (55)
18 (36)
Animals with malignant tumors0
2(11)
16 (33)
9(18)
aDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses converted to human equivalency doses using: HED = NOAELAm x (Body Weight Animal ^ Body Weight
Human)A(.25).
°Number of animals with observation, (corresponding percentage); calculated for this review.
Source: NCI (1979c).
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Table B.21. Reproduction Data for F0 Wistar Rats Treated with

Butylated Hydroxytoluene in Diet




Exposure Group, mg/kg-day
Parameter

0
25
100
500
No. of rats/dose group
Females
40
29
30
44

Males
39
29
30
44
Gestation rate (%)
88
95
93
95
No. of pups/litter
Mean
10.9
9.6
10.3
9.1a

After
8.0
8.0
8.0
7.9

standardization





At weaning
7.9
8.0
7.7
7.8
Body weight (g) of pupsb
At birth
5.9
5.9 (100)
5.7c
(97)
5.7
(97)

At Weaning
42.4
40.4° (95)
39.7d
(94)
25.3d
(60)
aArmitage-Cochran test for linear trend in proportions of litters with ten or more pups (p < 0.001)
conducted by the study authors.
bAverage of mean pup weight/litter, (percentage of control); calculated for this review.
Statistically significant by the (p < 0.05) Students /-test conducted by the study authors.
Statistically significant by the (p < 0.001) Students /-test conducted by the study authors.
Source: Olsenetal. (1986).
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Table B.22. Mean Body Weights of F1 Adult Wistar Rats Treated with
Butylated Hydroxytoluene in Diet for 141-144 Weeks"
Exposure Group,
mg/kg-day
No. of
Animals
Mean Body Weights (g) at Week
5
7
9
15
34
90
138
Male Rats
0
100
105
186
243
357
471
575
487
25
80
103
(98)
181b
(97)
244
(100)
350
(98)
453c
(96)
550c
(96)
450b
(92)
100
80
97d
(92)
181b
(97)
243
(100)
346b
(97)
447d
(95)
516d
(90)
433c
(89)
250e
99
83d
(79)
150d
(81)
216d
(89)
30 ld
(84)
385d
(82)
459d
(80)
413d
(85)
Exposure Group,
mg/kg-day
No. of
Animals
Mean Body Weights (g) at Week
5
7
9
15
34
90
138
Female Rats
0
100
86
140
176
231
277
344
313
25
79
86
(100)
139
(99)
176
(100)
227
(98)
268c
(97)
343
(100)
312
(100)
100
80
89b
(103)
139
(99)
176
(100)
226b
(98)
260d
(94)
319d
(93)
305
(97)
250e
99
68d
(79)
122d
(87)
159d
(90)
208d
(90)
247d
(89)
288d
(84)
281°
(90)
aAverage of mean body weights, (percentage of control); calculated fortius review.
bArmitage-Cochran test for linear trend in proportions of litters with ten or more pups (p £ 0.001)
conducted by the study authors.
Statistically significant by the (p < 0.05) Students t-test conducted by the study authors.
Statistically significant by the (p < 0.001) Students /-test conducted by the study authors.
e Rats in the high dose group were born to dams that were exposed to 500 mg/kg/day during gestation and
lactation but that the dose was reduced to 250 mg/kg/day in Fls that were exposed through feed after
weaning.
Source: Olsenetal. (1986).
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Table B.23. Mortality in F1 Adult Wistar Rats Treated with Butylated Hydroxytoluene in
Diet for 141-144 Weeks
Exposure
Group,
mg/kg-day
No. of
Animals
No. of Rats Dying During Specified Weeks"
0-90
91-104
105-113
114-118
119-126
127-132
133-140
Totalb
Males
0
100
20 (20)
10 (10)
13 (13)
8(8)
11(11)
10(10)
12(12)
84 (84)
25
80
8(10)
11(14)
6(8)
3(4)
13 (16)
11(14)
8(10)
60 (75)
100
80
8(10)
12 (15)
3(4)
2(3)
10 (13)
7(9)
11(14)
53 (66)
250
99
7(9)
7(19)
6(8)
4(5)
8(10)
13 (16)
10(13)
55 (56)
Females
0
100
16 (16)
15 (15)
18(18)
8(8)
11(11)
7(7)
8(8)
83 (83)
25
79
10 (13)
9(11)
4(5)
6(8)
13 (16)
10(13)
8(10)
60 (76)
100
80
5(6)
17 (21)
5(6)
5(6)
7(9)
9(11)
11(14)
59 (74)
250
99
9(9)
5(5)
11(11)
12 (12)
8(8)
5(5)
10(10)
60 (61)
aNumber of dead animals, (percentage of total animals); calculated for this review.
Calculated for this review from data reported in the study.
Source: Olsenetal. (1986).
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Table B.24. Serum Chemistry of F1 Adult Wistar Rats Treated with
Butylated Hydroxytoluene in Diet for 141-144 Weeks
Serum
Chemistry
Parameter
Exposure Group,
mg/kg-day
Cone, (nmol/liter serum) at week (of age)
9
19
43
108
Male Rats
Free
Cholesterol3
0
0.53 ± 0.02
0.64 ±0.04
0.72 ±0.03
1.13 ± 0.10

250
0.64 ± 0.04b
(121)
0.68 ±0.03
(106)
0.66 ±0.03
(92)
1.10 ±0.08
(97)
Total
Cholesterol3
0
2.05 ±0.09
2.31 ± 0.16
2.84 ±0.13
4.14 ±0.38

250
2.27 ±0.11
(110)
2.26 ±0.10
(98)
2.45 ± 0.08b
(86)
3.82 ±0.25
(92)
Phospholipids3
0
2.82 ±0.20
2.45 ±0.07
2.93 ±0.13
2.95 ±0.21

250
2.52 ±0.02
(89)
2.35 ±0.08
(96)
2.51 ± 0.09b
(86)
2.83 ±0.20
(96)
Triglycerides3
0
NR
1.67 ±0.20
1.85 ±0.22
1.76 ±0.21

250
NR
0.75 ± 0.08d
(45)
0.97 ± 0.12c
(52)
1.24 ±0.17
(70)
Serum
Chemistry
Parameter
Exposure Group,
mg/kg-day
Cone, (nmol/liter serum) at week (of age)
9
19
43
108
Female Rats
Free
Cholesterol3
0
0.58 ±0.02
0.68 ±0.03
0.75 ±0.03
0.93 ±0.06
250
0.82 ± 0.04d
(141)
0.83 ± 0.04c
(122)
0.92 ± 0.05c
(123)
0.81 ±0.04
(87)
Total
Cholesterol3
0
2.02 ±0.10
2.12 ± 0.10
2.72 ±0.13
3.21 ± 0.19
250
2.63 ± 0.1 ld
(131)
2.76 ± 0.1 ld
(130)
2.97 ±0.15
(109)
2.81 ±0.15
(88)
Phospholipids3
0
2.74 ±0.12
2.53 ±0.08
3.21 ±0.11
3.07 ±0.22
250
2.89 ±0.07
(105)
2.99 ± 0.09d
(118)
3.33 ±0.12
(104)
2.50 ± 0.14b
(81)
Triglycerides3
0
NR
1.28 ±0.15
2.02 ±0.17
3.42 ±0.38
250
NR
0.97 ±0.08
(76)
1.10 ± 0.12d
(55)
1.20 ± 0.10d
(35)
aMeans ± SE, (corresponding percentage of control); calculated for this review.
Statistically significant by the (p < 0.05) Students /-test conducted by the study authors.
Statistically significant by the (p < 0.01) Students /-test conducted by the study authors.
Statistically significant by the (p < 0.001) Students /-test conducted by the study authors.
Source: Olsenetal. (1986).
Notes: Notes: NR= Not Reported.
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Table B.25. Incidence of Hepatocellular Nodular Hyperplasia,

Hepatocellular Adenomas and Carcinomas in F1 Adult Wistar Rats Treated with
Butylated Hydroxytoluene in Diet for 141-144 Weeks



No. of Rats with Tumors

Exposure Group
(Human Equivalency
Dose, mg/kg-day) '1'
No. of
Animals
Nodular
Hyperplasia0
Adenomac
Carcinoma0
Total
Tumors'1
Male Rats

0(0)
100
2(2)
1(1)
1(1)
2(2)
25 (7.1)
80
0(0)
1(1)
0(0)
1(1)
100 (28)
80
2(2)
5(6)
1(1)
6(8)
250 (69)
99
2(2)
18e(18)
8f (8)
26 (26)g


No. of Rats with Tumors

Exposure Group
(HED, mg/kg-day)b
No. of
Animals
Nodular
Hyperplasia3
Adenoma3
Carcinoma3
Total
Tumors'1
Female Rats

0(0)
100
2(2)
2(2)
0(0)
2(2)
25 (6.4)
79
0(0)
3(4)
0(0)
3(4)
100 (25)
80
4(5)
6(8)
0(0)
6(8)
250 (62)
99
5(5)
12h(12)
21 (2)
14 (14)g
aDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses converted to human equivalency doses using:
HF.D = NOAELadj x (Body Weight Animal ^ Body Weight Human)A(.25).
°Number of animals with tumors, (corresponding percentage); calculated for this review.
dTotal tumors equals the sum of adenoma and carcinoma combined.
"Overall test for heterogeneity, p < 0.05; Test for trend, p < 0,01,
fOverall test for heterogeneity, p < 0.001; Test for trend, p < 0.001.
8 Statistically significant (p < 0.05) difference from controls by Chi-square test.
hOverall test for heterogeneity, not significant; Test for trend, p < 0.05.
'Overall test for heterogeneity, not significant; Test for trend, not significant.
Source: Olsenetal. (1986).
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Table B.26. Weeks During which Hepatocellular Adenomas and Carcinomas
were Detected in F1 Adult Wistar Rats Treated with Butylated Hydroxytoluene
in Diet for 141-144 Weeks
Dose Group
(HED, mg/kg-day)a
Age (week) at which tumors were detected
Adenomas
Carcinomas
Male Rats
0 mg/kg-day (0)
133
117
25 mg/kg-day (7.1)
119

100 mg/kg-day (28)
136, 139, 141, 143, 143
141
250 mg/kg-day (69)
115, 119, 125, 127, 138, 141, 141, 141,
141, 141, 141, 142, 142, 142, 142, 142,
143, 144
132, 141, 142, 142, 143, 143, 143, 143
Dose Group
(HED, mg/kg-day)a
Age (week) at which tumors were detected
Adenomas
Carcinomas
Female Rats
0 mg/kg-day (0)
117, 117

25 mg/kg-day (6.4)
132, 134, 143

100 mg/kg-day (25)
125, 129, 136, 142, 142, 143

250 mg/kg-day (62)
134, 135, 140, 140, 141, 141, 142, 142,
142, 142, 143, 143
141, 143
aDoses converted to human equivalency doses using:
HED = Dose x (Body Weight Animal ^ Body Weight Human)A(.25).
Source: Olsenetal. (1986).
Table B.27. Number of Tumor-Bearing F1 Adult Wistar Rats Treated
with Butylated Hydroxytoluene in Diet (Excludes Animals with


Hepatocellular Tumors) for 141-144 Weeks"



Males
Females

0
25
100
250
0
25
100
250
Dose Group
(HED,
mg/kg-day)b
mg/kg-
day (0)
mg/kg-
day
(7.1)
mg/kg-
day (28)
mg/kg-
day (69)
mg/kg-
day (0)
mg/kg-
day
(6.4)
mg/kg-
day (25)
mg/kg-
day (62)
No. of
100
80
80
99
100
79
80
99
Animals








Total tumor-
81 (81)
69 (86)
70 (88)
86 (87)
85 (85)
70 (89)
73 (91)
90 (91)
bearing rats








Animals with
malignant
tumors
36 (36)
28 (35)
32 (40)
48 (49)
21 (21)
27 (37)
17 (21)
35 (35)
Benign
66 (66)
55 (69)
54 (68)
70 (71)
77 (77)
67 (85)
69 (86)
78 (79)
tumors








One tumor
38 (38)
43 (54)
42 (53)
37 (37)
38 (38)
23 (26)
30 (38)
36 (36)
Two tumors
22 (22)
15(19)
17 (21)
24 (24)
27 (27)
29 (37)
24 (30)
25 (25)
Multiple
21 (21)
11(14)
11(14)
25 (25)
20 (20)
18 (23)
19 (24)
29 (29)
tumors








aNumber of animals with tumors, () -corresponding percentage; calculated for this review.
bDoses converted to human equivalency doses using: HED = Dose x (Body Weight Animal ^ Body Weight
Human)A(.25).
Source: Olsenetal. (1986).
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Table B.28. Occurrence of Nonneoplastic Lesions in F1 Adults Wistar Rats Treated
with Butylated Hydroxytoluene in Diet for 141-144 Weeks



Males
Females

0
25
100
250
0
25
100
250

mg/kg-
mg/kg-
mg/kg-
mg/kg-
mg/kg-
mg/kg-
mg/kg-
mg/kg-
Dose Group
day (0)
day
day (28)
day (69)
day (0)
day
day (25)
day (62)
(HED, mg/kg-day)a

(7.1)



(6.4)


No. of Animals
100
80
80
99
100
79
80
99
Heart








Fibrosis
72
40
44
36
43
22
25
9
Endocardiosis
1
4
1
2
-
-
-
2
Calcification
-
-
2
-
2
1
1
3
Liver








Fibrosis
-
-
1
-
1
4
1
3
Angiectasis
2
4
3
6
2
6
4
5
Eosinophilic necrosis
-
-
-
1
-
1
-
1
Basophilic areas
3
3
2
1
9
2
7
1
Focal cellular
6
7
14
8
1
7
11
16
enlargement








Cysts
1
1
6
17
7
2
1
9
Fatty metamorphosis
11
10
3
3
3
3
1
-
Peliosis
2
2
4
4
1
2
4
-
Bile-duct proliferation
1
2
5
12
5
5
2
4
Hemorrhage
2
-
-
1
-
-
1
-
aDoses converted to human equivalency doses using:
HED = Dose x (Body Weight Animal ^ Body Weight Human)A(.25).
Source: Olsenetal. (1986).
Notes: Quantitative statistics not provided for this data from the study and cannot be performed independently due to
lack of information.
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Table B.29. Survival of Male and Female B6C3Fi Mice after Oral Exposure

to Butylated Hydroxytoluene for 107 Weeks"

Exposure Group (Adjusted Daily Dose, mg/kg-day)b
Parameter
0 ppm (0)
3000 ppm (515)
6000 ppm (1029)
Male Mice
Sample size
20
50
50
Survival
12(60)
43 (86)
46 (92)

Exposure Group (Adjusted Daily Dose, mg/kg-day)b
Parameter
0 ppm
3000 ppm (518)
6000 ppm (1037)
Female Mice
Sample size
20
50
50
Survival
17 (85)
41 (82)
45 (90)
aNumber of animals surviving to the end of the study (corresponding percentage); reported by study authors.
bDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
Source: NCI (1979d).
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Table B.30. Selected Nonneoplastic and Neoplastic Pathology Findings
in Male and Female B6C3Fi Mice after Oral Exposure to
Butylated Hydroxytoluene for 107 Weeks
Parameter
Exposure group (Human Equivalency Dose,
mg/kg-day)ab
Male Mice
0 ppm (0)
3000 ppm (78)
6000 ppm (155)
Liver
Hepatocytomegalyc
0/20 (0)
9/48 (19)d
20/49 (41)d
Hepatocellular adenomac
2/20 (10)
11/48 (23)
7/49 (14)
Hepatocellular carcinomaec
9/20 (45)
12/48 (25)
6/49 (12)d
Angiosarcoma0
1/20 (5)
0/48 (0)
1/49 (2)
Peliosisc
0/20 (0)
34/48 (71)d
43/49 (88)d
Hepatocellular degeneration/necrosisc
2/20 (10)
34/48 (71)d
45/49 (92)d
Cytoplasmic vacuolatiorf
3/20 (15)
20/48 (42)d
22/49 (45)d
Lung
Alveolar/bronchiolar carcinoma0
5/20 (25)
12/50 (24)
7/49 (14)
Adenomac
2/20 (10)
9/50 (18)
10/49 (20)
Eye/lacrimal gland
Adenoma'"
0/20 (0)
0/50 (0)
4/50 (8)
Parameter
Exposure group (Human Equivalency Dose,
mg/kg-day)
Female Mice
0 ppm (0)
3000 ppm (78)
6000 ppm (155)
Liver
Hepatocytomegalyc
0/20 (0)
1/46 (2)
1/49 (2)
Hepatocellular adenomac
0/20 (0)
3/46 (7)
2/49 (4)
Hepatocellular carcinoma0
1/20 (5)
1/46 (2)
3/49 (6)
Angiosarcoma0
1/20 (5)
1/46 (2)
1/49 (2)
Peliosisc
0/20 (0)
0/46 (0)
0/49 (0)
Hepatocellular degeneration/necrosisc
0/20 (0)
0/46 (0)
0/49 (0)
Cytoplasmic vacuolatiotf
0/20 (0)
0/46 (0)
0/49 (0)
Lung
Alveolar/bronchiolar carcinoma0
1/20 (5)
4/46 (9)g
4/50 (8)
Adenomac
0/20 (0)
12/46 (26)g d
3/50 (6)
Eye/lacrimal gland
Adenoma'"
0/20 (0)
2/46 (4)
0/50 (0)
aDoses were converted from ppm to adjusted daily doses in mg/kg-day using the following formula:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
bDoses converted to human equivalency doses using:
HF.D = DoseADi x (Body Weight Animal ^ Body Weight Human)(0 25).
°NOS = Number of animals with observation total number of animals examined () -corresponding percentage.
Statistically significant (p < 0.05) from independent 72 test performed for this analysis.
"Significant negative dose-related trend by the Cochran-Armitage test performed by study authors.
Significant dose-related trend by the Cochran-Armitage test performed by study authors.
Significant difference between incidence of alveolar/bronchiolar carcinoma or adenoma in low dose females and
controls by Fisher exact test performed by study authors.
Source: NCI (1979d).
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Table B.31. Selected Tumor Incidence in Male and Female B6C3Fi Mice after Oral
Exposure to Butylated Hydroxytoluene for 107 Weeks
Parameter
Exposure Group (Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (78)
6000 ppm (155)
Male Mice
Animals in study
20
50
50
Animals examined histopathologically
20
50
49
Animals with primary tumors (%)c
17 (85)
39 (78)
32 (65)
Animals with benign tumors (%)c
4(20)
20 (40)
19 (39)
Animals with malignant tumors (%)c
16 (64)
32 (64)
19 (39)d
Parameter
Exposure Group (Human Equivalency Dose, mg/kg-day)ab
0 ppm (0)
3000 ppm (78)
6000 ppm (155)
Female Mice
Animals in study
20
50
50
Animals examined histopathologically
20
46
50
Animals with primary tumors (%)c
14 (70)
32 (70)
23 (46)
Animals with benign tumors (%)c
2(10)
22 (48)d
10 (20)
Animals with malignant tumors (%)c
13 (65)
16 (35)d
17 (34)d
aDoses converted to adjusted daily dose using the following formula: Dosc adj = Dose x Food Consumption per Day
x (1 Body Weight) x (Days Dosed ^Total Days).
bDoses converted to human equivalency doses using: HED = Doscadj x (Body Weight Animal ^ Body Weight
Human)(025).
°Number of animals with observation total number of animals examined, () -corresponding percentage.
Statistically significant (p <0.05) from independent 72 test performed for this analysis.
Source: NCI (1979d).
Table B.32. Average Final Body and Liver Weights in B6C3Fi Male Mice Without
Tumors Exposed to Butylated Hydroxytoluene by Diet for 104 Weeks
Tumor Type
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0% (0)
1% (1640)
2% (3480)
Final body weight (g)b
30.2 ±5.9
31.3 ±5.4
28.8 ±5.1
Liver weight (g)b
1.72 ±0.31
2.42 ± 0.43c
2.40 ± 0.73d
Relative liver weight
(% of body weight)b
5.8 ±0.7
7.9 ± 1.4C
8.2 ± 1.9C
aDoses were converted from % food, to ppm by dividing by 10,000 (1% = 10,000 ppm). Adjusted daily
doses were calculated using the following equation: DoseADj = Dose x Food Consumption per Day x (l
^-Body Weight) x (Days Dosed Total Days).
bMeans ± SD.
"Significantly different from controls at (p< 0.001) by Student's t- test conducted by study authors.
dSignificantly different from controls at (p< 0.01) by Student's t- test conducted by study authors.
Source: Inai et al. (1988).
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Table B.33. Incidences of Hepatocellular Lesions in B6C3Fi Male Mice Exposed to
Butylated Hydroxytoluene by Diet for 104 Weeks
Tumor Type
Exposure Group (Human Equivalency Dose, mg/kg-day)ab
0% (0)
1% (249)
2% (529)
No. of Foci of Cellular
Alteration
1/32 (3)
25/42c (60)
42/47c (89)
Adenoma
6/32 (19)
16/42 (38)
25/47c (53)
Carcinoma
7/32 (22)
11/42 (26)
8/47 (17)
Hemangioma
4/32 (13)
3/42 (7)
1/47 (2)
Angiosarcoma
0/32 (0)
0/42 (0)
1/47 (2)
aDoses were converted from % food, to ppm by dividing by 10,000 (1% = 10,000 ppm). Adjusted daily doses
were calculated using the following equation: DoseADj = Dose x Food Consumption per Day x (1 ^Body
Weight) x (Days Dosed ^Total Days).
bDoses were converted to human equivalency doses using the following formula: HED = Doscadj x (Body
Weight Animal ^ Body Weight Human)A(.25).
Significantly different from controls at (p< 0.05) by chi-square test conducted by study authors.
Source: Inai et al. (1988)
Table B.34. Liver and Body Weights in Wistar Rat Dams Exposed to Butylated
Hydroxytoluene via Diet for through Mating, Pregnancy and Lactation
Parameter
Exposure Group
0 mg/kg-day
500 mg/kg-day
750 mg/kg-day
1000 mg/kg-day
Number of dams
8
8
7
9
Terminal Body weights (g)"
294 ±7
281 ± 11 (96)
263 ± 7b (89)
234 ± 6b (80)
Liver weight (g)"
17.2 ± 1.0
24.7 ± 1.3b (144)
24.7 ± 0.6b (144)
22.6 ± 1.0b (131)
Relative liver weight
(liver/body weight %)"
5.81 ±0.22
8.77 ±0.25b (151)
9.39 ±0.23b (162)
9.66 ± 0.29b (166)
aMeans ± SE, ()-corresponding percentage of control calculated for this review.
bSignificantly different from control (p < 0.05) by Students /-test performed by study authors.
Source: McFarlane et al. (1997a).
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Table B.35. Litter Size, Survival, Body Weights, and Liver Weight of
Wistar Rat Pups (Fl) Exposed to Butylated Hydroxytoluene in Gestation, Lactation
Parameter
Exposure Group
0 mg/kg-day
500 mg/kg-day
750 mg/kg-day
1000 mg/kg-day
Number of dams
8
9
9
9
Pups/litter1
11.5± 1.0
11.4 ±0.8 (99)
11.0 ±0.7 (96)
10.0 ± 1.0 (87)
Dead or dying pups (within 4
days of birth)/ total no.
0/92
2/103
0/99
1/90
Litter weight at birth (g)"
77.3 ± 5.0
74.0 ±3.6 (96)
68.1 ±3.2 (88)
64.1 ±6.3 (83)
Pup weight at birth (g)"
6.83 ±0.21
6.60 ± 0.28 (97)
6.24 ±0.16 (91)b
6.46 ±0.12 (95)
Body weight of pups before
reduction (g)"
53.9 ±0.4
35.6 ± 2.0 (66)b
26.0 ± 0.9 (48)b
21.9 ±0.4 (41)b
Body weight of pups after
reduction (g)"
53.4 ±0.7
33.7 ±0.6 (63)b
26.5 ± 0.7 (50)b
21.5 ±0.3 (40)b
Liver weight of pups (g)"
2.18 ±0.08
1.57 ±0.15 (72)
1.02 ±0.09 (47)b
0.90 ± 0.09 (41)b
Relative liver weight of pups (g)"
4.02 ±0.07
3.85 ±0.20 (96)
3.95 ±0.12 (98)
3.95 ±0.24 (98)
aMeans ± SE ()-corresponding percentage of control calculated for this review.
bSignificantly different from control (p < 0.05) by Students t-test performed by study authors.
Source: McFarlane et al. (1997a).
Table B.36. Liver and Body Weights in Wistar Rat Dams Exposed to Butylated
Hydroxytoluene in Diet through Gestational Day 19-20
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
100 mg/kg-day
500 mg/kg-day
Terminal Body weights (g)a b
292 ±16
315 ± 10(108)
309 ± 12(106)
324 ±11 (111)
Liver weight (g)"
14.56 ± 1.34
13.73 ±0.70 (94)
14.45 ±0.64 (99)
17.18 ± 1.16 (118)
Relative liver weight
(liver/body weight %)ab
4.97 ±0.26
4.36 ±0.15 (88)
4.68 ± 0.08 (94)
5.28 ±0.24 (106)
aMeans ± SE ()-corresponding percentage calculated for this review.
bWeight of dams excluding the weight of fetuses and associated tissues.
Source: McFarlane et al. (1997b).
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Table B.37. Body Weights, Liver Weights and Enzyme Levels of Lactating and
Nonlactating Female Wistar Rats Exposed to Butylated Hydroxytoluene via Diet
Parameter
Exposure Group
0 mg/kg-day/
nonlactating
500 mg/kg-day/
nonlactating
0 mg/kg-day/
lactating
500 mg/kg-day/
lactating
Body weight (g)"
263 ±5
281 ±8 (107)
306 ± 10
317 ±14 (104)
Liver weight (g)"
9.15 ±0.61
13.23 ±0.62b (145)
16.95 ±0.28
23.89 ± 1.16b (141)
Liver/ body-weight ratio (g)"
3.47 ±0.19
4.70 ± 0.1 lb (135)
5.56 ±0.17
7.53 ±0.26b (135)
Glucose 6-phosphatase
(nmol/min/mg protein)3
29 ±2
22 ± 3 (76)
30 ±2
14 ± lb (47)
Total glutathione
(|imol/mg cytosolic protein)3
0.060 ± 0.005
0.066 ±0.003 (110)
0.051 ±0.002
0.020 ±0.001b (39)
Glutathione ^-transferase
(nmol/min/mg protein)3
0.94 ±0.18
2.02 ± 0.12b (215)
1.00 ±0.10
2.94 ± 0.22b (294)
Cytochrome P-450
(pmol/mg protein)3
0.339 ±0.014
0.558 ±0.070b (165)
0.273 ±0.010
0.461 ±0.030 (169)
Ethoxyresorufin ()-
deethylase
(pmol/min/mg protein)3
10.03 ± 1.41
9.66 ± 0.78 (96)
4.37 ±0.45
3.63 ±0.58 (83)
Pentoxyresorufin
O-depentylse
(pmol/min/mg protein)3
1.55 ± 0.15
100.16 ±5.79b (6462)
2.45 ±0.11
207.0 ±24.6b (8449)
aMeans ± SE, (corresponding percentage of corresponding nonlactating or lactating control); calculated for this
review.
bSignificantly different from control (p < 0.05) by Students /-test performed by study authors.
Source: McFarlane et al. (1997b).
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Table B.38. Litter Size, Body Weights, Liver Weights and Enzyme Levels of Wistar
Rat Pups (Fl) Exposed to Butylated Hydroxytoluene via Gestation (GD 19-20)
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
100 mg/kg-day
500 mg/kg-day
Pups/litter1
11.3 ± 0.8
11.0 ± 1.1 (97)
11.2 ±0.8 (99)
9.9 ± 1.2 (88)
Fetus weight (g)"
2.94 ±0.12
2.86 ±0.13 (97)
2.34 ± 0.07b (80)
2.71 ±0.09 (92)
Liver weight of fetuses (g)"
0.24 ±0.01
0.26 ±0.01 (108)
0.20 ±0.01 (83)
0.23 ± 0.02 (96)
Relative liver weight of
fetuses (g)"
8.08 ±0.27
8.43 ±0.24 (104)
8.43 ± 0.27 (104)
8.48 ±0.29 (105)
Glucose 6-phosphatase
(nmol/min/mg protein)3
2.43 ±0.75
2.59 ±0.36 (107)
1.42 ± 0.08b (58)
2.04 ±0.13 (84)
Total glutathione
(|imol/mg cytosolic protein)3
0.064 ± 0.005
0.065 ±0.008
(102)
0.074 ±0.005 (116)
0.072 ±0.008 (113)
Glutathione ^-transferase
(nmol/min/mg protein)3
0.187 ±0.025
0.226 ±0.023
(121)
0.195 ±0.010
(104)
0.212 ±0.010
(113)
Epoxide hydrolase
(nmol/min/mg protein)3
1.65 ±0.47
1.37 ±0.24 (83)
1.25 ±0.26 (76)
1.32 ±0.18(80)
aMeans ± SE; (corresponding percentage of control); calculated for this review.
bSignificantly different from control (p < 0.05) by Students /-test performed by study authors.
Source: McFarlane et al. (1997b).
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Table B.39. Liver, Body Weights, and Enzyme Levels in Wistar Rat Pups Exposed to
Butylated Hydroxytoluene via Gestation and Diet at Weaning (PND 21)
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
100 mg/kg-day
250 mg/kg-daya
Terminal Body weights
(g)b'
53.8 ±0.8
55.6 ±0.7 (103)
54.7 ± 1.3 (102)
46.2 ± 0.9 (86)
Liver weight (g)b
2.24 ± 0.07
2.29 ±0.07 (102)
2.48 ±0.12 (111)
2.40 ±0.13 (107)
Relative liver weight
(liver/body weight %)l:"
4.15 ±0.09
4.13 ±0.10 (100)
4.51 ±0.16 (109)
5.17 ±0.25c (125)
Glucose 6-phosphatase
(nmol/min/mg protein)b
47.7 ±4.1
46.7 ±3.7 (98)
39.1 ±3.9 (82)
36.9 ±3.4 (77)
Total glutathione
(|imol/mg cytosolic
protein)b
0.152 ±0.012
0.128 ±0.017 (84)
0.165 ±0.020 (109)
0.131 ±0.012 (86)
Glutathione ^-transferase
(nmol/min/mg protein)b
0.947 ±0.102
0.999 ±0.067 (105)
1.066 ±0.064 (113)
2.034 ±0.29c (215)
Cytochrome P-450
(nmol/mg protein)b
0.657 ±0.056
0.724 ±0.048 (110)
0.724 ±0.053 (110)
0.812 ±0.039 (124)
Ethoxyresorufin
O-deethylase
(pmol/min/mg protein)b
12.4 ±2.2
15.3 ±2.1 (123)
29.9 ± 7.6° (241)
24.1 ± 2.2° (194)
Benzphetamine
.Y-dcmcthvlase
(pmol/min/mg protein)b
4.58 ±0.57
5.34 ±0.47 (117)
9.27 ± 0.95° (202)
11.44 ± 1.69c (250)
Epoxide hydrolase
(nmol/min/mg protein)b
3.48 ±0.84
4.74 ±0.91 (136)
5.67 ± 1.05 (163)
12.81 ±2.05c (368)
aPups of dams receiving 500 mg/kg-day.
bMeans ± SE, (corresponding percentage); calculated for this review.
Significantly different from control (p < 0.05) by Students /-test performed by study authors.
Source: McFarlane et al. (1997b).
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Table B.40. Liver, Body Weights, and Enzyme Levels in Wistar Rat Pups Exposed to
Butylated Hydroxytoluene via Gestation and Diet at 4 Weeks Postweaning (PND 49)
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Terminal Body weights (g)b
284 ±7
294 ± 10 (104)
322 ±6 (113)
264 ± 9 (93)
Liver weight (g)b
14.1 ±0.3
14.7 ±0.5 (104)
16.6 ± 0.4° (118)
14.5 ±0.4 (103)
Relative liver weight
(liver/body weight %)l:"
4.98 ±0.10
5.02 ±0.10 (100)
5.19 ± 0.15° (104)
5.51 ± 0.14° (111)
Glucose 6-phosphatase
(nmol/min/mg protein)b
60.2 ±6.0
61.3 ±6.7 (102)
45.3 ± 3.4C (75)
43.6 ± 2.7C (72)
Total glutathione
(|imol/mg cytosolic protein)b
0.130 ±0.008
0.111 ±0.008
(85)
0.114 ±0.013 (87)
0.096 ± 0.007c (73)
Glutathione ^-transferase
(nmol/min/mg protein)b
1.73 ±0.11
1.89 ±0.10 (109)
2.48 ±0.14c (143)
2.74 ± 0.13° (158)
Cytochrome P-450
(nmol/mg protein)b
0.881 ±0.086
0.742 ±0.032
(84)
0.740 ± 0.037 (84)
0.866 ±0.061 (98)
Ethoxyresorufin
O-deethylase
(pmol/min/mg protein)b
8.91 ± 1.24
13 .04 ± 1.10°
(146)
12.31 ± 0.95° (138)
13.13 ± 1.65° (147)
Benzphetamine
.Y-dcmcthvlase
(pmol/min/mg protein)b
13.6 ± 1.4
10.8 ±0.8 (79)
10.6 ± 1.0 (78)
14.2 ± 1.4 (96)
Epoxide hydrolase
(nmol/min/mg protein)b
7.47 ± 1.22
8.93 ±0.92 (120)
11.10 ± 1.00c (149)
14.60 ± 1.70° (195)
aPups of dams receiving 500 mg/kg-day.
''Means ± SE, (corresponding percentage); calculated for this review.
Significantly different from control (p < 0.05) by Students t-test performed by study authors.
Source: McFarlane et al. (1997b).
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Table B.41. Liver, Body Weights, and Enzyme Levels in Wistar Rat Pups Exposed to
Butylated Hydroxytoluene via Gestation and Diet at 22 Weeks Postweaning (PND 175)
Parameter
Exposure Group
0 mg/kg-day
25 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Terminal Body weights (g)b'
652 ±21
631 ± 19(97)
601 ± 13 (92)
570 ± 17c (87)
Liver weight (g)b
22.25 ±0.95
21.25 ±0.67 (96)
22.27 ±0.55 (100)
23.73 ± 1.05 (107)
Relative liver weight
(liver/body weight %)l:"
3.41 ±0.10
3.39 ±0.12 (99)
3.71 ±0.12 (109)
4.15 ±0.10c (122)
Glucose 6-phosphatase
(nmol/min/mg protein)b
56.7 ±3.4
54.4 ±3.4 (96)
45.4 ± 3.3C (80)
43.6 ± 3.6C (77)
Total glutathione
(|imol/mg cytosolic protein)b
0.123 ±0.007
0.121 ±0.015
(98)
0.127 ±0.012 (103)
0.078 ±0.008c (63)
Glutathione ^-transferase
(nmol/min/mg protein)b
1.23 ±0.06
1.33 ±0.05 (108)
1.45 ±0.09 (118)
1.79 ±0.08c (146)
Cytochrome P-450
(nmol/mg protein)b
0.567 ±0.029
0.563 ±0.018
(99)
0.589 ±0.018 (104)
0.701 ±0.025c (124)
Ethoxyresorufin
O-deethylase
(pmol/min/mg protein)b
2.45 ±0.30
3.33 ±0.31 (136)
3.42 ±0.24c (140)
3.22 ±0.31 (131)
Benzphetamine
.Y-dcmcthvlase
(pmol/min/mg protein)b
10.21 ±0.70
9.62 ±0.51 (94)
10.02 ± 0.49 (98)
11.41 ±0.53 (112)
Epoxide hydrolase
(nmol/min/mg protein)b
8.15 ± 1.39
11.03 ± 1.76
(135)
14.02 ±2.70 (172)
18.26 ±3.06c (224)
aPups of dams receiving 500 mg/kg-day.
''Means ± SE, (corresponding percentage); calculated for this review.
Significantly different from control (p < 0.05) by Students t-test performed by study authors.
Source: McFarlane et al. (1997b).
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Table B.42. Summary of Data of Litters from F1 and F2 Generation Mice
Administered Butylated Hydroxytoluene



Exj
)osurc Group (Adjusted Daily Dose, mg/kg-day)a

0 %
0.015%
0.045%
0.135%
0.405%
Parameter
(0)
(29)
(88)
(263)
(790)
F1 generation
No. of litters
9
10
9
9
9
No. of pups
97
114
113
97
116
Litter size3
10.8 ±2.28
11.4 ± 1.43
12.6 ±2.19
10.8 ±3.70
12.9 ± 1.54


(106)
(117)
(100)
(119)
Litter weight (g)b
16.33 ±3.51
17.92 ±2.03
19.51 ±2.74
16.57 ±5.25
19.49 ±2.09


(110)
(119)
(101)
(119)
Sex ratio (male/female)
1.02 (49/48)
1.00 (57/57)
1.17(61/52)
2.03 (65/32)
1.17 (59/57)

F2
generation



No. of litters
10
9
9
10
10
No. of pups
117
99
106
115
109
Litter size3
11.7 ± 2.16
11.0 ± 2.12
11.8 ± 1.92
11.5 ±2.68
10.9 ±2.56


(94)
(101)
(98)
(93)
Litter weight (g)b
18.03 ±2.94
17.50 ±3.06
17.72 ± 1.78
17.67 ±3.05
16.92 ±3.41


(17.5)
(17.72)
(17.67)
(16.92)
Sex ratio (male/female)
1.09 (61/56)
1.02 (50/49)
1.16 (57/49)
1.35 (66/49)
0.70 (45/64)
aDoses were converted from % of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), and then ppm intake
in food is adjusted using
bEach value represents the mean ± SD, (corresponding percentage of controls is shown in parentheses); calculated
for this review.
the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
Source: Tanaka et al. (1993).
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Table B.43. Selected Behavioral Observations from F1 Generation Mice
Administered Butylated Hydroxytoluene
Parameter
Postnatal
Day
(PND)
Exposure Group (Adjusted Daily Dose, mg/kg-day)a
0%
(0)
0.015%
(29)
0.045%
(88)
0.135%
(263)
0.405%
(790)
Surface
Rightingb
7
1.47 ±0.325
1.84 ± 0.139c
(125)
1.69 ±0.248
(115)
1.43 ±0.400
(97)
1.64 ±0.274
(112)
Ambulation
(males)b'd
21
93.1 ±37.68
100.4 ± 47.66
(108)
49.6 ± 21.70c
(53)
79.1 ±66.01
(85)
71.3 ±48.14
(77)
Ambulation
(females)b'e
21
120.3 ± 53.42
145.8 ±29.48
(121)
101.8 ±52.42
(85)
125.7 ±68.03
(104)
111.2 ± 38.80
(92)
180° turn
(males)b'd
21
5.0 ± 1.85
5.7 ±2.36
(114)
3.8 ±2.49
(76)
3.8 ± 1.64
(76)
4.3 ±2.24
(86)
180° turn
(females)b'e
21
5.6 ±2.20
6.0 ± 1.41
(107)
4.8 ±2.38
(86)
5.7 ± 1.94
(102)
4.3 ± 1.00
(77)
aDoses were converted from % of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), and then ppm intake
in food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
''Each value represents the mean ± SD, (corresponding percentage of control); calculated for this review.
Significantly different from controls (p < 0.05) by unspecified methods.
dDifferent numbers of males were observed (8, 10, 8, 9, and 9; respectively).
"Different numbers of females were observed (8, 10, 8, 9, and 9; respectively).
Source: Tanaka et al. (1993).
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Table B.44. Selected Behavioral Observations from F2 Generation Mice

Administered Butylated Hydroxytoluene


Postnatal
Ex
posure Group (Adjusted Daily Dose, mg/kg-day)a

Day
0 %
0.015%
0.045%
0.135%
0.405%
Parameter
(PND)
(0)
(29)
(88)
(263)
(790)
Surface Rightingb
4
0.24 ±0.137
0.50 ±0.261
0.26 ± 0.264
0.48 ± 0.370c
0.45 ±0.593



(208)
(108)
(200)
(188)
Negative Geotaxisb
4
1.32 ±0.286
1.30 ±0.304
1.42 ±0.412
1.45 ±0.368
1.68 ± 0.422c



(98)
(108)
(110)
(127)
Ambulation
21
193.1 ±
121.7 ±
141.8 ±
149.3 ± 66.75
150.1 ±
(males)"

88.43
61.34 (63)
25.17c (73)
(77)
39.47 (78)
Ambulation
21
173.0 ±
160.2 ±
97.2 ±
177.9 ±46.72
161.8 ±
(females)be

67.87
42.61 (93)
36.05f (56)
(103)
52.58 (94)
180° turn (males)b'd
21
9.2 ±3.08
4.6 ± 2.35f
5.3 ± 2.55c
4.8 ± 2.86f
4.1 ±2.28f



(50)
(58)
(52)
(45)
180° turn (females)b e
21
6.8 ±4.32
6.4 ±4.42
3.4 ±2.30
6.6 ±3.57
5.3 ± 1.49



(94)
(50)
(97)
(78)
aDoses were converted from % of food to ppm by multiplying by 10,000 (1% = 10,000 ppm), and then ppm intake
in food is adjusted using the following equation:
DoseADi = Dose x Food Consumption per Day x (l -f- Body Weight) x (Days Dosed Total Days).
''Each value represents the mean ± SD, () -corresponding percentage calculated for this review.
Significantly different from controls (p < 0.05) by the Mann-Whitney U-test conducted by the study authors.
dDifferent numbers of males were observed (10, 9, 9, 10, and 10; respectively).
"Different numbers of females were observed (10, 9, 9, 10, and 10; respectively).
Significantly different from controls (p < 0.01) by the Mann-Whitney U-test conducted by the study authors.
Source: Tanaka et al. (1993).
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Table B.45. Behavioral Changes in F1 Swiss-Webster Mice Exposed
to 0.5% BHT from Gestation Through PND 42
Parameter
Measurement
Exposure Group (Adjusted Daily Dose, mg/kg-day)
Control
BHT
Mouse City Behaviors
Exploration
7.95 ±2.36
7.87 ± 3.11
Sleeping
0.58 ± 1.57
0.18 ± 0.70a
Grooming Self
1.05 ± 1.05
0.95 ± 1.31
Observation/Mouse
Session
Fighting
0.97 ±2.15
4.75 ± 8.52a
Climbing Screen
Mean base time (sec)
first 5 trials
5.86 ±0.21
5.42 ±0.61
Mean base time (sec)
second 5 trials
4.72 ± 0.23b
5.40 ±0.25
Mean climbing time
(sec) per trial
2.75 ± 1.84
2.86 ±0.32
Isolation-Induced
Aggression
% Fighters
31
62
Mean rank of latency to
onset of aggression
53.9
35.lb
Activity
Orientation reflex in
counts per hr per mouse
2744
2440
Psychomotor activity in
counts per hr per mouse
575
601
Statistically significant compared to control (p < 0.05) by the Wilcoxon 2-sample rank test conducted by the
study authors.
bStatistically significant compared to control (p < 0.005) by the Wilcoxon 2-sample rank test conducted by the
study authors.
Source: Stokes and Scudder (1974).
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APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE
CHRONIC p-RfD AND p-OSF
BENCHMARK DOSE CALCULATIONS FOR THE CHRONIC p-RfD
Modeling Procedure for Dichotomous Data
The BMD modeling of dichotomous data was conducted with EPA's BMDS
(version 2.1.2). For these data, all of the dichotomous models (i.e., Gamma, Multistage,
Logistic, Log-logistic, Probit, Log-probit, Weibull, and Quantal-linear models) available within
the software were fit using a BMR of 10% extra risk. Adequacy of model fit was judged based
on the x goodness-of-fit p-value (p > 0.1), magnitude of scaled residuals in the vicinity of the
BMR, and visual inspection of the model fit. Among all models providing adequate fit, the
lowest BMDL was selected if the BMDLs estimated from different models varied greater than
3-fold; otherwise, the BMDL from the model with the lowest AIC was selected as a potential
POD
Modeling Procedure for Continuous Data
The BMD modeling of continuous data was conducted with EPA's BMDS
(version 2.1.2). For these data (e.g., increased relative liver weight), all continuous models
available within the software were fit using a BMR of 10% relative risk. An adequate fit was
judged based on the % goodness-of-fit p-v alue (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 for
judging adequacy of model fit, a determination was made as to whether the variance across dose
groups was homogeneous. If a homogeneous 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
homogeneous 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 nonhomogeneous variance. If this nonhomogeneous variance model did not adequately fit
the data (i.e., Test 3; p-v alue < 0.1), the dataset was considered unsuitable for BMD modeling.
Among all models providing adequate fit, the lowest BMDL was selected if the BMDLs
estimated from different models varied greater than 3-fold; otherwise, the BMDL from the model
with the lowest AIC was selected as a potential POD from which to derive the RfD.
Focal Alveolar Histiocytosis in Rats Treated with BHT in The Diet For 105 Weeks
(NCI, 1979c)
All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data
of alveolar histiocytosis in F344 female rats fed BHT in the diet for 105 weeks (NCI, 1979c;
Table B.18) For the female rat data, all dose groups were included in the analysis (see Table C.l
and Figure C. 1). As assessed by the % goodness-of-fit statistic, the Logistic, Probit, Log-probit,
and Quantal-linear models adequately fit the data. The Probit model provided the best fit, as
assessed by AIC, for data from female rats (see Table C.l and Figure C.l). Estimated doses
associated with 10% extra risk and the 95% lower confidence limit on these doses (BMDio
values and BMDLio values, respectively) were 199 and 157 mg/kg-day in female rats.
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Table C.l. BMD Dose Response Modeling Results Based on the Incidence of Focal
Alveolar Histiocytosis in Female F344 Rats Fed BHT in the Diet for 105 Weeks.
Model
/>-value
AIC
BMD10
BMDL10
Gamma3
N/A
139.467
197
91
Multistage13
N/A
139.467
188
91
Logistic
0.8707
137.494
210
166
Log-logistic0
N/A
139.464
199
75
Probit
0.9295
137.475
199
157
Log-probitc
0.9022
137.482
219
155
Weibulf
N/A
139.467
194
91
aRestrict power >1.
bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported.
°Slope restricted to >1.
N/A = Not Applicable
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Probit Model with 0.95 Confidence Level
dose
20:50 12/29 2010
Figure C.l. Dose Response Modeling of Incidence Data for Focal Alveolar Histiocytosis
in Female F344 Rats Fed BHT in the Diet for 105 Weeks.
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Hepatocytomegaly in Mice Treated with BHT in the Diet for 107 Weeks (NCI, 1979d)
All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data
of hepatocytomegaly in male B6C3Fi mice fed BHT in the diet for 107 weeks (NCI, 1979d;
Table B.30) For these data, all dose groups were included in the analysis (see Table C.2 and
Figure C.2). As assessed by the % goodness-of-fit statistic, all available models adequately fit
the data. The Log-probit model provided the best fit, as assessed by AIC, for data from male
mice (see Table C.2 and Figure C.2). Estimated doses associated with 10% extra risk and the
95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively)
were 355 and 284 mg/kg-day in male mice.
Table C.2. BMD Dose Response Modeling Results Based on the Incidence of
Hepatocytomegaly in Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
Model
/>-value
AIC
BMD10
BMDL10
Gamma3
1
116.593
316
170
Multistage13
0.9999
116.593
295
170
Logistic
0.2392
118.79
471
375
Log-logistic0
1
116.593
324
143
Probit
0.2855
118.344
445
353
Log-probitc
0.9909
114.612
355
284
Weibulf
1
116.593
310
170
aRestrict power >1.
bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported.
°Slope restricted to >1.
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LogProbit Model with 0.95 Confidence Level
dose
20:55 12/29 2010
Figure C.2. Dose Response Modeling of Incidence Data for Hepatocytomegaly in
Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
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Liver Peliosis in Mice Treated with BHT in the Diet for 107 Weeks (NCI, 1979d)
All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data
of liver peliosis in male B6C3Fi mice fed BHT in the diet for 107 weeks (NCI, 1979d;
Table B.30) For these data, all dose groups were included in the analysis (see Table C.3 and
Figure C.3). As assessed by the % goodness-of-fit statistic, all available models besides Logistic
and Log-probit adequately fit the data. The Log-Logistic model was considered the best fit and
produced a BMDio and BMDLio of 73 and 14 mg/kg-day.
Table C.3. BMD Dose Response Modeling Results Based on the Incidence of
Liver Peliosis in Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
Model
/>-value
AIC
BMD10
BMDL10
Gamma3
0.8353
96.119
47
38
Multistage13
0.7527
89.744
45
36
Logistic
0.0047
108.056
137
103
Log-logisticc
1
97.765
73
14
Probit
0.0034
108.901
133
103
Log-probitc
0.9727
95.82
85
66
Weibulf
0.8353
96.119
47
38
aRestrict power >1.
bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported.
°Slope restricted to >1.
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Multistage Model with 0.95 Confidence Level
dose
13:29 06/21 2010
Figure C.3. Multistage Model for Male Liver Peliosis Data (NCI, 1979d)
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Liver Necrosis in Mice Treated with BHT in the Diet for 107 Weeks (NCI, 1979d)
All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data
of liver necrosis in male B6C3Fi mice fed BHT in the diet for 107 weeks (NCI, 1979d;
Table B.30). For these data, all dose groups were included in the analysis (see Table C.4 and
Figure C.4). As assessed by the % goodness-of-fit statistic, the log-logistics models adequately
fit the data. The Quantal-linear model provided the best fit, as assessed by AIC, for data from
male mice (see Table C.4 and Figure C.4). Estimated doses associated with 10% extra risk and
the 95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively)
were 142 and 16 mg/kg-day.
Table C.4. BMD Dose Response Modeling Results Based on the Incidence of Liver
Necrosis in Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
Model
/>-value
AIC
BMD10
BMDL10
Gamma3
N/A
104.129
65
37
Multistage13
N/A
96.88
52
34
Logistic
0.1565
104.149
117
88
Log-logisticc
N/A
104.129
142
16
Probit
0.1022
104.879
113
90
Log-probitc
N/A
104.129
132
65
Weibulf
N/A
104.129
59
37
aRestrict power >1.
bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported.
°Slope restricted to >1.
N/A = Not Applicable
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Multistage Model with 0.95 Confidence Level
dose
13:30 06/21 2010
Figure C.4. Dose Response Modeling of Incidence Data for Liver Necrosis in
Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
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Hepatic Cytoplasmic Vacuolation in Mice Treated with BHT in the Diet for 107 Weeks
(NCI, 1979d)
All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data
of hepatic cytoplasmic vacuolation in male B6C3Fi mice fed BHT in the diet for 107 weeks
(NCI, 1979d; Table B.30) For these data, all dose groups were included in the analysis (see
Table C.5 and Figure C.5). As assessed by the % goodness-of-fit statistic, all available models
adequately fit the data. The Log-logistic model provided the best fit, as assessed by AIC, for
data from male mice (see Table C.5 and Figure C.5). Estimated doses associated with 10% extra
risk and the 95% lower confidence limit on these doses (BMDio values and BMDLio values,
respectively) were 182 and 102 mg/kg-day.
Table C.5. BMD Dose Response Modeling Results Based on the Incidence of Hepatic
Cytoplasmic Vacuolation in Male B6C3Fi Mice Fed BHT in the Diet for 107 Weeks.
Model
/2/>-value
AIC
BMD10
BMDL10
Gamma3
0.2471
154.989
232
142
Multistage13
0.2464
154.797
234
143
Logistic
0.1489
155.794
356
256
Log-logisticc
0.3377
154.567
182
102
Probit
0.1547
155.726
344
246
Log-probitc
0.1040
156.301
400
252
Weibulf
0.2471
154.989
232
142
aRestrict power >1.
bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported.
°Slope restricted to >1.
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Log-Logistic Model with 0.95 Confidence Level
dose
13:32 06/21 2010
Figure C.5. Dose Response Modeling of Incidence Data for Hepatic Cytoplasmic
Vacuolation in Male B6C3F1 Mice Fed BHT in the Diet for 107 Weeks.
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Decreased Terminal Body Weight of F0 Wistar Rat Dams treated with BHT in the Diet for
14 Weeks (McFarlane et al., 1997a)
All available continuous models in BMDS (version 2.1.2) were fit to the decreased
terminal body weight data from Wistar rat dams exposed to BHT in the diet prior to and during
mating and throughout gestation and lactation (for a total of 14 weeks) (McFarlane et al., 1997a;
Table B.34). The Power model in BMDS provided an adequate fit to the data, and Test 2
(p = 0.2918) also indicated that using a constant variance model was appropriate for modeling
these data. Thus, all of the BMD modeling results shown in Table C.6 and Figure C.6 were
obtained from constant variance models. Estimated doses associated with 10% extra risk and the
95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively)
were 730 and 664 mg/kg-day.
Table C.6. BMD Modeling Results on Decreased Terminal Body Weight in

Rat Dams Exposed to BHT for 14 Weeks

Model
Test 2
Test 3
X p-Value
AIC
BMD10
BMDL10
Linear
0.2918
0.2918
<.0001
190.275
516
457
Polynomial
0.2918
0.2918
<0001
240.509
-9999
2123
Power
0.2918
0.2918
0.8433
168.491
730
664
Hill
0.2918
0.2918
N/A
170.498
729
664
N/A = Not Applicable
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Power Model with 0.95 Confidence Level
dose
21:00 12/29 2010
Figure C.6. Dose Response Modeling of Decreased Body Weight Data in
Wistar Rat Dams Fed BHT in the Diet for 14 Weeks.
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Increased Relative Liver Weight of Wistar Rat Dams Treated with BHT in the Diet for
14 Weeks (McFarlane et al., 1997a)
All available continuous models in BMDS (version 2.1.2) were fit to the increased
relative liver weight data from Wistar rat dams exposed to BHT in the diet prior to and during
mating and throughout gestation and lactation (for a total of 14 weeks) (McFarlane et al., 1997a;
Table B.34). No available model in BMDS provided an adequate fit to the data as Test 4 for all
models was less than 0.1. All of the BMD modeling results shown in Table C.7 were obtained
from constant variance models. Because all models for these data failed, a BMD output graph is
not provided.
Table C.7. BMD Modeling Results on Increased Relative Liver Weight in

Rat Dams Exposed to BHT for 14 Weeks

Model
Test 2
Test 3
X p-Value
AIC
BMD10
BMDL10
Linear
0.8389
0.7996
<.0001
-5.134
54
32
Polynomial
0.8389
0.7996
<0001
-5.134
54
32
Power
0.8389
0.7996
<0001
-5.134
54
32
Hill
0.8389
0.7996
<0001
-21.464
7.8 x 10"13
7.8 x 10"13
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Decreased Body Weight of Wistar Rat Pups Treated with BHT in the Diet for 3 Weeks
(McFarlane et al., 1997a)
All available continuous models in BMDS (version 2.1.2) were fit to the decreased
body-weight data from Wistar rat pups exposed to BHT in the diet throughout gestation and
lactation (for a total of 3 weeks) (McFarlane et al., 1997a; Table B.35). No available model in
BMDS provided an adequate fit to the data as Test 4 for all models was less than 0.1. All of the
BMD modeling results shown in Table C.8 were obtained from constant variance models.
Because all models for these data failed, a BMD output graph is not provided.
Table C.8. BMD Modeling Results on Decreased Body Weight in
Rat Pups Exposed to BHT for 3 Weeks
Model
Test 2
Test 3
X p-Value
AIC
BMDio
BMDL10
Linear
<.0001
<.0001
<.0001
213.523
150
145
Polynomial
<.0001
<0001
<0001
78.479
150
145
Power
<.0001
<0001
<0001
213.523
150
145
Hill
<.0001
<0001
<0001
213.523
150
0.000499
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APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE
ORAL SLOPE FACTOR
Model-Fitting Procedure for Cancer Incidence Data
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
Multistage-cancer model in the EPA benchmark dose software (BMDS) is fit to the incidence
data using the extra risk option. The Multistage-cancer model is run for all polynomial degrees
up to n - 1 (where n is the number of dose groups including control). An adequate model fit is
judged by three criteria: goodness-of-fit p-walue (p > 0.1), visual inspection of the dose-response
curve, and scaled residual at the data point (except the control) closest to the predefined
benchmark response (BMR). Among all the models providing adequate fit to the data, the
lowest bound of the BMD (BMDL) is selected as the point of departure when the difference
between the BMDLs estimated from these models is more than three-fold (unless it appears to be
an outlier); otherwise, the BMDL from the model with the lowest (Akaike Information Criterion)
AIC is chosen. In accordance with EPA (2000) guidance, BMDs and BMDLs associated with an
extra risk of 10% are calculated.
Model-Fitting Results for Hepatocellular Adenomas and Total Hepatocellular Tumors in
Wistar F1 Rats (Olsen et al., 1986)
Table B.25 shows the dose-response data on hepatocellular tumors in Wistar rats
administered BHT via diet for 141-144 weeks (Olsen et al., 1986). Modeling was performed
according to the procedure outlined above using BMDS version 2.1.2 with parameter restrictions
for rats based on the duration-adjusted HEDs shown in Table 2. Model predictions are shown in
Table 13. For incidence of hepatocellular adenomas in both male and female rats, the
multistage-cancer model provided an adequate fit (goodness-of-fit/>value > 0.1). The 2-degree
polynomial model yielded a BMDiohed value of 42 mg/kg-day with an associated 95% lower
confidence limit (BMDLiohed) of 30 mg/kg-day for male rats. The 1-degree polynomial model
yielded a BMDiohed value of 58 mg/kg-day with an associated 95% lower confidence limit
(BMDLiohed) of 36 mg/kg-day for female rats. The fit of the multistage-cancer models to the
hepatocellular adenoma incidence data for male and female rats is shown in Table 14 and
Figures D. 1-D.4. For incidence of total hepatocellular tumors in both male and female rats, the
2-degree polynomial model yielded a BMDiohed value of 41 mg/kg-day with an associated 95%
lower confidence limit (BMDLiohed) of 28 mg/kg-day for male rats. The 1-degree polynomial
model yielded a BMDiohed value of 49 mg/kg-day with an associated 95% lower confidence
limit (BMDLiohed) of 32 mg/kg-day for female rats.
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Multistage Cancer Model with 0.95 Confidence Level
dose
10:08 07/06 2011
Figure D.l. Multistage Cancer Model for Male Liver Adenoma Data (Olsen et al. [1986])
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Multistage Cancer Model with 0.95 Confidence Level
BMDL
Figure D.2. Multistage Cancer Model for Female Liver Adenoma Data (Olsen et al., 1986)
0	10	20	30	40	50	60	70
dose
10:09 07/06 2011
	I	I 1 1 1
Multistage Cancer
Linear extrapolation
BMD Lower Bound
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Dependent variable = Response
Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0.013896
Beta(1) = 0.000749719
Beta(2) = 5.02883e-005
Asymptotic Correlation Matrix of Parameter Estimates
Background	Beta(l)	Beta(2)
Background	1	-0.62	0.48
Beta (1)	-0.62	1	-0.97
Beta (2)	0.48	-0.97	1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0.0165429
0.000143576
5.96679e-005
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-93.4932
-93.74
-114.715
# Param's	Deviance	Test d.f.	P-value
4
3	0.493526	1	0.4824
1	42.4428	3	<.0001
AIC:
193.48
Goodness of Fit
Scaled
Dose	Est._Prob. Expected Observed	Size	Residual
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0.0000
7.1000
28.0000
69.0000
Chi^2 = 0.46
0.0165
0.0205
0.0653
0.2670
d.f. = 1
1.654 2.000	100
1.640 1.000	80
5.220 6.000	80
26.437 26.000	99
P-value = 0.4964
0.271
-0.505
0.353
-0.099
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
40.8353
28.2683
49.528
Taken together, (28.2683, 49.528 ) is a 90
interval for the BMD
two-sided confidence
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Multistage Cancer Slope Factor = 0.00353753
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Multistage Cancer Model with 0.95 Confidence Level
dose
10:07 07/06 2011
Figure D.4. Multistage Cancer Model for Female Total Hepatocellular Tumor Data
(Olsen et al., 1986)
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Multistage Cancer Model with 0.95 Confidence Level
dose
10:07 07/06 2011
Figure D.4. Multistage Cancer Model for Female Total Hepatocellular Tumor Data
(Olsen et al., 1986)
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