United States
Environmental Protection
1=1 m m Agency
EPA/690/R-16/004F
Final
9-01-2016
Provisional Peer-Reviewed Toxicity Values for
2-Mercaptobenzothiazole
(CASRN 149-30-4)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Elizabeth Oesterling Owens, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
SRC, Inc.
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PRIMARY INTERNAL REVIEWERS
Ghazi Dannan, PhD
National Center for Environmental Assessment, Washington, DC
Suryanarayana V. Vulimiri, BVSc, PhD, DABT
National Center for Environmental Assessment, Washington, DC
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this 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).
li
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 6
HUMAN STUDIES 15
Oral Exposures 15
Inhalation Exposures 15
ANIMAL STUDIES 17
Oral Exposures 17
Inhalation Exposures 30
OTHER DATA (GENOTOXICITY, ACUTE TESTS, OTHER EXAMINATIONS) 30
Genotoxicity 38
Acute Toxicity 39
Other Routes 39
Metabolism/Toxicokinetic Studies 41
Immunotoxicity 43
Neurotoxicity 43
DERIVATION 01 PROVISIONAL VALUES 44
DERIVATION OF ORAL REFERENCE DOSES 44
Derivation of a Subchronic Provisional Reference Dose (p-RfD) 45
Derivation of a Chronic Provisional Reference Dose (p-RfD) 50
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 52
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 52
MODE-OF-ACTION DISCI SSION 54
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 55
Derivation of a Provisional Oral Slope Factor (p-OSF) 55
APPENDIX A. SCREENING PROVISIONAL VALUES 59
APPENDIX B. DATA TABLES 60
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 72
APPENDIX D. REFERENCES 118
in
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFS
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2-MERCAPTOBENZOTHIAZOLE (CASRN 149-30-4)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the content of this PPRTV assessment should be directed to the EPA
Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
2-Mercaptobenzothiazole (MBT), CASRN 149-30-4, and its sodium (sodium MBT) and
zinc (zinc MBT) salts are primarily used in vulcanization processes as cure-rate accelerators for
both natural and synthetic rubber products. These compounds are also used as metal chelators,
corrosion inhibitors, and in ore flotation and veterinary drugs (RAPA Panel. 2003). The sodium
and zinc salts of MBT are registered fungicides, microbiocides, and bacteriostats; MBT itself
was registered as a pesticide active ingredient in 1956 (U.S. EPA. 1994). However, all pesticide
products containing MBT and zinc MBT have since been discontinued and only one product
(Vancide 51) containing sodium MBT is still registered for use in the United States (Keglev et
al.. 2014).
MBT is a solid that will exist partially as an anion in aqueous environments, based upon
its pKa of 6.93. As anions do not volatilize, volatilization from moist surfaces is not expected.
Volatilization from dry surfaces is also unlikely due to MBT's low vapor pressure. The capacity
of MBT to leach to groundwater or undergo runoff after a rain event would depend upon local
conditions, as displayed by the range of measured soil adsorption coefficients. In addition, the
MBT anion may complex with metal ions in the environment to form a less water-soluble
metal-anion complex (HSDB. 2010).
The sodium salt, sodium MBT, readily dissociates in water, as evidenced by its high
water solubility, to yield an MBT anion and sodium cation. This high solubility indicates that
sodium MBT would likely leach to groundwater or undergo runoff after a rain event. However,
as with the parent compound MBT, the MBT anion portion may complex with metal ions in the
environment to form a less water-soluble metal-anion complex. Conversely, zinc MBT, which
has much lower water solubility, is not expected to readily dissociate in water, but rather remain
associated as a metal-anion complex. In addition, zinc MBT's moderate water solubility
indicates that its propensity to leach to groundwater would be considerably less than that of the
sodium salt. Because both compounds are salts, volatilization is not expected to be an important
fate process. The empirical formulas for MBT, sodium MBT, and zinc MBT are C7H5NS2,
C?H4NNaS2, and (C?H4NS2)2Zn, respectively (see Figures 1-3). A table of physicochemical
properties is provided below (see Table 1).
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Figure 1. 2-Mercaptobenzothiazole Structure
Na+
Figure 2. Sodium 2-Mercaptobenzothiazole Structure
Figure 3. Zinc 2-Mercaptobenzothiazole Structure
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Table 1. Physicochemical Properties of MBT, Sodium MBT, and Zinc MBT
Property (unit)
MBT
(CASRN 149-30-4)
Sodium MBT
(CASRN 2492-26-4)
Zinc MBT
(CASRN 155-04-4)
Physical state
Solid3
Solid (hygroscopic and
prone to oxidation)3
Solid3
Boiling point (°C)
Decomposes >260"
ND
Decomposes >362a
Melting point (°C)
181a
>300b
337a
Density (g/cm3)
1.42a
ND
1.7a
Vapor pressure (mm Hg at
25°C)
<2.2 x 10 6 a
ND
ND
pH (unitless)
ND
10 (1% aqueous solution)
>11.5 (50% aqueous
solution)0
5.55 (1% aqueous
suspension)0
pKa (unitless)
6.93b
ND
ND
Solubility in water (mg/L at
25°C)
118 (atpH 7)a
>500,000a
90.9 (at 20°C)a
Octanol-water partition
coefficient (log Kow)
2.41a
-0.463
ND
Henry's law constant
(atm-m3/mol at 25°C)
4.1 x 10-9d
ND
ND
Soil adsorption coefficient
Koc (mL/g)
677-3,560 (measured
values in various soils)3
ND
ND
Atmospheric OH rate
constant (cm3/molecule-sec at
25°C)
40.6 x 10-12d
ND
ND
Atmospheric half-life (hr)
9.5d
ND
ND
Molecular weight (g/mol)
167.24
189.23
397.7
•'RAPA Panel (2003).
bU.S. EPA (2010).
CU.S. EPA (1994).
dU.S. EPA (2012c).
MBT = 2-mercaptobenzothiazole; ND = no data.
A summary of available toxicity values for MBT and its sodium and zinc salts from
U.S. EPA and other agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for MBT, Sodium MBT, and Zinc MBT
(CASRNs 149-30-4, 2492-26-4, and 155-04-4)
Source
(parameter)ab
Value
(applicability)
Notes
Reference
Noncancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST
NV
NA
U.S. EPA (201 la)
DWSHA
NV
NA
U.S. EPA (2012a)
ATSDR
NV
NA
ATSDR (2016)
IPCS
NV
NA
IPCS (2016);
WHO (2016)
Cal/EPA
NV
NA
Cal/EPA (2014):
Cal/EPA (2016a):
Cal/EPA (2016b)
OSHA
NV
NA
OSHA (2006);
OSHA (2011)
NIOSH
NV
NA
NIOSH (2015)
ACGIH
NV
NA
ACGIH (2015)
AIHA (WEEL)
5 mg/m3 (MBT)
8-hr TWA. Based on NOEL of 188 mg/kg in gavage
study, converted to inhalation exposure of 1,300 mg/m3;
TWA of 5 selected to protect against small potential for
carcinogenic activity.
AIHA (2013)
Cancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012a)
NTP
NV
NA
NTP (2014)
IARC
NV
NA
IARC (2015)
Cal/EPA
NV
NA
Cal/EPA (2011);
Cal/EPA (2016a):
Cal/EPA (2016b)
ACGIH
NV
NA
ACGIH (2015)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; AIHA = American Industrial
Hygiene Association; ATSDR = Agency for Toxic Substances and Disease Registry; Cal/EPA = California
Environmental Protection Agency; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health
Effects Assessment Summary Tables; IARC = International Agency for Research on Cancer; IPCS = International
Programme on Chemical Safety; IRIS = Integrated Risk Informational System; NIOSH = National Institute for
Occupational Safety and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health
Administration.
Parameters: WEEL = workplace environmental exposure level.
MBT = 2-mercaptobenzothiazole; NA = not applicable; NOEL = no-observed-effect level; NV = not available;
TWA = time-weighted average.
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Non-date-limited literature searches were conducted in February 2016 for studies relevant
to the derivation of provisional toxicity values for MBT (CASRN 149-30-4). Searches also
included names and CASRNs for the sodium and zinc salts of MBT (CASRNs 2492-26-4 and
155-04-4, respectively). The searches were conducted using U.S. EPA's Health and
Environmental Research Online (HERO) database of scientific literature. HERO searches the
following databases: PubMed, ToxLine (including TSCATS1), and Web of Science. The
following databases were searched outside of HERO for health-related values: ACGIH, ATSDR,
Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA Office of Water (OW), U.S. EPA
TSCATS2/TSCATS8e, NIOSH, NTP, and OSHA.
REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide overviews of the relevant noncancer and cancer databases
(respectively) for MBT. The tables include all potentially relevant repeat-dose, short-term-,
subchronic-, and chronic-duration studies, as well as reproductive and developmental toxicity
studies. Principal studies are identified in bold. The phrase "statistical significance," used
throughout the document, indicates ap-walue of < 0.05 unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for MBT (CASRN 149-30-4)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category"
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMCLb
LOAELb
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)b
Short-term
5 M/5 F, S-D rat,
0, 5,000, 10,000, 15,000,
Reduced body weight and increased
ND
NA
ND
Monsanto
NPR
range-finding study,
20,000, 25,000 ppm
relative and absolute liver weight in
(1989b)
diet, 4 wk
males and females were
ADD (M): 0, 425, 839,
accompanied by reduced food
1,232, 1,696, 2,143;
intake; these changes may have
ADD (F): 0, 432, 874,
resulted from poor palatability of the
1,320, 1,703, 2,058
diet, so effect levels were not
determined
Short-term
5 M/5 F, F344/N rat,
0, 156, 313,625, 1,250,
Reduced body-weight gain in males
ND
NA
ND
NTP (1988)
PR
gavage, 5 d/wk, 16 d
2,500 mg/kg
and females (1,875 mg/kg-d)
ADD: 0, 117, 235,469,
937.5, 1,875
Short-term
5 M/5 F, B6C3Fi
0, 188, 375, 750, 1,500,
Decreased survival, lethargy,
ND
NA
FEL:
NTP (1988)
PR
mouse, gavage, 5 d/wk,
3,000 mg/kg
prostration in females
1,125
16 d
ADD: 0, 141,281, 563,
1,125, 2,250
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Table 3A. Summary of Potentially Relevant Noncancer Data for MBT (CASRN 149-30-4)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Subchronic
12 M/12 F, S-D rat,
neurotoxicity study,
diet, 13 wk
0, 5,000, 15,000,
25,000 ppm
ADD (M): 0, 323.5,
991.1, 1,639.1;
ADD (F): 0, 384.4,
1,129.5, 1,920.4
Terminal body weight was reduced
by >11% in high-dose females;
however, reduced food intake was
also seen. FOB, motor activity, and
nervous system histopathology
results were not affected by
exposure. Small (3-5%) decreases
in brain weight, length, and width
were seen in males at
1,639.1 mg/kg-d, but not in
high-dose females. As the
body-weight change may have
resulted from poor palatability of the
diet, effect levels were not
determined
ND
NA
ND
Bio-Research
Laboratories
LTD (1990)
NPR
Subchronic
10 M/10 F, F344/N rat,
gavage, 5 d/wk, 13 wk
0,188, 375, 750,
1,500 mg/kg
ADD: 0,134, 268, 536,
1,071
Increased absolute and relative
liver weight (>10%) in females
and males
ND
14.8
(relative
liver
weight in
females)
134
NTP (1988)
PR,
PS
Subchronic
10 M/10 F, B6C3Fi
mouse, gavage, 5 d/wk,
13 wk
0, 94, 188, 375, 750,
1,500 mg/kg
ADD: 0, 67, 134, 268,
536, 1,071
Increased absolute and relative liver
weight (>10%) in females
ND
NA
67
FEL: 536
NTP (1988)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for MBT (CASRN 149-30-4)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Chronic
50 M/50 F, F344/N rat,
gavage, 5 d/wk, 103 wk
M: 0, 375, 750 mg/kg
F: 0, 188, 375 mg/kg
ADD (M): 0, 268, 536;
ADD (F): 0, 134, 268
Decreased survival, markedly
increased tumor incidences, and
forestomach lesions were seen in
male rats at >267.86 mg/kg-d; in
females, forestomach lesions and
increased tumor incidences were
seen at 267.86 mg/kg-d. Because
267.86 mg/kg-d was associated with
reduced survival in males and
tumors in males and females, it
cannot be identified as a LOAEL;
likewise, the lowest dose in females
was associated with a high tumor
incidence and thus cannot be
identified as a NOAEL
ND
NA
ND
NTP (1988)
(Not a
comprehensive
evaluation of
endpoints;
hematology,
clinical
chemistry, and
organ weights
were not
evaluated)
PR
Chronic
50 M/50 F, B6C3Fi
mouse, gavage, 5 d/wk,
103 wk
0, 375, 750 mg/kg
ADD: 0, 268, 536
Decreased survival of male and
female mice at 535.7 mg/kg-d,
beginning early in the study and in
the absence of tumors. An increased
incidence of hepatocellular
adenomas was seen in female mice
at 267.86 mg/kg-d, precluding the
identification of this dose as a
NOAEL despite the lack of
non-neoplastic effects
ND
NA
FEL: 536
NTP (1988)
(Not a
comprehensive
evaluation of
endpoints;
hematology,
clinical
chemistry, and
organ weights
were not
evaluated)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for MBT (CASRN 149-30-4)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Chronic
30 M/30 F (treated),
60 M/60 F (control),
Slc:ddY mouse, diet,
20 mo
0, 30, 120, 480,
1,920 ppm
ADD (M): 0, 3.60, 14.69,
57.90, 289.40;
ADD (F): 0, 3.61, 13.52,
58.82, 247.98
Increased kidney interstitial cell
infiltration at >57.90 mg/kg-d in
males. No effect levels identified
due to numerous study limitations
ND
NA
ND
Oeawa et al.
(1989): Garcia
(2004)
(Limited
information
available,
published in
Japanese with
limited
information in a
secondary
report)
NPR
Reproductive
28 M/28 F, S-D rat, diet,
at least 70 d prior to
mating, through
two generations
0, 2,500, 8,750,
15,000 ppm
ADD (M): 0, 172.1,
602.3, 1,033;
ADD (F): 0, 199.7,
699.0, 1,198
Increased incidence of basophilic
tubules of the renal cortex in
F0 males and increased relative liver
weight (12%) inFl male parents
ND
NA
172.1
Snrineborn
Laboratories
(1990b)
NPR
Developmental
0 MAS F, S-D rat,
gavage, GDs 6-15
0, 300, 600, 1,000, 1,500,
2,200
ADD: 0, 300, 600, 1,000,
1,500, 2,200
Decreased maternal survival
ND
NA
FEL:
2,200
Snrineborn
Laboratories
(1989c)
NPR
Developmental
0 M/5 F, NZW rabbit,
gavage, GDs 6-18
0, 150, 300, 600, 1,000,
1,500
ADD: 0, 150, 300, 600,
1,000, 1,500
Decreased maternal survival
(>600 mg/kg-d). Decreased
maternal body weight
(>600 mg/kg-d) and fetal body
weight (>150 mg/kg-d). Increased
clinical signs of toxicity in dams
(emaciation at >600 mg/kg-d)
Develop-
mental:
ND
NA
Maternal
FEL:600
Develop-
mental:
150
Soringboni
Laboratories
(1989b)
NPR
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Table 3A. Summary of Potentially Relevant Noncancer Data for MBT (CASRN 149-30-4)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Developmental
0 M/26 F, S-D rat,
gavage, GDs 6-15
0, 300, 1,200, 1,800
ADD: 0, 300, 1,200,
1,800
Clinical signs of toxicity and
decreased activity in dams;
increased postimplantation loss
Maternal:
1,200
Develop-
mental:
NA
NA
Maternal:
1,800
Develop-
mental:
300
Sorinebom
Laboratories
(1989e)
NPR
Developmental
0 M/20 F, NZW rabbit,
gavage, GDs 6-18
0, 50, 150, 300
ADD: 0, 50, 150, 300
No maternal or developmental
effects
Maternal
and
develop-
mental:
300
NA
ND
Sorinebom
Laboratories
(1989d)
NPR
2. Inhalation (mg/m3)a
ND
'Category (treatment/exposure duration: unless otherwise noted): Short-term = repeated exposure for >24 hours <30 days (U.S. EPA. 20021 long-term
(subchronic) = repeated exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in typically used laboratory animal species)
(U.S. EPA. 20021: chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in typically used laboratory animal species)
(U.S. EPA. 20021.
bDosimetry: Values are presented as ADDs (mg/kg-day) for oral noncancer effects. In contrast to other repeated exposure studies, values from animal gestational
exposure studies are not adjusted for exposure duration in calculation of the ADD.
°Notes: PS = principal study; PR = peer reviewed; NPR = not peer reviewed.
ADD = adjusted daily dose; BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit; F = female(s); FEL = frank
effect level; FOB = functional observational battery; GD = gestation day; LOAEL = lowest-observed-adverse-effect level; M = male(s);
MBT = 2-mercaptobenzothiazole; NA = not applicable; ND = no data; NOAEL = no-observed-adverse-effect level; NZW = New Zealand white; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for MBT (CASRN 149-30-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
and Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference (comments)
Notesb
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)a
Carcinogenicity
363 M/0 F,
occupational
epidemiology,
workers exposed at
least 6 mo during
1955-1984 (follow-up
through 2005)
Exposure categorized
as 0,0.1-1, 1-2.5,
2.5-6, or
6-20 mg/m3 based on
limited monitoring
data; individual
cumulative exposures
estimated as 0,
0.01-21.24,
21.25-63.74, or
>63.75 mg/m3-yr
Increased SMRs for colon (SMR = 232, 95%
CI = 100-457) and bladder (SMR = 374, 95%
CI = 162-737) cancers compared with national
mortality rates. Increased SRRs for cancer of
the bladder (SRR = 253, 95% CI = 131-441)
and multiple myeloma (4 cases, SRR = 465,
95% CI = 127-1,191) compared with national
cancer incidence rates. Significant (p < 0.05)
trends for increasing adjusted RR of colon
cancer and multiple myeloma with increasing
cumulative MBT exposure
NA
Sorahan and Pone
PR
(1993); Sorahan et al.
(2000); Sorahan (2009.
2008)
(Workers had potential
coexposure to other
chemicals including
or//zo-toluidine, aniline,
and PBN)
Carcinogenicity
1,059 M/0 F,
occupational
epidemiology,
workers exposed >1 d
between 1955-1977
(follow-up through
1996)
Exposure categorized
as 0, >0-0.5,
>0.5-2.0, >2.0-5.0,
or >5.0-20.0 mg/m3;
cumulative exposure
estimated as 0,
0.01-1.9, 2-7.9, or
8-129 mg/m3-yr
Increased SMR (SMR = 8.9, 95%
CI = 4.7-15.2) for bladder cancer in entire
MBT-exposed group (n = 600). In subgroup
with no possible coexposure to PAB (n = 270),
there were no bladder cancers. In subgroups
with likely or potential coexposure to PAB
(n = 89 and 511, respectively), bladder cancer
SMRs were elevated (SMRs = 27.1 and 4.3,
respectively). In the subgroup with potential
but unknown coexposure to PAB, SMR for
bladder cancer increased with cumulative
exposure to MBT (p = 0.04 for linear trend)
NA
Collins et al. (1999);
Strauss et al. (1993)
(An unknown
proportion of the MBT
group had coexposure to
PAB, and the highest
MBT exposures were
during the time when
PAB was also used in
the plant)
PR
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Table 3B. Summary of Potentially Relevant Cancer Data for MBT (CASRN 149-30-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
and Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference (comments)
Notesb
Animal
1. Oral (mg/kg-d)a
Carcinogenicity
50 M/50 F, F344/N
rat, gavage, 5 d/wk,
103 wk
M: 0,375,
750 mg/kg;
F: 0,188,375 mg/kg
HED (M): 0,64.3,
129;
HED (F): 0,32.2,
64.3
Statistically significantly increased
incidences of pituitary gland adenomas and
adrenal gland pheochromocytomas in
females, and statistically significantly
increased incidences of mesothelioma,
mononuclear cell leukemia, and tumors of
the pituitary gland, adrenal gland,
pancreas, preputial gland, and
subcutaneous tissue in males
8.91 (combined
tumors in
females)
NTP (1988)
PS, PR
Carcinogenicity
50 M/50 F, B6C3Fi
mouse, gavage,
5 d/wk, 103 wk
0, 375, 750 mg/kg
HED: 0, 37.5, 75.0
Statistically significantly increased incidence
of hepatocellular adenomas or carcinomas
(combined) in low-dose females
NA
NTP (1988)
PR
Carcinogenicity
18M/18F
C57BL/6 x C3H/Anf,
strain "X" and
18M/18F
C57BL/6 x AKR,
strain "Y" mouse,
MBT by gavage at
100 mg/kg-d from
PNDs 7-28 and diet
at 323 ppm thereafter,
18 mo
M: 0, 57.4;
F: 57.7 mg/kg-d
HED (M): 0, 8.04;
HED (F): 8.08
No statistically significant increase in
incidence of any tumor type
NA
Itines et al. (1969)
PR
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Table 3B. Summary of Potentially Relevant Cancer Data for MBT (CASRN 149-30-4)
Category
Number of
Male/Female, Strain,
Species, Study Type,
and Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference (comments)
Notesb
Carcinogenicity
18M/18F
C57BL/6 x C3H/Anf,
strain "X" and
18M/18F
C57BL/6 x AKR,
strain "Y" mouse,
zinc MBT by gavage
at 1,000 mg/kg-d from
PNDs 7-28 and diet
at 3,385 ppm
thereafter, 18 mo
M: 0, 252;
F: 253 mg/kg-d as
MBT equivalent
HED (M): 0, 35.3;
HED (F): 35.4
No statistically significant increase in
incidence of any tumor type
NA
limes et al. (1969)
PR
2. Inhalation (mg/m3)
ND
aDosimetry: The units for oral exposures are expressed as human equivalent doses (HEDs) in mg/kg-day. HED = animal dose (mg/kg-day) x (BWa ^ BWh)1/4.
bNotes: PS = principal study; PR = peer reviewed.
BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit; CI = confidence interval; F = female(s); HED = human
equivalent dose; M = male(s); MBT = 2-mercaptobenzothiazole; NA = not applicable; ND = no data; PAB = 4-aminobiphenyl; PBN = phenyl-p-naphthylamine;
PND = postnatal day; RR = relative risk; SMR = standardized mortality ratio; SRR = standardized rate ratio.
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HUMAN STUDIES
Oral Exposures
No studies have been identified.
Inhalation Exposures
No studies investigating the noncancer effects of exposure to MBT in humans have been
identified. Several studies investigated cancer morbidity and mortality, especially bladder
cancer, in two cohorts of rubber factory workers with MBT inhalation exposure. Sorahan and
colleagues (Sorahan. 2009. 2008; Sorahan et aL 2000; Sorahan and Pope. 1993) reported several
analyses of a cohort of 2,160 workers employed at least 6 months between 1955 and 1984 in a
chemical production facility in Ruabon, Wales, where vulcanization inhibitors and accelerants
and other rubber industry materials were produced. Strauss and colleagues (Collins et aL 1999;
Strauss et aL 1993) published two analyses of cancer mortality in 1,059 male workers employed
for at least 1 day between 1955 and 1977 at a similar manufacturing facility in Nitro, WV. The
studies of these cohorts suffer from several limitations that limit the ability to draw firm
conclusions regarding the association between MBT exposure and cancer in these workers.
These limitations included: (1) the numbers of workers with likely MBT exposure in both
cohorts were small (<600 workers each), and (2) the numbers of tumors observed were likewise
small. Also, the possibility of confounding is high. Both worker cohorts had potential exposure
to MBT, its derivatives, and other chemicals, including the known or suspected bladder
carcinogens, 4-aminobiphenyl (PAB), and phenyl-P-naphthylamine (PBN). MBT exposure
assessments for both cohorts were based on job-exposure matrices, using limited exposure
monitoring information, and both included workers who may have had very brief exposure. In
addition, data on tobacco use were not available for either cohort. Finally, follow-up studies of
the same cohort and studies of the different cohorts did not provide consistent findings, possibly
because the numbers of cases and the sizes of the cohorts were too small to yield stable results.
The most recent analyses of these cohorts (Sorahan. 2009. 2008; Collins et aL 1999) are
discussed here, as they provided the longest follow-up times and addressed the same cancer
endpoints as earlier analyses.
Sorahan (2008) and Sorahan (2009) evaluated cancer mortality and incidence in a
subcohort of the Wales factory workers consisting of those workers in job categories that had
probable exposure to MBT. Sorahan (2009) reported the results of all cancers, while Sorahan
(2008) reported the analysis of bladder cancer in particular. The subcohort of 363 workers (from
the entire cohort of 2,160) included 37 workers also believed to have had exposure to PBN,
24 workers believed to have had exposure to o-toluidine, and 8 workers believed to be exposed
to all three compounds (Sorahan et aL 2000). The subcohort of 363 workers studied by Sorahan
(2008) and Sorahan (2009) included 6 workers initially considered unexposed to MBT but
reclassified as exposed (Sorahan. 2008); earlier studies (Sorahan et aL 2000; Sorahan and Pope.
1993) reported the number of MBT-exposed workers as 357. Some members of the entire cohort
were believed to have been exposed to aniline; however, none of the studies indicated whether
there were job descriptions with exposure to both MBT and aniline. Exposures to MBT and its
derivatives were estimated as described in Sorahan and Pope (1993) and were categorized in this
study as zero exposure, very-low exposure (0.1-1 mg/m3), low exposure (1-2.5 mg/m3 or
<21.25 mg/m3-years), medium exposure (2.5-6 mg/m3 or 21.25-63.74 mg/m3-years), or high
exposure (6-20 mg/m3 or >63.75 mg/m3-years). Cancer morbidity and mortality rates in the
exposed cohort were compared to those observed in a control population of 1,797 unexposed
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workers in the same plant, as well as to those rates calculated from the general population of
England and Wales. Standardized mortality ratios (SMRs) and standardized ratio rates (SRRs)
were calculated using indirect standardization and Poisson regression analysis. Based on
national mortality rates, significant excess mortality for cancers of the colon (eight cases,
SMR = 232, 95% confidence interval [CI] = 100-457) and bladder (eight cases, SMR = 374,
95% CI = 162-737) were estimated. Based on national cancer incidence rates, significant excess
morbidity was estimated for cancer of the bladder (12 cases, SRR = 253, 95% CI = 131-441)
and multiple myeloma (four cases, SRR = 465, 95% CI = 127-1,191). Nonsignificant increases
in the SRRs for colon and lung cancers were also noted.
Using the internal comparison group (members of the cohort without MBT exposure),
significant (p < 0.05) trends for increasing adjusted (for age, calendar year, and exposure to other
compounds) relative risk (RR) with increasing cumulative MBT exposure were observed for
colon cancer and multiple myeloma (Sorahan. 2009). but not for lung cancer (Sorahan. 2009) or
bladder cancer (Sorahan. 2008). The adjusted RRs among those with greatest cumulative
exposure to MBT were 4.69 (95% CI = 1.38-15.90, three cases) for colon cancer and 20.57
(95% CI = 2.58-164, two cases) for multiple myeloma (Sorahan. 2009).
Both Collins et al. (1999) and Strauss et al. (1993) evaluated mortality in a cohort of
workers at the Nitro, WV facility; Collins et al. (1999) followed the cohort through
December 1996. A total of 600 out of the 1,059 workers were exposed to MBT. For workers
exposed to MBT, a detailed exposure assessment was performed, in which average annual air
concentrations were estimated for all jobs by an industrial hygienist using sampling data,
employee interviews, and company documents (Strauss et al.. 1993). To account for the
potential confounding of PAB exposure on bladder cancer mortalities reported in Collins et al.
(1999). Strauss et al. (1993) grouped workers exposed to MBT by their job category or time of
employment, in an effort to assess potential coexposure to PAB, using the following categories:
(1) workers with jobs with exposure to PAB (n = 89), (2) workers without jobs with exposure to
PAB but including workers with plant-wide jobs with potential PAB exposure (n = 511), and
(3) workers employed after PAB use was discontinued (n = 270, a subset of Group 2).
Cumulative MBT exposure was stratified as follows: no exposure, and 0.01-1.9, 2-7.9, and
8-129 mg/m3-years. SMRs were calculated, using the mortality experience of the white male
population of four counties within 20 miles of the facility as the referent rate.
SMRs for the entire cohort were reported; however, this group included workers without
MBT exposure (Collins et al.. 1999). SMRs for lung, prostate, and bladder cancer were
calculated for the 600 MBT-exposed workers. In those exposed to MBT, a significantly
increased SMR was observed for bladder cancer (SMR = 8.9, 95% CI = 4.7-15.2), while
prostate and lung cancer SMRs were not elevated. In subgroups with likely or possible
coexposure to PAB (1 and 2), bladder cancer SMRs were also significantly elevated
(SMR = 27.1, 95% CI = 11.7-53.4 and 4.3, 95% CI = 1.4-10.0, respectively). In the group of
270 workers with MBT exposure and no possible exposure to PAB (Group 3), there were no
bladder cancers. In Subgroup 2, the SMR for bladder cancer increased with cumulative exposure
to MBT (p = 0.04 for linear trend). Because an unknown proportion of this group had
coexposure to PAB, and the highest MBT exposures were during the time when PAB was also
used in the plant, Collins et al. (1999) concluded that the potential confounding made it difficult
to assess the risk of bladder cancer attributable to MBT exposure.
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MBT is well known to cause contact allergic dermatitis in humans exposed dermally, and
the compound is included in the standard patch test allergy panel (Diepgen et aL 2006; Baeret
al.. 1973). A large number of case reports and case series describing allergic dermatitis in
humans exposed to MBT are available, but they are not reviewed here because their usefulness
for deriving provisional oral and inhalation toxicity values is limited.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to MBT have been evaluated in
short-term-duration studies (Monsanto. 1989b; N I P. 1988). subchronic-duration studies (Bio-
Research Laboratories LTD. 1990; NTP. 1988). chronic-duration studies (Garcia. 2004; Ogawa
et al. 1989; NTP. 1988; Innes et al. 1969). reproductive toxicity studies (Springborn
Laboratories. 1990b). and developmental toxicity studies (Springborn Laboratories. 1989b. c,
d, e).
Short-Term-Duration Studies
Monsanto (1989b)
In an unpublished range-finding study (Monsanto, 1989b), groups of Sprague-Dawley
(S-D) rats (five/sex/group) were administered MBT (97.6% purity) at target concentrations of 0,
5,000, 10,000, 15,000, 20,000, or 25,000 ppm in the diet, for approximately 4 weeks. Average
daily test material intakes estimated by the study authors were 0, 425, 839, 1,232, 1,696, or
2,143 mg/kg-day, respectively, in males and 0, 432, 874, 1,320, 1,703, or 2,058 mg/kg-day,
respectively, in females. All animals were observed twice daily for mortality and moribundity.
Detailed observations for clinical signs of toxicity, and recording of body weights and food
consumption, were performed weekly. All animals were sacrificed at the end of the exposure
period and examined for grossly visible external and internal abnormalities. The liver was
collected from each animal and weighed; no other organs were weighed. Statistical analyses
were conducted, including Dunnett's test and Bartlett's test.
No unscheduled deaths were reported by Monsanto (1989b). There were no test
substance-related clinical signs during the exposure period. Effects of exposure on body weight
were first noted during Week 1 in males and females. Cumulative body-weight gains over the
4-week period were statistically significantly reduced in males at >1,232 mg/kg-day (increasing
with dose from 17-21% lower than controls) and in females at 1,703 mg/kg-day (26—41%). The
decreases in cumulative body-weight gain were correlated with statistically significant reductions
in food consumption in males (11-12%) and females (11%) at the same doses. Terminal body
weights were biologically significantly decreased (10—12% lower than controls) in males
exposed to >1,696 mg/kg-day, but only in females exposed to 1,703 mg/kg-day (and not
2,058 mg/kg-day). No effects of treatment on food efficiency (weight gain as a function of food
intake) were observed in males or females. Relative liver weights were biologically significantly
increased in males (17—28% higher than controls) and in females (15—30%) at all doses.
Absolute liver weights were biologically significantly increased in males at all doses except
1,232 mg/kg-day (11-15%) and in females at >874 mg/kg-day (14—19%). The greater increases
in relative liver weight compared to absolute liver weight may have been due, in part, to reduced
body weight. No test substance-related macroscopic abnormalities were seen at necropsy;
microscopic examinations were not performed. Because the reduced body weight and increased
liver weight were accompanied by reduced intake of food containing the test compound, these
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changes may have resulted from poor palatability of the test material. Consequently, adverse
effect levels were not determined for this study.
NTP (1988)
NTP (1988) conducted two range-finding studies in groups of F344/N rats and
B6C3Fi mice (five/sex/group) administered MBT (96-97% purity), 5 days/week, for 16 days
(12 doses over 16 days). In the first study, groups of rats and mice were administered 0, 156,
313, 625, 1,250, or 2,500 mg/kg of MBT in corn oil by gavage. Adjusted daily doses (ADDs)1
are 0, 117, 235, 469, 937.5, or 1,875 mg/kg-day, respectively. In the second study, B6C3Fi mice
were administered 0, 188, 375, 750, 1,500, or 3,000 mg/kg in corn oil by gavage. ADDs are 0,
141, 281, 563, 1,125, or 2,250 mg/kg-day, respectively. All animals in both studies were
observed twice daily and weighed on Days 1, 8, and 15. All animals were sacrificed at the end
of exposure. Hematology, clinical chemistry, and organ weights were not measured. Histology
was performed on all vehicle control and high-dose (1,875 mg/kg-day) male rats, one rat exposed
to 235 mg/kg-day, and one high-dose female rat (1,875 mg/kg-day). Histology was not
performed on any mice in either study.
In rats, no chemical-related deaths occurred. Mean body-weight gains were lower
(6-7 g, 8-14%) in high-dose rats (1,875 mg/kg-day) of both sexes compared to controls. No
chemical-related lesions were reported in rats.
Results for mice from the first study were not reported due to "an excessive number of
gavage accidents." In the second study, 4/5 male and 5/5 female high-dose mice
(2,250 mg/kg-day) and 4/5 female mice administered 1,125 mg/kg-day died; lethargy and
prostration were reported after the first gavage dosing of these mice (>1,125 mg/kg-day).
Terminal body weight and body-weight gains were not different between control and treated
mice. No chemical-related lesions were reported in the mice. Due to the changes in
body-weight gain and survival, NTP (1988) chose doses of 0, 94 (mice only), 188, 375, 750, or
1,500 mg/kg for the subchronic-duration study. Based on these data in mice, 1,125 mg/kg-day is
a frank effect level (FEL) for mortality (4/5 female mice died).
Subchronic-Duration Studies
Bio-Research Laboratories LTD (1990)
In an unpublished neurotoxicity study, groups of S-D rats (12/sex/group) were exposed to
MBT (purity not available2) at dietary concentrations of 0, 5,000, 15,000, or 25,000 ppm for
13 weeks (Bio-Research Laboratories LTD, 1990). Average test material intakes estimated by
the study authors were 0, 323.5, 991.1, or 1,639.1 mg/kg-day, respectively, in males and 0,
384.4, 1,129.5, or 1,920.4 mg/kg-day, respectively, in females. All animals were examined at
least twice daily for mortality and clinical signs of toxicity. Results of detailed clinical
observations and body weights were recorded weekly. Food consumption was measured once
weekly for the control, mid-dose, and high-dose groups and twice weekly for the low-dose
group. All animals were subjected to a functional observational battery (FOB) prior to exposure;
at 1, 6, and 24 hours after initiation of the test diets; and again on study Days 7, 14, 35, 64, and
91. Qualitative FOB parameters evaluated included the following: observations in the chamber
1 ADDs were calculated by multiplying by 12 doses and dividing by 16 days
(e.g., 156 mg/kg x (12 16) =117 mg/kg-day).
2According to the table of contents, pp. C341-C617 contained purity information; however, these pages were
missing from the available copy of the document.
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(body position, locomotor activity, bizarre behavior [static], tremors, twitches, convulsions,
piloerection, respiratory rate/pattern, and defecation), handling observations (vocalization, pupil
size, lacrimation, salivation, urinary staining, diarrhea, body tone, abdominal tone, pinna reflex,
corneal reflex, extensor thrust, tail/toe pinch, and auricular startle), observations in an arena
(defecation, gait, bizarre behavior [movement], and limb rotation), observations at the edge of an
arena (positional passivity), surface observations (olfactory response, visual placing, and air
righting reflex), and observations under the cage (urination). Quantitative FOB parameters
included forelimb and hindlimb grip strength and hindlimb splay. All animals were examined
for effects on motor activity prior to initiation, and on study Days 29, 63, and 90.
At scheduled necropsy, six animals/sex/group from the control and high-dose groups
were anaesthetized and perfused for neuropathological evaluations; brain weight, length, and
maximum width were recorded. The following tissues were examined microscopically in the
selected animals: brain (forebrain, center of the cerebrum, midbrain, cerebellum and pons, and
medulla oblongata), spinal cord (cervical and lumbar swelling), skeletal muscle
(e.g., gastrocnemius), and peripheral nervous system (sciatic nerve, lumbar dorsal root ganglion
and fibers, lumbar ventral root fibers, cervical dorsal root ganglion and fibers, cervical ventral
root fibers, sural nerve, tibial nerve, and Gasserian ganglion). No other organs were weighed or
examined microscopically. The remaining six animals/sex/group were examined for gross
pathological changes. Statistical analysis of continuous endpoints employed analysis of variance
(ANOVA) followed by Dunnett's test (when variances were not significantly different by
Bartlett's test) or Kruskal-Wallis test followed by Dunn's test (when variances differed).
Repeated measures analysis was used for motor activity counts, and quantal data were analyzed
using Fisher's exact test (with alpha adjusted for multiple comparisons).
No mortalities occurred during the study, and there were no treatment-related clinical
signs (Bio-Research Laboratories LTD, 1990). Mean body weights were statistically
significantly decreased in males at 991.1 mg/kg-day on Days 1 and 7; in high-dose males and
mid- and high-dose females, mean body weights were statistically significantly decreased
throughout most of the study. Terminal body weights were 9% lower than controls in high-dose
males, 7% lower in mid-dose females, and 11% lower than controls in high-dose females. Mean
food consumption was statistically significantly decreased in males during Weeks 1 (mid and
high doses) and 13 (high dose only); in females, statistically significant decreases in food
consumption were noted at the mid dose during Weeks 1 and 5 and at the high dose during
Weeks 1, 2, and 5-9). The food intake differences from control were as high as 20% on isolated
occasions; in high-dose females, the decrease was between 8 and 13% during Weeks 5-9. No
test substance-related effects on FOB or motor activity parameters were observed. Statistically
significant decreases in brain weight (5% lower than controls), length (3%), and width (3%) were
observed in high-dose males (lower dose groups were not examined). These decreases in brain
measures corresponded to lower terminal body weights; there were no differences in relative
brain weight between control and high-dose males. In females, no statistically significant
differences in brain weight, length, or width occurred at any dose. There were no
exposure-related effects on nervous system tissue histopathology. As with the 4-week dietary
study in rats conducted by Monsanto (1989b)., it is plausible that the body-weight decrements
seen in this study were related to the decreases in food consumption and poor palatability.
Further, the biological significance of small (3—5%) differences in brain morphometry is
uncertain. Thus, effect levels were not determined for this study.
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NTP (1988)
NTP (1988) conducted a 13-week study in rats and mice to evaluate the effects of
repeated administration of MBT (96-97% purity) and to determine the doses to be used in the
2-year studies. Groups of 10 male and 10 female F344/N rats and B6C3Fi mice were
administered 0, 94 (mice only), 188, 375, 750, or 1,500 mg/kg of MBT in corn oil by gavage,
5 days/week, for 13 weeks. ADDs (from 5-7 days) are 0, 67 (mice only), 134, 268, 536, or
1,071 mg/kg-day, respectively. All animals were observed for mortality or moribundity twice
daily and weighed weekly. Hematology and clinical chemistry were not evaluated. At sacrifice
at the end of exposure, gross necropsies were performed on all animals excluding those that had
been cannibalized or had undergone extreme autolysis. Liver weights were recorded, but no
other organ weights were obtained. Histopathology examinations were performed on some, but
not all, animals from all dose groups. The following tissues were examined: adrenal glands,
brain, colon, esophagus, eyes (if grossly abnormal), gall bladder (mice only), heart, kidneys,
liver, lungs and bronchi, mammary glands, mandibular or mesenteric lymph nodes, pancreas,
parathyroids, pituitary gland, prostate and testes or ovaries and uterus, salivary glands, small
intestine, spleen, spinal cord, sternebrae or femur or vertebrae including marrow, stomach,
thymus, thyroid gland, trachea, and urinary bladder. Any tissues with gross lesions or masses
were also examined.
NTP (1988) reported that no compound-related deaths occurred among rats. Gavage
error deaths included one male in the 268-mg/kg-day group and two in the 1,071-mg/kg-day
group, and one female in the 134-mg/kg-day group and two in the 536-mg/kg-day group.
Clinical signs of toxicity consisting of irritable behavior that increased in severity with
increasing dose were observed (incidences not reported). The study authors suggested that this
behavior reflected resistance to gavage; however, as the clinical signs in rats were more
pronounced at higher doses, and clinical signs suggestive of neurotoxicity were seen in the
13-week study in mice (described below), a compound-related neurotoxic response cannot be
ruled out. Necropsy body weights were slightly lower (3-9%) than controls in males at doses
>268 mg/kg-day and in females at all dose groups, but the difference was <10% in all groups
(see Table B-l). Body-weight gain also decreased with increasing dose (7—17%) compared to
controls. Relative liver weights were statistically significantly increased (14-36%) relative to
controls) in both male and female rats of all treated groups (see Table B-l). Absolute
liver-weight increases (>10%) occurred in all dose groups in females (18-27% higher than
controls) and males (15—38% higher than controls). The study authors reported that no
treatment-related gross or microscopic lesions were seen (data not shown). Based on >10%
increased relative and absolute liver weights in male and female rats, a
lowest-observed-adverse-effect level (LOAEL) of 134 mg/kg-day (the lowest dose tested) is
identified for this study. A no-observed-adverse-effect level (NOAEL) is not identified.
In mice, 5/10 males and 7/10 females from the 1,071-mg/kg-day group, as well as
2/10 females from the 536-mg/kg-day group, died prematurely (see Table B-2) (NTP. 1988).
Two of the deaths, apparently in the high-dose group3, were due to gavage error. The cause(s) of
the remaining deaths was not reported, but is assumed to be related to treatment, especially given
the observation of clinical signs including clonic seizures, lacrimation, and salivation in these
'The text in the results section of the NTP (1988) 13-week study in mice only mentioned the high-dose deaths, and
reported that two of the deaths were due to gavage error. The summary table in that section (see Table 22) also
shows deaths of two females in the 750-mg/kg (535.7 mg/kg-day adjusted dose) group.
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two dose groups. At 268 and 536 mg/kg-day, lethargy and rough coats were reported
(incidences not reported). Terminal body weights were slightly lower than controls in male mice
receiving doses >268 mg/kg-day, but the differences from control were <10% (4-6%) and were
not statistically significant. Female mice had >10% increases in absolute liver weight at doses
greater than or equal to 536 mg/kg-day (13-22% compared to control) and in relative liver
weight (11-28%) compared to controls) at all doses (see Table B-2). Absolute liver-weight
increases >10% were observed at doses >134 mg/kg-day, and relative liver-weight increases
>10%) were observed in the highest dose group (1,071 mg/kg-day) in males. No
treatment-related gross or microscopic lesions were observed (data not shown). These data
indicate that 536 mg/kg-day is a FEL for mortality (2/10 females died). Based on the >10%
increases in relative liver weight in female mice at all doses, a LOAEL of 67 mg/kg-day (lowest
dose tested) is identified for this study. No NOAEL is identified.
Chronic-Duration and Carcinogenicity Studies
NTP fl988)
A chronic toxicity and carcinogenicity study of MBT (purity 96-97%) was conducted in
rats and mice (N TP. 1988). Groups of 50/sex F344/N rats and B6C3Fi mice were administered
MBT in corn oil by gavage at doses of 0, 188 (female rats only), 375, or 750 (male rats and both
sexes of mice) mg/kg for 5 days/week for 103 weeks. The respective ADDs, calculated as the
product of the gavage dose and 5/7 days per week, were 0, 134, 268, or 536 mg/kg-day. The
animals were observed twice daily for mortality and moribundity, and clinical signs were
recorded once a week. Body weights were obtained weekly for the first 12 weeks and monthly
thereafter until study termination. Hematology, clinical chemistry, and organ weights were not
evaluated. All animals, including those that were found dead, were evaluated for gross and
microscopic pathology; tissues examined for histopathology included: adrenal glands, brain,
colon, esophagus, eyes (if grossly abnormal), gall bladder (mice only), heart, kidneys, liver,
lungs and bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas,
parathyroids, pituitary gland, prostate and testes or ovaries and uterus, salivary glands, small
intestine, spleen, spinal cord, sternebrae or femur or vertebrae including marrow, stomach,
thymus, thyroid gland, trachea, and urinary bladder. Three statistical analyses were conducted,
including life table analysis, incidental tumor analysis, and unadjusted analyses (Fisher's exact
and Cochran-Armitage linear trend tests). Life table and incidental tumor analyses, which adjust
for intercurrent mortality, were applied to the male rat data to account for survival differences
among groups. Life table analysis assumes that all tumors observed in animals dying before the
end of the study were "fatal," and incidental tumor analysis assumes that all tumors observed
before the end of the study were "incidental" and unrelated to the cause of death.
A statistically significant (at both doses), dose-dependent decrease in survival was seen in
treated male rats beginning in Week 83 (cumulative incidences of nonaccidental deaths prior to
termination, including moribund sacrifices, were 8/50, 28/50, and 29/49 in control, low-dose, and
high-dose males); survival of treated females did not differ from controls, but was low in all
groups (nonaccidental deaths prior to termination were 21/49, 18/49, and 25/50 in control,
low-dose, and high-dose females) ( N TP. 1988). Deaths of female rats occurred throughout the
study, while male deaths primarily occurred after at least 60 weeks on study. The study authors
reviewed individual animal data and noted that most of the rats that died prematurely had tumors.
Body weights of males and females were higher than (not statistically significant), or did not
differ from, controls. The only clinical sign noted was lethargy after dosing; the study authors
did not report incidences of this finding or any further details. Table B-3 shows selected
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non-neoplastic effects. Statistically significantly increased incidences of non-neoplastic lesions
in the forestomach (ulcers, inflammation, epithelial hyperplasia, and hyperkeratosis) were
observed in males of both dose groups. Incidences of non-neoplastic forestomach lesions also
were increased in treated females, although the incidences were lower than in males, and only
the incidence of ulcers in the high-dose group was statistically significantly different from the
control incidence (see Table B-3). NTP (1988) reported an increase in the mean severity score
for nephropathy (observed in all male rats and >75% of female rats, including controls) in
exposed male rats. Severity scores of 3.4 (moderate-severe) were recorded in male rats exposed
to 268 and 536 mg/kg-day vs. 2.3 (mild-moderate) in control males. NTP (1988) also noted that
renal pelvic epithelial cell hyperplasia and tubular cell hyperplasia were observed in treated male
rats and not in controls, although the incidences (1-4 rats in groups of 49-50) were not
statistically significantly different from controls. Determination of effect levels in this study is
complicated by the statistically significant reductions in survival and statistically significantly
increased incidences of multiple tumor types (discussed further below) at 268 and
536 mg/kg-day in male rats. In female rats, the high dose of 268 mg/kg-day might be considered
a LOAEL based on an increased incidence of forestomach ulcers; however, because this dose
was associated with reduced survival in males and tumors in both males and females, it cannot
be used as the LOAEL. Although non-neoplastic effects were not seen in females at
134 mg/kg-day, this dose cannot be identified as a NOAEL due to the confounding effect of
tumors seen at this dose.
Increased incidences of neoplastic lesions were reported in a number of rat tissues
(see Table B-4). Pairwise comparisons by life table analysis showed statistically significantly
increased incidences of adrenal gland pheochromocytoma (with or without malignant
pheochromocytoma), pancreatic acinar cell adenoma, and preputial gland adenoma (and
adenoma or carcinoma) in male rats at both doses. Pairwise comparisons by life table analysis
showed statistically significantly increased incidences of mononuclear cell leukemia and
pituitary gland adenoma in male rats at the low dose but not the high dose. At the high dose, the
incidences of subcutaneous fibroma and subcutaneous fibroma, neurofibroma, sarcoma, or
fibrosarcoma (combined) were statistically significantly increased in male rats when analyzed by
life table test; however, NTP (1988) stated that the incidences were not statistically significant by
the incidental tumor test. A statistically significant trend by life table or incidental tumor test
was reported in male rats for incidence of mesotheliomas, pancreatic acinar cell adenoma,
adrenal gland pheochromocytoma, preputial gland adenoma or carcinoma, and subcutaneous
fibroma, neurofibroma, sarcoma, or fibrosarcoma (combined). NTP (1988) reported the
incidence did not exceed the historical control range for corn oil vehicle animals for
mesothelioma and preputial gland adenoma (and adenoma or carcinoma); however, these lesions
were statistically significantly different from concurrent controls. In female rats, exposure to the
high dose resulted in statistically significantly increased incidences of pituitary gland adenoma
and adenoma or carcinoma (combined) and adrenal gland pheochromocytoma; pituitary and
adrenal gland tumor incidences at the low dose were not statistically significantly elevated, but
the incidences were very similar to the high-dose incidences (see Table B-4). NTP (1988)
concluded that under the conditions of the rat study, there was "some evidence of carcinogenic
activity" among male rats (based on increased incidences of mononuclear cell leukemia,
pancreatic acinar cell adenomas, adrenal gland pheochromocytomas, and preputial gland
adenomas or carcinomas), and "some evidence of carcinogenic activity" among female F344/N
rats (based on increased incidences of adrenal gland pheochromocytomas and pituitary gland
adenomas).
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A total of six high-dose male mice and four high-dose females died during Week 13 after
they were accidentally dosed twice in a 16-hour period; these animals were not included in the
statistical analysis of survival after Week 12. After excluding the accidental deaths, analysis of
survival among high-dose females showed statistically significantly reduced survival (22/46
survived to termination, compared with 30/44 controls,/* = 0.004). The deaths of high-dose
female mice occurred early in the study; the first high-dose female death occurred during Week 3
on study, and deaths began to occur regularly after Week 16. Statistical analysis of survival of
treated male mice showed no difference from controls; however, Kaplan-Meier survival curves
showed that a number of high-dose male mice died early in the study (there were
10 nonaccidental deaths between Weeks 7 and 47). After the first year, survival of high-dose
males stabilized such that survival to termination was not statistically different from controls.
NTP (1988) reported that lung hemorrhage and congestion were seen in many of the mice that
died prematurely and that tumors were not seen in these animals. Table B-5 summarizes the
incidences of nonaccidental deaths in male and female mice. As with the rats, mice were
observed to be lethargic after dosing (incidence of this effect not reported). NTP (1988)
observed dose-dependent decreases (4-14% lower than controls) in mean body weights among
male mice between Weeks 3 and 64; subsequently, dosed males regained body weight such that
terminal body weights were comparable to controls. Among females, mean body weights of the
high-dose animals were lower than controls between Weeks 42 and 90, but the difference did not
exceed 6%. Mean body weights of the low-dose females were comparable to those of controls
throughout the study period. Minimal to mild bronchopneumonia occurred in all groups of mice,
including controls, at incidences between 24 and 49%; this effect was attributed to Sendai virus
infection based on serology in sentinel animals. Based on the minimal to mild severity of the
bronchopneumonia, it is unlikely that the viral infection contributed to the early deaths, although
individual animal data on this endpoint are not available to investigate this relationship or the
potential relationship between the bronchopneumonia and the lung hemorrhage and congestion.
No treatment-related non-neoplastic lesions were seen in males or females at any dose. The high
dose in this study (536 mg/kg-day) is considered to be a FEL based on decreased survival of
mice beginning early in the study and in the absence of tumors. As discussed further below, the
low dose (268 mg/kg-day) was associated with an increased incidence of hepatocellular
adenomas in female mice and thus cannot be considered a NOAEL, despite the lack of
non-neoplastic effects.
Despite the early mortality in high-dose mice, NTP (1988) concluded that the final
survival rates were sufficient to allow evaluation of potential carcinogenicity. No evidence of
treatment-related neoplastic lesions were seen in male mice. In low-dose females, the incidence
of hepatocellular adenoma or carcinoma was statistically significantly increased; however, the
incidence at the high dose was not different from controls (see Table B-5). NTP (1988) noted
that the absence of tumors at the high dose may have been due to the decreased survival in this
group, as the premature deaths occurred early in the study, and hepatocellular tumors tend to
appear late in mice. NTP (1988) concluded that, under the conditions of the study, there was "no
evidence of carcinogenic activity" in male B6C3Fi mice, but "equivocal evidence of
carcinogenic activity" in female B6C3Fi mice based on the increased incidence of hepatocellular
adenomas or carcinomas (combined).
Garcia (2004); Ogawaeial (1989)
Ogawa et al. (1989) conducted a 20-month chronic toxicity study of MBT (purity
unknown) in mice (Slc:ddY), which was published in Japanese. Only the abstract from Ogawa
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et al. (1989) was available in English and a summary of the study was published by Garcia
(2004). The information below comes from the Ogawa et al (1989) abstract and the summary
by Garcia (2004). Groups of 30 mice per sex (treated) and 60 control mice per sex received 0,
30, 120, 480, or 1,920 ppm MBT in the diet for 20 months. Garcia (2004) calculated daily doses
of 0, 3.60, 14.69, 57.90, or 289.40 mg/kg-day, respectively, in males and 0, 3.61, 13.52, 58.82, or
247.98 mg/kg-day, respectively, in females. Interim sacrifices were conducted at 6 and
12 months, leaving groups of 6-14 male mice and 7-15 female mice for sacrifice and evaluation
at the end of the 20-month exposure. Based on information provided by Garcia (2004).
toxicological evaluations in the study included clinical signs, body weight, food consumption,
hematology (red blood cell [RBC] and white blood cell [WBC] counts, hemoglobin [Hb],
hematocrit [Hct], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH],
mean corpuscular hemoglobin concentration [MCHC], and platelets), serum chemistry (total
protein, albumin, albumin:globulin ratio, blood urea nitrogen [BUN], total cholesterol, alkaline
phosphatase [ALP], alanine aminotransferase [ALT], and aspartate aminotransferase [AST]), and
histopathology of the lungs, liver, and kidneys.
Garcia (2004) did not discuss mortality in the summary of Ogawa et al. (1989). Based on
the review, decreased body-weight gain was observed in highest-dose males throughout the
study. No treatment-related effects were seen in hematological parameters (Garcia. 2004). An
increased rate of cell infiltration in the interstitium of the kidneys (cell type not provided) was
reported in male mice at 57.90 and 289.40 mg/kg-day at the end of the study; however, this
observation may reflect a spurious finding because (1) there was no clear dose-response at the
12-month sacrifice; (2) the incidences did not increase (and at some doses, actually decreased)
between the 12- and 20-month sacrifices; (3) the numbers of animals per group were small
(5-10) at each sacrifice; and (4) control animals had highly variable incidence of interstitial cell
infiltration of the kidneys (7-60%), suggesting high incidence of spontaneous lesions. No
treatment-related increase in non-neoplastic histopathology of the lung or liver, nor tumor
incidence in the lung, liver, or kidneys was seen. Effect levels for this study cannot be identified
because available information is limited to an abstract and a secondary report.
Innes et al. (1969)
In a study examining the potential carcinogenicity of 120 compounds, two hybrid strains
of mice (C57BL/6 x C3H/Anf, strain "X" and C57BL/6 x AKR, strain "Y") were given MBT
(purity not specified; Captax formulation) via gavage (in 0.5% gelatin) at the estimated
maximum tolerated dose (MTD) of 100 mg/kg from Postnatal Day (PND) 7 to weaning at
4 weeks of age. The daily dose was not adjusted for weight gain during the gavage exposure
period. From these animals, groups of 18 mice/sex/dose were selected to continue exposure to
MBT in the diet for 17 additional months (total exposure duration 18 months) at a concentration
of 323 ppm; this concentration was selected to yield the same 100-mg/kg dose based on food
intake and body weight at 4 weeks of age. A separate group of mice received
zinc mercaptobenzothiazole (Zetax) at 1,000 mg/kg in 0.5% gelatin until 4 weeks of age and then
3,385 ppm zinc MBT in the diet for 17 months. Crude estimates of the time-weighted average
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(TWA) daily doses4 using default food intake and body weight from chronic-duration studies are
57.4 mg/kg-day for males and 57.7 mg/kg-day for females for MBT administration and
252 mg/kg-day for males and 253 mg/kg-day for females (as equivalent dose of MBT) for
zinc MBT administration. Human equivalent doses (HEDs) of 8.04 and 8.08 mg/kg-day were
calculated for male and female mice exposed to MBT from these dose estimates using the
mouse:human dosimetric adjustment factor (DAF) of 0.14 based on the animal :human
body-weight (BW1 4) ratio recommended by U.S. EPA (2011b). HEDs of 35.3 and
35.4 mg/kg-day are calculated for male and female mice administered zinc MBT. All animals
were sacrificed at -18 months of age. The study authors reported no statistically significant
increase in tumor incidences in either the MBT or the zinc MBT groups, but quantitative results
were not given.
Reproductive Studies
Sprinsborn Laboratories (1990b)
In a two-generation reproduction toxicity study, Springborn Laboratories (1990b)
administered MBT (98.5 and 98.2% purity; no further information regarding impurities was
reported) in the diet to groups of 28 male and female S-D (Crl:CD®COBS®BR) rats at
concentrations of 0, 2,500, 8,750, or 15,000 ppm for at least 70 days prior to cohabitation, during
cohabitation, and until scheduled sacrifice. ADDs estimated from default body weight and food
intake values for S-D rats in a chronic-duration study (U.S. EPA. 1988) are 0, 172.1, 602.3, or
1,033 mg/kg-day for males and 0, 199.7, 699.0, or 1,198 mg/kg-day for females, respectively.5
After birth, litters (Fl) were raised until Lactation Day (LD) 21, when selected weanlings were
administered MBT in the diet at the same doses as their parents. F0 and Fl parents were
observed for clinical signs of toxicity, morbidity, or mortality daily. Body weights and food
intake were measured weekly for F0 and Fl parental males for the duration of the study, and for
females prior to copulation and at intervals during gestation and lactation. Parameters related to
reproductive function, including precoital interval, copulation, fertility indices, pregnancy
percentage, and gestation length, were evaluated. F0 and Fl parents were sacrificed and
necropsied after weaning of their offspring (Fl and F2, respectively). The liver, kidneys, and
testes or ovaries of all F0 and Fl parents were weighed. Selected tissues and organs of the F0
and Fl parents were examined microscopically in the control and high-dose groups (1,033 or
1,198 mg/kg-day). In addition, microscopic examinations of the kidneys of F0 parents of the low
and mid-range doses, and the kidneys and livers of Fl parents of the low and mid doses were
performed. Offspring viability was determined daily in both the Fl and F2 generations. The
study authors examined Fl and F2 offspring on LDs 0, 4, 7, 14, and 21 and recorded body
weights on LDs 1, 4, 7, 14, and 21. On LD 4, litter size was reduced to eight offspring (four
each males and females, if possible). Moribund and culled pups were sacrificed and necropsied,
and the surviving F2 offspring were sacrificed and necropsied on LD 21. Statistical analysis of
4For each compound, the dose administered by gavage is assumed to remain constant during the 3-week
administration period. The dietary concentration is converted to an ADD using male and female default values for
food intake and body weight in B6C3Fi mice (data on the tested strains were not available) in chronic-duration
studies (U.S. EPA. 19881. The doses administered by gavage and diet are time weighted (3 weeks via gavage and
75 weeks via diet) to yield the estimated ADDs. The equivalent dose of MBT is calculated from the dose of
zinc MBT by multiplying by the ratio of the molecular weights of the two compounds (167.24 g/mol:397.7 g/mol).
5Body weight and food consumption data are not provided in the study report. The ADDs are calculated using
default values for food intake and body weight in male and female S-D rats in chronic-duration studies (U.S. EPA.
1988). For example, 2,500 ppm MBT = 2,500 mg/kg MBT x (0.036 kg/day 0.523 kg) = 172.1 mg/kg-day.
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continuous data employed ANOVA and Dunnett's test; pup sex ratios and pup viability were
evaluated using the x2 test.
Survival of F0 and F1 parents was not affected by exposure to MBT (Springborn
Laboratories. 1990b). Clinical signs, litter sizes, and pup viability were comparable for control
and treated animals of all generations. Metrics of reproductive function were also similar in the
control and treated animals in both generations. Body weight was decreased in several treated
groups but the change was <10% compared to controls. In F0 and F1 parental males, body
weights were statistically significantly lower than controls at 602.3 and 1,033 mg/kg-day
throughout most of the study; at these doses, statistically significant decreases of 6-9% were
recorded at the last measurement. High-dose F0 females also exhibited statistically significantly
decreased body weight, accompanied frequently by decreased food intake, from Week 3 through
the premating period, on Gestation Day (GD) 20, on LDs 1 and 14, and during Weeks 19 and 20.
At 699.0 mg/kg-day, female F0 rats exhibited sporadic decreases in body weight during the
premating period and Week 20. Terminal body weights in F0 females were statistically
significantly lower than controls at 699.0 and 1,198 mg/kg-day (5 and 7% respectively). In
F1 females, statistically significant reductions in body weight were seen consistently at all doses
in the premating period and in the mid- and high-dose groups during gestation and lactation;
terminal body weights were statistically significantly lower only at the mid and high doses
(6-7%)). Statistically significantly decreased food intake was noted occasionally in F0 and
F1 males and females at >602.3 or 699.0 mg/kg-day.
Necropsy body weights were not reported; as noted earlier, terminal body weights
(Week 20 in F0 animals and Week 38 in F1 animals) were statistically significantly reduced
(5-9%o lower than controls) in both F0 and F1 male and female parents exposed to 602.3 or
699.0 or 1,033 or 1,198 mg/kg-day (Springborn Laboratories. 1990b) (see Table B-6). In
F0 male and female parents, relative liver weights were statistically and biologically significantly
increased at 602.3 or 699.0 and 1,033 or 1,198 mg/kg-day (12-17%) compared with controls),
while absolute liver weights were increased by <7% compared with controls (not statistically
significantly), suggesting that the relative weight change may reflect body-weight reductions. In
F1 male and female parents, statistically and biologically significant increases in relative liver
weight at the mid and high doses (15—33%) were accompanied by absolute liver-weight
increases of 8-22%, suggesting that in these animals, the relative weights were not primarily
attributable to body-weight reductions. F1 male parents also exhibited statistically and
biologically significantly increased relative liver weight (12%>) accompanied by an absolute
liver-weight increase of 9% at the low dose of 172.1 mg/kg-day. The liver-weight changes are
consistent with the histopathology findings of statistically significantly increased incidences of
hepatocyte hypertrophy in male and female F1 parents at 602.3 or 699.0 and 1,033 or
1,198 mg/kg-day, with greater incidences in males (see Table B-7).
In male F0 and F1 parents, relative kidney weights were statistically significantly
increased at 602.3 and 1,033 mg/kg-day (15—20%), and absolute kidney weights were increased
by 8-10%o at these doses (statistically significant only in high-dose F1 males; see Table B-6).
Relative kidney weights were increased >10% in the mid- and high-dose groups of F0 and
F1 female parents (10-15%); these changes were accompanied by increases in absolute kidney
weight of 3-7%o and were likely influenced by body-weight reductions. Treatment-related
histopathological changes were observed in kidneys (primarily in males) of F0 and F1 parental
rats. Statistically significantly increased incidences of cortical tubular basophilia were observed
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at all doses in F0 males and at 1,033 mg/kg-day in F1 males, but not in females (see Table B-7).
Statistically significantly increased incidences of brown pigment in the lumen and epithelial cells
of the proximal convoluted tubules were observed at 602.3 and 1,033 mg/kg-day in F0 and
F1 males and at 1,198 mg/kg-day in F1 females; the study authors suggested that the presence of
brown pigment in the lumen reflected excretion via the kidney rather than a toxic effect on the
kidney, but provided no information to support this hypothesis. The study authors also reported
increased incidences of alpha 2u-globulin (a2u-g) inclusions in epithelial cells of the proximal
convoluted tubules in male rats. However, the study authors' conclusion was based on
hematoxylin-eosin staining rather than immunohistochemistry, which provides more rigorous
evidence; further, there were no other signs of a2u-g accumulation (e.g., granular casts,
exfoliation of cells into the tubular lumen, increased mitotic figures) (Frazier et al.. 2012; U.S.
EPA, 1991).
Body weights of F1 pups were statistically significantly reduced at 602.3 or 699.0 and
1,033 or 1,198 mg/kg-day on LD 14 (8 and 14% lower than controls, respectively) and LD 21
(12 and 21%, respectively; see Table B-8). In F2 pups, statistically significant body-weight
reductions were observed at all doses on LD 14 (9, 9, and 14% lower than controls in the low-,
mid-, and high-dose groups) and LD 21 (9, 12, and 21% lower than controls). A LOAEL of
172.1 mg/kg-day (the lowest dose tested) is identified based on increased incidence of basophilic
tubules in male F0 rats and a >10% increase in relative liver weight in male F1 rats.
Developmental Studies
Springborn Laboratories (1989c)
Springborn Laboratories (1989c) conducted a dose range-finding study for a
developmental study in groups of timed-mated female S-D rats (six/group) exposed to MBT
(98%) purity) suspended in corn oil via gavage at doses of 0, 300, 600, 1,000, 1,500, or
2,200 mg/kg-day once daily on GDs 6-15. The control group received the vehicle only. During
the study, maternal animals were examined for mortality twice daily and for clinical signs of
toxicity once daily. Maternal body weight was measured on GDs 0, 6, 9, 12, 16, and 20.
Maternal body-weight gain was determined for GDs 0-6, 6-9, 9-12, 12-16, 16-20, 6-16, and
0-20. All surviving dams were sacrificed on GD 20, and cesarean sections were performed. A
maternal necropsy was performed that included examination of external surfaces, orifices, and
viscera, and determination of pregnancy status. The number of viable and nonviable fetuses,
number of early and late resorptions, and number of corpora lutea were recorded. The fetuses
were weighed, sexed, and examined externally for morphological abnormalities.
Decreased survival was reported following exposure to 2,200 mg/kg-day. Two rats died
before GD 20 (on GD 10 and GD 11), and lesions were noted in their gastrointestinal tracts.
Clinical signs of toxicity were reported in rats of all dose groups and included urine staining,
dark material around eyes, nose, and mouth, salivation, and chin dragging. During GDs 6-9,
maternal body-weight gain was decreased in rats exposed to 1,500 and 2,200 mg/kg-day (6-19 g
loss compared with 3 g gain in controls), but were not statistically different than control by the
end of gestation (<4% decrease from control). No differences were observed between the control
and treatment groups for the other maternal and developmental toxicity parameters examined.
Due to excessive maternal mortality at 2,200 mg/kg-day, Springborn Laboratories (1989c) chose
dosage levels of 300, 1,200, and 1,800 mg/kg-day for the subsequent teratology study
(Springborn Laboratories. 1989e). Based on these data, 2,200 mg/kg-day is a FEL for mortality
(2/6 rats died).
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Springborn Laboratories (1989b)
Springborn Laboratories (1989b) conducted a dose range-finding study for a
developmental study in groups of artificially inseminated New Zealand white (NZW) rabbits
(five/group) were exposed to MBT (98% purity) suspended in 1% methylcellulose via gavage at
doses of 0, 150, 300, 600, 1,000, or 1,500 mg/kg-day once daily on GDs 6-18. During the study,
maternal animals were examined for mortality twice daily and for clinical signs of toxicity once
daily. Maternal body weight was measured on GDs 0, 6, 9, 12, 15, 19, 24, and 29. Maternal
body-weight gain was determined for GDs 0-6, 6-9, 9-12, 12-15, 19-24, 24-29, 6-19, 0-29,
and 19-29. All surviving does were sacrificed on GD 29, and cesarean sections were performed.
Maternal necropsy was performed on animals that died prior to GD 29. The number of viable
and nonviable fetuses, number of early and late resorptions, and number of corpora lutea were
recorded. The fetuses were weighed and examined externally for morphological abnormalities.
Decreased survival was reported at doses >600 mg/kg-day (1/5 deaths on GD 27 at
600 mg/kg-day, 3/5 deaths on GDs 17-20 at 1,000 mg/kg-day, 5/5 deaths on GDs 11-12 at
1,500 mg/kg-day). Abortions were reported in one rabbit in each of the 600- and
1,000-mg/kg-day groups on GDs 27 and 14, respectively. None of the rabbits in the 1,000- and
1,500-mg/kg-day groups survived until scheduled cesarean section. Clinical signs of toxicity
were reported at all treatment levels, including reduced defecation, emaciation
(>600 mg/kg-day), and labored breathing (1,500 mg/kg-day). Terminal body-weight decreased
>10% in does administered >600 mg/kg-day, with dose-dependent decreases in body weight in
all treatment groups. Body-weight gain was less than controls in all treatment groups throughout
the majority of gestation but was comparable to controls in the 150- and 300-mg/kg-day groups
at end of gestation (GDs 19-29). Two does administered 300 mg/kg-day had severe
body-weight losses during gestation. Intrauterine survival was decreased in the 600-mg/kg-day
group due to increased early and late resorptions and postimplantation loss. Fetal weights were
dose-dependently decreased >10% in all treatment groups (-14, -16, and —61% in 150, 300, and
600 mg/kg-day, respectively). No biologically significant differences in fetal external
abnormalities were observed between the control and treatment groups examined. Due to
excessive maternal and developmental toxicity at 600 mg/kg-day, Springborn Laboratories
(1989b) chose doses of 50, 150, and 300 mg/kg-day for the subsequent teratology study. Based
on decreased fetal weight, a developmental LOAEL (lowest dose tested) of 150 mg/kg-day is
identified. Based on increased maternal mortality, 600 mg/kg-day is identified as a FEL
(1/5 rabbits died).
Springborn Laboratories (1989e)
Springborn Laboratories (1989e) evaluated teratogenic effects of MBT in groups of
timed-mated female S-D rats (26/group) exposed to MBT (98% purity) suspended in corn oil via
gavage at doses of 0, 300, 1,200, and 1,800 mg/kg-day once daily on GDs 6-15. The control
group received the vehicle only. Dose estimates were calculated by the study authors using the
mean body weight of vehicle control animals on GD 0 (i.e., 0.281 kg). Analytical measurement
indicated that dosing formulations were within ±10% of nominal concentrations. During the
study, maternal animals were examined for mortality twice daily and for clinical signs of toxicity
once daily. Maternal body weight and food consumption were measured on GDs 0, 6, 9, 12, 16,
and 20. All surviving dams were sacrificed on GD 20, and cesarean sections were performed.
Maternal necropsy examinations included an evaluation of external surfaces, orifices, and
viscera, and a thorough uterine examination. The number of viable and nonviable fetuses,
number of early and late resorptions, and number of corpora lutea were recorded. Uteri with no
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evidence of implants were examined macroscopically for the detection of early embryonic
deaths. The fetuses were weighed, sexed, and examined externally for morphological
abnormalities. Approximately one-half of the fetuses were examined for visceral abnormalities
and one-half were examined for skeletal abnormalities. Statistical analyses of continuous
endpoints was done using ANOVA followed by Dunnett's test. The Mann-Whitney U test was
used to assess postimplantation losses and numbers of dead fetuses and resorptions, while the
X2 test was used for sex ratio. Incidences of malformations and variations were evaluated using
Fisher's exact test. Developmental endpoints were evaluated using both fetuses and litters as the
experimental units of analysis.
No treatment-related maternal mortalities were observed (Sprinuborn Laboratories.
1989e). One maternal death occurred in the 1,800-mg/kg-day group on GD 16 and was
attributed to a congenital anomaly (umbilical hernia), which was aggravated by pregnancy and
not considered to be treatment-related. Maternal survival was 100% in the other dose groups.
Clinical signs of toxicity in maternal animals were seen at the 1,200- and 1,800-mg/kg-day doses
(see Table B-9). The most prevalent signs were seen postdosing, and included salivation, dark
material around the mouth, and urine staining. A decrease in activity was also observed
postdosing in several rats administered 1,800 mg/kg-day. During GDs 6-9, body-weight gain of
maternal animals at 1,800 mg/kg-day was statistically significantly decreased (loss of 9 g
compared with gain of 3 g in controls) and food consumption was decreased by 27% relative to
controls during this time. However, the decrease in mean maternal body weight on GD 9 was
small (4%> less than controls at the high dose). After GD 9, increased food consumption was
seen at 1,200 and 1,800 mg/kg-day (11 and 14% higher than controls, respectively, during
GDs 9-12), and mean body weights of treated rats were not statistically or biologically
significantly different from controls (see Table B-10).
Springborn Laboratories (1989e) reported pregnancy percentages of 92.3, 88.5, 96.2, and
84.6%) at 0, 300, 1,200, and 1,800 mg/kg-day, respectively. Mean postimplantation loss per litter
was increased, primarily due to increases in early resorptions, at >300 mg/kg-day
(see Table B-l 1). Springborn Laboratories (1989e) reported that the mean numbers of
postimplantation losses per litter were within the laboratory historical control range
(range: 0.6-1.4; mean: 0.9) for rats of the same strain at 300 (mean of 1.3 per litter) and 1,200
(1.3 per litter) mg/kg-day, but was well above the range at 1,800 mg/kg-day (1.7 per litter).
However, the concurrent controls did not exhibit any abnormal toxicity and were used for
comparison. No statistically significant or biologically significant differences were observed
between the control and treatment groups for the other maternal and developmental toxicity
parameters examined. EPA identifies a maternal LOAEL of 1,800 mg/kg-day and a NOAEL of
1,200 mg/kg-day, based on clinical signs of toxicity and decreased activity. Based on increased
postimplantation loss compared to the concurrent control group, the developmental LOAEL
(lowest dose tested) is 300 mg/kg-day.
Springborn Laboratories (1989d)
Springborn Laboratories (1989d) also evaluated developmental toxicity of MBT in
rabbits. Artificially inseminated NZW rabbits (20/group) were exposed to MBT (98% purity)
suspended in 1% methylcellulose via gavage at doses of 0, 50, 150, or 300 mg/kg-day once daily
on GDs 6-18. The control group received the vehicle only. Dose estimates were calculated
using the mean body weight of vehicle control animals on GD 0 (i.e., 4.413 kg). Analytical
measurement indicated that dosing formulations were within ±8% of nominal concentrations.
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During the study, maternal animals were examined for mortality twice daily and for clinical
signs of toxicity once daily. Maternal body weight was measured on GDs 0, 6, 9, 12, 15, 19, 24,
and 29, and food consumption was measured daily. Maternal body-weight gain and food
consumption (calculated as g/animal-day and g/kg-day) were determined for GDs 0-6, 6-9,
9-12, 12-15, 15-19, 19-24, 24-29, 6-19, and 0-29. All does were sacrificed on GD 29, and
cesarean sections were performed. For maternal necropsy examinations, the thoracic,
abdominal, and pelvic cavities were opened, the viscera examined, and the livers weighed. The
numbers of viable and nonviable fetuses, early and late resorptions, and corpora lutea were
recorded. Uteri with no macroscopic evidence of implants were examined for the detection of
early embryonic deaths. The fetuses were weighed, sexed, and examined for external, visceral,
and skeletal abnormalities. Statistical analyses of continuous endpoints were done using
ANOVA followed by Dunnett's test. The Mann Whitney U test was used to assess
postimplantation losses and numbers of dead fetuses and resorptions, while the %2 test was used
for sex ratio. Incidences of malformations and variations were evaluated using Fisher's exact
test. Developmental endpoints were evaluated using both fetuses and litters as the experimental
units of analysis.
There were no treatment-related maternal mortalities or clinical signs of toxicity among
the rabbits (Springborn Laboratories. 1989d). One doe in the 150-mg/kg-day group died on
GD 13 due to trauma associated with intubation. During GDs 15-19, a statistically
nonsignificant reduction in maternal body-weight gain occurred at 300 mg/kg-day (<10%
relative to the control group). No other effects on body weight nor effects on food consumption,
were observed. At necropsy, no macroscopic abnormalities were observed that were considered
to be test substance-related. Statistically nonsignificant increases in absolute liver weights
occurred at 150 and 300 mg/kg-day (4-7% higher than controls) and relative liver weight at
300 mg/kg-day (4% higher than control). No statistically significant or biologically relevant
differences were observed between the control and treatment groups for any of the other
maternal or developmental toxicity parameters examined. Given the small magnitude of changes
in body weight and liver weight at 300 mg/kg-day, these effects are not considered as the basis
for determining a LOAEL. Thus, the highest dose tested of 300 mg/kg-day is identified as the
NOAEL for maternal and developmental toxicity.
Inhalation Exposures
No studies evaluating the effects of MBT in animals after repeated inhalation exposure
have been located in the available literature.
OTHER DATA (GENOTOXICITY, ACUTE TESTS, OTHER EXAMINATIONS)
Table 4 provides an overview of genotoxicity studies of MBT. Other supporting studies
on MBT are discussed afterward, including:
• acute-duration oral and inhalation studies,
• studies using other routes of exposure,
• an acute immunotoxicity study,
• an acute neurotoxicity study, and
• metabolism/toxicokinetic studies.
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella strains
TA98, TA100,
TA1535, TA1537,
and TA1538
3, 10, 30, 100,
300 ng/plate;
Second test with
TA98 and TA1538 at
100, 250, 300, 450,
600 ng/plate
Plate incorporation assay. Toxicity observed at doses
>333 ng/plate.
Pliarmakon
Research
International
(1984a. 1984b)
Mutation
Salmonella strains
TA98, TA100,
TA1535, TA1537,
and TA1538
0.1, 1.0, 10, 100,
500 ng/plate
Plate incorporation assay.
Litton Bionetics
(1976)
Mutation
Salmonella strains
TA98, TA100,
TA1535, and
TA1537
0,3.3, 10,33, 100,
333, 1,000, 3,333,
10,000 ng/plate
(TA98 also tested at
200, 400, 500, 600,
700,
10,000 ng/plate)
Plate incorporation assay. Strain TA98 had equivocal and
weakly positive results at doses >333 |ig/platc in some
trials with metabolic activation, but other trials at same
doses were negative. In addition, slight toxicity seen at
doses >333 |ig/platc: precipitation seen at >3,333 |ig/platc.
NTP (1988);
Zeieer et al. (1987)
Mutation
Salmonella strains
TA98, TA100,
TA1535, TA1537,
and TA1538
Without activation:
32, 100, 320, 500,
1,000 ng/plate
With activation:
100 ng/plate
These data provided in a handwritten report without any
information on methods.
Goodyear Tire &
Rubber Company
(1985)
Mutation
Salmonella strains
TA98, TA100,
TA1535, and
TA1537
0.1, 1, 10,
100 ng/plate
Plate incorporation assay. Toxicity was observed at
1,000 ng/plate.
Goodyear Tire &
Rubber Company
(1985)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Mutation
Salmonella strains
TA98, TA100,
TA1535, and
TA1537
8-200 ng/plate
NT
Plate incorporation assay.
Crebelli et al.
(1985)
Mutation
Salmonella (strains
not reported)
NR
Study details not provided; MBT exhibited a statistically
nonsignificant mutagenic response at nontoxic
concentrations (toxic concentration not reached). It is
unclear whether results were with or without metabolic
activation.
Doiuier et al.
(1983)
DNA damage
(SOS/umu)
Salmonella strain
TA1535/pSK1002
Without S9: up to
19 iig/mL (LC50)
With S9: up to
20 ng/mL (LC50)
MBT did not induce DNA damage at concentrations less
than or equal to the LC50 with or without metabolic
activation; higher concentrations were not tested.
Ye et al. (2014)
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
Saccharomyces
cerevisiae strain D4
0.1, 1.0, 10, 100,
500 ng/plate
—
—
Plate incorporation assay.
Litton Bionetics
(1976)
Genotoxicity studies in mammalian cells in vitro
Mutation
Mouse lymphoma
cells (L5178Y)
Without S9: 0, 3.75,
15, 40, 60, 80, 100,
150 ng/mL
WithS9:0, 3.75, 5,
15, 20, 40, 50, 60,
80, 100, 120,
150 ng/mL
±
±
MBT was mutagenic only at concentrations that were
highly cytotoxic with or without metabolic activation.
Mutant frequency was increased 1.8-8.7-fold without
metabolic activation (at <10% relative survival) and
1.7-2.7-fold with metabolic activation (at 7-20% relative
survival). Cytotoxicity (survival <50% of controls) was
observed at >3.75 ng/mL without metabolic activation and
>20 |ig/mL with metabolic activation. Observed effects are
likely due to cytotoxicity rather than genotoxicity.
Litton Bionetics
(1985)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Mutation
Mouse lymphoma
cells (L5178Y)
Without S9: 30, 40,
50, 60, 80, 100, 120,
150 ng/mL
With S9: 1.25,2.50,
4, 5, 6, 7.5, 8, 10, 12,
15, 16, 20 ng/mL
+
Mutagenic with metabolic activation at >5 |ig/mL.
Cytotoxicity (survival <50% of controls) was observed at
>40 |ig/mL without metabolic activation and at >7.5 ng/mL
with metabolic activation.
NIP (1988)
Mutation
Mouse lymphoma
cells (L5178Y)
Without S9: 0, 30,
40, 50, 60, 80, 100,
150 ng/mL
With S9: 0, 4, 5, 6, 8,
10, 12, 16, 20 ng/mL
+
A statistically significant 2-3-fold increase in mutant
frequency was observed at >5 |ig/mL with metabolic
activation. Cytotoxicity (survival <50% of controls) was
observed at >40 ng/mL without metabolic activation and at
>8 ng/mL with metabolic activation.
Mvhretal. (1990)
Mutation
(HGPRT)
CHO cells
Without S9: 1, 5, 10,
30, 50 ng/mL
With S9: 10, 25, 75,
150, 300 ng/mL
Cytotoxicity was observed at doses >100 |ig/mL without
activation and at 1,000 |ig/mL with activation; at
333.33 ng/mL with activation, survival relative to controls
was 17%.
Pliarmakon
Research
International
(1984c)
Mutation
(HGPRT)
Chinese hamster V79
cells
0, 50, 100,
300 |ig/mL
NT
MBT was not cytotoxic at the highest concentration.
Do tine r et al.
(1983)
SCE
CHO cells
Without S9: 0, 12.5,
14.9, 20.1,
24.8 ng/mL
With S9: 0, 99.2,
247.5,351.6, 401.6,
445.3, 501.5, 502.3,
750 iig/mL
+
A statistically significant 23-37% increase in SCEs was
observed after exposure to >351.6 |ig/mL in the presence of
metabolic activation; SCEs were not induced without
metabolic activation. No cells survived at concentrations
of 24.8 |ig/mL without metabolic activation or
>502.3 |ig/mL with metabolic activation.
NIP (1988)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
CA
CHO cells
Without S9: 0, 10,
14.9, 19.9,
30.1 ng/mL
With S9: 0, 351.8,
373.5, 399, 400.8,
425,450, 451 ng/mL
+
The percentage of cells with CAs was statistically
significantly increased by 8-26% at >351.8 |ig/mL with
metabolic activation; CAs were not induced without
metabolic activation. No cells survived at highest
concentrations evaluated (30.1 ng/mL without metabolic
activation, 500.5 ng/mL with metabolic activation in
Trial 1, 450 |ig/mL with metabolic activation in Trial 2).
NIP (1988)
CA
Chinese hamster
lung cells
Without S9: 0, 200,
400 iig/mL
With S9: 0, 200, 400,
600 ng/mL
±
Results with metabolic activation were judged as
inconclusive at 400 ng/mL (5-10% total CA frequency).
Results without metabolic activation were judged as
negative (<5% total CA frequency). Cytotoxicity was
observed at 400 ng/mL without activation and 600 ng/mL
with S9 activation.
Matsuoka et al.
(2005)
Micronucleus
test
Human A549 lung
carcinoma cells
0,3.13,6.25, 12.5,
25, 50, 100 ng/mL
—
NT
Cytotoxicity at the highest tested concentration was 15%.
Ye et al. (2014)
Micronucleus
test
Human MGC-803
gastric carcinoma
epithelial cells
0,3.13,6.25, 12.5,
25, 50, 100 ng/mL
NT
Cytotoxicity at the highest tested concentration was 29%.
Ye et al. (2014)
Cell
transformation
Balb/3T3 cells
10.5-63 ng/mL
NT
Cytotoxicity (survival <50% of controls) was observed at
doses >31.3 ng/mL.
Goodyear Tire &
Rubber Company
(1985)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Genotoxicity studies—in vivo
Dominant
lethal
mutagenicity
S-D rats
(28 M/group), dosed
in diet for 13 wk,
then mated for 2 wk
to untreated females;
females sacrificed on
GD 13 for evaluation
of numbers of
embryonic deaths
and viable embryos
0, 2,500, 8,750,
15,000 ppm in diet
(0, 220, 770,
1,300 mg/kg-d)
No statistically significant or dose-related increase in
embryonic deaths (no dominant lethal effect). In treated
males, statistically significantly reduced food consumption
and body weights were seen at >8,750 ppm. Significantly
reduced body-weight gain occurred at 2,500 ppm during
Wk 1-3 and 7-8.
Snrineborn
Laboratories
f 1989a)
Mouse bone
marrow CA
assay
Swiss albino mice
(four/group; sex not
reported) were
administered a single
i.p. injection of
zinc MBT in cotton
seed oil and
sacrificed 36 hr later
for evaluation of
CAs in bone marrow
0, 480, 960, 1,920 ng
zinc MBT/20 g (24,
48, 96 mg/kg)
CA incidence was comparable between mice exposed to
MBT and controls.
Mohanan et al.
(2000)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
MN
CD-I mice
(four/sex/group)
were administered a
single i.p. injection
of MBT and
sacrificed 30 or 48 hr
later. Additional
groups
(four/sex/group)
were given
two injections at 0
and 24 hr, and
sacrificed 48 or 72 hr
later; bone marrow
was extracted and
scored for
polychromatic
erythrocytes with
MN
300 mg/kg per
injection
No statistically significant increase in MN was seen in any
group.
Pliarmakon
Research
International
Q984d. 1984e)
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Table 4. Summary of MBT Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
DNA binding
F344 rats (10/sex)
were given a single
gavage dose of
[2-14C]-labelled
MBT and sacrificed
8 hr later;
radioactivity was
measured in DNA
collected from
various tissues
375 mg/kg
Little radioactivity was detected in tissues; <0.6% in liver
and <0.03% in remaining tissues. Based on radioactivity in
DNA, low binding to DNA was measured: 1.5-3.0 pmol
MBT/mg DNA in the liver and 0.15-0.76 pmol MBT/mg
DNA in bone marrow. No DNA binding was detected in
pancreatic, adrenal, or pituitary tissue. Covalent binding
index (calculated as |imol chemical bound/mol
DNA) (mmol chemical applied/kg body weight) was very
low (1-3 in the liver; compared with a covalent binding
index value of 6,000 for strong hepatocarcinogen,
dime thy lnitro samine).
Brewster et al.
(1989); Monsanto
f 1989a)
a+ = positive; - = negative; ± = inconclusive.
CA = chromosomal aberrations; CHO = Chinese hamster ovary; DNA = deoxyribonucleic acid; GD = Gestation Day; i.p. = intraperitoneal; LC50 = median lethal
concentration; M = male(s); MBT = 2-mercaptobenzothiazole; MN = micronuclei; NR = not reported; NT = not tested; S-D = Sprague-Dawley; SCE = sister chromatid
exchange.
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Genotoxicity
The potential genotoxicity of MBT has been evaluated in several in vitro studies and a
limited number of in vivo studies. In general, the data indicate that MBT is not mutagenic.
Limited in vitro data suggest that MBT may cause clastogenic effects in mammalian cells;
however, findings are inconsistent among studies and cell types, and in vivo data were negative.
MBT was not mutagenic in Salmonella typhimurium strains with or without metabolic
activation (Ye et al.. 2014; N I P. 1988; Zeiger et aL 1987; ('rebelli et al.. 1985; Goodyear Tire &
Rubber Company, 1985; Pharmakon Research International, 1984a, b; Donner et al., 1983;
Litton Bionetics. 1976) or Saccharomyces cerevisiae strain D4 (Litton Bionetics, 1976). Urine
obtained from 72 male workers employed in a tire factory who were exposed to MBT (along
with numerous other chemicals) was also not mutagenic in S. typhimurium (('rebel li et al, 1985).
In mammalian cells, MBT was mutagenic in mouse L5178Y lymphoma cells, but typically only
at concentrations associated with cytotoxicity (Myhretal, 1990; NTP, 1988; Litton Bionetics,
1985). MBT was not mutagenic in Chinese hamster ovary (CHO) cells with or without
metabolic activation or Chinese hamster V79 cells without metabolic activation (Pharmakon
Research International, 1984c; Donner et al, 1983). In vivo, MBT did not induce dominant
lethal mutations in male rats exposed to dietary doses up to 1,300 mg/kg-day for 13 weeks prior
to mating (Sprineborn Laboratories, 1990a, 1989a).
Chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) were statistically
significantly increased by 8-37% in CHO cells exposed to MBT with metabolic activation
(NTP, 1988). However, in Chinese hamster lung cells, evidence for CAs following metabolic
activation was inconclusive (Matsuoka et al, 2005). Neither CAs nor SCEs were induced in
either cell type without metabolic activation (Matsuoka et al.. 2005; NTP, 1988). Micronuclei
(MN) were not induced in human A549 lung carcinoma cells or MGC-803 gastric carcinoma
epithelial cells without metabolic activation; cells were not tested with metabolic activation (Ye
et al, 2014). In vivo, neither CAs nor MN were induced in mouse bone marrow following a
single intraperitoneal (i.p.) injections of zinc MBT at doses up to 96 mg/kg or MBT at
300 mg/kg, respectively (Mohanan et al, 2000; Pharmakon Research International, 1984d, e).
MBT did not induce deoxyribonucleic acid (DNA) damage in S. typhimurium strain
TA1535/pSK1002 (Ye et al, 2014). No evidence of DNA binding was observed in various rat
tissues following a single gavage dose of [2-14C]-labelled MBT (Brewster et al, 1989;
Monsanto. 1989a).
MBT exposure at concentrations of 10.5-63 |ag/m L did not induce morphological
transformation of Balb/3T3 cells in vitro; cytotoxicity (survival <50% of controls) was observed
at doses >31.3 |ig/mL (Goodyear Tire & Rubber Company, 1985).
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Acute Toxicity
Industrial Bio-Test Laboratories6 (IB T Labs. 1977c) exposed single female albino rats to
doses of 0, 30, 100, 300, 1,000, 3,000, or 10,000 mg/kg MBT (purity not reported) in corn oil.
The only death during the 14-day observation period was the rat exposed to 10,000 mg/kg.
There were no clinical signs or gross necropsy findings at sublethal doses (IB I' Labs. 1977c).
Younger Laboratories (1975a, 1975b) estimated oral median lethal dose (LD50) values of
3,800 mg/kg (95% CI = 3,530-4,100 mg/kg) or 2,830 mg/kg (95% CI = 2,690-2,970 mg/kg) in
S-D albino rats exposed to MBT powder or Thiotax MBT (respectively) and observed for
14 days. The oral rat LD50 for zinc MBT in corn oil was 7,500 mg zinc MBT/kg
(95%) CI = 6,820-8,250 mg zinc MBT/kg) under the same conditions (Younger Laboratories.
1975a). Arthur D Little (1977) reported an oral LD50 of 5,000 mg/kg in Swiss albino CaFl mice
exposed to MBT by gavage and observed for 7 days. Guess and O'l.earv (1969) reported an oral
LD50 of 2,000 mg/kg (95%o CI = 1,798-2,225 mg/kg) in male white mice observed for 72 hours
after dosing. Dow Chemical Co (1961) observed no deaths when two rats received a single oral
dose of 3,980 mg/kg MBT (Captax) in corn oil; autopsy findings indicated liver and kidney
injury.
IB T Labs (1977a) exposed Charles River rats by whole-body inhalation to heated dust of
MBT (1,270 mg/m3) for 4 hours and observed them for 14 days to assess acute lethality. None
of the 10 rats (five/sex) died. Ptosis (eyelid drooping) and hypoactivity lasting about an hour
were observed after the third hour of exposure; there were no effects on body weight or gross
necropsy findings. When three rats (strain and sex not reported) were exposed via inhalation to a
saturated atmosphere of MBT (concentration estimated by authors as 104 ppm) for 7 hours, there
were no deaths or clinical signs, but liver and kidney damage were seen at autopsy (Dow
Chemical Co. 1961).
Other Routes
The acute lethality of dermal exposure to MBT was assessed in individual male albino
rabbits exposed to doses of 300, 1,000, or 3,000 mg/kg applied as an aqueous slurry to abraded
skin; none of the three rabbits died (IB I' Labs, 1977b). No deaths were observed when
New Zealand rabbits were exposed to dermal applications of MBT, Thiotax MBT, or zinc MBT
in corn oil at doses up to 7,940 mg/kg (Younger Laboratories. 1975a. b, 1974).
IB I' Labs (1977b) reported that at MBT doses >1,000 mg/kg, erythema, mild edema, and
slight desquamation of the skin at the application site were noted. Dermal hyperemia and
exfoliation were noted in rat skin after 3-10 dermal applications of undiluted MBT or MBT in
10% Dowanol DPM (Dipropylene glycol methyl ether) (Dow Chemical Co, 1961). Younger
Laboratories (1975a. 1975b. 1974) reported that MBT, Thiotax MBT, and zinc MBT were not
6 A total of 618 of 867 nonacute toxicity studies conducted by Industrial Bio-Test Laboratories (including subacute,
subchronic-duration, carcinogenicity, reproductive toxicity, genotoxicity, and neurotoxicity studies) were found to
be invalid during a post hoc audit program conducted by U.S. EPA and the Canadian Health and Welfare
Department (OECD. 20071. Discrepancies and deficiencies were also identified in acute studies, but the focus of the
investigation was on repeated exposure studies that formed the basis of regulatory decisions. The laboratory closed
in 1978. OECD (2007) outlined specific criteria for using data generated by Industrial Bio-Test Laboratories, and
recommended rejecting a study when either a regulatory or internal audit revealed problems impacting the reliability
of the findings, or when the findings of unaudited studies are inconsistent with data collected later by reputable
laboratories. OECD (2007) recommended that studies that have not been audited should be used with caution and
only as weak evidence if supported by later data from reputable laboratories. No information was available on
internal or external auditing of this study.
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irritating to the skin of rabbits when applied as a finely ground sample moistened with water at a
dose of 500 mg for 24 hours.
The LD50 for MBT administered intraperitoneally to male white mice (observed for
72 hours) was 437 mg/kg (95% CI = 415-461 mg/kg) (Guess and O'I.earv. 1969). Signs of
toxicity in animals exposed to doses >335 mg/kg included marked peripheral vasodilation,
salivation, and prolonged clonic and tonic seizures, suggesting central nervous system (CNS)
stimulation. Pretreatment with pentobarbital (25 mg/kg, i.p.) blocked the convulsive attacks and
salivation induced by high doses of MBT (550 mg/kg). When male white mice were given
repeated daily i.p. injections of one-fourth and one-eighth the LD50 (-110 and 55 mg/kg-day) for
1 week, there were no clinical signs of toxicity or effects on body weight or gross necropsy
findings (Guess and O'I.earv. 1969). However, the authors reported severe histopathological
liver damage in the form of extensive necrosis and disruption of anatomic organization, cloudy
swelling, accumulation of cytoplasmic granules, fatty infiltration and degeneration, rupture of
cell walls, and profound changes in nuclei in the high-dose mice (histopathology was not
examined in low-dose mice). Diffuse, cloudy swelling of the renal tubules was also noted in
exposed mice (Guess and O'I.earv. 1969). Functional damage to the liver following MBT
administration (110 mg/kg, i.p.) was confirmed by prolongation of sleeping time in a
hexobarbital narcosis study.
In a dose range-finding study for an in vivo micronucleus assay, groups of four CD-I
mice (two/sex) were given i.p. injections of MBT at doses of 16.6, 50, 166.6, 500, or
1,666.6 mg/kg and observed for 48 hours (Pharmakon Research International. 1984d. e). All
mice exposed to 1,666.6 mg/kg died, as did both females dosed with 500 mg/kg. Clinical signs
were seen in the males exposed to 500 mg/kg, including decreased muscle tone and activity as
well as ptosis. Some animals exposed to 50 and 166.6 mg/kg were noted to have decreased
muscle tone; no signs of toxicity were seen at 16.6 mg/kg. The study authors noted the
following clinical signs after a single injection of 300 mg/kg MBT: prostration, hypoactivity,
hyperpnea, ptosis, tremors, and loss of righting in some animals; no deaths occurred at this dose
(Pharmakon Research International, 1984d, e).
In a developmental toxicity test, MBT in dimethyl sulfoxide (DMSO) was administered
subcutaneously, on GDs 6-14 or 6-15 (AKR mice only), to pregnant BL6, C3H, and AKR mice
at 464 mg/kg and to C3H mice at 300 mg/kg (Bionetics. 1968). Dams were allowed to litter, and
offspring were evaluated at birth and PND 8, then sacrificed. In high-dose C3H mice,
statistically significantly decreased fetal weight and crown-rump length (compared to controls)
were observed; these effects were not seen in AKR or BL6 mice. Increased incidences of
abnormal fetuses were seen in the BL6 and C3H strains exposed to 464 mg/kg, although a repeat
experiment with BL6 mice failed to confirm these findings. Group sizes were small in this study
(6-13 litters), limiting the reliability of the information. There was an increase in maternal liver
weight (relative to body weight) in BL6 (both experiments), C3H (300 mg/kg), and AKR mice
administered 200 mg/kg MBT intraperitoneally to rats on GDs 1-15 Hardin et al. (1981). The
study authors reported no treatment-related histopathological effects in maternal tissues (brain,
heart, lungs, liver, spleen, kidneys, adrenals, and ovaries) and no maternal toxicity (evaluated as
reduced body-weight gain or statistically significant changes in two or more organ weights). No
fetal toxicity (reduced pre or postimplantation survival or fetal body weight or length) nor
teratogenesis (grossly visible external or internal malformations) was observed. MBT
(200 mg/kg in sunflower oil, purity not reported) was injected into the stomach of female albino
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rats (11-25/group) either (1) before the beginning of pregnancy on the first and third days of
estrus or (2) on GDs 4 and 1 1 (Aleksandrov. 1982). The rats were sacrificed on GD 19.
Administration of MBT before pregnancy resulted in increased duration of the estrous cycle
(5.7 ± 0.3 days) compared to controls (4.4 ± 0.3 days), decreased speed to the onset of
conception (1.6 ± 0.2 cycles) compared to controls (1.0 cycles), and increased postimplantation
embryonic mortality ("index of mutagenicity"). Administration before or during pregnancy
resulted in decreased number of corpora lutea and living fetuses and increased total percent
embryonic mortality. A 23% decrease in fetal weight was also reported.
Injection of MBT or zinc MBT into 3-day-old chick embryos did not affect viability or
malformations in one study (Korhonen et aL 1982). A subsequent study (Korhonen et aL 1983)
reported that MBT (dose not specified) induced a 20% incidence of malformed embryos.
Metabolism/Toxicokinetic Studies
A single study provides most of the available information on toxicokinetics of MBT. El
Dareer et al. (1989) examined the disposition of MBT administered by oral, intravenous (i.v.),
and dermal exposure routes in rats and dermal exposure in guinea pigs. F344 rats of both sexes
were dosed daily by gavage for 14 days with unlabeled MBT at 0.509 mg/kg-day prior to a
single gavage dose of 0.503 mg/kg of [14C]-MBT. In i.v. exposure experiments, male and female
rats were given single i.v. doses of 0.602 mg/kg [14C]-MBT. In dermal exposure experiments,
male and female rats and female Hartley guinea pigs were topically exposed to [14C]-MBT at an
approximate dose of 36.1 [j,g/animal; the exposed area was occluded and remained unwashed for
96 hours.
After oral exposure, absorption was rapid and essentially complete in rats. Radioactivity
was detected in the blood and plasma at the first time point measured (8 hours postdosing), and
90.7-101% of the administered radioactivity was excreted in the urine within 96 hours (El
Dareer et al.. 1989). MBT was widely distributed through the body; in rats sacrificed 8 hours
after dosing, the highest levels of radioactivity were measured in the kidneys, thyroid, and liver.
The study authors calculated half-lives for plasma and blood (see Table 5). The half-lives of the
alpha (distribution) phase were similar for plasma and whole blood, while the beta (elimination)
phases were much longer for whole blood than for plasma. To explore whether binding to
erythrocytes explained the longer elimination phase, the study authors fractionated erythrocytes
from exposed rats and showed that the radioactivity was primarily associated with erythrocyte
cell membranes. El Dareer et al. (1989) suggested that this association might result from
disulfide formation between MBT and sulfhydryl groups on the membrane, and that such binding
might persist until erythrocyte turnover. The long elimination phase for plasma was postulated
to result from binding to plasma proteins or erythrocyte lysis.
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Table 5. Half-Lives (h) for Alpha (distribution) and Beta (elimination) Phases in Rats
Exposed to 14C-MBT by Gavage3
Whole Blood
Plasma
Alpha
Beta
Alpha
Beta
Males
4.7
6,000
4.12
312
Females
8.56
5,180
3.73
79.6
aEl Dareer et al. (1989).
MBT = 2-mercaptobenzothiazole.
Experiments in rats intravenously dosed showed similar plasma and whole blood
elimination phases, similar tissue distribution, and urinary excretion (El Dareer et al.. 1989). In
the dermal exposure experiments, guinea pigs absorbed a greater percentage of the MBT than
rats (33.4 vs. 16.1-17.5%), but the urinary excretion levels were similar (91% of the absorbed
dose was excreted in urine in male rats, 94% in female rats, and 98% in female guinea pigs).
Metabolites of MBT excreted in rat urine within 8 hours after gavage dosing were
analyzed by high performance liquid chromatography and spectrophotometry (El Dareer et al,
1989). Only two metabolites were identified, and unchanged MBT was not detected in urine.
The major metabolite (relative quantities were not reported) was determined to be a
thioglucuronide of MBT, and the second was a sulfonic acid derivative of MBT (El Dareer et al..
1989). In a study of guinea pigs exposed derm ally to MBT, glucuronide and sulfate derivatives
of MBT were reportedly detected in urine [Nagamatsu et al. (1979) as cited in El Dareer et al.
(1989)1.
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Immunotoxicity
Ahuia et al. (2009) tested the sensitizing potential of MBT in a biphasic local lymph node
assay using dermal and oral experimental protocols. Female Balb/c mice were sensitized via
3 days of dermal exposure to concentrations of 3, 10, or 30% or three daily gavage doses of 1,
10, or 100 mg/kg. The animals in both groups were subsequently challenged with auricular
applications of 3, 10, or 30% concentrations of MBT on Days 15-17. At the end of the
challenge, ear thickness was measured, auricular lymph nodes were weighed, and lymph node
cells were counted and assayed by flow cytometry for lymphocyte subtype counts. In the dermal
study, statistically significant increases in total lymphocyte cell count and lymph node weights,
and a statistically significant decrease in CD8+ cells, were observed at the low and mid MBT
exposure concentrations, but not at the highest concentration. In the oral study, a statistically
significant increase in total cell count was observed at 1 mg/kg (but not at higher doses), and
increased lymph node weight was observed at 10 mg/kg. The study authors concluded that MBT
is a mild to moderate allergen with sensitizing potential after oral and dermal exposure (Ahuia et
al.. 2009).
Neurotoxicity
Indications of neurotoxicity have been reported in several studies; however, consistent
evidence for neurotoxic mechanisms or effects is not available. As discussed in the "Other
Routes" section, acute i.p. administration of MBT (>335 mg/kg) induced CNS stimulation,
which was blocked by pretreatment with pentobarbital (Guess and O'Leary, 1969). Other studies
described in the "Oral Exposures" section of this document report lethargy following dosing,
salivation, irritable behavior increasing with dose, and resistance to gavage. Additionally, a
repeat-dose neurotoxicity study in S-D rats did not find evidence of altered motor activity or
FOB parameters following dietary MBT exposure for 13 weeks (up to 1,920.4 mg/kg-day) (Bio-
Research Laboratories LTD, 1990).
Bio-Research Laboratories LTD (1989) conducted an acute oral neurotoxicity study in
groups of 12 male and 12 female S-D rats. Single doses of 0, 500, 1,250, or 2,750 mg/kg MBT
(purity not reported) in corn oil were administered by gavage. Before dosing and during the
14-day observation period, the animals were examined in a FOB (1, 6, and 24 hours after dosing
and on Days 7 and 14). Motor activity was evaluated before exposure and at 12 hours after
dosing. All animals were necropsied at the end of the observation period. One female rat dosed
with 2,750 mg/kg MBT died 12 hours after exposure. Males in the high-dose group and all
dosed females exhibited muzzle staining and increased incidences of salivation. Decreased
vocalization was noted in males exposed to >1,250 mg/kg when observed 1 and 6 hours after
dosing, and increased urinary staining was noted in high-dose females 24 hours after dosing.
Motor activity was statistically significantly reduced in females at doses >1,260 mg/kg and in
high-dose males; grip strength and hindlimb splay were not affected by exposure. There were no
notable gross necropsy findings. The study authors suggested that the observed effects may be
related to nonspecific acute toxicity rather than indicating neurotoxicity.
Intraperitoneal injection of 300 mg/kg MBT in mice (strain and sex not specified)
resulted in reduced noradrenaline (60% less than controls) 1 and 2 hours after dosing, as well as
increased dopamine (24% higher) when assayed 2 hours after dosing (Johnson et al., 1970). The
animals were observed to be "extremely depressed," with markedly reduced spontaneous motor
activity after dosing. Marked ptosis occurred after 2 hours. By 4 hours after dosing, the animals
behaved as the controls (Johnson et al, 1970). The study authors also reported that MBT
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noncompetitively inhibited bovine dopamine ^-hydroxylase in vitro and postulated that chelation
of copper ion (which is critical to the enzyme activity) was the mechanism by which the
catecholamine levels were altered in vivo.
DERIVATION OF PROVISIONAL VALUES
Tables 6 and 7 present summaries of noncancer and cancer references values,
respectively.
Table 6. Summary of Noncancer Reference Values for MBT (CASRN 149-30-4)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
POD
Method
POD
(HED)
UFc
Principal
Study
Subchronic p-RfD
(mg/kg-d)
Rat/female
Increased relative
liver weight
4 x 10-2
BMDLio
3.56
100
NTP (1988)
Chronic p-RfD
(mg/kg-d)
Rat/female
Increased relative
liver weight
4 x 1(T3
BMDLio
3.56
1,000
NTP (1988)
Subchronic p-RfC
(mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
BMDLio = 10% benchmark dose lower confidence limit; HED = human equivalent dose;
MBT = 2-mercaptobenzothiazole; NDr = not determined; POD = point of departure; p-RfC = provisional reference
concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor.
Table 7. Summary of Cancer Reference Values for MBT (CASRN 149-30-4)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
Rat/female
Combined tumors
1.1 X 10-2
NTP (1988)
p-IUR (mg/m3)-1
NDr
MBT = 2-mercaptobenzothiazole; NDr = not determined; p-IUR = provisional inhalation unit risk;
p-OSF = provisional oral slope factor.
DERIVATION OF ORAL REFERENCE DOSES
Studies of MBT that are potentially relevant to the derivation of provisional reference
dose (p-RfD) values include a short-term-duration range-finding study in rats exposed via the
diet (Monsanto. 1989b). short-term-duration range-finding studies in rats and mice exposed by
gavage (N I P. 1988). a subchronic neurotoxicity study in rats exposed via the diet (Bio-Research
Laboratories LTD. 1990). subchronic and chronic toxicity studies in rats and mice exposed by
gavage (NTP. 1988). a two-generation reproductive toxicity study in rats exposed via the diet
(Springborn Laboratories. 1990b). and developmental toxicity studies in rats and rabbits exposed
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by gavage (Springborn Laboratories. 1989b. c, d, e). Subchronic and chronic p-RfDs were
derived based on the available studies.
Derivation of a Subchronic Provisional Reference Dose (p-RfD)
The subchronic-duration study of adult rats exposed to MBT by gavage is considered the
principal study for derivation of the subchronic p-RfD (NIP. 1988). The critical effect from this
study is increased relative liver weight in female rats.
The subchronic toxicity studies conducted by NTP (1988) report administration of MBT
by gavage to F344/N rats and B6C3Fi mice (10/sex/dose) for 13 weeks. These studies are
included in a peer-reviewed technical report conducted according to Good Laboratory Practice
(GLP) standards with adequate numbers of dose groups, appropriate dose spacing, sufficient
group sizes, and quantitation of results to describe dose-response relationships for the critical
effects. However, uncertainty remains due to the lack of comprehensive endpoint evaluation;
hematology, clinical chemistry, urinalysis, and organ-weight measurements for organs other than
liver were not performed or reported. Short-term exposure studies in rats and mice were not
selected as principal studies because of the brief exposure durations (16-28 days). The
subchronic exposure neurotoxicity study by Bio-Research Laboratories LTD (1990) reported
reduced body weight that occurred in the context of reduced food intake potentially resulting
from poor palatability of the test substance in the diet and was thus not considered as the
principal study. Among the remaining studies, the most sensitive effects were increased absolute
and relative liver weight in male rats and female rats and mice (NTP. 1988) (see Table 8).
Reproductive and developmental studies were not selected as principal studies because effects
resulted from higher doses than the dose producing organ-weight changes in adult rats reported
by NTP (1988). In the two-generation reproductive study in rats (Springborn Laboratories.
1990b). a LOAEL of 172.1 mg/kg-day was identified based on increased incidence of basophilic
tubules in male F0 rats and a >10% increase in relative liver weight in male F1 rats, consistent
with effects observed by NTP (1988). In the developmental toxicity studies in rats, a
developmental LOAEL of 300 mg/kg-day was identified for an increase in postimplantation loss
(Springborn Laboratories, 1989e). In the developmental toxicity range-finding study in rabbits, a
developmental LOAEL (lowest dose tested) of 150 mg/kg-day was identified due to >10%
decreased fetal weight; however, the follow-up developmental study did not replicate this fetal
toxicity with larger sample sizes and a NOAEL of 300 mg/kg-day was identified for maternal
and developmental toxicity in rabbits (Springborn Laboratories. 1989b. d).
The subchronic-duration studies conducted by NTP (1988) reported biologically
significant (i.e., >10% compared to control) increases in absolute and relative liver weight at all
treatment doses in rats and biologically significant increases in relative liver weight at all doses
and in absolute liver weight at doses >536 mg/kg-day in mice (see Table 8). Few studies
measured organ weight following MBT exposure; however, the three other studies that examined
liver weight also reported increased relative liver weight in exposed rats and rabbits (Springborn
Laboratories. 1990b; Monsanto. 1989b; Springborn Laboratories. 1989d). The reproductive
study in rats by Springborn Laboratories (1990b) reported increased absolute and relative liver
weights in F0 and F1 male and female parents. These organ-weight changes were accompanied
by histopathological findings of increased incidences of hepatocellular hypertrophy. Liver
damage and weight changes have also been observed following exposure to MBT via other
routes (i.e., subcutaneous, i.p.) and exposure durations (i.e., acute) (Guess and O'I.earv. 1969;
Bionetics. 1968; Dow Chemical Co. 1961). The LOAEL and lowest dose tested of
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134 mg/kg-day for increased absolute and/or relative liver weight in male and female rats and
LOAEL of 67 mg/kg-day for increased relative liver weight in female mice are lower than all
other LOAEL values in the database and were selected for benchmark dose (BMD) modeling
(see Table 9).
Table 8. Selected Non-neoplastic Endpoints in F344 Rats and B6C3Fi Mice Exposed by
Gavage to MBT for 13 Weeksa'b
Male Rats
Average daily dose
(mg/kg-d)
0
134
268
536
1,071
Number of animals
10
10
9
10
8
Absolute liver weight
(mg)
13,593 ±2,121
15,661 ±793
(+15%)
15,861 ± 1,712
(+17%)
18,742 ±2,631**
(+38%)
16,759 ±2,660**
(+23%)
Relative liver weight
(mg/g)
38.4 ±6.07
43.9 ± 1.87*
(+14%)
47.2 ±2.79**
(+23%)
54.8 ±5.08**
(+43%)
51.3 ±5.42**
(+34%)
Female Rats
Average daily dose
(mg/kg-d)
0
134
268
536
1,071
Number of animals
10
9
10
8
10
Absolute liver weight
(mg)
6,606 ± 795
7,818 ±814**
(+18%)
8,027 ± 688**
(+22%)
7,988 ±591**
(+21%)
8,413 ±652**
(+27%)
Relative liver weight
(mg/g)
31.8 ±3.28
39.3 ±3.53**
(+24%)
39.9 ±2.99**
(+25%)
41.8 ± 2.81**
(+31%)
43.2 ±2.61**
(+36%)
Female Mice
Average daily dose
(mg/kg-d)
0
67
134
268
536
1,071
Number of animals
10
10
10
10
8
3°
Absolute liver weight
(mg)
1,129 ±242
1,237 ± 123
(+9.6%)
1,238 ±113
(+9.7%)
1,232 ± 124
(+9.1%)
1,281 ± 126
(+13%)
1,383 ± 96
(+22%)
Relative liver weight
(mg/g)
42.9 ±7.71
48.6 ±5.03
(+13%)
47.9 ±3.61
(+12%)
47.8 ±3.74
(+11%)
49.2 ±4.70
(+15%)
54.7 ±3.45**
(+28%)
"NT IP (1988).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
Statistically significant mortality (7/10).
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
**Statistically significantly different from control atp< 0.01, as reported by the study authors (Dunnett's test).
MBT = 2-mercaptobenzothiazole.
Potential points of departure (PODs) from NTP (1988) were modeled using the EPA's
Benchmark Dose Software (BMDS, Version 2.6) (see Table 8). The results are summarized in
Table 9. A benchmark response (BMR) of 10% relative deviation (RD) from the control mean
was used. Appendix C contains details of the modeling. No model fit was achieved with the
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data on absolute and relative liver weight in male rats, absolute liver weight in female rats, or
absolute and relative liver weight in female mice due to the nonlinearity of the dose response.
Modeling of the data on relative liver weight in female rats yielded a 10% benchmark dose lower
confidence limit (BMDLio) estimate of 14.82 mg/kg-day.
Table 9. Potential Subchronic PODs in F344 Rats and B6C3Fi Mice Exposed by Gavage to
MBT for 13 Weeks3
NOAEL
LOAEL
Animal PODb
POD (HED)°
Endpoint
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
Male Rats
Absolute liver weight
ND
134
LOAEL = 134
LOAEL (HED) = 32.2
Relative liver weight
ND
134
LOAEL = 134
LOAEL (HED) = 32.2
Female Rats
Absolute liver weight
ND
134
LOAEL = 134
LOAEL (HED) = 32.2
Relative liver weightd
ND
134
BMDLio = 14.8
BMDLio (HED) = 3.56
Female Mice
Absolute liver weight
268
536
NOAEL = 268
NOAEL (HED) = 37.5
Relative liver weight
ND
67
LOAEL = 67
LOAEL (HED) = 9.4
aNTP (1988).
bBMD modeling results are described in detail in Appendix C.
cPOD (HED) = Animal POD (mg/kg-day) x DAF of 0.24 for rats or 0.14 for mice (U.S. EPA. 2011b).
dChosen as the critical effect for derivation of the subchronic p-RfD.
BMD = benchmark dose; BMDLio = 10% benchmark dose lower confidence limit; DAF = dosimetric adjustment
factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
MBT = 2-mercaptobenzothiazole; ND = no data; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose.
In Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA, 2011b), the Agency endorses a hierarchy of approaches to derive
human equivalent oral exposures from data from laboratory animal species, with the preferred
approach being physiologically based toxicokinetic modeling. Other approaches may include
using some chemical-specific information, without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, EPA endorses BW3'4 as a default to extrapolate toxicologically
equivalent doses of orally administered agents from all laboratory animals to humans for the
purpose of deriving an RfD under certain exposure conditions. More specifically, the use of
BW3/4 scaling for deriving an RfD is recommended when the observed effects are associated
with the parent compound or a stable metabolite, but not for portal-of-entry effects.
A validated human physiologically based pharmacokinetic (PBPK) model for MBT is not
available for use in extrapolating doses from animals to humans. In addition, liver-weight
changes are not a portal-of-entry effect. Therefore, scaling by BW3/4 is relevant for deriving
HED for this effect.
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Following U.S. EPA (2011b) guidance, all potential PODs in rats and mice are converted
to a HED through the application of a DAF7 derived as follows:
DAF = (BWa1/4 - BWh1/4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a reference BWa of 0.25 kg for rats and 0.025 kg for mice, and a reference BWh of
70 kg for humans (U.S. EPA. 1988), the resulting DAFs are 0.24 and 0.14 for rats and mice,
respectively. Each POD candidate is multiplied by the appropriate species-specific DAF to
obtain POD (HED) (see Table 9).
Absolute and relative liver-weight changes in male and female rats were biologically
significant at the lowest dose tested (i.e., LOAEL = 134 mg/kg-day); thus, no NOAEL was
identified in these animals. Relative liver-weight changes in female mice were also observed at
the lowest dose tested (LOAEL = 67 mg/kg-day); however, increases in absolute liver weight in
these animals were not biologically significant until higher dose levels
(LOAEL = 536 mg/kg-day). This multiple dose level difference in the LOAEL between absolute
and relative liver-weight changes in female mice lends uncertainty to the biological significance
of the relative-weight change in female mice. The lowest POD (HED) following subchronic
exposure to MBT is increased relative liver weight in female rats
(BMDLio [HED] = 3.56 mg/kg-day), which was the only potential POD that resulted in a
suitable BMDS model fit. This POD (HED) allows for a better representation of the full range of
the dose-response for liver-weight changes in rats compared to the other potential PODs.
Therefore, it is concluded that the BMDLio (HED) for relative liver weight in female rats is
protective of other effects observed following MBT exposure. Based on the consistency and
coherence in effects on the liver across species and sexes and biological significance of the
changes, the BMDLio (HED) for relative liver-weight increases in female rats
(3.56 mg/kg-day) is selected as the POD for derivation of the subchronic p-RfD.
Subchronic p-RfD = BMDLio (HED) UFc
= 3.56 mg/kg-day -M00
= 4 x 10"2 mg/kg-day
The composite uncertainty factor (UFc) for the subchronic p-RfD for MBT is 100, as
summarized in Table 10.
7 As described in detail in Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 201 lb), rate-related processes scale across species in a manner related to both the direct
(BWm) and allometric scaling (BW3/4) aspects such that BW3'4 ^ BW1 1 = BW ' converted to a
DAF = BWa"4 - BWi,1'4.
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Table 10. Uncertainty Factors for the Subchronic p-RfD for MBT
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following MBT exposure. The toxicokinetic
uncertainty has been accounted for by calculating a HED through application of a DAF as outlined
in the EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of the
Oral Reference Dose (U.S. EPA. 2011b).
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of MBT in humans.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database. The available
subchronic- and chronic-duration toxicity studies for MBT are not comprehensive. However, the
database includes a two-seneration reproductive toxicity study in rats (Snrineborn Laboratories.
1990b). developmental toxicity studies in rats and rabbits (Sprineborn Laboratories. 1989d. el and
subchronic-duration and acute neurotoxicity studies in rats (Bio-Research Laboratories LTD.
1990. 1989s). all via the oral route.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because the POD is from a subchronic-duration study.
UFC
100
Composite UF = UFA x UFH x UFD x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent
dose; MBT = 2-mercaptobenzothiazole; POD = point of departure.
Confidence in the subchronic p-RfD for MBT is medium as explained in Table 11.
Table 11. Confidence Descriptors for the Subchronic p-RfD for MBT
Confidence Categories
Designation
Discussion
Confidence in principal
study
M
Confidence in the t>rincit>al study is medium. The study bv NTP (1988) is
a well-conducted, peer-reviewed, GLP-compliant study of oral exposure to
MBT; however, hematology, clinical chemistry, urinalysis, and
organ-weight measurements for organs other than liver were not assessed.
Confidence in database
M
Confidence in the subchronic database is medium. The database includes
subchronic-duration studies in rats and mice, a two-generation
reproductive toxicity study in rats, developmental toxicity studies in rats
and rabbits, and subchronic and acute neurotoxicity studies in rats.
However, the available subchronic-duration studies were not
comprehensive and lacked hematology, clinical chemistry, urinalysis, and
organ-weight measurements for organs other than liver.
Confidence in
subchronic p-RfD:i
M
The overall confidence in the subchronic p-RfD for MBT is medium.
aThe overall confidence cannot be greater than the lowest entry in a table.
GLP = Good Laboratory Practice; M = medium; MBT = 2-mercaptobenzothiazole; p-RfD = provisional reference
dose.
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Derivation of a Chronic Provisional Reference Dose (p-RfD)
After evaluation of the available oral exposure studies, it was determined that the same
critical effect and principal study as the subchronic p-RfD was adequate for deriving a chronic
p-RfD. The chronic-duration studies of MBT administration conducted by NTP (1988) cannot
be used to derive noncancer toxicity values because effect levels could not be determined for
these studies due to the occurrence of tumors at the tested doses (see study summary for
additional details). The chronic-duration study by NTP (1988) did report increased incidence of
forestomach lesions in male and female rats at 268 mg/kg-day. Males had increased ulcers,
inflammation, epithelial hyperplasia, and hyperkeratosis, and females had increased ulcers.
However, no changes in the forestomach were observed in the sub chronic-duration study, and
liver weight was not evaluated in the chronic-duration study. Therefore, there is uncertainty
whether a POD based on the lesions in the forestomach would be protective against the
potentially more sensitive changes in the liver. In addition, a greater body of evidence supports
the changes in the liver compared to the forestomach as discussed in the "Derivation of a
Subchronic Provisional Reference Dose" section. Thus, it is concluded that the BMDLio (HED)
for relative liver weight in female rats is protective of other effects observed following MBT
exposure. Based on the consistency and coherence in effects on the liver across species and
sexes and biological significance of the changes, the BMDLio (HED) for relative liver-weight
increases in female rats (3.56 mg/kg-day) is selected as the POD for derivation of the
chronic p-RfD.
The chronic p-RfD was derived as follows:
Chronic p-RfD = BMDLio (HED) UFc
= 3.56 mg/kg-day ^ 1,000
= 4 x 10"3 mg/kg-day
Table 12 summarizes the UFc for the chronic p-RfD for MBT.
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Table 12. Uncertainty Factors for the Chronic p-RfD for MBT
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic
or toxicodynamic differences between rats and humans following MBT exposure. The
toxicokinetic uncertainty has been accounted for by calculating a HED through
application of a DAF as outlined in the EPA's Recommended Use of Body Weight4 as
the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011b).
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human
variability in susceptibility in the absence of quantitative information to assess the
toxicokinetics and toxicodynamics of MBT in humans.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database. The
available subchronic- and chronic-duration toxicity studies for MBT are not
comprehensive. However, the database includes a two-generation reproductive toxicity
studv in rats (Snrineborn Laboratories. 1990b). developmental toxicity studies in rats and
rabbits (Soriiigbom Laboratories. 1989d. e). and subchronic-duration and acute
neurotoxicity studies in rats (Bio-Research Laboratories LTD. 1990. 1989). all via the
oral route. However, only the NTP (1988) studies were Deer reviewed: the remaining
studies were unpublished and not peer reviewed.
UFl
1
A UFl of 1 is applied because POD is a BMDL.
UFS
10
A UFS of 10 is applied to account for the extrapolation from less than chronic exposure.
UFC
1,000
Composite UF = UFA x UFH x UFD x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent
dose; MBT = 2-mercaptobenzothiazole; POD = point of departure.
Confidence in the chronic p-RfD for MBT is medium as explained in Table 13.
Table 13. Confidence Descriptors for the Chronic p-RfD for MBT
Confidence Categories
Designation
Discussion
Confidence in principal
study
M
Confidence in the t>rincit>al study is medium. The study bv NTP (1988) is
a well-conducted, peer-reviewed, GLP-compliant study of oral exposure to
MBT; however, hematology, clinical chemistry, urinalysis, and
organ-weight measurements for organs other than liver were not assessed.
Confidence in database
M
Confidence in the chronic database is medium. The database includes
chronic-duration studies in rats and mice, a two-generation reproductive
toxicity study in rats, developmental toxicity studies in rats and rabbits,
subchronic-duration studies in rats and mice, and acute neurotoxicity
studies in rats. However, the available chronic-duration studies were not
comprehensive and lacked hematology, clinical chemistry, urinalysis, and
organ-weight measurements.
Confidence in chronic
p-RfDa
M
The overall confidence in the chronic p-RfD for MBT is medium.
aThe overall confidence cannot be greater than the lowest entry in a table.
GLP = Good Laboratory Practice; M = medium; MBT = 2-mercaptobenzothiazole; p-RfD = provisional reference
dose.
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DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies identifying non-neoplastic effects of MBT in humans or animals exposed by
inhalation have been identified in the literature reviewed, precluding derivation of RfCs.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Tables 14 and 15 provide the cancer weight-of-evidence (WOE) descriptors for oral and
inhalation exposure to MBT, respectively.
Table 14. Cancer WOE Descriptor for Oral Exposure to MBT
Possible WOE
Descriptor
Designation
Route of Entry
(oral, inhalation,
or both)
Comments
"Carcinogenic to
Humans "
NS
NA
The available data do not support this.
"Likely to Be
Carcinogenic to Humans"
Selected
Oral
NTP (1988) conducted carcinogenicity studies in
rats and mice and concluded that there was some
evidence of carcinogenicity in male rats based on
statistically significant increases in the incidences
of mononuclear cell leukemia, pancreatic acinar
cell adenomas, adrenal gland
pheochromocytomas, and preputial gland
adenomas or carcinomas. NTP indicated that
there was some evidence of carcinogenicity in
female rats based on statistically significant
increases in the incidences of adrenal gland
pheochromocytomas and pituitary gland
adenomas. For mice, NTP concluded that there
was no evidence of carcinogenicity in male mice
and equivocal evidence of carcinogenicity in
female mice based on increased incidences of
hepatocellular adenomas or carcinomas.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
The available data do not support this, as multiple
tumor types were observed in 2 sexes and species of
animals (male and female rats and female mice)
CNTP. 1988s).
"Inadequate Information
to Assess Carcinogenic
Potential"
NS
NA
The available data do not support this. Data are
available demonstrating increased tumor incidence
in animals (NTP. 1988).
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
The available data do not support this.
MBT = 2-mercaptobenzothiazole; NA = not applicable, NS = not selected; NTP = National Toxicology Program;
WOE = weight of evidence.
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Table 15. Cancer Weight-of-Evidence Descriptor for Inhalation of MBT
Possible WOE
Descriptor
Designation
Route of Entry
(oral, inhalation,
or both)
Comments
"Carcinogenic to
Humans "
NS
NA
The available data do not support this. The available
epidemiological studies suffer from a number of
limitations that preclude robust conclusions
regarding carcinogenic potential (see text for detail).
There are no carcinogenicity studies in animals
exposed by inhalation.
"Likely to Be
Carcinogenic to Humans "
NS
NA
The available data do not support this. The available
epidemiological studies suffer from a number of
limitations that preclude robust conclusions
regarding carcinogenic potential (see text for detail).
There are no carcinogenicity studies in animals
exposed by inhalation.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
The available data do not support this. The available
epidemiological studies suffer from a number of
limitations that preclude robust conclusions
regarding carcinogenic potential (see text for detail).
There are no carcinogenicity studies in animals
exposed by inhalation.
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Inhalation
Several epidemiological studies investigated
cancer morbidity and mortality in 2 cohorts of
rubber factory workers with MBT inhalation
exDOSure (Sorahan. 2009. 2008; Sorahan et al..
2000; Collins et al.. 1999; Sorahan and Pone.
1993; Strauss et al.. 1993). These studies
suggested potential associations between MBT
and bladder cancer, colon cancer, and multiple
myeloma. However, the studies of these cohorts
suffer from a number of limitations that preclude
robust conclusions regarding carcinogenic
potential (see text for detail). There are no
carcinogenicity studies in animals exposed by
inhalation.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
The available data do not support this.
MBT = 2-mercaptobenzothiazole; NA = not applicable, NS = not selected; WOE = weight of evidence.
In accordance with U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment, oral
exposure to MBT is "Likely to be Carcinogenic to Humans. " There are no human data on the
potential carcinogenicity of MBT by oral exposure. The carcinogenicity of MBT has been tested
in three chronic-duration studies of rats and mice exposed orally (NTP. 1988; Innes et ai, 1969).
Chronic exposure (103 weeks) to MBT via gavage resulted in statistically significantly increased
incidences of tumors in male and female rats and female mice, including statistically significant
increases in the incidences of mononuclear cell leukemia, pituitary gland adenomas, pancreatic
acinar cell adenomas, mesothelioma, adrenal gland pheochromocytomas, fibroma, neurofibroma,
sarcoma, or fibrosarcomas, and preputial gland adenomas or carcinomas in male rats (NTP.
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1988). Statistically significant increases in the incidences of adrenal gland pheochromocytomas
and pituitary gland adenomas occurred in female rats, and increased incidences of hepatocellular
adenomas or carcinomas occurred in female mice (N I P. 1988). In the rat study, NTP (1988)
concluded that there was some evidence of carcinogenicity in males based on statistically
significant increases in the incidences of mononuclear cell leukemia, pancreatic acinar cell
adenomas, adrenal gland pheochromocytomas, and preputial gland adenomas or carcinomas.
NTP (1988) also concluded that there was some evidence of carcinogenicity in females based on
statistically significant increases in the incidences of adrenal gland pheochromocytomas and
pituitary gland adenomas. In the mouse study, NTP concluded that there was no evidence of
carcinogenicity in male mice and equivocal evidence of carcinogenicity in female mice based on
increased incidences of hepatocellular adenomas or carcinomas. Increased incidence of tumors
was not reported in mice exposed to MBT or zinc MBT for 18 months (Innes et ai, 1969);
however, quantitative results were not presented.
As stated in the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005),
supporting data to conclude that a chemical is "Likely to Be Carcinogenic to Humans " includes
"an agent that has tested positive in animal experiments in more than one species, sex, strain,
site, or exposure route, with or without evidence of carcinogenicity in humans." Based on this
example and the data available in male and female rats and mice, exposure to MBT is "Likely to
Be Carcinogenic to Humans. "
In accordance with U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment, the
database for inhalation exposure to MBT provides "Inadequate Information to Assess
Carcinogenic Potential. " No chronic-duration animal inhalation studies of MBT were
identified. Several epidemiological studies investigated cancer morbidity and mortality in two
cohorts of rubber factory workers with MBT inhalation exposure (Sorahan. 2009, 2008; Sorahan
et ai, 2000; Collins et ai, 1999; Sorahan and Pope. 1993; Strauss et ai, 1993). These studies
suggested potential associations between MBT and bladder cancer, colon cancer, and multiple
myeloma. As discussed earlier, the studies of these cohorts suffer from a number of limitations.
They include:
• The numbers of workers with likely MBT exposure in both cohorts were small
(<600 workers each), and the numbers of cancers observed were also small.
• Both worker cohorts had potential exposure to MBT, its derivatives, and other chemicals,
including the known or suspected bladder carcinogens, PAB and PBN.
• MBT exposure assessments for both cohorts were based on job-exposure matrices, using
limited exposure monitoring information.
• Data on tobacco use were not available for either cohort, raising the possibility of
confounding.
• Follow-up studies of the same cohort and studies of the different cohorts did not provide
consistent findings, possibly because the numbers of cases and the sizes of the cohorts
were too small to yield stable results.
MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005) define mode of
action (MO A) ".. .as a sequence of key events and processes, starting with interaction of an agent
with a cell, proceeding through operational and anatomical changes, and resulting in cancer
formation." Examples of possible modes of carcinogenic action for any given chemical include
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"mutagenicity, mitogenesis, programmed cell death, cytotoxicity with reparative cell
proliferation, and immune suppression" (pp. 1-10).
The MOA for carcinogenic effects of MBT is not known. In general, available data
indicate that MBT is not mutagenic. Limited in vitro data suggest that MBT may cause
clastogenic effects in mammalian cells; however, findings are inconsistent between studies and
cell types, and in vivo data were negative (see "Genotoxicity" section for more details). No
other information suggesting potential MOA(s) for MBT carcinogenicity is available. Thus, a
linear approach to derivation of provisional cancer potency values is applied as recommended by
the Guidelines for Carcinogenic Risk Assessment (U.S. EPA. 2005).
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of a Provisional Oral Slope Factor (p-OSF)
The 2-year carcinogenicity studies in rats and mice conducted by NTP (1988) were
selected as the principal studies for the development of a p-OSF. This study was conducted in
accordance with GLP principles, the results are peer reviewed, and the study meets the standards
of study design and performance with respect to the number of animals used, the examination of
potential toxicity endpoints, and the presentation of information.
Statistically significant increases in the incidences of mononuclear cell leukemia,
pituitary gland adenomas, pancreatic acinar cell adenomas, mesothelioma, adrenal gland
pheochromocytomas, fibroma, neurofibroma, sarcoma, or fibrosarcomas and preputial gland
adenomas or carcinomas in male rats; significant increases in the incidences of adrenal gland
pheochromocytomas and pituitary gland adenomas in female rats; and increased incidences of
hepatocellular adenomas or carcinomas in female mice were reported (NTP. 1988). Several
tumor types observed at statistically significantly increased incidence were not considered for
deriving the p-OSF. NTP (1988) noted that a number of tumor types observed in male rats did
not exhibit monotonic dose-response relationships. As shown in Table B-4, the incidences of
mononuclear cell leukemia, pituitary gland adenomas, and pancreatic acinar cell adenomas in
male rats suggested inverted U-shaped dose-response relationships (incidences declined from the
low to the high dose, often to incidences at or below the control values). Survival of male rats
was statistically significantly reduced by treatment; however, NTP (1988) indicated that the lack
of a monotonic dose-response relationship was not likely to be attributable to increased
mortality, because comparable numbers of male rats in the low- and high-dose groups were at
risk at the end of the study. In addition, significant dose-related trends were not seen in life table
or incidental tumor tests (accounting for mortality) for mononuclear cell leukemia or pituitary
gland adenomas in male rats. While pancreatic acinar cell adenomas exhibited a statistically
significant trend by life table test, adjusted incidence rates (4.5, 45.7, and 23% in control,
low-dose, and high-dose groups) reported by NTP (1988) also did not exhibit a dose-response
relationship, indicating that adjustment for mortality would not yield a monotonic dose-response
relationship. Therefore, based on the lack of dose-response relationship, these tumor incidence
data were not used for BMD modeling. Similarly, while low-dose female mice exhibited
statistically significantly increased incidence of liver tumors, the dose-response was not
monotonic and there was no significant dose-related trend by either life table or incidental tumor
test (NTP, 1988); thus, this tumor type was not used. The remaining data for tumors occurring in
male and female rats were selected for BMD modeling and are provided in Table 16.
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Table 16. Incidences of Selected Neoplastic Lesions in F344/N Rats Exposed to MBT by
Gavage for 103 Weeks"
Male Rats
HED, mg/kg-d
0
64.3
129
Mesothelioma
0/50t
2/50 (4%)
3/50 (6%)
Adrenal gland
Pheochromocytoma
18/50+ (36%)
25/50* (50%)
22/49* (45%)
Pheochromocytoma or malignant pheochromocytoma
18/50+ (36%)
27/501*# (54%)
24/491*# (49%)
Preputial gland
Adenoma
0/50+
4/50* (8%)
4/50* (8%)
Adenoma or carcinoma
1/50+ (2%)
6/50* (12%)
5/50* (10%)
Subcutaneous tissue
Fibroma
2/50+ (4%)
3/50 (6%)
6/50* (12%)
Fibroma, neurofibroma, sarcoma, or fibrosarcoma
3/50+ (6%)
6/50 (12%)
7/50* (14%)
Female Rats
HED, mg/kg-d
0
32.2
64.3
Pituitary gland
Adenoma
15/49+ (31%)
24/50 (48%)
25/501*# (50%)
Adenoma or adenocarcinoma
16/49+ (33%)
24/50 (48%)
25/501*# (50%)
Adrenal gland, pheochromocytoma
1/50+ (2%)
5/50 (10%)
6/50* (12%)
•'N' T'P ( 1988).
* Statistically significantly different from concurrent control at p< 0.05 based on life table test performed by the
study authors.
"Statistically significantly different from concurrent control at p< 0.05 based on incidental tumor test performed by
the study authors.
Statistically significant (p < 0.05) dose-related trend by life table or incidental tumor analysis or both.
HED = human equivalent dose; MBT = 2-mercaptobenzothiazole.
The incidences of the various tumor types were modeled using BMDS, and the modeling
results are presented in Table 17. Based on the BMD modeling results, the calculated cancer
slope factors for the various tumor types in male and female rats were calculated and also
presented in Table 17. Because treatment with MBT produced multiple types of tumors in
different tissues within a single study (i.e., within a single sex and species), the overall oral
cancer slope factor for MBT exposure was derived based on the incidence data for combined
tumors assuming that different tumor types are independent from each other. The overall tumor
incidence was fit with the MSCombo multiple tumor model (BMDS, Version 2.6;
see Appendix C for details), and the estimated BMDio, BMDLio, and calculated cancer slope
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factors are presented in Table 17.8 This modeling provides an estimate of composite risk for
developing any combination of tumors at any site within a single study. Modeling procedures
and results are described in detail in Appendix C.
Prior to modeling, all doses were converted to HEDs using BW3/4 scaling, as described
below.
Dose (HED) = Dose x (BWa/BWh)14
where:
Dose = average daily animal dose of MBT
BWa = reference animal body weight9
BWh = 70 kg, reference human body weight (U.S. EPA, 1988)
Using a BWa of 0.25 kg for rats and a BWh of 70 kg for humans, the resulting default
DAF is 0.24 (U.S. EPA, 2011b. 2005).
8Because no software is currently available to estimate composite risk from multiple tumor types and adjust for
group differences in survival, data for tumor incidence in male rats were also modeled using a Poly-3
survival-adjusted number at risk to account for decreased survival. Modeling the observed incidence and Poly-3
weighted number give a BMDLio (HED) = 10.8, which is higher than the BMDLio (HED) of 8.91 for combined
tumors in female rats.
9Time-weighted body weight was not reported by study authors or calculable from reported study data. Default
animal body weights (0.25 kg for rats and 0.025 kg for mice) were used in calculating HED values.
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Table 17. Comparison of BMD Model Results for Possible Tumor Endpoints for
Derivation of the p-OSFa
Tumor Endpoint
Model Type
Goodness-of-Fit
/>-Valucb
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Potential p-OSF
(mg/kg-d)1
Female Rats
Adrenal gland
pheochromocytoma
Multistage cancer
(1st order)
0.5478
55.0
30.5
3.3 x 10~3
Pituitary gland
adenoma or
adenocarcinoma
Multistage cancer
(1st order)
0.4873
21.0
10.9
9.2 x 10~3
Combined tumors
MSCombo
15.2
8.91
1.1 x 10"2
Male Rats
Mesothelioma
Multistage cancer
(1st order)
1.000
333
147
6.8 x 10-4
Adrenal gland
pheochromocytoma or
malignant
pheochromocytoma
Multistage cancer
(1st order)
0.1991
51.1
23.0
4.3 x lO-3
Preputial gland
adenoma or carcinoma
Multistage cancer
(1st order)
0.2107
118
62.5
1.6 x 10~3
Subcutaneous tissue
fibroma, neurofibroma,
sarcoma, or
fibrosarcoma
Multistage cancer
(1st order)
0.7217
142
63.7
1.6 x 10~3
Combined tumors
MSCombo
24.9
15.0
6.7 x 10-3
aNTP (1988).
bValues >0.05 meet conventional goodness-of-fit criteria.
BMD = benchmark dose; BMDio = 10% benchmark dose; BMDLio = 10% benchmark dose lower confidence limit;
HED = human equivalent dose; p-OSF = provisional oral slope factor.
Among all of the modeled tumor types, the lowest POD
(BMDLio [HED] = 8.91 mg/kg-day) was obtained in modeling of the incidence of combined
tumors in female rats. The MOA by which MBT induces tumors is not known. In the absence
of definitive information, a linear approach is used to obtain the slope from the POD. The
p-OSF of 1.1 x 10"2 (mg/kg-day)"1 was derived as follows:
p-OSF = BMR -h BMDLio (HED)
= 0.1 ^ 8.91 mg/kg-day
= 1.1 x 10"2 (mg/kg-day)"1
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APPENDIX A. SCREENING PROVISIONAL VALUES
No screening values for 2-mercaptobenzothiazole are identified.
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APPENDIX B. DATA TABLES
Table B-l. Absolute and Relative Liver Weights in F344 Rats Exposed by Gavage to MBT
for 13 Weeksa'b
Male Rats
Average daily dose,
mg/kg-d
0
134
268
536
1,071
Number of animals
10
10
9
10
8
Necropsy body weight
(g)
355 ± 19.1
357 ±8.5
(+0.6%)
336 ±23.1
(-5.4%)
342 ±31.5
(-3.7%)
325 ±26.8*
(-8.5%)
Absolute liver weight
(mg)
13,593 ±2,121
15,661 ±793
(+15%)
15,861 ± 1,712
(+17%)
18,742 ±2,631**
(+38%)
16,759 ± 2,660**
(+23%)
Relative liver weight
(mg/g)
38.4 ±6.07
43.9 ± 1.87*
(+14%)
47.2 ±2.79**
(+23%)
54.8 ±5.08**
(+43%)
51.3 ±5.42**
(+34%)
Female Rats
Average daily dose,
mg/kg-d
0
134
268
536
1,071
Number of animals
10
9
10
8
10
Necropsy body weight
(g)
208 ± 15.9
200 ± 15.6
(-3.8%)
201 ± 11.3
(-3.4%)
191 ±8.8*
(-8.2%)
195 ± 12.1
(-6.3%)
Absolute liver weight
(mg)
6,606 ± 795
7,818 ±814**
(+18%)
8,027 ± 688**
(+22%)
7,988 ±591**
(+21%)
8,413 ±652**
(+27%)
Relative liver weight
(mg/g)
31.8 ±3.28
39.3 ± 3.53**
(+24%)
39.9 ±2.99**
(+25%)
41.8 ± 2.81**
(+31%)
43.2 ±2.61**
(+36%)
•'N'TP (1988).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
**Statistically significantly different from control atp< 0.01, as reported by the study authors (Dunnett's test).
MBT = 2-mercaptobenzothiazole.
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Table B-2. Mortality and Liver Weights in B6C3Fi Mice Exposed by Gavage to MBT for
13 Weeksa'b
Male Rats
Average daily dose,
mg/kg-d
0
67
134
268
536
1,071
Mortality
0/10
0/10
0/10
0/10
0/10
5/10* (50%)
Necropsy body weight (g)
36.7 ±2.8
37.0 ±2.6
(+0.8%)
37.7 + 3.1
(+2.7%)
35.1 + 3.4
(-4.4%)
34.4 + 2.0
(-6.3%)
35.2 + 2.8
(-4.1%)
Absolute liver weight (mg)
1,821 ±213
1,942 ± 208
(+7%)
2,034+ 184
(+12%)
1,855 + 231
(+2%)
1,809+ 115
(-1%)
2,090 + 184
(+15%)
Relative liver weight
(mg/g)
49.6 ±4.34
52.5 ± 4.02
(+5.8%)
54.0 + 3.23*
(+8.9%)
52.8 + 3.51
(+6.5%)
52.6 + 3.15
(+6.0%)
59.5 + 3.94**
(+20%)
Female Rats
Average daily dose,
mg/kg-d
0
67
134
268
536
1,071
Mortality
0/10
0/10
0/10
0/10
2/10 (20%)
7/10* (70%)
Necropsy body weight (g)
26.2 ± 1.3
25.5±1.3
(-2.7%)
25.9+ 1.7
(-1.1%)
25.8+ 1.3
(-1.5%)
26.1 + 1.3
(-0.4%)
25.3 + 0.3
(-3.4%)
Absolute liver weight (mg)
1,129 ±242
1,237 ± 123
(+9.6%)
1,238+ 113
(+9.7%)
1,232+ 124
(+9.1%)
1,281 + 126
(+13%)
1,383 + 96
(+22%)
Relative liver weight
(mg/g)
42.9 ±7.71
48.6 + 5.03
(+13%)
47.9 + 3.61
(+12%)
47.8 + 3.74
(+11%)
49.2 + 4.70
(+15%)
54.7 + 3.45**
(+28%)
•'N' T'P ( 1988).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
**Statistically significantly different from control atp< 0.01, as reported by the study authors (Dunnett's test).
MBT = 2-mercaptobenzothiazole.
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Table B-3. Nonaccidental Deaths and Incidences of Selected Non-neoplastic Lesions in
F344/N Rats Exposed to MBT by Gavage for 103 Weeks3
Male Rats
Average daily dose, mg/kg-d
0
134
268
536
Nonaccidental deaths'3
8/50 (16%)
NA
28/50** (56%)
29/49** (59%)
Forestomach
Ulcer
0/50
NA
5/50* (10%)
5/49* (10%)
Inflammation
0/50
NA
11/50* (22%)
14/49* (29%)
Epithelial hyperplasia
1/50 (2%)
NA
12/50* (24%)
17/49* (35%)
Hyperkeratosis
0/50
NA
12/50* (24%)
17/49* (35%)
Kidney
Nephropathy
50/50 (100%)
NA
50/50 (100%)
50/50 (100%)
Nephropathy severity (mean)
2.3
NA
3.4
3.4
Pelvis epithelial hyperplasia
0/50
NA
4/50 (8%)
1/49 (2%)
Tubule focal hyperplasia
0/50
NA
3/50 (6%)
3/49 (6%)
Female Rats
Average daily dose, mg/kg-d
0
134
268
536
Nonaccidental deaths'3
21/49 (43%)
18/49 (37%)
25/50 (50%)
NA
Forestomach
Ulcer
0/49
3/50 (6%)
5/50* (10%)
NA
Inflammation
2/49 (4%)
4/50 (8%)
7/50 (14%)
NA
Epithelial hyperplasia
1/49 (2%)
4/50 (8%)
1/50 (2%)
NA
Hyperkeratosis
1/49 (2%)
4/50 (8%)
1/50 (2%)
NA
Kidney
Nephropathy (severity not reported)
38/49 (76%)
42/50 (84%)
41/50 (82%)
NA
aNTP (1988).
bExcludes animals accidentally killed.
* Significantly different from control at p< 0.05 based on Fisher's exact test performed for this review.
**Significantly different from control atp< 0.01 based on life table test performed by the study authors.
MBT = 2-mercaptobenzothiazole; NA = not available.
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Table B-4. Incidences of Selected Neoplastic Lesions in F344/N Rats Exposed to MBT by
Gavage for 103 Weeks"
Male Rats
HED, mg/kg-d
0
64.3
129
Historical control incidence in
NTP studies (corn oil gavage)
Mononuclear cell leukemia
7/50 (14%)
16/50* (32%)
3/50 (6%)
202/1,450 (14%)
Range: 1/50-14/50
Pituitary gland adenoma
14/50
(28%)
21/50* (42%)
12/48 (24%)
344/1,411 (8%)
Range: 5/50-19/50
Pancreatic acinar cell adenoma
2/50+ (4%)
13/50*#
(26%)
6/49* (12%)
80/1,381 (8%)
Range: 0/50-14/50
Mesothelioma
0/50+
2/50 (4%)
3/50 (6%)
55/1,450 (4%)
Range: 0/50-6/50
Adrenal gland
Pheochromocytoma
18/50+
(36%)
25/50* (50%)
22/49* (45%)
338/1,442 (23%)
Range: 2/50-20/49
Pheochromocytoma or malignant
pheochromocytoma
18/50+
(36%)
27/50*#
(54%)
24/49*#
(49%)
347/1,442 (24%)
Range: 2/50-20/49
Preputial gland
Adenoma
0/50+
4/50* (8%)
4/50* (8%)
30/1,450 (2%)
Range: 0/50-7/50
Adenoma or carcinoma
1/50+ (2%)
6/50* (12%)
5/50* (10%)
65/1,450 (4.5%)
Range: 0/50-9/50
Subcutaneous tissue
Fibroma
2/50+ (4%)
3/50 (6%)
6/50* (12%)
93/1,450 (6.4%)
Range: 0/50-6/50
Fibroma, neurofibroma, sarcoma,
or fibrosarcoma
3/50+ (6%)
6/50 (12%)
7/50* (14%)
126/1,450 (8.7%)
Range: 1/50-8/50
Kidney
Transitional cell papilloma
0/50
1/50 (2%)
1/49 (2%)
1/1,488 (0.07%)
Range: NR
Transitional cell carcinoma
0/50
1/50 (2%)
0/49
NR
Tubular cell adenoma
0/50
1/50 (2%)
1/50 (2%)
3/1,488 (0.2%)
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Table B-4. Incidences of Selected Neoplastic Lesions in F344/N Rats Exposed to MBT by
Gavage for 103 Weeks"
Female Rats
HED, mg/kg-d
0
32.2
64.3
Historical control incidence in
NTP studies (corn oil gavage)
Pituitary gland
Adenoma
15/49*
(31%)
24/50 (48%)
25/50*#
(50%)
520/1,407 (37%)
Range: 9/50-27/49
Adenoma or adenocarcinoma
16/49*
(33%)
24/50 (48%)
25/50*#
(50%)
561/1,407 (39.9%)
Range: 11/50-30/49
Adrenal gland, pheochromocytoma
1/50+ (2%)
5/50 (10%)
6/50* (12%)
82/1,443 (5.7%)
Range: 0/50-7/50
"NTP (1988).
* Significantly dilferent from concurrent control at p< 0.05 based on life table test performed by the study authors.
"Significantly different from concurrent control at p< 0.05 based on incidental tumor test performed by the study
authors.
Significant (p < 0.05) dose-related trend by life table or incidental tumor analysis or both.
HED = human equivalent dose; MBT = 2-mercaptobenzothiazole; NR = not reported; NTP = National Toxicology
Program.
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Table B-5. Incidence of Nonaccidental Deaths Prior to Termination and Neoplastic
Lesions in B6C3Fi Mice Exposed by Gavage to MBT for 103 Weeksa'b
Male Mice
Average daily dose, mg/kg-d (HED)
0
268
(HED = 37.5)
536
(HED = 75.0)
Mortality
11/50 (22%)
17/50 (34%)
14/44 (32%)
Hepatocellular adenoma or carcinoma (combined)
16/49 (33%)
21/50 (42%)
14/50 (28%)
Female Mice
Average daily dose, mg/kg-d (HED)
0
268
(HED = 37.5)
536
(HED = 75.0)
Mortality
13/50° (26%)
10/49 (22%)
24/46** (52%)
Hepatocellular adenoma or carcinoma (combined)
4/50 (8%)
12/49* (24%)
4/50 (8%)
"NTP (1988).
bExcludes animals accidentally killed.
°Excludes two female vehicle control mice that died during termination period.
* Significantly different from control at p< 0.05 based on life table test performed by the study authors.
**Significantly different from control atp< 0.01 based on life table test performed by the study authors.
HED = human equivalent dose; MBT = 2-mercaptobenzothiazole.
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Table B-6. Absolute and Relative Kidney and Liver Weights in F0 and F1 S-D Parents
Exposed to MBT in the Diet"
Male Rats
Parameter
Exposure group, mg/kg-d (n = 28)
0
172.1
602.3
1,033
F0 parents
Terminal body weight (Wk 20) (g)
577 ±52.1
561 ±45.8
(-3%)
540 ±48.4*
(-6%)
530 ±60.3*
(-8%)
Absolute kidney weight (g)
4.28 ± 0.448b
4.39 ±0.442
(+3%)
4.69 ±0.470
(+10%)
4.70 ±0.549
(+10%)
Relative kidney weight (g/100 g)
0.743 ±0.0681
0.783 ± 0.0573
(+5%)
0.870 ±0.0689*
(+17%)
0.891 ±0.0819*
(+20%)
Absolute liver weight (g)
22.85 ±2.914
22.22 ± 2.734
(-3%)
23.91 ±2.609
(+5%)
24.56 ±3.249
(+7%)
Relative liver weight (g/100 g)
3.956 ±0.3412
3.955 ±0.3288
(0%)
4.429 ±0.3002*
(+12%)
4.636 ± 0.3355*
(+17%)
F1 parents
Terminal body weight (Wk 38) (g)
627 ± 59.7
609 ± 52.5
(-3%)
584 ±54.9*
(-7%)
572 ± 70.0*
(-9%)
Absolute kidney weight (g)
4.55 ± 0.511
4.67 ±0.483
(+3%)
4.92 ± 0.667
(+8%)
4.98 ±0.594*
(+9%)
Relative kidney weight (g/100 g)
0.720 ± 0.0707
0.761 ±0.0782
(+6%)
0.831 ±0.0682*
(+15%)
0.862 ± 0.0566*
(+20%)
Absolute liver weight (g)
22.77 ±3.027
24.77 ±3.394
(+9%)
26.33 ± 3.365*
(+16%)
27.74 ±4.391*
(+22%)
Relative liver weight (g/100 g)
3.591 ±0.3104
4.018 ±0.4010*
(+12%)
4.451 ± 0.3339*
(+24%)
4.783 ±0.3447*
(+33%)
Female Rats
Parameter
Exposure group, mg/kg-d (n = 28)
0
199.7
699.0
1,198
F0 parents
Terminal body weight (Wk 20) (g)
354 ±32.3
347 ±32.4
(-2%)
336 ±21.8*
(-5%)
329 ±22.6*
(-7%)
Absolute kidney weight (g)
2.87 ±0.301
2.97 ±0.296
(+3%)
3.00 ±0.319
(+5%)
3.07 ±0.299
(+7%)
Relative kidney weight (g/100 g)
0.812 ±0.0559
0.860 ±0.0753
(+6%)
0.892 ±0.0735*
(+10%)
0.935 ± 0.0862*
(+15%)
Absolute liver weight (g)
15.84 ± 1.989
15.77 ± 1.834
(0%)
16.92 ± 1.633
(+7%)
16.96 ±2.144
(+7%)
Relative liver weight (g/100 g)
4.471 ±0.399
4.549 ±0.4051
(+2%)
5.033 ±0.3028*
(+13%)
5.149 ±0.4241*
(+15%)
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Table B-6. Absolute and Relative Kidney and Liver Weights in F0 and F1 S-D Parents
Exposed to MBT in the Diet"
F1 parents
Terminal body weight (Wk 38) (g)
324 ±28.3
312 ±20.3
(-4%)
304 ±28.3*
(-6%)
302 ±21.3*
("7%)
Absolute kidney weight (g)
2.74 ±0.311
2.82 ±0.178
(+3%)
2.87 ±0.310
(+5%)
2.82 ±0.251
(+3%)
Relative kidney weight (g/100 g)
0.837 ±0.0739
0.894 ± 0.0666*
(+7%)
0.936 ±0.0649*
(+12%)
0.942 ±0.0659*
(+13%)
Absolute liver weight (g)
13.31 ± 1.912
13.73 ± 1.953
(+3%)
14.35 ± 1.694
(+8%)
14.80±1.648
(+11%)
Relative liver weight (g/100 g)
4.071 ±0.5640
4.342 ±0.49
(+7%)
4.678 ±0.4617*
(+15%)
4.932 + 0.4149*
(+21%)
"Spriiigbom Laboratories (1990b).
bValues expressed as mean ± standard deviation (% change compared with control); % change
control = [(treatment mean - control mean) + control mean] x 100.
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
MBT = 2-mercaptobenzothiazole; S-D = Sprague-Dawley.
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Table B-7. Incidence of Histopathological Findings in the Kidneys and Liver of F0 and
F1 S-D Parents Exposed to MBT in the Diet"
Male Rats
Exposure Group, mg/kg-d
Parameter
0
172.1
602.3
1,033
F0 parents
a2u-g inclusions, tubules
4/28 (14%)
10/28* (36%)
8/28 (29%)
13/28* (46%)
Kidney, basophilic tubules, cortex
2/28 (7%)
9/28* (32%)
10/28* (36%)
13/28* (46%)
Kidney, pigment, brown
0/28
0/28
12/28* (43%)
17/28* (61%)
F1 parents
a2u-g inclusions, tubules
4/28 (14%)
13/28* (46%)
10/28* (36%)
14/28* (50%)
Kidney, basophilic tubules, cortex
0/28
3/28(11%)
4/28 (14%)
10/28* (36%)
Kidney, pigment, brown
0/28
0/28
13/28* (46%)
20/28* (71%)
Liver, hepatocyte hypertrophy
0/28
1/28 (4%)
22/28* (79%)
23/28* (82%)
Female Rats
Exposure Group, mg/kg-d
Parameter
0
199.7
699.0
1,198
F0 parents
Kidney, basophilic tubules, cortex
0/28
0/28
0/28
0/28
Kidney, pigment, brown
0/28
0/28
1/28 (4%)
4/28 (14%)
F1 parents
Kidney, basophilic tubules, cortex
1/28 (4%)
0/28
1/28 (4%)
2/28 (7%)
Kidney, pigment, brown
0/28
0/28
0/28
6/28* (21%)
Liver, hepatocyte hypertrophy
0/28
0/28
5/28* (18%)
10/28* (36%)
aSpriiigbom Laboratories (1990b).
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
a2u-g = alpha 2u-globulin; MBT = 2-mercaptobenzothiazole; S-D = Sprague-Dawley.
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Table B-8. Body Weights in F1 and F2 S-D Pups Exposed to MBT via Lactation
(PNDs 1-21) and in the Diet (PNDs 15-21)a
Parameter
Maternal Dose, mg/kg-d (n = 23 or 27)
0
199.7
699.0
1,198
F1 pups
LD 7
16.8 ± 1.93
16.2 ±2.17 (-4%)
15.7 ± 2.50 (-7%)
15.1 ± 1.74 (-10%)
LD 14
34.7 ±2.45
33.4 ±3.06 (-4%)
31.9 ±3.30* (-8%)
29.8 ±3.07* (-14%)
LD 21
56.1 ±3.89
53.0 ±4.87 (-6%)
49.1 ±4.70* (-12%)
44.4 ±4.52* (-21%)
F2 pups
LD 7
16.0 ± 1.75
14.9 ± 1.65 (-7%)
15.3 ± 1.78 (-4%)
14.8 ± 1.55 (-10%)
LD 14
34.4 ±2.59
31.4 ±2.62* (-9%)
31.2 ±2.33* (-9%)
29.3 ±2.12* (-14%)
LD 21
55.6 ±4.18
50.6 ±3.63* (-9%)
49.0 ±4.11* (-12%)
44.2 ±3.01* (-21%)
"Spriiigbom Laboratories (1990b).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) control mean] x 100.
* Statistically significantly different from control at p< 0.01, as reported by the study authors (Dunnett's test).
LD = lactation day; MBT = 2-mercaptobenzothiazole; PND = postnatal day; S-D = Sprague-Dawley.
Table B-9. Occurrence of Selected Clinical Signs in Pregnant Rats Exposed by Gavage to
MBT on GDs 6-15a
Exposure Group, mg/kg-d
Parameter
0
300
1,200
1,800
Daily observations1"
Urine stain
10(8)
0(0)
6(3)
35(11)
Dark material around nose
2(2)
3(3)
1(1)
5(4)
Dark material around mouth
0
0
0
8(5)
Postdose observations'"
Salivation
0
0
38(17)
35 (15)
Urine stain
0
0
7(5)
4(4)
Dark material around mouth
0
1(1)
15 (12)
10 (9)
Activity decreased
0
0
0
7(5)
aSpringbom Laboratories (1989e).
bData reported as total number of occurrences (number of animals with at least one occurrence).
GD = gestation day; MBT = 2-mercaptobenzothiazole.
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Table B-10. Summary of Weight and Food Consumption Data for Pregnant Rats Exposed
by Gavage to MBT on GDs 6-15a
Parameter
Exposure Group, mg/kg-d
0
300
1,200
1,800
Mean body weight
(g)b
DO
281 ± 19.3
279 ± 15.9 (-0.7%)
277 ± 14.9 (-1.4%)
277 ± 13.2 (-1.4%)
D 6
318 ± 21.9
314 ± 18.7 (-1.3%)
315 ± 16.0 (-0.9%)
317 ± 18.0 (-0.3%)
D 9
322 ±21.4
321 ± 16.5 (-0.3%)
322 ± 18.2 (0)
308 ± 18.2* (-4.3%)
D 12
340 ±23.7
342 ± 16.5 (+0.6%)
341 ± 19.7 (+0.3%)
333 ±28.0 (-2.1%)
D 16
367 ±29.4
372 ± 18.7 (+1.4%)
366 ± 22.8 (-0.3)
364 ± 24.7 (-0.8%)
D 20
435 ±40.9
445 ± 22.6 (+2.3%)
433 ±28.3 (-0.5)
429 ±32.7 (-1.4%)
Mean food intake
(g/kg-d)b
D 0-6
83 ±6.5
84 ±5.9 (+1.2%)
85 ± 5.2 (+2.4%)
87 ± 5.6 (+4.8%)
D 6-9
52 ± 5.1
54 ± 5.3 (+3.8%)
52 ± 8.4 (0)
38 ± 10.9** (-27%)
D 9-12
57 ±4.8
61 ± 4.6 (+7.0%)
63 ±5.6* (+11%)
65 ± 15.5** (+14%)
D 12-16
57 ±6.6
59 ±3.7 (+3.5%)
59 ±5.2 (+3.5%)
61 ± 9.6 (+7.0%)
D 16-20
74 ±6.1
72 ± 3.1 (-2.7%)
72 ± 3.9 (-2.7%)
74 ± 5.3 (0)
"Springbom Laboratories (1989e).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean)/control mean] x 100.
* Significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
**Significantly different from control atp< 0.01, as reported by the study authors (Dunnett's test).
GD = gestation day; MBT = 2-mercaptobenzothiazole.
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Table B-ll. Selected Developmental Effects in Rats Exposed by Gavage to MBT on
GDs 6-15a
Parameter
Exposure Group, mg/kg-d
0
300
1,200
1,800
Number of females gravid
24 (92.3%)
23 (88.5%)
25 (96.2%)
22 (84.6%)
Corpora luteab
18.0 ±3.4
17.8 ± 2.2 (-1%)
17.6 ± 2.6 (-2%)
18.8 ±2.1 (+4%)
Implantation sitesb
14.8 ±4.0
16.6 ± 1.4 (+12%)
15.8 ±3.2 (+7%)
16.6 ±3.2 (+12%)
Preimplantation lossb
3.2 ±5.4
1.2 ± 1.7 (-63%)
1.8 ±3.2 (-44%)
2.2 ±3.8 (-31%)
Postimplantation lossbc
0.8 ±0.9
1.3 ±0.9* (+63%)
1.3 ± 1.2 (+63%)
1.7 ± 1.7* (+113%)
Early resorptions'3
0.8 ±0.9
1.3 ±0.9* (+63%)
1.3 ± 1.2 (+63%)
1.6 ± 1.7 (+100%)
Late resorptions'3
0
0
0
0 ± 0.2 (n = 1)
Dead fetuses
0
0
0
0
Viable fetuses'3
14.0 ±3.8
15.3 ± 2.0 (+9%)
14.5 ± 3.2 (+4%)
14.9 ±3.1 (+6%)
Sexb
Male
7.7 ±2.8
7.9 ± 2.3 (+3%)
7.6 ± 2.5 (-1%)
7.1 ±2.7 (-8%)
Female
6.3 ±2.5
7.3 ± 2.4 (+16%)
6.9 ± 2.7 (+10%)
7.8 ±3.0 (+24%)
Fetal weight (g)b
3.7 ±0.5
3.6 ±0.2 (-3%)
3.7 ±0.2 (0)
3.5 ±0.3 (-5%)
aSpriiigbom Laboratories (1989e).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
^Historical control range in the laboratory: 0.6-1.4 per litter; mean: 0.9 per litter.
* Significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
GD = gestation day; MBT = 2-mercaptobenzothiazole.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING OF NONCANCER ENDPOINTS
As discussed in the body of the report in the "Derivation of Oral Reference Doses"
section, the endpoints selected for benchmark dose (BMD) modeling were increased relative and
absolute liver weight in male and female rats and female mice exposed to
2-mercaptobenzothiazole (MBT) for 13 weeks by gavage (N I P. 1988). The incidence data used
in the modeling are shown in Tables 8 and C-l.
Table C-l. Selected Non-neoplastic Endpoints in F344 Rats and B6C3Fi Mice Exposed by
Gavage to MBT for 13 Weeksa'b
Male Rats
Average daily dose,
mg/kg-d
0
134
268
536
1,071
Number of animals
10
10
9
10
8
Absolute liver weight
(mg)
13,593 ±2,121
15,661 ±793
(+15%)
15,861 ± 1,712
(+17%)
18,742 ±2,631**
(+38%)
16,759 ±2,660**
(+23%)
Relative liver weight
(mg/g)
38.4 ±6.07
43.9 ± 1.87*
(+14%)
47.2 ±2.79**
(+23%)
54.8 ±5.08**
(+43%)
51.3 ±5.42**
(+34%)
Female Rats
Average daily dose,
mg/kg-d
0
134
268
536
1,071
Number of animals
10
9
10
8
10
Absolute liver weight
(mg)
6,606 ± 795
7,818 ±814**
(+18%)
8,027 ± 688**
(+22%)
7,988 ±591**
(+21%)
8,413 ±652**
(+27%)
Relative liver weight
(mg/g)
31.8 ±3.28
39.3 ± 3.53**
(+24%)
39.9 ±2.99**
(+25%)
41.8 ±2.81**
(+31%)
43.2 ±2.61**
(+36%)
Female Mice
Average daily dose,
mg/kg-d
0
67
134
268
536
1,071
Number of animals
10
10
10
10
8
3°
Absolute liver weight
(mg)
1,129 ±242
1,237 ± 123
(+9.6%)
1,238 ±113
(+9.7%)
1,232 ± 124
(+9.1%)
1,281 ± 126
(+13%)
1,383 ± 96
(+22%)
Relative liver weight
(mg/g)
42.9 ±7.71
48.6 ±5.03
(+13%)
47.9 ±3.61
(+12%)
47.8 ±3.74
(+11%)
49.2 ±4.70
(+15%)
54.7 ±3.45**
(+28%)
•'N'TP (1988).
bValues expressed as mean ± standard deviation (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
Statistically significant mortality (7/10).
* Statistically significantly different from control at p< 0.05, as reported by the study authors (Dunnett's test).
**Statistically significantly different from control atp< 0.01, as reported by the study authors (Dunnett's test).
MBT = 2-mercaptobenzothiazole.
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MODELING PROCEDURE FOR CONTINUOUS DATA
BMD modeling of continuous noncancer data was conducted with the EPA's Benchmark
Dose Software (BMDS, Version 2.5). For these data, all continuous models available within the
software were fit using a benchmark response (BMR) of 10% extra risk or 1 standard deviation
(SD). Adequacy of model fit was judged based on the %2 goodness-of-fit p-value (p> 0.1),
magnitude of the scaled residuals at the data point (except the control) closest to the predefined
BMR (absolute value <2.0), and visual inspection of the model fit. In addition to these three
criteria forjudging the 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 the 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<0. 1), the data set was considered unsuitable
for BMD modeling. In the cases where no best model was found to fit to the data, a reduced data
set without the high-dose group was further attempted for modeling and the result was present
along with that of the full data set. Among all of the models providing adequate fit, the
benchmark dose lower confidence limit (BMDL) from the model with the lowest Akaike's
information criterion (AIC) was selected as a potential point of departure (POD) when BMDL
values were sufficiently close. Otherwise, the lowest BMDL was selected as a potential POD.
Model Predictions for Increased Absolute Liver Weight in Male Rats
The procedure outlined above was applied to the data on increased absolute liver weight
in male rats (see Table C-l) (N I P. 1988). Neither the constant nor the nonconstant variance
models in the BMDS provided adequate fit to the variance data; thus, these data were not
suitable for BMD modeling (see Table C-2).
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Table C-2. Modeling Results for Increased Absolute Liver Weights in Male F344 Rats
Administered MBT via Gavage for 13 Weeksa'g
Model
Test for Significant
Difference />-Valueb
Variance
/>-Valuce
Means
/j-Value'
Scaled Residuals
for Dose Groupd
AIC
Constant variance, all doses
Exponential (Model 2)e
<0.0001
0.008339
0.0003809
2.952
784.1144
Exponential (Model 3)e
<0.0001
0.008339
0.0003809
2.952
784.1144
Exponential (Model 4)e
<0.0001
0.008339
0.02882
0.1036
774.9049
Exponential (Model 5)e
<0.0001
0.008339
0.009944
0.6077
776.4563
Hill6
<0.0001
0.008339
0.006453
0.399
777.230503
Lineal
<0.0001
0.008339
0.000508
2.88
783.507998
Polynomial (2-degree/
<0.0001
0.008339
0.000508
2.88
783.507994
Polynomial (3-degree/
<0.0001
0.008339
0.0005077
2.88
783.509001
Power6
<0.0001
0.008339
0.000508
2.88
783.507994
Nonconstant variance, all doses
Linearf
<0.0001
0.006591
0.002715
0.212
780.506214
Constant variance, highest dose dropped
Lineal
<0.0001
0.006376
0.4309
0.986
633.007758
Nonconstant variance, highest dose dropped
Lineal
<0.0001
0.003587
0.5179
1.08
633.585511
•'N' T'P ( 1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD and the largest residual at any dose.
Tower restricted to >1.
Coefficients restricted to be negative.
gNo model was selected. Neither the constant nor nonconstant variance models provide adequate fit to the variance
data using either the full or reduced dataset.
AIC = Akaike's information criterion; BMD = benchmark dose; MBT = 2-mercaptobenzothiazole.
Model Predictions for Increased Relative Liver Weight in Male Rats
The procedure outlined above was applied to the data on increased relative liver weight in
male rats (see Table C-l) (NTP. 1988). Neither the constant nor the nonconstant variance
models in the BMDS provided adequate fit to the variance data; thus, these data were not
suitable for BMD modeling (see Table C-3).
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Table C-3. Modeling Results for Increased Relative Liver Weights in Male F344 Rats
Administered MBT via Gavage for 13 Weeksa'g
Model
Test for Significant
Difference />-Valueb
Variance
/>-Valuce
Means
/j-Value'
Scaled Residuals
for Dose Groupd
AIC
Constant variance, all doses
Exponential (Model 2)e
<0.0001
0.003799
<0.0001
3.396
218.362
Exponential (Model 3)e
<0.0001
0.003799
<0.0001
3.396
218.362
Exponential (Model 4)e
<0.0001
0.003799
0.02857
0.3174
198.9066
Exponential (Model 5)e
<0.0001
0.003799
0.0288
0.5853
198.5753
Hill6
<0.0001
0.003799
0.01187
0.463
200.126363
Lineal
<0.0001
0.003799
<0.0001
0.833
216.60979
Polynomial (2-degree/
<0.0001
0.003799
<0.0001
0.833
216.60979
Polynomial (3-degree/
<0.0001
0.003799
<0.0001
0.833
216.60979
Power6
<0.0001
0.003799
<0.0001
0.833
216.60979
Nonconstant variance, all doses
Linearf
<0.0001
0.001502
<0.0001
1.02
216.517628
Constant variance, highest dose dropped
Linearf
<0.0001
0.001962
0.7012
0.625
155.886575
Nonconstant variance, highest dose dropped
Linearf
<0.0001
0.0007209
0.629
0.607
157.73721
•'N1TP ( 1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD and the largest residual at any dose.
Tower restricted to >1.
Coefficients restricted to be negative.
gNo model was selected. Neither the constant nor nonconstant variance models provide adequate fit to the variance
data using either the full or reduced dataset.
AIC = Akaike's information criterion; BMD = benchmark dose; MBT = 2-mercaptobenzothiazole.
Model Predictions for Increased Absolute Liver Weight in Female Rats
The procedure outlined above was applied to the data on increased absolute liver weight
in female rats (NTP. 1988) (see Table C-1). The constant variance model provided adequate fit
to the variance data (p > 0.1). With the constant variance model applied, the Exponential
(Models 4 and 5) and Hill models provided adequate fit to the means data. BMDL values for
models providing adequate fit differed by four-fold. However, the ratio of BMD to BMDL
values is very large (>100-fold) for all models that provide an adequate fit. For this reason, the
data are not suitable for BMD modeling (see Table C-4).
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Table C-4. Modeling Results for Increased Absolute Liver Weights in Female F344 Rats
Administered MBT via Gavage for 13 Weeks"
Model
BMD
BMDLio
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
AIC
Constant variance, all doses
Exponential (Model 2)e
626.618
448.444
0.0001553
0.8587
0.00171
680.7429
Exponential (Model 3)e
626.618
448.444
0.0001553
0.8587
0.00171
680.7429
Exponential (Model 4)e
56.8777
0.228986
0.0001553
0.8587
0.4105
669.3949
Exponential (Model 5)e
56.8776
0.23809
0.0001553
0.8587
0.4105
669.3949
Hill6
42.4377
3.06 x IO"5
0.0001553
0.8587
0.5348
668.865905
Lineal
585.717
406.056
0.0001553
0.8587
0.002113
680.292823
Polynomial (2-degree/
585.662
406.158
0.0001553
0.8587
0.002117
680.288636
Polynomial (3-degree/
585.671
405.964
0.0001553
0.8587
0.002109
680.296611
Power6
585.661
406.158
0.0001553
0.8587
0.002117
680.288635
•'N' T'P (1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD and the largest residual at any dose.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); MBT - 2-mercaptobenzothiazole.
Model Predictions for Increased Relative Liver Weight in Female Rats
The procedure outlined above was applied to the data on increased relative liver weight in
female rats (N I P. 1988) (see Table C-1). Table C-5 summarizes the BMD modeling results.
The constant variance model provided adequate fit to the variance data. With the constant
variance model applied, the Exponential (Models 4 and 5) and Hill models provided adequate fit
to the means data (p> 0.1). BMDLs for models providing adequate fit were sufficiently close
(differed by less than two to threefold), so the model with the lowest AIC was selected (Hill).
Visual inspection of the shape of the Hill dose-response curve in the low-dose region appears to
be reasonable. Thus, the BMDLio of 14.8 mg/kg-day from this model is selected for this
endpoint (see Figure C-l and the BMD text output for details).
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Table C-5. Modeling Results for Increased Relative Liver Weights in Female F344 Rats
Administered MBT via Gavage for 13 Weeks"
Model
BMD
BMDLio
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
AIC
Constant variance, all doses
Exponential (Model 2)e
483.317
374.461
<0.0001
0.8968
<0.0001
179.8442
Exponential (Model 3)e
483.317
374.461
<0.0001
0.8968
<0.0001
179.8442
Exponential (Model 4)e
52.3996
27.9336
<0.0001
0.8968
0.2475
157.6713
Exponential (Model 5)e
52.3996
27.9336
<0.0001
0.8968
0.2475
157.6713
Hill6
36.2984
14.8244
<0.0001
0.8968
0.5774
155.97703
Lineal
434.701
327.334
<0.0001
0.8968
<0.0001
178.674032
Polynomial (2-degree/
434.7
327.334
<0.0001
0.8968
<0.0001
178.674032
Polynomial (3-degree/
434.701
327.334
<0.0001
0.8968
<0.0001
178.674032
Power6
434.701
327.334
<0.0001
0.8968
<0.0001
178.674032
•'N' T'P (1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals at doses closest to BMD and the largest residual at any dose.
Tower restricted to >1.
Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); MBT - 2-mercaptobenzothiazole.
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Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hill
46
44
42
40
38
36
34
32
30
BMDL
BMD
28
0
200
400
600
800
1000
dose
09:09 02/25 2016
Figure C-l. Hill Model for Increased Relative Liver Weight in Female Rats Administered
MBT via Gavage for 13 Weeks (NTP. 1988)
Text Output for Hill Model for Increased Relative Liver Weight in Female Rats
Administered MBT via Gavage for 13 Weeks (NTP, 1988)
Hill Model. (Version: 2.17; Date: 01/28/2013)
Input Data File: C:/Users/bowens/BMDS2601/Data/hil_Continuousl_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS2601/Data/hil_Continuousl_Opt.pit
Thu Feb 25 09:09:01 2016
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*dose^n/(k^n + dose^n)
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
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Power parameter restricted to be greater than 1
A constant variance model is fit
Total number of dose groups = 5
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 9.37036
rho = 0 Specified
intercept = 31.8
v = 11.4
n = 1.19055
k = 166.52
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho -n
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha intercept v k
alpha 1 -5e-009 1.4e-007 9.7e-008
intercept -5e-009 1 -0.54 0.32
v 1.4e-007 -0.54 1 0.52
k 9.7e-008 0.32 0.52 1
the user,
Parameter Estimates
Interval
Variable
Limit
alpha
12.0371
intercept
33.6661
v
14.9781
n
k
198.327
Estimate
8.57154
31.8439
12.0427
1
100.975
Std. Err.
1.76817
0.929732
1.49767
NA
49.6702
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
5.10598
30.0216
9.10735
3.62317
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
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0
10
31.8
31.8
3.28
2.93
-0.0474
134 .3
9
39.3
38 .7
3.53
2.93
0.596
2 67.9
10
39.9
40.6
2.99
2.93
-0.745
535 .7
8
41.8
42
2.81
2.93
-0.171
1071
10
43.2
42.8
2 . 61
2.93
0.379
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^2
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log(likelihood)
-73.439239
-72.897270
-73.439239
-73.988515
-98.834106
# Param's
6
10
6
4
2
AIC
158.878478
165.794540
158.878478
155.977030
201.668212
Explanation of Tests
Test 1:
Test
Test
Test
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test
-2*log(Likelihood Ratio) Test df
p-value
Test
Test
Test
Test
51.8737
1.08394
1.08394
1.09855
<.0001
0.8968
0.8968
0.5774
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
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The p-value for Test 3 is greater than . 1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect =
0.1
Risk Type
Relative deviation
Confidence level
0. 95
BMD
36.2984
BMDL
14.8244
Model Predictions for Increased Absolute Liver Weight in Female Mice
The procedure outlined above was applied to the data on increased absolute liver weight
in female mice (N I P. 1988) (see Table C-1). The constant variance model did not provide
adequate fit to the variance data (p< 0.1). The nonconstant variance model did provide an
adequate fit to the variance data (p> 0.1). The Exponential (2- and 3-degree), Hill, Linear,
Polynomial (2- and 3-degree), and Power models provided adequate fit to the means data.
However, visual inspection of the plots showed the Exponential (2- and 3-degree), Linear,
Polynomial, and Power models did not provide a good fit to the data in the low dose range. In
addition, the ratio of BMD to BMDL values provided by the Hill model is very large
(>100-fold). Removing the high-dose group did not improve the model fit. For these reasons,
the data were determined to be not suitable for BMD modeling (see Table C-6).
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Table C-6. Modeling Results for Increased Absolute Liver Weights in Female Mice
Administered MBT via Gavage for 13 Weeks"
Model
BMD
BMDLio
Test for Significant
Difference />-Valueb
Variance
/>-Valuce
Means
/>-Valuce
AIC
Constant variance, all doses
Exponential (Model 2)6
636.589
391.993
0.03112
0.04899
0.6167
566.0242
Exponential (Model 3)e
636.589
391.993
0.03112
0.04899
0.6167
566.0242
Exponential (Model 4)e
458.073
2.43592
0.03112
0.04899
0.4967
567.7505
Exponential (Model 5)e
458.072
1.92029
0.03112
0.04899
0.4967
567.7505
Hill6
166.825
0.00093185
0.03112
0.04899
0.5739
567.359494
Lineal
615.628
361.926
0.03112
0.04899
0.6261
565.97082
Polynomial (2-degree/
615.634
361.926
0.03112
0.04899
0.6261
565.97082
Polynomial (3-degree/
615.642
361.926
0.03112
0.04899
0.6261
565.97082
Power6
615.64
361.926
0.03112
0.04899
0.6261
565.97082
Nonconstant variance, all doses
Exponential (Model 2)d
672.57
464.652
0.03112
0.9393
0.1024
562.7552
Exponential (Model 3)d
672.571
464.652
0.03112
0.9393
0.1024
562.7552
Exponential (Model 4)d
426.345
1.5383
0.03112
0.9393
0.07711
563.8785
Exponential (Model 5)d
426.345
1.60125
0.03112
0.9393
0.07711
563.8785
Hille
55.6028
1.07 x 10-12
0.03112
0.9393
0.232
561.324452
Lineal
646.418
430.352
0.03112
0.9393
0.109
562.599204
Polynomial (2-degree)6
646.418
430.352
0.03112
0.9393
0.109
562.599204
Polynomial (3-degree)6
646.418
430.352
0.03112
0.9393
0.109
562.599204
Power6
646.418
430.352
0.03112
0.9393
0.109
562.599204
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Table C-6. Modeling Results for Increased Absolute Liver Weights in Female Mice
Administered MBT via Gavage for 13 Weeks"
Model
BMD
BMDLio
Test for Significant
Difference />-Valucb
Variance
/>-Valuce
Means
/>-Valuce
AIC
Nonconstant variance, high dose dropped
Exponential (Model 2)d
654.21
340.709
0.06175
0.889
0.05397
533.3498
Exponential (Model 3)d
654.21
340.709
0.06175
0.889
0.05397
533.3498
Exponential (Model 4)d
68.5973
0.114258
0.06175
0.889
0.8062
528.1367
Exponential (Model 5)d
34.0961
0.112673
0.06175
0.889
0.491
530.1801
Hill6
63.5382
3.10 x IO"5
0.06175
0.889
0.8195
528.103897
Lineal
645.758
325.109
0.06175
0.889
0.05658
533.244022
Polynomial (2-degree)6
645.755
325.109
0.06175
0.889
0.05658
533.244022
Polynomial (3-degree)6
645.755
325.109
0.06175
0.889
0.05658
533.244022
Power6
645.756
325.109
0.06175
0.889
0.05658
533.244022
"NTP (1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dPower restricted to >1.
"Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); MBT - 2-mercaptobenzothiazole.
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Model Predictions for Increased Relative Liver Weight in Female Mice
The procedure outlined above was applied to the data on increased relative liver weight in
female mice (N I P. 1988) (see Table C-1). The constant variance model did not provide an
adequate fit to the variance data (p< 0.1). The nonconstant variance model did provide an
adequate fit to the variance data (p> 0.1), but not the means data (p< 0.1). After dropping the
high-dose group and applying the nonconstant variance model, the Exponential (Models 4 and 5)
and Hill models provided adequate fit to the means data. BMDLs for models providing adequate
fit differed by more than 100-fold. In addition, the ratio of BMD to BMDL values is very large
(>100-fold) for all models that provide an adequate fit. For these reasons, the data were not
suitable for BMD modeling (see Table C-7).
Table C-7. Modeling Results for Increased Relative Liver Weights in Female Mice
Administered MBT via Gavage for 13 Weeks3
Model
BMD
BMDLio
Test for Significant
Difference />-Valueb
Variance
/>-Valuce
Means
/>-Valuce
AIC
Constant variance, all doses
Exponential (Model 2)d
580.301
384.207
0.006477
0.09094
0.1836
223.6164
Exponential (Model 3)d
580.301
384.207
0.006477
0.09094
0.1836
223.6164
Exponential (Model 4)d
459.826
2.99338
0.006477
0.09094
0.1088
225.4588
Exponential (Model 5)d
459.826
2.5459
0.006477
0.09094
0.1088
225.4588
Hill6
55.7046
0.000271543
0.006477
0.09094
0.2188
223.827617
Lineal
558.964
354.726
0.006477
0.09094
0.1869
223.568261
Polynomial (2-degree)6
558.964
354.726
0.006477
0.09094
0.1869
223.568261
Polynomial (3-degree)6
558.964
354.726
0.006477
0.09094
0.1869
223.568261
Power6
558.964
354.726
0.006477
0.09094
0.1869
223.568261
Nonconstant variance, all doses
Exponential (Model 2)d
587.092
432.887
0.006477
0.701
0.06265
221.0346
Exponential (Model 3)d
587.092
432.887
0.006477
0.701
0.06265
221.0346
Exponential (Model 4)d
494.184
1.81331
0.006477
0.701
0.03298
222.8339
Exponential (Model 5)d
494.183
2.1962
0.006477
0.701
0.03298
222.8339
Hill6
41.3349
1.07 x 10-12
0.006477
0.701
0.09193
220.53908
Lineal
561.733
399.693
0.006477
0.701
0.065
220.944482
Polynomial (2-degree)6
561.733
399.693
0.006477
0.701
0.065
220.944482
Polynomial (3-degree)6
564.197
399.698
0.006477
0.701
0.03138
222.944257
Power6
561.733
399.693
0.006477
0.701
0.065
220.944482
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Table C-7. Modeling Results for Increased Relative Liver Weights in Female Mice
Administered MBT via Gavage for 13 Weeks"
Model
BMD
BMDLio
Test for Significant
Difference /j-Valucb
Variance
/j-Value'
Means
/>-Valuce
AIC
Nonconstant variance, high dose dropped
Exponential (Model 2)d
710.433
369.19
0.02619
0.5628
0.03248
211.5112
Exponential (Model 3)d
710.433
369.19
0.02619
0.5628
0.03248
211.5112
Exponential (Model 4)d
41.711
0.102377
0.02619
0.5628
0.9504
204.841
Exponential (Model 5)d
41.7109
0.159579
0.02619
0.5628
0.9504
204.841
Hill6
25.1861
5.36 x 10~13
0.02619
0.5628
0.9601
204.820687
Lineal
701.798
354.147
0.02619
0.5628
0.03428
211.39192
Polynomial (2-degree)6
701.798
354.147
0.02619
0.5628
0.03428
211.39192
Polynomial (3-degree)6
701.797
354.147
0.02619
0.5628
0.03428
211.39192
Power6
701.798
354.147
0.02619
0.5628
0.03428
211.39192
"NTP (1988).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dPower restricted to >1.
"Coefficients restricted to be negative.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); MBT - 2-mercaptobenzothiazole.
MODELING OF CANCER ENDPOINTS
As discussed in the body of the report in the "Derivation of a Provisional Oral Slope
Factor" section, the tumor types selected for BMD modeling were mesothelioma, adrenal gland
pheochromocytoma or malignant pheochromocytoma, preputial gland adenoma or carcinoma,
and subcutaneous tissue fibroma, neurofibroma, sarcoma, or fibrosarcoma in male rats (NTP.
1988), and adrenal gland pheochromocytoma and pituitary gland adenoma or adenocarcinoma in
female rats exposed to MBT for 2 years by gavage (N TP. 1988). The incidence data used in the
modeling are shown in Table C-8.
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Table C-8. Incidences of Selected Neoplastic Lesions in F344/N Rats Exposed to MBT by
Gavage for 103 Weeks"
Male Rats
HED, mg/kg-db
0
64.3
129
Mesothelioma
0/50t
2/50 (4%)
3/50 (6%)
Adrenal gland pheochromocytoma or malignant pheochromocytoma
18/50+ (36%)
27/50*# (54%)
24/49*# (49%)
Preputial gland adenoma or carcinoma
1/50+ (2%)
6/50* (12%)
5/50* (10%)
Subcutaneous tissue fibroma, neurofibroma, sarcoma, or
fibrosarcoma
3/50+ (6%)
6/50 (12%)
7/50* (14%)
Female Rats
HED, mg/kg-db
0
32.2
64.3
Pituitary gland adenoma or adenocarcinoma
16/49+ (33%)
24/50 (48%)
25/50*# (50%)
Adrenal gland, pheochromocytoma
1/50+ (2%)
5/50 (10%)
6/50* (12%)
"NTP (1988).
bGavage doses were adjusted for continuous exposure by multiplying the administered gavage dose by
(5/7) days/week and converted into HEDs using BW3/4 scaling.
* Significantly different from concurrent control at p< 0.05 based on life table test performed by the study authors.
"Significantly different from concurrent control at p< 0.05 based on incidental tumor test performed by the study
authors.
Significant (p < 0.05) dose-related trend by life table or incidental tumor analysis or both.
BW = body weight; HED = human equivalent dose; MBT = 2-mercaptobenzothiazole.
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence is as follows. The
Multistage cancer model in EPA's BMDS, Version 2.5 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: (1) goodness-of-fit p-walue (p < 0.1), (2) visual inspection of the dose-response curve,
and (3) scaled residual at the data point (except the control) closest to the predefined BMR.
Among all of the models providing adequate fit to the data, the benchmark dose lower
confidence limit (BMDL) for the model with the lowest AIC is selected as POD when BMDL
values were within a factor of 2-3. When BMDL values from models providing adequate fit
varied more than two or threefold, the lowest BMDL was selected as a potential POD. In
accordance with U.S. EPA (2012b) guidance, benchmark dose (BMD) and BMDL values
associated with an extra risk of 10% are calculated.
Model Predictions for Mesothelioma in Male Rats
The procedure outlined above was applied to the data for incidence of mesothelioma in
male rats (see Table C-8). The software converged on the 1-degree model, which provided
adequate fit (p > 0.05); thus, it was selected as the best-fitting model (see Table C-9). The
BMDio (HED) and BMDLio (HED) values from this model were 198 and 103 mg/kg-day,
respectively. Figure C-2 shows the model fit to the data.
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Table C-9. Modeling Results for Mesothelioma in Male F344/N Rats Exposed to MBT by
Gavage for 103 Weeks"
Model
DF
x2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(1-degree)0'1
2
0.09
0.9546
-0.177
41.5816
197.752
102.662
Multistage cancer
(2-degree)°
2
0.09
0.9546
-0.177
41.5816
197.752
102.662
Multistage cancer
(3-degree)0
2
0.09
0.9546
-0.177
41.5816
197.752
102.662
•'N1TP ( 1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation
0.15
o
0
it
<
c
o
tj
ra
0.05
BMDL
3MD
0
50
100
150
200
dose
15:09 03/18 2016
Figure C-2.1-Degree Multistage Cancer Model for Mesothelioma in Male Rats
Administered MBT via Gavage for 104 Weeks (NTP, 1988)
Text Output for Multistage Cancer Model for Mesothelioma in Male Rats Administered
MBT via Gavage for 103 Weeks (NTP. 1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous_Opt.pit
Fri Mar 18 15:20:11 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
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Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0032923
Beta(1) = 0.000481135
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)
Beta (1) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 NA
Beta(1) 0.000532791 0.000238304 6.57237e-005
0.000999858
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-19.7456
-19.7908
-21.9217
# Param's Deviance Test d.f.
3
1 0.0904085 2
1 4.35226 2
P-value
0.9558
0.1135
AIC:
41.5816
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 50.000 0.000
64.2860 0.0337 1.684 2.000 50.000 0.248
128.6000 0.0662 3.311 3.000 50.000 -0.177
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Chi^2 = 0.09 d.f. = 2 P-value = 0.9546
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 197.752
BMDL = 102.662
BMDU = 740.797
Taken together, (102.662, 740.797) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.000974069
Model Predictions for Adrenal Gland Pheochromocytoma or Malignant
Pheochromocytoma in Male Rats
The procedure outlined above was applied to the data for incidence of adrenal gland
pheochromocytoma or malignant pheochromocytoma in male rats (see Table C-8). The software
converged on the 1-degree model, which provided adequate fit (p > 0.05); thus, it was selected as
the best-fitting model (see Table C-10). The BMDio (HED) and BMDLio (HED) values from
this model were 51.1 and 23.0 mg/kg-day, respectively. Figure C-3 shows the model fit to the
data.
Table C-10. Modeling Results for Adrenal Gland Pheochromocytoma or Malignant
Pheochromocytoma in Male F344/N Rats Exposed to MBT by Gavage for 103 Weeks"
Model
DF
X2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(l-degree)cd
1
1.65
0.1991
1.043
207.891
51.1241
23.0143
Multistage cancer
(2-degree)°
1
1.65
0.1991
1.043
207.891
51.1241
23.0143
Multistage cancer
(3-degree)0
1
1.65
0.1991
1.043
207.891
51.1241
23.0143
•'N' T'P (1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.7
0.6
0.5
o
0
it
<
c
.o
o
ra
0.4
0.3
BMD
0.2
0
40
60
80
100
120
dose
09:54 02/25 2016
Figure C-3.1-Degree Multistage Cancer Model for Adrenal Gland Pheochromocytoma in
Male Rats Administered MBT via Gavage for 104 Weeks (NTP, 1988)
Text Output for Multistage Cancer Model for Adrenal Gland Pheochromocytoma or
Malignant Pheochromocytoma in Male Rats Administered MBT via Gavage for 103 Weeks
(NTP. 1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous2_Opt.(d)
Gnuplot Plotting File:
C:/Users/bowens/BMDS2 601/Data/msc_Dichotomous2_Opt.pit
Thu Feb 25 09:54:35 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
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The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.4 04 65 6
Beta(1) = 0.00176225
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.73
Beta (1) -0.73 1
Parameter Estimates
Interval
Variable
Limit
Background
0.521525
Beta(1)
0.00502919
Estimate
0.390834
0.00206088
95.0% Wald Confidence
Std. Err. Lower Conf. Limit Upper Conf.
0.0666806 0.260142
0.00151448 -0.000907443
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param'
-101.122 3
-101.946 2
-102.873 1
Deviance Test d.f.
1.64683
3.50083
P-value
0.1994
0.1737
AIC:
207.891
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.3908 19.542 18.000 50.000 -0.447
64.2860 0.4664 23.321 27.000 50.000 1.043
128.6000 0.5327 26.100 24.000 49.000 -0.601
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Chi^2 =1.65 d.f. = 1 P-value = 0.1991
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 51.12 41
BMDL = 23.0143
BMDU = 6.54 617e+007
Taken together, (23.0143, 6.54617e+007) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00434512
Model Predictions for Preputial Gland Adenoma or Carcinoma in Male Rats
The procedure outlined above was applied to the data for incidence of preputial gland
adenoma or carcinoma in male rats (see Table C-8). The software converged on the 1-degree
model, which provided adequate fit (p > 0.05); thus, it was selected as the best-fitting model
(see Table C-l 1). The BMDio (HED) and BMDLio (HED) values from this model were 118 and
62.5 mg/kg-day, respectively. Figure C-4 shows the model fit to the data.
Table C-ll. Modeling Results for Preputial Gland Adenoma or Carcinoma in Male
F344/N Rats Exposed to MBT by Gavage for 103 Weeks"
Model
DF
X2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(l-degree)c,d
1
1.57
0.2107
-0.67
84.4896
117.939
62.4815
Multistage cancer
(2-degree)°
1
1.57
0.2107
-0.67
84.4896
117.939
62.4815
Multistage cancer
(3-degree)0
1
1.57
0.2107
-0.67
84.4896
117.939
62.4815
•'N IP (1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.25
0.2
0.15
o
0
it
<
c
.o
o
ra
0.05
0
20
40
80
100
dose
09:59 02/25 2016
Figure C-4.1-Degree Multistage Cancer Model for Preputial Gland Adenoma or
Carcinoma in Male Rats Administered MBT via Gavage for 104 Weeks (NTP, 1988)
Text Output for Multistage Cancer Model for Preputial Gland Adenoma or Carcinoma in
Male Rats Administered MBT via Gavage for 103 Weeks (NTP, 1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous3_Opt.(d)
Gnuplot Plotting File:
C:/Users/bowens/BMDS2 601/Data/msc_Dichotomous3_Opt.pit
Thu Feb 25 09:59:43 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
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Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.04102 9
Beta(1) = 0.000662118
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.63
Beta (1) -0.63 1
Parameter Estimates
Interval
Variable
Limit
Background
0.0755413
Beta(1)
0.00180592
95.0% Wald Confidence
Estimate Std. Err. Lower Conf. Limit Upper Conf.
0.0264206 0.025062 -0.0227001
0.00089335 0.000465605 -1.9218e-005
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-39.5024
-40.2448
-41. 8154
# Param's
3
2
1
Deviance Test d.f.
1.48486
4.6261
P-value
0.223
0. 09896
AIC:
84.4896
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0264 1.321 1.000 50.000 -0.283
64.2860 0.0808 4.038 6.000 50.000 1.018
128.6000 0.1321 6.604 5.000 50.000 -0.670
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Chi^2 = 1.57 d.f. = 1 P-value = 0.2107
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 117.939
BMDL = 62.4815
BMDU = 1507.44
Taken together, (62.4815, 1507.44) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00160047
Model Predictions for Subcutaneous Tissue Fibroma, Neurofibroma, Sarcoma, or
Fibrosarcoma in Male Rats
The procedure outlined above was applied to the data for incidence of subcutaneous
tissue fibroma, neurofibroma, sarcoma, or fibrosarcoma in male rats (see Table C-8). The
software converged on the 1-degree model, which provided adequate fit (p > 0.05); thus, it was
selected as the best-fitting model (see Table C-12). The BMDio (HED) and BMDLio (HED)
values from this model were 142 and 63.7 mg/kg-day, respectively. Figure C-5 shows the model
fit to the data.
Table C-12. Modeling Results for Subcutaneous Tissue Fibroma, Neurofibroma, Sarcoma,
or Fibrosarcoma in Male F344/N Rats Exposed to MBT by Gavage for 103 Weeks"
Model
DF
x2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(l-degree)cd
1
0.13
0.7217
-0.175
104.01
142.3
63.7278
Multistage cancer
(2-degree)°
1
0.13
0.7217
-0.175
104.01
142.3
63.7278
Multistage cancer
(3-degree)0
1
0.13
0.7217
-0.175
104.01
142.3
63.7278
•'N'TP (1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.25
0.2
0.1
0.05
0
Multistage Cancer
Linear extrapolation
BMD"
0 20 40 60 80 100 120 140
dose
10:05 02/25 2016
Figure C-5.1-Degree Multistage Cancer Model for Subcutaneous Tissue Fibroma,
Neurofibroma, Sarcoma, or Fibrosarcoma in Male Rats Administered MBT via Gavage for
103 Weeks (NTP. 1988)
Text Output for Multistage Cancer Model for Subcutaneous Tissue Fibroma,
Neurofibroma, Sarcoma, or Fibrosarcoma in Male Rats Administered MBT via Gavage for
103 Weeks (NTP. 1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous4_Opt.(d)
Gnuplot Plotting File:
C:/Users/bowens/BMDS2 601/Data/msc_Dichotomous4_Opt.pit
Thu Feb 25 10:05:38 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
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The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0667121
Beta(1) = 0.000691636
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.68
Beta (1) -0.68 1
Parameter Estimates
Interval
Variable
Limit
Background
0.129828
Beta (1)
0.00179534
95.0% Wald Confidence
Estimate Std. Err. Lower Conf. Limit Upper Conf.
0.0637767 0.0337003 -0.00227464
0.000740409 0.000538242 -0.000314526
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-49.9428
-50. 0052
-50.9233
# Param's
3
2
1
Deviance Test d.f.
0.124811
1.96108
P-value
0.7239
0.3751
AIC:
104.01
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0638 3.189 3.000 50.000 -0.109
64.2860 0.1073 5.365 6.000 50.000 0.290
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128.6000 0.1488 7.440 7.000 50.000 -0.175
Chi^2 = 0.13 d.f. = 1 P-value = 0.7217
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 142.3
BMDL = 63.7278
BMDU = 2.10827e+008
Taken together, (63.7278, 2.10827e+008) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00156917
Model Predictions for MS_ Combo-Multiple Tumor Model for All Tumor Types in
Male Rats
MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data
for mesothelioma, adrenal gland pheochromocytoma or malignant pheochromocytoma, preputial
gland adenoma or carcinoma, and subcutaneous tissue fibroma, neurofibroma, sarcoma, or
fibrosarcoma in male rats. For each tumor type, the best-fitting Multistage model (i.e., the
degree of polynomial setting) was maintained in the MSCombo model run. The calculated
combined tumor BMDLio (HED) based on the MS Combo model is 15.0 mg/kg-day.
Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Male Rats
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Fri Mar 18 15:18:23 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomous.dax
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Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0032923
Beta(1) = 0.000481135
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)
Beta (1) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 * * *
Beta(1) 0.000532791 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-19.7456
-19.7908
-21.9217
0.0904085
4 .35226
0.9558
0.1135
AIC:
41.5816
Log-likelihood Constant
16.99398096819764
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 50.000 0.000
64.2860 0.0337 1.684 2.000 50.000 0.248
128.6000 0.0662 3.311 3.000 50.000 -0.177
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Chi^2 = 0.09 d.f. = 2 P-value = 0.9546
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 197.752
BMDL = 102.662
BMDU = 740.797
Taken together, (102.662, 740.797) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.000974069
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Fri Mar 18 15:18:23 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomous2.dax
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.4 04 65 6
Beta(1) = 0.00176225
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Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.7 6
Beta (1) -0.76 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.390834 * * *
Beta(1) 0.00206088 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-101.122
-101.946
-102.873
1.64683
3.50083
0.1994
0.1737
AIC:
207.891
Log-likelihood Constant
94 . 615324369879303
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
64.2860
128.6000
Chi^2 =1.65
0.3908
0.4664
0.5327
d.f. = 1
19.542 18.000 50.000
23.321 27.000 50.000
26.100 24.000 49.000
P-value = 0.1991
-0.447
1. 043
-0.601
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 51.12 41
BMDL = 23.0143
BMDU = 6.54 617e+007
Taken together, (23.0143, 6.54617e+007) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00434512
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MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Fri Mar 18 15:18:23 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomous3.dax
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.04102 9
Beta(1) = 0.000662118
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.81
Beta (1) -0.81 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.0264206 * * *
Beta(1) 0.00089335 * * *
* - Indicates that this value is not calculated.
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Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-39.5024
-40.2448
-41. 8154
1.48486
4.6261
0.223
0. 09896
AIC:
84.4896
Log-likelihood Constant
35.059609165698149
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
64.2860
128.6000
0.0264
0.0808
0.1321
1.321
4.038
6. 604
1.000
6.000
5.000
50.000
50.000
50.000
-0.283
1. 018
-0.670
Chi^2 = 1.57
d.f. = 1
P-value = 0.2107
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 117.939
BMDL = 62.4815
BMDU = 1507. 44
Taken together, (62.4815, 1507.44) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00160047
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Fri Mar 18 15:18:23 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
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Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomous4.dax
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0667121
Beta(1) = 0.000691636
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.77
Beta (1) -0.77 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.0637767 * * *
Beta(1) 0.000740409 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-49.9428
-50. 0052
-50.9233
0.124811
1.96108
0.7239
0.3751
AIC:
104.01
Log-likelihood Constant
44.884053510893828
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0638 3.189 3.000 50.000 -0.109
64.2860 0.1073 5.365 6.000 50.000 0.290
128.6000 0.1488 7.440 7.000 50.000 -0.175
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Chi^2 =0.13
d.f. = 1
P-value = 0.7217
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
142 .3
63.7278
1. 71842e+008
Taken together, (63.7278, 1.71842e+008) is a 90
interval for the BMD
Multistage Cancer Slope Factor = 0.00156917
two-sided confidence
**** Start of combined BMD and BMDL Calculations.****
Combined Log-Likelihood -211.98 62 98 08735791
Combined Log-likelihood Constant 191.55296801466895
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0. 95
24.9231
14.9871
Multistage Cancer Slope Factor = 0.00667239
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Model Predictions for Adrenal Gland Pheochromocytoma in Female Rats
The procedure outlined above was applied to the data for incidence of adrenal gland
pheochromocytoma in female rats (see Table C-8). Table C-13 summarizes the BMD modeling
results. Both the 1- and 2-degree Multistage cancer models converged on the same model,
yielding a BMDio (HED) and BMDLio (HED) of 55.0 and 30.5 mg/kg-day, respectively
(see Figure C-6 and the BMD text output for details).
Table C-13. Modeling Results for Increased Incidence of Adrenal Gland
Pheochromocytoma in Female F344/N Rats Exposed to MBT by Gavage for 103 Weeks"
Model
DF
x2
/2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(l-degree)cd
1
0.36
0.5478
-0.327
83.3553
55.0149
30.5228
Multistage cancer
(2-degree)c
1
0.36
0.5478
-0.327
83.3553
55.0149
30.5228
•'N'TP (1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. Both models provided adequate fit to the data. The Multistage cancer (2-degree) converged upon
the Multistage cancer (1-degree), so the Multistage cancer (1-degree) was selected.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.25
0.2
0.15
o
0
it
<
c
.o
o
ra
0.05
BMD
0
10
20
40
50
60
dose
10:49 02/25 2016
Figure C-6.1-Degree Multistage Cancer Model for Adrenal Gland Pheochromocytoma in
Female Rats Administered MBT via Gavage for 104 Weeks (NTP, 1988)
Text Output for 1-Degree Multistage Cancer for Adrenal Gland Pheochromocytoma in
Female Rats Administered MBT via Gavage for 104 Weeks (NTP, 1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous cancer_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous
cancer_Opt.pit
Thu Feb 25 10:49:22 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
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Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0301108
Beta(1) = 0.00167512
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.55
Beta (1) -0.55 1
Parameter Estimates
Interval
Variable
Limit
Background
0.0652852
Beta(1)
0.00362317
95.0% Wald Confidence
Estimate Std. Err. Lower Conf. Limit Upper Conf.
0.0226243 0.0217662 -0.0200367
0.00191513 0.000871465 0.000207087
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-39.5024
-39.6776
-41. 8154
# Param's Deviance Test d.f. P-value
3
2 0.350555 1 0.5538
1 4.6261 2 0.09896
AIC:
83.3553
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0226 1.131 1.000 50.000 -0.125
32.2300 0.0811 4.056 5.000 50.000 0.489
64.2860 0.1358 6.792 6.000 50.000 -0.327
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Chi^2 = 0.36 d.f. = 1 P-value = 0.5478
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 55.0149
BMDL = 30.5228
BMDU = 248.014
Taken together, (30.5228, 248.014) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00327624
Model Predictions for Pituitary Gland Adenoma or Adenocarcinoma in Female Rats
The procedure outlined above was applied to the data for incidence of pituitary gland
adenoma or adenocarcinoma in female rats (see Table C-8). Table C-14 summarizes the BMD
modeling results. Both the 1- and 2-degree Multistage cancer models converged on the same
model, yielding a BMDio (HED) and BMDLio (HED) of 21.0 and 10.9 mg/kg-day
(see Figure C-7 and the BMD text output for details).
Table C-14. Modeling Results for Increased Incidence of Pituitary Gland Adenoma or
Adenocarcinoma in Female F344/N Rats Exposed to MBT by Gavage for 103 Weeks"
Model
DF
x2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio (HED)
(mg/kg-d)
BMDLio (HED)
(mg/kg-d)
Multistage cancer
(1-degree)0'1
1
0.48
0.4873
0.564
204.937
20.9922
10.8656
Multistage cancer
(2-degree)c
1
0.48
0.4873
0.564
204.937
20.9922
10.8656
•'N' T'P (1988).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. Both models provided adequate fit to the data. The Multistage cancer (2-degree) converged upon
the Multistage cancer (1-degree), so the Multistage cancer (1-degree) was selected.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected BMR; BMDL = 95% lower confidence limit on the BMD (subscripts denote
BMR: i.e ., io - exposure concentration associated with 10% extra risk); DF - degree(s) of freedom; HED - human
equivalent dose; MBT = 2-mercaptobenzothiazole.
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.6
0.5
0.4
0.3
0.2
BMDL
BMD
0
10
20
30
40
50
60
dose
10:52 02/25 2016
Figure C-7. 1-Degree Multistage Cancer Model for Pituitary Gland Adenoma or
Adenocarcinoma in Female Rats Administered MBT via Gavage for 104 Weeks (NTP,
1988)
Text Output for 1-Degree Multistage Cancer Model for Pituitary Gland Adenoma or
Adenocarcinoma in Female Rats Administered MBT via Gavage for 104 Weeks (NTP,
1988)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS2601/Data/msc_Dichotomous_Opt.pit
Thu Feb 25 10:52:08 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
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The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.35 05 6
Beta(1) = 0.00463603
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.7
Beta (1) -0.7 1
Parameter Estimates
Interval
Variable
Limit
Background
0.469719
Beta(1)
0.0105221
Estimate
0.342175
0.00501904
95.0% Wald Confidence
Std. Err. Lower Conf. Limit Upper Conf.
0.065075 0.21463
0.00280774 -0.000484026
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-100.228
-100.468
-102.064
# Param's
3
2
1
Deviance Test d.f.
0. 481126
3.67297
P-value
0.4879
0.1594
AIC:
204.937
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.3422 16.767 16.000 49.000 -0.231
32.2300 0.4404 22.021 24.000 50.000 0.564
64.2860 0.5236 26.179 25.000 50.000 -0.334
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Chi^2 = 0.48 d.f. = 1 P-value = 0.4873
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 2 0.9922
BMDL = 10.8656
BMDU = 2 63.023
Taken together, (10.8656, 263.023) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.00920333
Model Predictions for MS_ Combo-Multiple Tumor Model for All Tumor Types in
Female Rats
MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data
for adrenal gland pheochromocytoma and pituitary gland adenoma or adenocarcinoma in female
rats. For each tumor type, the best-fitting Multistage model (i.e., the degree of polynomial
setting) was maintained in the MSCombo model run. The calculated combined tumor
BMDLio (HED) based on the MS Combo model is 8.91 mg/kg-day. This BMDLio (HED) is
used as the POD to derive the provisional oral slope factor (p-OSF).
Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Female
Rats
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Thu Feb 25 11:11:37 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomous.dax
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Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.35 05 6
Beta(1) = 0.00463603
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.75
Beta (1) -0.75 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.342175 * * *
Beta(1) 0.00501904 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-100.228
-100.468
-102.064
# Param's Deviance Test d.f.
3
2 0.481126 1
1 3.67297 2
P-value
0.4879
0.1594
AIC:
204.937
Log-likelihood Constant
93.741309121153364
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.3422 16.767 16.000 49.000 -0.231
32.2300 0.4404 22.021 24.000 50.000 0.564
64.2860 0.5236 26.179 25.000 50.000 -0.334
Chi^2 = 0.48 d.f. = 1 P-value = 0.4873
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Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 2 0.9922
BMDL = 10.8656
BMDU = 2 63.023
Taken together, (10.8656, 263.023) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00920333
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS2601\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 601\Data\multi_test.plt
Thu Feb 25 11:11:37 2016
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = Dichotomouscancer.dax
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0301108
Beta(1) = 0.00167512
Asymptotic Correlation Matrix of Parameter Estimates
115
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Background Beta(l)
Background 1 -0.78
Beta (1) -0.78 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.0226243 * * *
Beta(1) 0.00191513 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-39.5024
-39.6776
-41. 8154
# Param's Deviance Test d.f. P-value
3
2 0.350555 1 0.5538
1 4.6261 2 0.09896
AIC:
83.3553
Log-likelihood Constant
35.059609165698149
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0226 1.131 1.000 50.000 -0.125
32.2300 0.0811 4.056 5.000 50.000 0.489
64.2860 0.1358 6.792 6.000 50.000 -0.327
Chi^2 = 0.36 d.f. = 1 P-value = 0.5478
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 55.0149
BMDL = 30.5228
BMDU = 248.014
Taken together, (30.5228, 248.014) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00327624
116 2-Mercaptobenzothiazole
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**** Start of combined BMD and BMDL Calculations.****
Combined Log-Likelihood -140.14592704037494
Combined Log-likelihood Constant 12 8.80091828 685153
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 15.1944
BMDL = 8.9079
Multistage Cancer Slope Factor = 0.011226
117 2-Mercaptobenzothiazole
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