United States
kS^laMIjk Environmental Protection
^J^iniiil m11 Agency
EPA/690/R-10/024F
Final
11-08-2010
Provisional Peer-Reviewed Toxicity Values for
Tributyl phosphate
(CASRN 126-73-8)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Alan J. Weinrich, CIH, CAE
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Debdas Mukerjee, Ph.D.
National Center for Environmental Assessment, Cincinnati, OH
Angela Howard, Ph.D.
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300)

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	11
BACKGROUND	1
HISTORY	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	2
INTRODUCTION	2
REVIEW 01 PERTINENT DATA	3
HUMAN STUDIES	3
Oral Exposure	3
Inhalation Exposure	3
ANIMAL STUDIES	4
Oral Exposure	4
Subchronic Studies	4
Chronic Studies	13
Reproductive and Developmental Studies	17
Inhalation Exposure	22
Subchronic Studies	22
OTHER STUDIES	22
Acute/Short-term Toxicity	22
Other Routes	23
Neurotoxicity	23
Genotoxicity	23
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL	24
RfDs FOR TRIBUTYL PHOSPHATE	24
SUBCHRONIC p-RfD DERIVATION	28
CHRONIC p-RfD DERIVATION	29
FEASIBILITY OF DERIVING SUBCHRONIC AND CHRONIC PROVISIONAL RfCs FOR
TRIBUTYL PHOSPHATE	31
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR TRIBUTYL PHOSPHATE	32
WEIGHT-OF-EVIDENCE DESCRIPTOR	32
MODE-OF-ACTION DISCUSSION	32
Urinary Bladder Tumors	32
Key Events	33
Strength, Consistency, Specificity of Association	33
Dose-response Concordance	33
Temporal Relationships	35
Biological Plausibility and Coherence	35
Conclusions	35
Hepatocellular Adenomas	35
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK	36
Oral Exposure	36
Inhalation Exposure	37
REFERENCES	37
APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING FOR ORAL SLOPE FACTOR
	43
APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR RAT LETHALITY	45
l

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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMD
benchmark dose
BMCL
benchmark concentration lower bound 95% confidence interval
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
TRIBUTYL PHOSPHATE (CASRN 126-73-8)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	EPA's Integrated Risk Information System (IRIS)
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program
3)	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR);
~	California Environmental Protection Agency (CalEPA) values; and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may 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), or OSRTI.
INTRODUCTION
Tributyl phosphate is not listed in IRIS (U.S. EPA, 2008), the Drinking Water Standards
and Health Advisory list (U.S. EPA, 2006), the Health Effects Assessment Summary Tables
(U.S. EPA, 1997), or the Chemical Assessments and Related Activities (CARA) list
(U.S. EPA 1991, 1994). Occupational exposure limits, expressed as 8-hour time-weighted
averages, include an Occupational Safety and Health Administration (OSHA, 2008) permissible
exposure limit of 5 mg/m3, a National Institute of Occupational Safety and Health
"3
(NIOSH, 2005) recommended exposure limit of 2.5 mg/m and an American Conference of
Governmental Industrial Hygienists (ACGIH, 2001, 2007) threshold limit value (TLV) of
"3
0.2 ppm (2.2 mg/m ), based in part on analogy to triphenyl phosphate, to minimize potential for
headache, nausea, and irritation. Neither the Agency for Toxic Substances and Disease Registry
(ATSDR) nor the International Agency for Research on Cancer (IARC) has published documents
on tributyl phosphate toxicity or carcinogenicity (ATSDR, 2008; IARC, 2008). The National
Toxicology Program (NTP, 2008) has not performed toxicity or carcinogenicity assessments for
tributyl phosphate and this compound was not on the 11th Report on Carcinogens. The following
reviews, which did not derive toxicity values, also were consulted:
•	World Health Organization (WHO, 1991) Environmental Health Criteria Document
for tributyl phosphate.
•	United Nations Organization for Economic Cooperation and Development
(OECD, 2001) SIDS.
•	A published review of tributyl phosphate toxicity (Bisesi, 2001).
To identify toxicological information pertinent to the derivation of provisional toxicity
values for tributyl phosphate, literature searches initially were conducted in May 2007 and
updated in June 2009 using the following databases: MEDLINE, TOXLINE, BIOSIS, TSCATS,
CCRIS, DART/ETIC, GENETOX, HSDB, and Current Contents (prior 6 months). Except
where noted, the literature searches were not limited by date.
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REVIEW OF PERTINENT DATA
HUMAN STUDIES
Oral Exposure
No studies regarding the oral toxicity of tributyl phosphate in humans were located.
Inhalation Exposure
Among the summary sources consulted, ACGIH (2001) reported unpublished
information suggesting that workers exposed to 15 mg/m tributyl phosphate complained of
nausea and headache and WHO (1991) concluded that airborne exposure to tributyl phosphate
caused irritation of human skin, eyes, and respiratory tract.
Reape (1982) conducted a field study to evaluate neurotoxicity endpoints in industrial
workers who were exposed to aryl phosphates for an average of 13.3 years. In 1974, modern
industrial hygiene controls were installed in the plant to decrease exposure to airborne
substances. In 1982, personal and area airborne tributyl phosphate concentrations in the plant
ranged from 1 to 15 ppb (0.01 to 0.16 mg/m3), depending on the work area. Other airborne
contaminants present in the work areas included triaryl phosphate, dichlorobenzene, other aryl
and alkyl phosphates, and other organics; exposures to these contaminants ranged from
undetectable to 143 ppb. No air concentration or exposure data were available prior to 1974,
when worker exposures probably were much higher because it preceded installation of modern
exposure control measures. Observations from clinical neurological examinations,
measurements of nerve conduction velocity, and personal interviews of exposed workers were
not different from the general population. Reape (1982) concluded there was no apparent
association between chronic exposure of workers to these low concentrations of aryl phosphates
and development of neurological health effects. However, because former workers who left
employment before the study dates were not evaluated, it is unclear whether this conclusion is
justified. Former workers who experienced symptoms might have self-selected themselves into
different workplaces.
In a follow-up study, 12 workers exposed to air contaminants containing tributyl
phosphate and other compounds were evaluated for serum monocyte counts, as determined by
monocyte nonspecific esterase staining activity (Mandel et al., 1989). Exposure concentrations
and length of exposure to tributyl phosphate for these workers were not reported. Monocyte
counts were similar between the general population and workers exposed to tributyl phosphate.
Keegan et al. (2009) reviewed extensive data on exposures to potential chemical warfare
agents and their surrogates, in United Kingdom tests of these agents. Significant exposures to
tributyl phosphate in 1959-1960 were documented. While this ongoing epidemiological study
appears to be a potential source of relevant human data on tributyl phosphate, such data were not
available at this writing.
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ANIMAL STUDIES
Oral Exposure
Subchronic Studies
There have been several subchronic studies of tributyl phosphate in rats. In one study,
Cascieri et al. (1985; FMC Corporation, 1985) treated Sprague-Dawley rats (15/gender/dose;
approximately 44 days of age) with 0, 8, 40, 200, 1000, or 5000 ppm of tributyl phosphate
(purity >99%) in the diet for 13 weeks. The doses calculated for this review from body weight
and food consumption data reported in the study were 0, 1,3, 14, 68, and 360 mg/kg-day for
males and 0, 1, 3, 16, 81, and 423 mg/kg-day for females. Mortality and clinical signs were
evaluated daily and body weights and food consumption measured weekly. Comprehensive
hematology and serum chemistry measurements were conducted in five rats/gender/dose at the
interim sacrifice of 45 days and at terminal sacrifice. At terminal sacrifice, ophthalmology;
brain, heart, liver, kidney, gonad, and adrenal weights; gross necropsy on all major tissues and
organs; and histology on all major tissues and organs in the control and high-dose groups, and
liver and urinary bladders in all groups also were evaluated.
The treatment had no adverse effects with respect to mortality, ophthalmology, interim
hematology, or gross necropsy (FMC Corporation, 1985; Cascieri et al., 1985). With the
exception of measurements in the brain among 200- and 1000-ppm male rats exposed for
13 weeks, cholinesterase activities (see Table 1) did not vary significantly from controls. The
45-day plasma activities were significantly higher in females at 1 mg/kg-day and 13-week brain
values in males at 14 and 68 mg/kg-day; however, no dose-related trends were apparent.
Abdominogenital staining was observed sporadically in males and females fed 1000 or
5000 ppm. Body-weight gain was significantly (p < 0.01) lower than controls in both high-dose
males (25%) and females (30%). Food consumption was significantly (p < 0.05) decreased
throughout the study in the high-dose males (8—14%) and during Weeks 3-5 in the high-dose
females (9—12%). Table 2 shows the significant changes observed at termination in hematology,
clinical chemistry, organ weights, and histopathology. A significant increase in activated partial
thromboplastin (APTT) time was observed in the high-dose males at termination. Alanine
aminotransferase (ALT) concentrations were 32% above controls in high-dose males at interim
evaluation and 41% elevated in high-dose females at termination. Serum Gamma-glutamyl
transpeptidase (GGT) activity was significantly increased in males at 1000 and 5000 ppm and
females at 5000 ppm at the interim sampling time, and in high-dose males and females at
termination. At terminal sacrifice, significant increases were observed in serum cholesterol
among the high-dose females and in albumin and calcium concentrations among the high-dose
males. Significant increases in liver/brain weight ratios were observed in the 1000- and
5000-ppm males and in the 5000-ppm females, but no histological abnormalities were observed
in any organ other than the urinary bladder of the treated rats. Generalized transitional-cell
hyperplasia was observed in the urinary bladders of all 1000- and 5000-ppm males and in 8/9 of
the 5000-ppm females; this effect was not observed in rats in lower dose groups. No neoplastic
lesions were observed in any tissue examined microscopically. This study identified a NOAEL
of 200 ppm (14 mg/kg-day) for urinary bladder hyperplasia in male rats treated with tributyl
phosphate in the diet for 13 weeks. The associated LOAEL was 1000 ppm (68 mg/kg-day) at
which 10/10 male rats exhibited urinary bladder hyperplasia.
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Table 1
. Acetylcholinesterase Activities from a 13-Week Rat Dietary Studya



PPM in
Diet

45-Day Treatment
13-Week Treatment
Pretest
0
8
40
200
1000
5000
0
8
40
200
1000
5000
Males (mg/kg-day)
0
1
3
14
68
360
0
1
3
14
68
360
RBC
1.69 U/ml
1.86b±
1.78 ±
1.58 ±
1.62 ±
1.82 ±
1.78 ±
1.58 ±
1.66 ±
1.60 ±
1.68 ±
1.62 ±
1.74 ±


0.76
0.13
0.13
0.10
0.28
0.23
0.18
0.33
0.37
0.25
0.30
0.21
Plasma
0.63 U/ml
0.48 ±
0.48 ±
0.48 ±
0.47 ±
0.40 ±
0.40 ±
0.58 ±
0.56 ±
0.54 ±
0.58 ±
0.48
0.48 ±


0.08
0.08
0.05
0.13
0.07
0.10
0.08
0.15
0.09
0.13
±0.08
0.05
Brain
13.38 U/g
13.18 ±
12.74 ±
13.24 ±
13.78
12.80
12.68
11.20 ±
11.88
11.66
12.06° ±
12.12°
11.68


1.02
0.34
0.68
±0.84
±0.66
±0.77
0.99
±0.40
±0.59
0.29
±0.36
±0.46
Females (mg/kg-day)
0
1
3
16
81
423
0
1
3
16
81
423
RBC
1.55 U/ml
1.32 ±
1.52 ±
1.36 ±
1.36 ±
1.50 ±
1.55 ±
1.66
1.60 ±
1.62 ±
1.56 ±
1.66 ±
1.76 ±


0.22
0.30
0.18
0.24
0.26
0.13
±0.23
0.16
0.23
0.33
0.18
0.34
Plasma
0.92 U/ml
1.48 ±
2.38 ±
1.96 ±
1.84 ±
1.38 ±
1.37 ±
2.34 ±
2.48 ±
2.36 ±
1.72 ±
2.12 ±
1.92 ±


0.39
0.63
0.66
0.58
0.62
0.46
0.44
0.54
0.60
0.08
0.66
0.42
Brain
14.14 U/g
13.04 ±
11.24 ±
13.07 ±
12.86
13.07
12.68
12.34 ±
11.82
12.20
12.15 ±
12.10 ±
12.20


2.22
1.32
1.19
± 1.45
±3.05
± 1.51
0.42
±0.83
± 1.08
0.73
0.87
±0.42
"FMC Corporation, 1985
bMean ± standard deviation
°Significantly different from controls (p < 0.05)
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Table 2. Significant Changes at Study Termination in Rats Fed Tributyl Phosphate in the Diet for 13 Weeks3
Dietary Concentration
Control
8 ppm
40 ppm
200 ppm
1000 ppm
5000 ppm
Males
Dose
0 mg/kg-day
1 mg/kg-day
3 mg/kg-day
14 mg/kg-day
68 mg/kg-day
360 mg/kg-day
Hematology
APPT
22.60 ± 1.194b
22.20 ± 1.204
22.60 ± 2.460
22.40 ± 2.460
19.10 ± 1.917
28.40 ± 7.266°
Clinical Chemistry
GGT (U/L)
0.84 ±0.097
0.80 ±0.136
00.69 ±0.133
0.71 ±0.140
0.62 ±0.045
2.28 ±0.419°
Albumin (g/dL)
3.94 ±0.261
3.96 ±0.219
4.04 ±0.344
4.10 ±0.224
4.14 ±0.207
4.42 ± 0.227°
Calcium(mg/dL)
9.80 ±0.255
9.84 ±0.182
9.94 ±0.152
9.90 ±0.212
9.96 ±0.404
10.20 ±0.158°
Organ Weights
Liver/brain weight (%)
677.420
784.214
735.063
692.429
799.001°
904.973°
Histopathology1
Transitional cell hyperplasia urinary bladder
(incidence)
0/10
0/10
0/10
0/10
10/10e
10/10°
Females
Dose
0 mg/kg-day
1 mg/kg-day
3 mg/kg-day
16 mg/kg-day
81 mg/kg-day
423 mg/kg-day
Hematology
APPT
16.13 ± 1.887
15.20 ± 1.891
17.16 ± 1.872
16.50 ±0.707
15.70 ±2.864
15.60 ±2.043
Clinical Chemistry
ALT(U/L)
19.40 ± 1.517
21.80 ±2.864
24.60 ±5.505
24.60 ±2.881
22.00 ±5.385
27.40 ± 3.435°
GGT (U/L)
1.17 ±0.304
1.49 ±0.275
1.56 ±0.360
1.94 ±0.735
1.22 ±0.213
3.23 ± 1.211°
Cholesterol (mg/dL)
32.00 ±5.958
32.00 ± 6.042
30.60 ±7.956
32.80 ± 10.378
38.00 ± 9.722
51.20 ± 16.829°
Organ Weights
Liver/brain weight (%)
457.904
450.962
473.965
481.110
495.054
531.468°
Histopathologyd
Transitional cell hyperplasia urinary bladder
(incidence)
0/10
0/10
0/10
0/10
0/10
8/9°
aFMC Corporation, 1985; Cascieri et al., 1985
bMean ± standard deviation
°Significantly different from control, p < 0.05
Statistical tests on incidence data conducted for this review using Fisher's exact test.
><0.01
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Effects of tributyl phosphate on the urinary bladder were studied in more detail by
Arnold et al. (1997) and the Bayer (1996). Groups of 10 male Sprague-Dawley rats were fed
diets containing 0, 200, 700, or 3000 ppm of tributyl phosphate (purity 99.7%) for 10 weeks.
The doses, calculated by the study authors from body weight and food consumption data in the
study, were 0, 15, 53, and 230 mg/kg-day in the control, low-, mid-, and high-dose groups,
respectively. Another group received 3000 ppm of tributyl phosphate and 12,300 ppm of
ammonium chloride to evaluate the effect of urinary acidification. A final group received
3000 ppm of tributyl phosphate for 10 weeks followed by a 10-week recovery period. Clinical
observations were made weekly, as were body weight and food consumption measurements.
Urinalysis (pH, total protein, creatinine, calcium, phosphorus, magnesium, and osmolality) was
performed on samples collected during Week 11 from all groups and during Week 21 of the
high-dose and control recovery groups; scanning electron microscopy (SEM) of the urine also
was performed. Upon sacrifice during Week 11 or 21, the animals were necropsied and the
bladder and were kidneys weighed. The bladder was examined under SEM and subjected to
immunohistochemical analysis for bromodeoxyuridine (BRdU) labeling as a measure of cell
proliferation.
There were no significant differences in food consumption between groups, but body
weights were significantly (p < 0.05) decreased (-10% based on graphical presentation of data)
in the high-dose tributyl phosphate groups, both with and without ammonium chloride
(Arnold et al., 1997; Bayer, 1996). Body weights for rats in the recovery group returned to the
level of the control rats during the recovery phase. Urine chemistry was similar in treated and
control animals apart from a slight, but statistically significant, decrease in osmolality and
creatinine concentrations in high-dose animals. Scanning electron microscopic examination of
the urine showed no treatment-related crystalluria, urinary precipitate, or calculi.
In the bladder, a statistically significant increase in simple hyperplasia was observed in
the mid- and high-dose groups, as well as the group receiving both ammonium chloride and
tributyl phosphate (see Table 3) (Arnold et al., 1997; Bayer, 1996). Papillary and nodular
hyperplasia also was observed in these three groups, but the increased incidence was significant
only in the high-dose group. Focal necrosis of the bladder epithelium, with erosion, ulceration,
and hemorrhage into the lumen, was observed in the mid- and high-dose groups, as well as
inflammation associated with ulceration in the high-dose group. Classification of the bladder
changes using SEM categories (1-5, with Categories 3-5 considered abnormal) confirmed the
findings; bladders of 10/10 rats exposed at 700 ppm and 9/10 exposed to 3000 ppm were
classified as either Category 4 or 5. Treatments to acidify the urine did not totally inhibit the
proliferative response in the bladder epithelium, but it did cause the effects of 10 weeks exposure
to 3000 ppm to be less severe. Examination of the recovery group showed the bladder changes
to be reversible, with no significant changes being observed upon light or scanning electron
microscopy of the epithelia in the treated and control recovery groups. However, an increased
fibrosis of the submucosa was observed in the recovery animals, possibly representing scar tissue
formed during treatment-related ulceration. This study identifies a NOAEL of 200 ppm
(15 mg/kg-day) for urinary bladder cell hyperplasia in male rats treated with tributyl phosphate
in the diet for 10 weeks. The associated LOAEL is 700 ppm (53 mg/kg-day) at which 8/10 rats
exhibited urinary bladder hyperplasia.
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Table 3. Effects on the Urinary Bladder of Male Rats Fed Tributyl Phosphate
in the Diet for 10 Weeksa

Week 11 Sacrifice
Week 20 Sacrifice
(Recovery Groups)
Control
200 ppm
700 ppm
3000 ppm
Control
3000 ppm
0
mg/kg-day
15
mg/kg-day
53
mg/kg-day
230
mg/kg-day
0
mg/kg-day
230
mg/kg-day
Absolute bladder
weight (g)
0.124 ±0.010b
0.129 ±0.009
0.155 ±0.011
0.218 ±0.027c
0.155 ±0.014
0.227 ± 0.01 lc
Bladder/body
weight (g/kg)
0.227 ±0.016
0.238 ±0.019
0.297 ± 0.020
0.446 ± 0.052°
0.235 ± 0.020
0.357 ±0.027c
Simple
hyperplasia of
bladder
0/10
0/10
8/10d
10/10d
0/9
2/8
Papillary/nodular
hyperplasia of
bladder
0/10
0/10
2/10
6/10d
0/9
0/8
Fibrosis of
submucosa
No data
No data
No data
No data
0/9
6/8 d
BRdU Labeling
Index
0.20 ±0.03
0.34 ±0.16
0.48 ±0.12
1.81 ± 0.30e
0.12 ±0.02
0.08 ±0.01
aArnold et al., 1997; Bayer, 1996
bMean ± standard error
Significantly different from control, p < 0.05
dp < 0.01 by Fisher's exact test conducted for this review
"Arnold et al. (1997) reported that this increase was statistically significant when compared with controls but did not
report a p-valuc
Earlier feeding studies in rats were conducted by Oishi et al. (1980, 1982). Male Wistar
rats (10-11/dose; 5 weeks of age) were treated with 0, 0.5, or 1% tributyl phosphate
(purity >97%) in the diet for 10 weeks (Oishi et al., 1980). Using initial and final body weight
and mean food consumption data from the study, the following doses were calculated for this
review: 0, 425, and 870 mg/kg-day. Body weights and food and water consumption were
measured daily. Upon sacrifice, blood was collected for coagulation time measurements and
serum chemistry, including ALT, aspartate aminotransferase (AST), alkaline phosphatase (ALP),
total protein, glucose, blood urea nitrogen (BUN), cholesterol, and electrolytes; organ weights
(brain, liver, and kidneys) were recorded; and acetylcholinesterase activity in brain, liver, and
serum was measured. Gross necropsy and histopathology evaluation were not conducted. Both
terminal body weights and food consumption were significantly (p < 0.05) decreased in the low-
(17% and 15%, respectively) and high- (31% and 24%, respectively) dose groups. Significant
increases in relative brain (15-33% higher than controls) and kidney (10-21% higher) weights
were observed in both dose groups, despite significant decreases in the absolute weights of both
organs (5—16% decreases) at these doses, indicating that the relative organ weight increases were
secondary to body weight reductions. Relative liver weights also were significantly increased
(32-56%)) at both doses, with no significant change in absolute liver weights, indicating that the
relative liver weight changes also were a function of body weight differences. Significant serum
protein and cholesterol increases were observed in high-dose animals. Significant increases in
serum BUN and blood coagulation time and decreases in serum glucose concentrations were
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observed in both treated groups while activities of serum ALT, AST, and ALP were decreased in
the low-dose group, only. Brain cholinesterase activity was significantly increased in both dose
groups, though increases in serum and liver cholinesterase activity were not significant. With the
exceptions of ALT, ALP, and brain cholinesterase, the observed changes appeared to be
dose-related. This study identified a LOAEL of 0.5% (425 mg/kg-day) for 15-17% decreased
body-weight gains, clinical chemistry effects and increased blood coagulation times in rats
treated in the diet for 10 weeks; no NOAEL is identified in this study.
In a subsequent study by these researchers, male Wistar rats (5 weeks of age) were
treated with 0 (18 animals) or 0.5% (8 animals) tributyl phosphate (purity unknown) in the diet
for 9 weeks (Oishi et al., 1982). Based on initial and final body weight data from the study and
food consumption data from the previous study (Oishi et al., 1980), the doses calculated for this
review were 0 and 417 mg/kg-day. The following parameters were used to assess toxicity:
body-weight gains; hematology, including prothrombin and APTT time, red (RBC) and white
blood cell (WBC) counts, hemoglobin (Hgb) concentrations, hematocrit (Hct), and mean
corpuscular volumes (MCV); serum chemistry (total protein, BUN, cholesterol, ALT, AST,
ALP, cholinesterase, bile acids, and electrolytes), organ weights (liver, kidneys, spleen, and
testes), and histology of the liver, kidney, and spleen. Final body weights of rats treated with
tributyl phosphate were significantly (p < 0.05) lower than controls (11%); food consumption
data were not reported. BUN concentrations were significantly increased (16% higher than
control) in treated rats. Absolute and relative liver weights were significantly (p < 0.05)
increased (16% and 32% higher than controls, respectively) in treated rats; other organ weight
changes were secondary to body weight decreases in treated animals. No other effects were
observed. Oishi et al. (1982) identified a LOAEL of 0.5% (417 mg/kg-day) for an 11% decrease
in body weights and increased serum BUN concentrations among male rats treated for 9 weeks;
no NOAEL is identified in this study.
Laham et al. (1985) conducted a gavage study in rats. Sprague-Dawley rats
(12/gender/dose; average body weights 206-294 g) were treated by gavage with 0 or 200 mg/kg
of tributyl phosphate (purity 98.4%), 5 days/week, for 18 weeks (Laham et al., 1985). An
additional group received similar treatment with 300 mg/kg for 6 weeks, followed by 350 mg/kg
for 12 weeks. The time-weighted average doses were 0, 143, and 238 mg/kg-day for the control,
low- and high-dose groups, respectively. Clinical examinations were conducted daily and body
weights recorded weekly. Upon sacrifice, blood was collected for hematology (RBC and WBC
counts, Hct, MCV, Hgb, mean corpuscular hemoglobin concentration, and serum chemistry
(RBC acetylcholinesterase, albumin, bilirubin, BUN, cholesterol, creatinine, glucose, total
protein, triglycerides, GGT, AST, ALT, ALP, alpha-hydroxybutyrate dehydrogenase, lactate
dehydrogenase [LDH], creatinine phosphokinase, amylase, and electrolytes). Organ weights
(heart, kidneys, liver, lungs, ovaries, testes, and spleen) were recorded and gross necropsy
performed; histology examinations (six rats/gender/dose) were made on weighed organs as well
as the adrenals, brain, intestines, stomach, thyroid, and urinary bladder.
The treatment had no adverse effects on clinical signs or hematology findings
(Laham et al., 1985). High-dose males had significantly (p < 0.001) decreased body weights
(terminal body weights were 14% lower than controls) and significantly increased relative
kidney weights (19% higher than controls). High-dose females had significantly decreased RBC
acetylcholinesterase activity (8% below controls) and significantly increased relative kidney
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weight, absolute and relative liver weight, and absolute spleen weight (9, 29, 28, and 24% above
controls, respectively). All low- and high-dose rats had diffuse hyperplasia of the urinary
bladder epithelium and subepithelial capillaries; severity ranged from mild, in females, to
moderate, in males (see Table 4). Focal nodular epithelial hyperplasia was observed in urinary
bladders of high-dose males and females and low-dose males. Slight mononuclear cell
infiltration was observed in urinary bladders of one low-dose and one high-dose male. Capillary
hyperplasia and mononuclear cell infiltration were each observed in the urinary bladders of
single female control animals. No other treatment-related gross or histopathological
abnormalities were observed. This study identified a LOAEL of 143 mg/kg-day for urinary
bladder hyperplasia in all male and female rats treated by gavage for 18 weeks; no NOAEL is
identified in this study.
Table 4. Incidence and Severity of Bladder Changes in

Rats Exposed to Tributyl Phosphate by Gavage for 18 Weeksa


Male
Female

Control
143
248 mg/kg-
Control
143
248 mg/kg-


mg/kg-day
day

mg/kg-day
day
Transitional Cell Hyperplasia
Diffuse
0/6b
6/6 (+++)
6/6 (+++)
0/6
6/6 (++)
6/6 (++)
Nodular
0/6
5/6 (++)
6/6 (++)
0/6
0/6
1/6 (+)
Capillary Hyperplasia
0/6
6/6 (+++)
6/6 (+++)
1/6 (+)
6/6 (++)
6/6 (++)
Mononuclear Cell Infiltration
0/6
1/6 (+)
1/6 (+)
1/6 (+)
0/6
0/6
Edema
0/6
2/6 (+)
0/6
0/6
0/6
0/6
'Laham ct al.. 1985
incidence (severity; + = slight, ++ = mild, +++ = moderate)
Healy et al. (1995; Bio-Research Laboratories, 1991) treated 12 Sprague-Dawley
rats/gender/dose (age 46-50 days) by gavage with 0, 32.5, 100, or 325 mg/kg-day of tributyl
phosphate (>99% pure) in corn oil, 7 days/week, for 13 weeks. The following parameters were
used to assess toxicity: mortality, clinical signs, body-weight gains, food consumption, motor
activity tests (Days 28, 62, and 92) and qualitative and quantitative functional observation
battery (FOB) for neurobehavioral changes (1, 6, and 24 hours following first dosing, and
Days 7, 14, 35, 63, and 91). Gross necropsy was performed on major tissues and organs of all
groups at study termination and histopathological evaluation of neurological tissues, including
brain (several areas), spinal cord (several levels), gastrocnemius muscle, and peripheral
structures of the nervous system, was performed on six animals in each of the control and
high-dose groups. Healy et al. (1995; Bio-Research Laboratories, 1991) observed salivation, a
typical, early cholinergic sign of organophosphate toxicity (Costa, 2008), among
•	the low-dose rats in one male on each of only 4/89 days of observations and in one to
three females per day on 15/89 days;
•	the mid-dose rats in two to twelve males and females, most with slight to moderate
degree, on all but two of the earliest days of observation; and
•	almost all the high-dose rats, with moderate to severe degree, on every day of
observation.
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Early in the study, the salivation occurred postdosing, but it also was observed predosing during
the second and third months on study, probably a residual effect from the prior day treatment,
according to Healy et al. (1995; Bio-Research Laboratories, 1991). Early deaths occurred in two
males and one female in the mid-dose groups and in three males and four females in the
high-dose groups. Healy et al. (1995; Bio-Research Laboratories, 1991) reported that the clinical
and pathological findings indicated that at least some of these deaths resulted from aspiration of
saliva into the lungs. It is believed that the deaths resulted from a combination of all three of the
following factors:
•	Increased salivation caused by the cholinergic response to TBP.
•	Cholinergic respiratory inhibition (Gallo and Lawryk, 1991; Lotti, 2001).
•	The gavage treatment, itself.
The rats most likely would have survived if they had not been intubated for gavage. Because the
gavage treatment method was a constant while the other two factors were cholinergic responses
to TBP ingestion, it is concluded that the dose-dependent frequency of deaths were indicators of
severity of the cholinergic response rather than frank effects caused by TBP toxicity.
Muzzle staining was observed in mid- and high-dose females and in high-dose males.
Urogenital staining was observed in some high-dose animals during Week 1 of treatment;
two high-dose females also had red-colored urine. Incidences of these clinical signs were not
reported. Body weights were significantly (p < 0.05) decreased from Day 14 of the study in
high-dose males and from Day 35 in females. Based on graphical presentation of body weight
data, terminal body weights were approximately 20% and 10% below controls in high-dose
males and females, respectively. Body weights were lower than controls in 100 mg/kg-day
females throughout the latter half of the study, but the difference did not reach statistical
significance. No difference in body weights was observed in low-dose animals of either gender
or in mid-dose males. Food consumption was significantly (p < 0.01) decreased in high-dose
animals during the first week of treatment. The treatment had no adverse effects on qualitative
or quantitative FOB assessments, motor activity tests, or gross pathology (including brain
weight, length, or width). Histopathological examination of neurological tissues revealed no
abnormalities. This study identifies a NOAEL of 32.5 mg/kg-day and a LOAEL of
100 mg/kg-day for deaths (3/24) that apparently resulted from aspiration of TBP-contaminated
saliva. Conclusions based on clinical signs of toxicity (salivation and muzzle staining) in rats
treated by gavage for 13 weeks were less clear; 32.5 mg/kg-day might have been a LOAEL for
the rarely observed cholinergic salivation or the next higher dose, 100 mg/kg-day, might be
considered a LOAEL for this frequently observed sign.
Bio/dynamics Inc. (1991a) conducted a subchronic study in mice. CD-I mice
(15/gender/dose; approximately 42 days of age) were treated with 0, 500, 2000, or 8000 ppm of
tributyl phosphate (purity 99.1%) in the diet for 13 weeks. The doses, calculated for this review
from body weight and food consumption data in the study, were 0, 96, 383, or 1479 mg/kg-day
for males and 0, 119, 462, or 1769 mg/kg-day for females. Daily examinations for mortality and
clinical signs were made and measures of body weight and food consumption were recorded
weekly. Blood was collected for hematology (Hct, RBC, WBC, reticulocyte count, platelet
count, differential leukocyte count, erythrocyte morphology) and serum chemistry (AST, ALT,
creatinine, ALP, albumin, calcium, phosphorous) at interim sacrifice of five mice/gender/dose
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after 1 month and on all survivors at termination. At termination, ophthalmology, organ weights
(brain, heart, liver, kidneys, gonads, and adrenals), gross necropsy (all major tissues and organs)
and histology (all major tissues and organs in control and high-dose groups; gross lesions,
epididymides, kidneys, lungs, testes, liver, and urinary bladders in all groups) were evaluated.
The treatment had no adverse effects with respect to mortality, clinical signs, or
ophthalmology (Bio/dynamics Inc., 1991a). In high-dose animals, food consumption was
significantly decreased, and both males and females lost weight over the first week of treatment.
Overall body-weight gains were decreased (20-29%) in high-dose males and females. Estimates
of weekly body-weight gains also were reduced in mid-dose males sporadically during the
treatment period. At study termination, body weights of mid- and high-dose males and
high-dose females were 97, 99, and 93%, respectively, of controls. Significant changes observed
at study termination are shown in Table 5. Slight—but significant—decreases in hematocrit and
erythrocyte counts were observed in high-dose females. A significant increase in platelet counts
was observed in high-dose females at 30 days, but not at termination. Significant increases in
serum calcium and albumin concentrations were observed at 30 days (in females, only albumin
increased) and, at study termination, in high-dose males and females. At study termination,
significant increases were observed in ALT and ALP in high-dose males and in ALT in
high-dose females. The absolute liver weights, the liver/body weight ratios, and the liver/brain
weight ratios all were significantly increased in mid- and high-dose males and females. Gross
enlargement of the liver was observed in all high-dose animals, in one mid-dose male, and in one
mid-dose female. Brown or tan discoloration of the liver was observed in 7 of 20 high-dose
animals and in 1 mid-dose male. Incidences of centrilobular hepatocyte hypertrophy were
significantly increased (p < 0.05 by Fisher's exact test conducted for this review) in mid- and
high-dose males and in high-dose females (see Table 5); incidences of urinary bladder epithelial
hyperplasia were significantly increased (p < 0.01) in mid- and high-dose animals of both
genders. The severity of both endpoints was characterized as minimal or slight (mid-dose group)
and slight or moderate (high-dose group). This study identified a NOAEL of 500 ppm
(96 mg/kg-day) and a LOAEL of 2000 ppm (383 mg/kg-day) for centrilobular hepatocyte
hypertrophy (with changes in liver enzymes and gross evidence of toxicity) in mice treated in the
diet for 13 weeks. The data for urinary bladder hyperplasia were less clear, demonstrating an
80%) (8/10) response rate at 2000 ppm (383 mg/kg-day) and a 10%> (1/10) response rate at
500 ppm (96 mg/kg-day). Because the 10%> response at 96 mg/kg-day was not significantly
greater than the 0/10 response among controls, it is unclear whether this dose should be
considered a LOAEL or a NOAEL.
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Table 5. Significant Changes at Study Termination in
Mice Fed Tributyl Phosphate in the Diet for 13 Weeksa
Dietary tributyl phosphate
Control
500 ppm
2000 ppm
8000 ppm
Males
mg/kg-day
0
96
383
1479
Clinical Chemistry
ALT(IU/L)
40 ± 16b
54 ±41
51 ± 19
133 ± 192d
ALP (IU/L)
56 ± 10
56 ± 10
57 ± 15
84 ± 26e
Albumin (g/dL)
3.2 ±0.3
3.2 ±0.2
3.0 ±0.1
3.9 ± 0.2e
Calcium (mg/dL)
8.4 ±0.1
8.5 ±0.3
8.5 ±0.3
9.3 ± 0.3e
Organ Weights
Absolute liver weight (g)
1.68 ±0.16
1.85 ±0.15
2.10 ± 0.15e
3.75 ± 0.36e
Liver/body weight (x 100)
5.41 ±0.49
5.65 ±0.41
6.82 ± 0.29e
12.17 ±0.87e
Liver/brain weight
3.56 ±0.35
3.73 ±0.35
4.30 ± 0.32e
8.01 ±0.60e
Histopathology0
Centrilobular hepatocyte
hypertrophy
3/10
0/10
8/10d
10/10e
Urinary bladder hyperplasia
0/10
1/10
8/10e
10/10e
Females
mg/kg-day
0
119
462
1769
Hematology
Hct (%)
41.8 ± 1.8
41.7 ±2.7
40.5 ± 1.1
38.7 ± 1.9e
RBC (106/hL)
8.60 ± 0.42
8.55 ±0.46
8.45 ±0.38
8.12 ± 0.34d
Clinical Chemistry
ALT(IUZL)
34 ±7
26 ±6
71 ±53
71 ±34e
Albumin (g/dL)
3.6 ±0.2
3.3 ±0.7
3.3 ±0.3
4.3 ± 0.3e
Calcium (mg/dL)
8.8 ±0.2
8.6 ±0.2
8.7 ±0.3
9.6 ± 0.4e
Organ Weights
Absolute liver weight (g)
1.33 ±0.11
1.36 ±0.13
1.57 ± 0.1 le
2.63 ±0.19e
Liver/bodyweight (/100)
5.30 ±0.27
5.50 ±0.31
6.47 ± 0.34e
11.49 ±0.60e
Liver/brain weight
2.73 ±0.34
2.76 ±0.21
3.19 ± 0.3le
5.52 ± 0.38e
Histopathology0
Centrilobular hepatocyte
hypertrophy (incidence)
0/10
0/10
3/10
10/10e
Urinary bladder hyperplasia
(incidence)
0/10
0/10
9/10e
10/10e
aBio/dynamics Inc., 1991a
bMean ± standard deviation
Statistical analysis of incidence data conducted for this review using Fisher's exact test
dp < 0.05
><0.01
Chronic Studies
Auletta et al. (1998a) supplied Sprague-Dawley rats (50/gender/dose) with diets
containing 0, 200, 700, or 3000 ppm of tributyl phosphate (purity 99.7%) for 2 years.
Auletta et al. (1998a,b) estimated the mean actual intake of tributyl phosphate as 0, 8.9, 32.5, or
143.3 mg/kg-day for males and 0, 11.6, 42.0, or 181.5 mg/kg-day for females in the 0-, 200-,
700-, and 3000-ppm groups, respectively. Body weights and food consumption were measured
weekly for the first 13 weeks and monthly thereafter. Hematology analysis (RBC, WBC,
differential leukocyte count, and erythrocyte morphology) was performed at 12, 18, and
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24 months, and urinalysis (pH, occult blood, and sediment examination) was performed at
3 weeks and at 3, 6, 12, and 18 months. All surviving animals were sacrificed at 24 months and
received a full gross examination. Histopathological examinations were carried out on a
comprehensive set of organs and tissues in high-dose animals, controls, and any rats found dead
or sacrificed early, and on target organs (kidneys, liver, urinary bladder, and any tissues with
gross lesions) from all groups.
Body-weight gains were significantly (p < 0.05) decreased in high-dose rats compared
with controls; mean terminal body weights were 19-20% lower in high-dose rats compared with
controls. The only clinical sign attributable to tributyl phosphate exposure was increased red
discoloration of the urine in some high-dose males (incidences were 2/50, 3/50, 3/50, and
14/50 from control through high-dose). Survival, hematology, and urinalysis parameters were
similar in control and treated animals. The only significant nonneoplastic finding was a
dose-related increase in the incidence and severity of urinary bladder hyperplasia, as shown in
Table 6. No other nonneoplastic lesions associated with dietary administration of tributyl
phosphate were observed. This study identified a LOAEL for urinary bladder hyperplasia of
700 ppm (32.5 mg/kg-day for males and 42 mg/kg-day for females) and a NOAEL of 200 ppm
(8.9 mg/kg-day for males and 11.6 mg/kg-day for females).
Incidences of urinary bladder tumors in 3000-ppm male and female rats were elevated
compared with control incidences (Auletta et al., 1998a). Table 6 shows the incidences of
bladder tumors in all groups. All of the carcinomas were described as transitional cell
carcinomas, except for one in the high-dose-male group, which showed a marked squamous cell
component. Historical control incidences for urinary bladder transitional cell carcinoma in
control Sprague-Dawley rats from the testing laboratory were 1/857 in males and 0/779 in
females. Rats with urinary bladder papillomas were reported also "frequently" to have had
hyperplasia. In contrast, it was not possible to determine the presence or absence of hyperplasia
in rats with urinary bladder carcinomas, because most of the epithelium was involved in the
malignancy.
In the chronic mouse study, Auletta et al. (1998b) supplied CD-I mice (50/gender/dose)
with diets containing 0, 150, 1000, or 3500 ppm of tributyl phosphate (purity 99.7%) for
18 months. Auletta et al. (1998b) estimated the mean actual intake of tributyl phosphate as 0,
28.9, 169, or 585 mg/kg-day for males and 0, 24.1, 206, or 711 mg/kg-day for females in the 0-,
150-, 1000-, and 3500-ppm groups, respectively. Body weights and food consumption were
measured weekly for the first 13 weeks and monthly thereafter. Hematology (RBC, WBC, and
differential leukocyte count and erythrocyte morphology) was performed for half of the animals
at 12 months and for all animals prior to the 18-month sacrifice. All animals received a full
gross examination at death or sacrifice. Full histopathological examinations (see list for rat study
described previously) were carried out on high-dose and control mice and on target organs
(kidneys, liver, lung, urinary bladder, and any tissues with gross lesions) from all groups.
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Table 6. Incidence of Urinary Bladder Lesions in Rats


Fed Tributyl Phosphate in the Diet for 2 Yearsa

Dietary Tributyl
Phosphate
Control
200 ppm
700 ppm
3000 ppm
Males
mg/kg-day
0
8.9
32.5
143.3
Hyperplasia
Trace
1/50
0/50
7/49
6/42°
Mild
0/50
1/50
2/49
8/42°
Moderate
1/50
1/50
2/49
1/42°
Severe
1/50
1/50
1/49
2/42°
Total hyperplasia
3/50
3/50
12/49b
17/42c'd
Neoplasia
Papilloma
0/50
0/50
2/49
23/49d
Squamous cell
0/50
0/50
0/49
1/49
carcinoma




Transitional cell
0/50
0/50
0/49
6/49d
carcinoma




Total neoplasms
0/50
0/50
2/49
30/49d
Females
mg/kg-day
0
11.6
42.0
181.5
Hyperplasia
Trace
0/50
0/50
4/49
12/47°
Mild
0/50
1/50
0/49
13/47°
Moderate
1/50
0/50
0/49
3/47c
Severe
0/50
0/50
1/49
1/47°
Total hyperplasia
1/50
1/50
5/49
29/47°'d
Neoplasia
Papilloma
0/50
0/50
1/49
1 l/49d
Squamous cell
0/50
0/50
0/49
0/49
carcinoma




Transitional cell
0/50
0/50
0/49
2/49
carcinoma




Total neoplasms
0/50
0/50
1/49
13/49
aAuletta et al., 1998a
bSignificantly different from control by Fisher's exact test conducted for this review, p < 0.05
The number of animals at risk for hyperplasia (denominator) was adjusted to eliminate animals with bladder
carcinoma, as hyperplasia could not be evaluated in these animals (see text).
d/?<0.01
No clinical effects of tributyl phosphate were observed in any group. Survival of the
high-dose males was significantly lower than the mid-dose males but was not significantly
reduced compared with controls. Survival of all treated groups was within that of historical
controls in the testing laboratory. A significant decrease in weight gain was observed in
high-dose animals over the study; terminal body weights were 10% lower than controls in both
males and females in this group. There were no hematological changes associated with tributyl
phosphate administration. Significant, dose-related increases in absolute liver weights,
liver/body weight ratios, and liver/brain weight ratios were observed in mid- and high-dose
animals of both genders (see Table 7). Nonneoplastic lesions were not observed in the liver or
other tissues. Auletta et al. (1998b) identified the low-dose of 150 ppm (28.9 mg/kg-day in
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males and 24.1 mg/kg-day in females) as a NOAEL for chronic toxicity. The mid- and
high-dose levels were associated with significant increases in absolute and relative liver weight
(see Table 7) but without any liver histopathology. Serum chemistry was not analyzed in this
study. A 3-month dietary study in CD-I mice reported increased serum activities of liver
enzymes, elevated liver weights, and hepatocyte hypertrophy in mice exposed to dietary
concentrations of 2000 or 8000 ppm of tributyl phosphate (Bio/dynamics Inc., 1991a).
Auletta et al. (1998b) speculated that the lack of hepatocyte hypertrophy in the 18-month mouse
study might have been due to the development of tolerance with chronic administration.
Because the changes in liver weight were not associated with other toxicological correlates, this
endpoint was not considered adverse for the purpose of identifying a LOAEL. The high dose
from this study (585 mg/kg-day in males and 711 mg/kg-day in females) was considered a
LOAEL for decreased body weight and the mid dose (169 and 206 mg/kg-day in males and
females, respectively) was considered a NOAEL.
Table 7. Changes in Absolute and Relative Liver Weight in CD-I Mice Fed
Tributyl Phosphate in the Diet for 18 Monthsa
Dietary tributyl
phosphate
Control
150 ppm
1000 ppm
3500 ppm
Male
mg/kg-day
0
28.9
169
585
Liver weight (g)
1.737 ±0.372b
1.856 ±0.657
2.105 ±0.717c
2.451 ±0.917c
Liver/body weight
(xl00)
5.37 ± 1.25
5.63 ±2.03
6.29 ± 1.79°
8.01 ±2.60c
Liver/brain weight
3.25 ±0.86
3.51 ± 1.24
3.98 ± 1.37°
4.68 ± 1.67°
Female
mg/kg-day
0
24.1
206
711
Liver weight (g)
1.483 ±0.255
1.552 ±0.234
1.878 ±0.930d
2.055 ±0.338c
Liver/body weight
(xl00)
5.20 ±0.69
5.59 ±0.75
6.56 ± 2.48°
7.65 ± 0.92°
Liver/brain weight
2.82 ±0.46
2.87 ±0.42
3.61 ± 1.85°
4.00 ± 0.79°
aAuletta et al., 1998b
bMean ± standard deviation
><0.01
dSignificantly different from control, p < 0.05
There was a significant increase in the incidence of liver adenomas (see Table 8) in
high-dose males (Auletta et al., 1998b). Historical incidences for hepatocellular adenomas in
control male CD-I mice from the testing laboratory ranged from 2/59 to 10/60. Incidences of
hepatocellular carcinomas were not significantly increased in treated mice compared with
controls. Exposed groups showed no other increased incidences of neoplastic lesions when
compared with the control group.
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Table 8. Incidence of Liver Tumors in CD-I Mice Fed Tributyl Phosphate
in the Diet for 18 Monthsa,b

0 ppm
150 ppm
1000 ppm
3500 ppm
Male (mg/kg-day)
0
28.9
169
585
Hepatocellular carcinoma
4/50
4/50
3/50
3/50
Hepatocellular adenoma
3/50
6/50
7/50
10/50°
Female (mg/kg-day)
0
24.1
206
711
Hepatocellular carcinoma
0/50
0/50
0/50
0/50
Hepatocellular adenoma
0/50
0/50
1/50
2/50
aAuletta et al., 1998b
bIncludes animals that died during the treatment period
Significantly different from control, p < 0.03
Reproductive and Developmental Studies
Two reproductive toxicity studies were located for tributyl phosphate. In a
one-generation range-finding study, CD rats (10/gender/dose) were treated in the diet with 0,
100, 300, 1500, or 5000 ppm of tributyl phosphate (99.7% pure) for 2 weeks premating and
during breeding periods (SOCMA, 1991; Tyl et al., 1997). Using allometric values for body
weight and food consumption (U.S. EPA, 1988), the doses were calculated for this review to be
0, 9, 26, 129, or 431 mg/kg-day for male rats and 0, 10, 29, 147, or 490 mg/kg-day for female
rats. Male rats were sacrificed following breeding and gross necropsy was performed. Female
rats received additional treatment during gestation, lactation, and weaning, and they were
sacrificed on Postnatal Day 21 for gross necropsy. Thus, males and females received
approximately 4 and 10 weeks of treatment, respectively. F1 generation pups were culled to
5/gender/litter/dose at Postnatal Day 4 and were sacrificed on Postnatal Day 21 for histological
examination of urinary bladders of both genders and testes of males.
Tyl et al. (1997) reported that the high dose was associated with "profoundly reduced
body weight and weight gain" in parental animals, but details were not provided in either report.
Gross necropsy of adults revealed changes in the urinary bladder (thickening, hyperemia, and a
soft, velvety texture) in males treated with 1500 ppm and in both genders treated with 5000 ppm,
but the incidences were not reported. Prior to scheduled sacrifice, one male weanling in the
1500-ppm group and two male and two female weanlings in the 5000-ppm groups died.
Tyl et al. (1997) also reported reduced pup weight at 1500 ppm, but neither report provided data
or statistical analysis of this endpoint. The incidences of hyperplasia of the urinary bladder
epithelium were significantly increased over controls in female weanlings of the 1500- and
5000-ppm groups and in male weanlings in the 5000-ppm group (see Table 9). The severity of
hyperplasia was dose-related and ranged from minimal to moderate. Because testicular lesions
were observed in only one high-dose male pup, Tyl et al. (1997) concluded that "no unequivocal
treatment-related lesions" were observed in the testes of male weanlings. No further information
regarding experimental method or results was provided in either report. This study identified a
NOAEL of 100 ppm (9 and 10 mg/kg-day in males and females, respectively). It is unclear
whether the 300-ppm dose (26 and 29 mg/kg-day in males and females, respectively) was a
LOAEL or a NOAEL: the 1/10 response rate is not statistically significant, but it could have
been biologically significant for urinary bladder effects in female weanling and adult male rats,
as well as reduced pup weights. Prior to sacrifice, one male weanling 1500 ppm (147 mg/kg-day
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maternal dose) died. Tyl et al. (1997) attributed the weanling mortality at the next higher dose
(5000 ppm) to tributyl phosphate treatment. However, in the definitive study (described below),
no treatment-related fetal or weanling mortality was observed when larger groups of CD rats
were exposed to dietary concentrations up to 3000 ppm. Thus, it is not clear whether the death
of the single male weanling in the 1500-ppm group of the range-finding study was related to
treatment.
Table 9. Incidence of Urinary Bladder Hyperplasia in Weanling Rats Exposed
In Utero and Via Lactation to Dietary Tributyl Phosphate3

Urinary Bladder
Hyperplasia
Control
100 ppm
300 ppm
1500 ppm
3000 ppm
Maternal mg/kg-day
0
10
29
147
490
Male
Minimal
0/10
1/10
1/10
1/9
2/8
Mild
0/10
0/10
0/10
0/9
4/8
Moderate
0/10
0/10
0/10
0/9
1/8
Total
0/10
1/10
1/10
1/9
7/8b
Female
Minimal
1/10
0/10
0/10
5/10
0/8
Mild
0/10
0/10
0/10
2/10
6/8
Moderate
0/10
0/10
0/10
0/10
2/8
Total
1/10
0/10
0/10
7/10b
8/8b
aSOCMA, 1991; Tyl et al., 1997
bSignificantly different from control by Fisher's exact test conducted for this review, p < 0.01
In the definitive two-generation reproductive toxicity study (Tyl et al., 1997), weanling
CD rats (30/gender/dose), designated the F0 generation, were administered tributyl phosphate
(99.7% pure) in the diet at 0, 200, 700, or 3000 ppm beginning 10 weeks prior to a 3-week
mating period (females were randomly mated with males from the same dose group). Exposure
to tributyl phosphate was continued throughout the entire study period. Pups (F1 generation)
were counted, sexed, and examined grossly on Postnatal Days (PND) 1, 4, 7, 14, and 21. Litters
were randomly culled to a maximum of eight (with as equal a gender ratio as possible) on
PND 4. At weaning on PND 21, 30 F1 weanlings/gender/group were randomly selected as
parents of the F2 generation and the remaining weanlings were examined externally, sacrificed,
and 10/gender/group necropsied. Parental F0 animals also were necropsied: males after mating,
females after weaning the F1 litters. F1 weanlings continued to consume the F0 diets of their
parents for 11 weeks before mating. The F1 parents and the F2 offspring were then treated as
described above for the F0 and F1 generations. F0 and F1 parental necropsy included gross
examination and histopathological examination of any gross lesions and the following tissues
from high-dose and control animals: pituitary, ovaries, testes, vagina, uterus, epididymides,
seminal vesicles, prostate, urinary bladder, kidneys, and liver. Gross lesions, urinary bladders,
male kidneys, and female livers of mid- and low-dose animals also received microscopic
examinations. F1 and F2 weanlings also received a gross examination and gross lesions were
preserved, but the lesions were not microscopically examined. The incidence of
treatment-related histopathological findings in parental animals was not analyzed statistically.
Tyl et al. (1997) calculated tributyl phosphate consumption for each interval based on body
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weight and food consumption data. Table 10 presents the approximate ranges of tributyl
phosphate doses for males and females.
Table 10. Rat Doses in a 2-Generation Reproductive Toxicity Study
of Tributyl Phosphatea
Generation
Gender
Period of Study
Approximate doses (mg/kg-day)
200 ppm
700 ppm
3000 ppm
F0
Males
Prebreeding
11-18
38-65
160-264
Females
Prebreeding
12-17
41-60
178-266
Gestation
13
47
214
Lactation
26
94
404
F1
Males
Prebreeding
10-21
36-72
166-328
Females
Prebreeding
13-21
44-70
193-316
Gestation
13
45
217
Lactation
31
107
502
aTyl et al., 1997
F0 and F1 males and females in the 3000-ppm group showed significant (p < 0.01)
reductions in body weight and body-weight gain over all treatment periods, as did F1 females in
the 700-ppm group over most of the treatment periods. Data were presented graphically;
however, body weight decrements appeared to be at least 10% following the prebreeding period
in both genders exposed at the high dose. No reproductive effects (mating and fertility indices,
gestational length, or histology of the reproductive organs) attributable to tributyl phosphate
exposure were observed in parental animals from the F0 or F1 generations. There were no
effects of treatment at any dose on total or live litter size, gender ratio, or pre- or postnatal loss
for F1 or F2 litters. F1 and F2 pup body weights were consistently and significantly (p < 0.05)
reduced at 3000 ppm. At 700 ppm, the F2 pup weights (per litter) were significantly reduced on
PND 1 and 21, and at 200 ppm F2 pup weights (per litter) were significantly reduced at PND 14.
There were no treatment-related clinical observations for pups during lactation and no
treatment-related necropsy findings for pups that died during lactation or were necropsied at
weaning. For adults, there were no treatment-related gross lesions in F0 or F1 animals in any of
the groups. Histologically, both male and female F0 and F1 rats exhibited urinary bladder
epithelial hyperplasia in the 700-ppm (incidence 16-22%) and 3000-ppm (incidence 100%)
groups. Urinary bladder hyperplasia also was observed in one of the F0 males, two of the
F1 males, and in two of the F0 females of the 200-ppm group. Renal pelvic epithelial
hyperplasia was observed in F0 and F1 males of the 3000-ppm group but not in males from the
other treatment groups. Hepatic centrilobular hypertrophy was observed in F0 and F1 females of
the 700- and 3000-ppm groups but not in females from the 200-ppm group. The incidences of
liver and kidney lesions are reported in Table 11. Although identification of a clear NOAEL and
LOAEL from these data is complicated by the ranges of doses received by the animals, these
findings suggest that tributyl phosphate is not a specific reproductive toxicant in the rat. These
findings also confirm the appearance of urinary bladder hyperplasia at dietary concentrations of
700 ppm and above, and possibly as low as 200 ppm.
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The developmental effects of tributyl phosphate have been studied in rats and rabbits. In
one rat study, Noda et al. (1994) treated pregnant Wistar rats (20 females/dose) by gavage with
0, 62.5, 125, 250, or 500 mg/kg-day of tributyl phosphate (purity 99.7%) in olive oil on
Gestation Day 7-17 and then sacrificed on Gestation Day 20. Parameters used to assess
maternal toxicity were mortality; clinical signs; body-weight gain; food consumption; liver,
kidney, spleen, and gravid uterus weights; and gross necropsy. The parameters used to assess
developmental toxicity include pregnancy rates; early and late resorptions; and fetal viability,
body weights, and external, skeletal, and visceral malformations. Data were analyzed using the
litter as the statistical unit.
Table 11. Incidence (as percent) of Treatment-Related Histopathological Findings in
Two-Generation Rat Reproductive Toxicity Study of Dietary Tributyl Phosphate3

Control
200 ppm
700 ppm
3000 ppm
Urinary bladder hyperplasia
F0 males
0.0b
3.3
75.9
100
F0 females
0.0
6.7
70.0
100
F1 males
0.0
7.1
53.3
100
F1 females
0.0
0.0
70.0
100
Liver centrilobular hypertrophy
F0 males
0.0
NA
NA
0.0
F0 females
0.0
0.0
10.0
93.3
F1 males
0.0
NA
NA
0.0
F1 females
0.0
0.0
3.3
83.3
Renal pelvic epithelial hyperplasia
F0 males
0.0
0.0
0.0
6.7
F0 females
0.0
NA
NA
0.0
F1 males
0.0
0.0
0.0
33.3
F1 females
0.0
NA
NA
0.0
"Tyletal., 1997
bPercent of animals affected; 29-30 animals examined per gender per group per generation.
Treatment produced a dose-related decrease in maternal body-weight gain and food
consumption (Noda et al., 1994). Body weights and body-weight gains adjusted for uterine
weights were significantly (p < 0.01) lower than controls in the 250- and 500-mg/kg-day groups.
Body weights were 7 and 9% lower than controls in the 250- and 500-mg/kg-day groups, but
adjusted body-weight gains were reduced by 39 and 75%, respectively. In addition, based on
data presented graphically, body weights in the 125-mg/kg-day group were significantly lower
than controls during the last 4 days of gestation. Piloerection and wetting of abdominal hair with
urine and salivation were observed in two-thirds of females during treatment with
500 mg/kg-day; these signs disappeared after the end of treatment. Transient salivation was
observed in only one female of the 250 mg/kg-day group. Absolute kidney and gravid uterus
weights were unaffected by treatment but significantly increased absolute liver weights
(7%> higher than controls) and decreased spleen weights (11%) were seen in the 500-mg/kg-day
group. No significant difference between groups was observed for the numbers of corpora lutea,
implants, living fetuses, incidence of dead or resorbed fetuses, gender ratio, or body weight of
living fetuses of either gender. One case of malformation was observed in the 125-mg/kg-day
group (conjoined twins with 3 forelimbs and 4 hindlimbs). The incidence of rudimentary lumbar
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rib was significantly increased among fetuses of the 500-mg/kg-day group (34 fetuses from
14 dams affected vs. 6 fetuses from 3 dams in the control group). No treatment-related increase
in visceral anomalies was observed. Developmental toxicity in the form of a significant increase
in rudimentary lumbar ribs was observed at a LOAEL of 500 mg/kg-day with a NOAEL of
250 mg/kg-day. This study also identified a LOAEL of 125 mg/kg-day for reduced maternal
body weight, and a malformed fetus, and a NOAEL of 62.5 mg/kg-day.
In an earlier rat study, Bio/dynamics Inc. (1991b) treated pregnant Sprague-Dawley rats
(24 females/dose; 64 days of age) by gavage with 0, 188, 375, or 750 mg/kg-day of tributyl
phosphate (purity 100%) in corn oil on Gestation Days 6-15 and then were sacrificed on
Gestation Day 20. Parameters used to assess maternal toxicity were mortality, clinical signs,
body-weight gain, food consumption, liver weight, and gross necropsy. Parameters measured to
assess developmental toxicity were pregnancy rates, early and late resorptions, and fetal
viability, body weights, and external visceral and skeletal variations and malformations.
High-dose dams had a significant increase in mortality rate (7/24). Bio/dynamics Inc. (1991b)
considered six of the seven deaths to be related to treatment, although the physiological cause of
these deaths was not reported. A dose-related decrease in maternal body-weight gain was
observed. After adjustment for uterine weights, total body-weight gains (during Gestation
Day 6-20) were 25, 43, and 87% lower than controls in the low-, mid-, and high-dose dams
(p < 0.01). Food consumption on Gestation Days 6-11 was significantly decreased in
mid- (12%) and high-dose (37%) dams. A dose-related increase in clinical signs (salivation,
yellow staining of the skin, red anogenital stains, wetness of ventral abdominal area, excessive
lacrimation) was observed in dams of all dose groups. A significant, dose-related increase in
relative liver weight was observed in all treatment groups, with no change in absolute liver
weight; this effect was probably related to the observed decrease in body-weight gain, rather than
a treatment-related effect on the liver. No abnormalities were observed following gross necropsy
of dams. The treatment had no adverse effects with respect to pregnancy rate, number of corpora
lutea, implantations, early or late resorptions, fetal viability, gender ratio, external or visceral
abnormalities, or skeletal malformations. Fetal weight was significantly (p < 0.01) decreased at
the high-dose. The incidence of fetuses with one or more ossification variations was
significantly (p < 0.01) increased in all treatment groups (69, 86, 91, and 94% in control through
high dose); these developmental delays were considered to be mild in the low- and mid-dose
groups (some were in the range of historical controls) but were extensive in the high-dose group.
This study identifies a LOAEL of 188 mg/kg-day for slight maternal (decreased body-weight
gain, clinical signs) and developmental toxicity (increased incidence of ossification variations)
and a FEL of 750 mg/kg-day for maternal mortality; no NOAELs are identified in this study.
Bio/dynamics Inc. (1991c) treated pregnant New Zealand White rabbits
(18 females/dose) by gavage with 0, 50, 150, or 400 mg/kg-day of tributyl phosphate (purity
99.7%) in corn oil on Gestation Days 6-18 and then sacrificed on Gestation Day 30. Evaluations
to assess maternal toxicity included mortality, clinical signs, body-weight gain, food
consumption, liver weights, gross necropsy, and histology of gross abnormalities. Evaluations to
assess developmental toxicity included pregnancy rates and early and late resorptions, and fetal
viability, body weights, and external, visceral, and skeletal variations and malformations. The
treatment had no adverse effects with respect to clinical signs, relative liver weights, pregnancy
rates, abortion, premature delivery, preimplantation loss, fetal viability, body weights, gender
distribution or external, visceral or skeletal abnormalities; however, two high-dose females
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died—one from handling errors and one from unknown causes. Body weight gain on Gestation
Days 6-9 was 15, -9, -15, and -34 g in control, low-, mid-, and high-dose females, respectively.
The loss of weight in the high-dose group was significantly greater than in controls. After
correction for uterine weight, all groups lost weight over the gestation period and there were no
treatment-related differences. Nonsignificant decreases in food consumption (9-21%) from
Day 7-14 of gestation and nonsignificant increases in number of resorptions/female and ratio of
resorptions/implants were observed in high-dose females. Bio/dynamics Inc. (1991c) considered
these statistically nonsignificant changes to be suggestive of a treatment-related effect in
high-dose females. This study identifies a NOAEL of 150 mg/kg-day and a LOAEL of
400 mg/kg-day for slightly reduced maternal weight gain during Gestation Day 6-9 and for
nonsignificant increases in number of fetal resorptions. Because the increase in fetal resorptions
was not statistically significant, it is unclear whether the high dose represents a LOAEL or
NOAEL for developmental toxicity.
Inhalation Exposure
Subchronic Studies
A study by Kalinina (1971), summarized in a review by the Bayer (1994), reported that
rats and rabbits (number, strain, and gender not specified) exposed to 13.6 mg/m3 of airborne
tributyl phosphate 5 hours/day, 5 days/week, for 4 months, showed a reduction in cholinesterase
activity to 33% after 3 months of exposure. Although the report did not specify, this appeared to
represent activity compared to that of either the untreated animals or the treated animals prior to
exposure. There also were unspecified effects on physiological and biochemical parameters of
the liver. Cholinesterase activity returned to normal in the post exposure period. In the same
study, exposure to lower concentrations (5.1 mg/m3 for rats and 4.8 mg/m3 for rabbits), for a
similar period of time, had no effect on cholinesterase activity. Although no further details of
this study were available, 4.8 and 5.1 mg/m3 appear to represent NOAELs in rats and rabbits,
respectively, for reduced cholinesterase activity.
OTHER STUDIES
Acute/Short-term Toxicity
In shorter-term studies, gross lesions of the urinary bladder were observed in male rats
treated with 1500 ppm in the diet for 4 weeks (SOCMA, 1991). The dose calculated for this
review was 129 mg/kg-day based on body weight and food consumption values from EPA, 1988.
Slightly decreased body-weight gain was observed in male mice treated in the diet with
5000 ppm for 4 weeks (dose calculated by Bio/dynamics Inc., 1990 was 803 mg/kg-day).
Decreased body-weight gain, 20% mortality, and renal tubular damage were observed in rats
treated by gavage with 130 mg/kg-day for 1 month (Mitomo et al., 1980). Degenerative changes
in seminiferous tubules (1 of 4 rats), hematological and serum chemistry effects, decreased
spleen weight, decreased rate of nerve conduction, and histological evidence of toxicity to the
peripheral nervous system were observed in rats treated by gavage with 0.42 ml/kg-day
(410 mg/kg-day) for 14 days (Laham et al., 1983; Laham and Long, 1984).
"3
Eller (1937) reported a 6-hour inhalation LC50 of 1359 mg/m in rats and a 4-5 hour LC50
of 2500 mg/m3 in cats. WHO (1991) reported single-dose oral LD50s in rats ranging from
approximately 1400 mg/kg (Johannsen et al., 1977; Mitomo et al., 1980) to approximately
3000 mg/kg (Dave and Lidman 1978; Eastman Kodak, 1986); in mice of 400-800 mg/kg
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(Eastman Kodak, 1986) and 900-1240 mg/kg (Mitomo et al., 1980); and in chickens of
1800 mg/kg (Johannsen et al., 1977).
Other Routes
The pneumotoxic effects of tributyl phosphate were assessed using biochemical markers
of damage in bronchoalveolar lavage fluid (BALF) (Salovsky et al., 1998). A group of 30 male
Wistar rats was treated intratracheally with 5 L of a 20% mixture of tributyl phosphate in
«-dodecane. A similar group of 30 rats was used as controls, but Salovsky et al. (1998) did not
state if these animals received an intratracheal instillation of //-dodecane or were untreated; six
animals from each group were sacrificed on posttreatment days 1, 3, 7, 14, and 28. The right
lungs were homogenized and the supernatant used for biochemical analysis, the left lungs were
lavaged and the BALF was used for cell counting and biochemical analysis. Analyses included
(1) for BALF: total cell number, lactate dehydrogenase activity, and total protein content; (2) for
serum: cholinesterase activity; and 3) for lung homogenate: superoxide dismutase activity,
catalase activity, glutathione peroxidase activity, glutathione reductase activity, cholinesterase
activity, and malondialdehyde content. Treated animals showed significant increases in cell
number, protein content, and lactate dehydrogenase activity of BALF and significant reductions
in serum cholinesterase activity and the activities of superoxide dismutase, glutathione
peroxidase, and glutathione reductase in lung homogenate on Day 1 compared with controls.
The decrease in superoxide dismutase continued to be significant up until Day 7, but significant
differences were not seen in the other enzyme activities at later time points. A single exposure to
tributyl phosphate appeared to induce moderate, but transient, injury to the lungs and produced
only mild inhibition of cholinesterase.
Neurotoxicity
Several studies were designed specifically to investigate the neurological effects of
tributyl phosphate. In a neurophysiology study by Laham et al. (1983), Sprague-Dawley rats
(10/gender/dose) were treated by gavage with 0, 0.28, or 0.42 ml/kg-day of tributyl phosphate for
14 consecutive days; the corresponding doses were 0, 273, or 410 mg/kg-day. High-dose males
had significantly decreased conduction velocity in the caudal nerve, and both genders had
histological evidence of toxicity to the sciatic nerve (retraction of Schwann cell processes
surrounding unmyelinated fibers) at the high-dose. This study identifies a NOAEL of
273 mg/kg-day and a LOAEL of 410 mg/kg-day for neurotoxicity in rats treated by gavage for
14 days.
Other neurotoxicity studies were conducted on hens. White Leghorn hens given two oral
doses of 1500 mg/kg of tributyl phosphate (the oral LD50 in hens, as determined in a preliminary
trial), separated by a 21-day interval, did not show behavioral or neuropathological evidence of
delayed neurotoxicity (Carrington et al., 1990). In the same study, a single dose of 1500 mg/kg
did not inhibit brain neurotoxic esterase in hens. A similar study was conducted by
Johannsen et al. (1977). They reported that administration of two oral doses of 1840 mg/kg of
tributyl phosphate to White Leghorn hens, separated by a 21-day interval, did not produce
behavioral or neuropathological evidence of delayed neurotoxicity.
Genotoxicity
A series of genotoxicity studies of tributyl phosphate have produced primarily negative
results. Tributyl phosphate was negative for genotoxicity in the following bacterial mutagenicity
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studies: a mutagenicity assay with the TA98, TA100, TA1535, TA1537, and TA1538 strains of
S. typhimurium, both with and without metabolic activation (Microbiological Associates, 1977);
a mutagenicity assay with the TA98, TA100, TA1535, and TA1537 strains of S. typhimurium,
both with and without metabolic activation (Bayer, 1985); a mutagenicity assay with the
hisC117, hisG46, hisD3052, TA1530, TA1531, TA1532, and TA1534 strains of S. typhimurium',
and a mutagenicity assay with WP2 isogenic strains of Escherichia coli (Hanna and Dyer, 1975).
A single Russian bacterial mutagenicity study (Gafieva and Chudin, 1986) reported positive
results in the TA1535 and TA1538 strains of S. typhimurium.
Tributyl phosphate also was negative in two in vitro assays with mammalian cells: a
CHO/HGPRT mutation assay, both with and without metabolic activation (Microbiological
Associates, 1990a), and a cytogenetics assay with Chinese hamster ovary cells, both with and
without metabolic activation (Microbiological Associates, 1990b). A recessive lethal mutation
test in the Oregon-R strain of Drosophila melanogaster (Hanna and Dyer, 1975) and an in vivo
cytogenetics bone marrow assay in rats (Microbiological Associates, 1990c) also gave negative
results.
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL
RfDs FOR TRIBUTYL PHOSPHATE
The database for tributyl phosphate includes several well conducted subchronic and
chronic toxicity studies in rats and mice, as well as a number of reproductive and developmental
toxicity studies in rats and rabbits. Table 12 summarizes the NOAELs and LOAELs from all the
subchronic oral studies that were of adequate quality for p-RfD derivation and Table 13
summarizes the chronic data. Short-term studies (<4 weeks duration) are not included because
adequate studies of subchronic duration identifying lower LOAELs are available.
As Tables 12 and 13 indicate, urinary bladder hyperplasia in male rats was observed at
lower doses than other endpoints. This effect has been observed in several subchronic dietary
studies (Bayer, 1996; Arnold et al., 1997; FMC Corporation, 1985; Cascieri et al., 1985), in a
subchronic gavage study (Laham et al., 1985), in a multigeneration reproductive toxicity study
(Tyl et al., 1997), and in a chronic dietary study (Auletta et al., 1998a). Urinary bladder
hyperplasia also was observed in mice of both genders exposed to tributyl phosphate for
13 weeks (Bio/dynamics Inc., 1991a). In addition, gross lesions of the urinary bladder were
observed in male and female rats exposed to tributyl phosphate in the diet (SOCMA, 1991;
Tyl et al., 1997). Thus, the animal data identify urinary bladder hyperplasia as an effect of
repeated oral exposure to tributyl phosphate. However, because several studies reported tumors
in this same tissue, it is possible that bladder hyperplasia is a preneoplastic or precursor effect.
An analysis of the mode of carcinogenic action for the formation of bladder tumors is conducted
in this assessment. The results of that analysis indicate that the key events in the hypothesized
mode of action of tributyl phosphate-induced bladder neoplasms are not well established, but the
available data suggest tributyl phosphate may induce regenerative cell proliferation in response
to epithelial damage in the bladders of rats. For this reason, bladder hyperplasia has not been
selected as the endpoint for derivation of the p-RfD, anticipating that the provisional oral slope
factor will be protective of this effect.
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Table 12. Summary of Oral Noncancer Dose-Response Information from Subchronic Studies Suitable for RfD Derivation
Species
Route
Dose (mg/kg-day)
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses at the LOAEL
Reference
Rat
Diet
0, 1, 3, 14, 68, 360 (m);
0, 1, 3, 16, 81, 423 (f)
14
68
(10/10 incidence)
Urinary bladder transitional cell hyperplasia3 in
males
FMC Corporation, 1985;
Cascieri et al., 1985
Rat
Diet
0, 15, 53, 230 mg/kg-day
for 10 weeks
15
53
(8/10 incidence)
Urinary bladder hyperplasia3 in males
Arnold et al., 1997; Bayer,
1996
Rat
Diet
0, 425, 870 mg/kg-day for
10 weeks
NA
425
Increased brain cholinesteraseb; decreased body-
weight gain, clinical chemistry effects, increased
blood coagulation time
Oishietal., 1980
Rat
Diet
0, 417 mg/kg-day, 9 wks
NA
417
Decreased body weight and increased BUN
Oishi et al., 1982
Rat
Gavage
0, 143, 238 mg/kg-day
(adjusted for continuous
exposure) for 18 weeks
NA
143
(6/6 incidence)
Urinary bladder hyperplasia3
Lahametal., 1985
Rat
Gavage
0, 32.5, 100,
325 mg/kg-day 7
days/week for 13 weeks
NA
32.5 (rare &
transient)
100 (frequent &
persistent)
Clinical signs of cholinergic toxicity (salivation);
muzzle staining, alopecia
Healy et al., 1995;
Bio-Research Laboratories,
1991
Rat
Gavage
0, 32.5, 100,
325 mg/kg-day
7 days/week for 13 weeks
32.5
100
(3/24 incidence)
Animal deaths, apparently from aspiration of saliva
exacerbated by gavage treatment
Healy et al., 1995;
Bio-Research Laboratories,
1991
Mouse
Diet
0, 96, 383,
1479 mg/kg-day (m) and
0, 119, 462,
1769 mg/kg-day (f) for
13 weeks
NA
96°
(1/10 incidence)
383
(8/10 incidence)
Urinary bladder hyperplasia3
Bio/dynamics Inc., 1991a
Mouse
Diet
0, 96, 383,
1479 mg/kg-day (m) and
0, 119, 462,
1769 mg/kg-day (f) for
13 weeks
96
383
(8/10 incidence)
Centrilobular hepatocyte hypertrophy (with
supporting evidence for liver effects)
Bio/dynamics Inc., 1991a
aApparently a preneoplastic lesion
Statistically significant but no dose-related increase in brain cholinesterase
°No statistically significant difference from control response (0/10)
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Table 13. Summary of Oral Noncancer Dose-Response Information from
Chronic and Developmental Studies Suitable for RfD Derivation
Species
Gender
Dose (mg/kg-day)
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses at the LOAEL
Reference
Rat
Diet
M/F
0, 8.9, 32.5,
143.3 mg/kg-day (m) and
0, 11.6, 42,
181.5 mg/kg-day (f) for
2 years
8.9 (m)
11.6(f)
32.5 (m)
12/49 incidence
42(f)
5/49 incidence
Urinary bladder hyperplasia3
Auletta et al., 1998a
Mouse
Diet
M/F
0, 28.9, 169,
585mg/kg-day (m) and 0,
24.1, 206, 711 mg/kg-day
(f) for 18 months
169 (m)
206(f)
585 (m)
711(f)
Decreased body weight
Auletta et al., 1998b
Rat
Developmental
Gavage
F
0, 62.5, 125, 250,
500 mg/kg-day on
GD 7-17
62.5 (maternal)
250 (developmental)
125 (maternal)
500 (developmental)
Reduced weight gain in dams; increased
incidence rudimentary lumbar ribs in
offspring
Nodaetal., 1994
Rat
Developmental
Gavage
F
0, 188, 375,
750 mg/kg-day on
GD 6-15
NA
188 (maternal and
developmental)
Clinical signs (salivation, staining of
skin, red anogenital staining, wetness of
abdominal area, lacrimation) in dams;
dose-related delays in skeletal
ossification
Bio/dynamics Inc., 1991b
Rabbit
Developmental
Gavage
F
0, 50, 150,
400 mg/kg-day on
GD 6-18
150 (maternal)
400 (fetal)
400 (maternal)
NA (fetal)
Significant body weight loss
Bio/dynamics Inc., 1991c
Rat 2-
generation
Reproductive
Diet study
M/F
10-21, 36-72, and
160-328 mg/kg-day (m),
and 12-31, 41-107,
178-502 mg/kg-day (f)

10-3 lb
(3.3-6.7% incidence,
F0; 7.1% incidence,
F1 males)
36-107
Urinary bladder hyperplasia3 (both
genders and both generations)
Tyletal., 1997
"Apparently a preneoplastic lesion
bNot a statistically significant difference from the control response (0%)
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Evidence of peripheral neurotoxicity was observed in rats treated by gavage with
410 mg/kg-day of tributyl phosphate for 14 days (Laham et al., 1983). No evidence for delayed
neurotoxicity was observed in hens exposed orally to 1500-1840 mg/kg, over a 21-day period
(Johannsen et al., 1977; Carrington et al., 1990).
In a more comprehensive 13-week gavage study in rats, Healy et al. (1995;
Bio-Research Laboratories, 1991) observed increased salivation among a majority of the rats
treated with 100 mg/kg-day and occasionally at 32.5 mg/kg-day. Although Healy et al. (1995)
did not classify salivation as resulting from neurotoxicity, it is a classic sign of a cholinergic
response (Costa, 2008; Lotti, 2001; Rhone-Poulenc, 1992; Union Carbide, 1971;
U.S. EPA, 1993). In this same study, 3/24 rats died following treatment with lOOmg/kg-day and
7/24 died following treatment with 325 mg/kg-day. However, based on the observations of
Healy et al. (1995; Bio-Research Laboratories, 1991) and cholinergic response data reported by
Gallo and Lawryk (1991), and Lotti (2001), it is concluded that the deaths are attributable to
aspiration of saliva, resulting from the salivation response in concert with respiratory inhibition
typical of cholinergic toxicity, and exacerbated by the gavage treatment.
Other studies also reported salivation responses following treatment with TBP.
Noda et al. (1994) reported salivation in two-thirds of pregnant rats following gavage treatment
with 500 mg/kg-day on gestational Days 7-17, in only 1/20 at 250 mg/kg-day, and among no rats
treated with lower doses. Bio/dynamics Inc. (1991b) reported a dose-related increase in clinical
signs including salivation in all dose groups of pregnant rats (0, 188, 375, or 750 mg/kg-day via
gavage).
Bio/dynamics Inc. (1991c) reported no developmental effects in rabbits treated with
doses up to 400 mg/kg-day. Several other oral developmental toxicity studies found no evidence
of selective toxicity to the fetus in rats. Developmental effects always were accompanied by
maternal toxicity in rats. Noda et al. (1994) found developmental toxicity in the form of a
significant increase in rudimentary lumbar ribs at 500 mg/kg-day of tributyl phosphate.
Bio/dynamics Inc. (1991b) observed slight developmental toxicity in the form of ossification
variations in rats at 188 mg/kg-day of tributyl phosphate and Tyl et al. (1997) observed
developmental toxicity in the form of reduced weight of offspring in rats exposed to 3000 ppm
(214-217 mg/kg-day) of tributyl phosphate during a 2-generation reproductive study. No
exposure-related effects on reproductive performance or reproductive organ histology were
found in the 2-generation rat study. However, developmental and reproductive endpoints are not
critical for tributyl phosphate because maternal systemic toxicity was evident at much lower
doses than those at which developmental or reproductive effects have been reported.
Urinary hyperplasia is not chosen as the critical effect for derivation of p-RfDs because
this endpoint was likely to be a preneoplastic effect. However, cholinergic effects, including
salivation, were reported in several rat studies; salivation is recognized as an early effect of
organophosphate insecticide toxicity (Costa, 2008). The subchronic rat study by
Healy et al. (1995; Bio-Research Laboratories, 1991) reported a dose-related increase in
salivation among male and female rats gavaged with tributyl phosphate, beginning with
occasional effects at 32.5 mg/kg-day. Although Healy et al. (1995; Bio-Research Laboratories,
1991) reported deaths among 3/24 rats at the next higher dose (100 mg/kg-day), these deaths are
attributed to aspiration of contaminated saliva. Based on the following data, these deaths are
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attributed, in part, to the gavage administration and considered indicators of increased severity of
the cholinergic salivation response. Another gavage study using doses averaging up to
238 mg/kg-day for 18 weeks (Laham et al., 1985) reported no mortality. In addition, dietary
studies using much higher doses of tributyl phosphate, including the following, reported no
increased mortality:
•	Rats, fed up to 230 mg/kg-day for 10 weeks (Arnold et al., 1997; Bayer, 1996).
•	Rats, fed up to 423 mg/kg-day for 13 weeks (FMC Corporation, 1985;
Cascieri et al., 1985).
•	Rats, fed up to 870 mg/kg-day for 10 weeks (Oishi et al., 1980, 1982).
•	Mice fed up to 1769 mg/kg-day for 13 weeks (Bio/dynamics Inc., 1991a).
•	Rats fed up to 181.5 mg/kg-day for 2 years (Auletta et al., 1998a).
•	Female mice fed 711 mg/kg-day for 18 months, although males fed 585 mg/kg-day
exhibited a slight, statistically insignificant increase in mortality
(Auletta et al., 1998b).
SUBCHRONIC p-RfD DERIVATION
The subchronic Healy et al. (1995; Bio-Research Laboratories, 1991) rat gavage data
demonstrating dose-related increased salivation and deaths is selected as the key study for the
subchronic p-RfD. The following potential points of departure (POD) are identified from these
data:
•	32.5 mg/kg-day is a LOAEL for occasional salivation.
•	A BMDL5 of 14.6 mg/kg-day, calculated from the data revealing deaths from
aspiration of saliva among 0/24 male and female rats treated with 32.5 mg/kg-day,
3/24 rats at 100 mg/kg-day, and 7/24 rats at 325 mg/kg-day (see Appendix B). The
BMDLio is not considered as a potential POD because the BMDLi0s calculated by all
models were greater than the LOAEL (see Table B-l).
Because the mode of action is known, lethality from aspiration of saliva is considered as a
surrogate for increased severity of the cholinergic salivation response. Assuming application of
the same uncertainty factors (UF) for parameters other than for the POD and rounding to one
significant figure, two potential PODs were compared as follows:
•	LOAEL of 32.5 mg/kg-day divided by an UFL of 10 = 3.25 mg/kg-day.
•	BMDL5 of 14.6 mg/kg-day divided by an UFL of 1 = 14.6 mg/kg-day.
Other UFs being equal, the LOAEL of 32.5 mg/kg-day would result in a lower p-RfD and is
selected as the POD for deriving a subchronic p-RfD.
A composite UF of 1000 is applied to the LOAEL POD to calculate a subchronic p-RfD
as follows:
Subchronic p-RfD = LOAEL UF
= 32.5 mg/kg-day ^ 1000
= 0.0325 mg/kg-day or 3 x 10"2 mg/kg-day
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The composite UF of 1000 is composed of the following:
•	An UF of 10 has been applied for extrapolating from a LOAEL to a NOAEL for
estimating the POD.
•	An UF of 10 has been applied for interspecies extrapolation to account for
potential pharmacokinetic and pharmacodynamic differences between rats and
humans.
•	An UF of 10 for intraspecies differences has been applied to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans.
•	No database uncertainty factor is required. The toxicological database for oral
tributyl phosphate included high quality chronic and subchronic bioassays in two
species, four developmental toxicity studies in two species, a multigeneration
reproduction study, and a subchronic neurotoxicity study. Although there were
multigeneration reproductive toxicity data in only one species, available
information suggested that systemic maternal toxicity occurred at lower doses
than reproductive or developmental effects. Although the critical study
(Healy et al., 1995; Bio-Research Laboratories, 1991) demonstrated infrequent
effects at the lowest dose tested and a death that apparently was treatment-related
occurred at the next higher dose, which was only a factor of 3 higher than the
POD dose, other studies (Noda et al., 1994; Bio/dynamics Inc., 1991b)
demonstrated higher NOAELs and LOAELs for similar effects. Therefore, it is
concluded that additional data are unlikely to result in a lower POD or subchronic
p-RfD.
Confidence in the principal study (Healy et al., 1995; Bio-Research Laboratories, 1991)
is medium. This was a subchronic oral toxicity study that used 12 animals/gender/dose exposed
to a range of doses and measured sensitive endpoints in the species that appeared to be most
sensitive. However, it does not identify a NOAEL. Confidence in the database is
moderate-to-high. While the animal database contained high quality studies on a variety of
endpoints and in multiple species, there were no human data or mechanistic information to
determine whether the critical endpoint observed in animal species also is relevant to humans.
Confidence in the subchronic p-RfD is, therefore, medium-to-high.
CHRONIC p-RfD DERIVATION
The 2-year rat bioassay by Auletta et al. (1998a) has been considered as a potential
principal study for p-RfD derivation because it identified the lowest chronic LOAEL within the
array of animal data. However, the critical effect—urinary bladder hyperplasia—seems likely to
be a preneoplastic effect, in light of tumors reported in the same tissue in rats and mice
(Auletta et al., 1998a,b). There is uncertainty associated with whether or not this effect is a
precursor to tumor formation, and as such, this effect was not chosen for derivation of the p-RfD.
Auletta et al. (1998b) also identified an 18-month NOAEL of 169 mg/kg-day for
decreased body-weight gain in mice. The Bio/dynamics Inc. (1991c) developmental study
demonstrates a similar 13-day NOAEL of 150 mg/kg-day for weight loss in pregnant rabbits
while the Noda et al. (1994) data identify an 11-day NOAEL of 62 mg/kg-day and a LOAEL of
125 mg/kg-day for reduced weight gain in pregnant rats. The only chronic study
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(Bio/dynamics Inc., 1991b) that demonstrated the subchronic critical effect (cholinergic
salivation) identifies a LOAEL of 188 mg/kg-day for that effect. When considering these data in
concert with the subchronic data, the subchronic LOAEL of 32.5 mg/kg-day, used as the
subchronic POD, also is selected as the POD for deriving the chronic p-RfD.
The subchronic LOAEL of 32.5 mg/kg-day for salivation in male and female rats
(Healy et al., 1995; Bio-Research Laboratories, 1991) is used as the POD for calculation of the
chronic p-RfD because it is lower than other potential PODs. A composite UF of 3000, as
discussed above, is applied to the POD to calculate a chronic p-RfD as follows:
Chronic p-RfD = Subchronic LOAEL UF
= 32.5 mg/kg-day 3000
= 0.011 mg/kg-day or 1 x 10" mg/kg-day
The composite UF of 3000 is composed of the following:
•	An UFl of 10 has been applied for using a LOAEL POD.
•	An UFs of 3 is applied for using subchronic data to derive a chronic p-RfD. The
chronic data demonstrated the salivation effect only at higher doses, and LOAELs
for other effects were at similar or higher doses. This suggests that no additional
UF would be necessary for application of the subchronic POD. However, a
review of the detailed data (Bio-Research Laboratories, 1991,
p. B124-131 [tables 67 & 68]) notes a gradual increase in frequency of this effect
during the progression of the 13-week study, at the middle and high doses.
Although the biology of cholinergic responses suggests it is unlikely for the
critical effect to continue to increase in severity, an UF of 3 was applied for using
a subchronic POD to derive the chronic p-RfD.
•	An UFa of 10 has been applied for interspecies extrapolation to account for
potential pharmacokinetic and pharmacodynamic differences between rats and
humans.
•	An UFh of 10 for intraspecies differences has been applied to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans.
•	No database uncertainty factor is required. The toxicological database for oral
tributyl phosphate included high quality chronic and subchronic bioassays in two
species, four developmental toxicity studies in two species, a multigeneration
reproduction study, and a subchronic neurotoxicity study. Although there were
multigeneration reproductive toxicity data in only one species, available
information suggested that systemic maternal toxicity occurred at lower doses
than reproductive or developmental effects. Although the critical study
(Healy et al., 1995; Bio-Research Laboratories, 1991) demonstrated infrequent
effects at the lowest dose tested and a death that apparently was treatment-related
occurred at the next higher dose, which was only a factor of 3 higher than the
POD dose, other studies (Noda et al., 1994; Bio/dynamics Inc., 1991b)
demonstrated higher NOAELs and LOAELs for similar effects. Therefore, it is
concluded that additional data are unlikely to result in a lower POD or subchronic
p-RfD.
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Confidence in the principal study (Healy et al., 1995; Bio-Research Laboratories, 1991)
is medium. This was a subchronic oral toxicity study that used 12 animals/gender/dose exposed
to a range of doses and measured sensitive endpoints in the species that appeared to be most
sensitive. However, it does not identify a NOAEL. Confidence in the database is medium.
While the animal database contained high quality studies on a variety of endpoints and in
multiple species, there were no human data or mechanistic information to determine whether the
critical endpoint observed in animal species also is relevant to humans. In addition, the critical
study was only of subchronic duration. Confidence in the chronic p-RfD is, therefore, medium.
FEASIBILITY OF DERIVING SUBCHRONIC AND CHRONIC PROVISIONAL
RfCs FOR TRIBUTYL PHOSPHATE
No human inhalation studies suitable for derivation of subchronic or chronic p-RfCs for
tributyl phosphate have been located. Human data were limited to epidemiological studies of
workers exposed to mixed compounds, including tributyl phosphate, and animal studies that
examined only acute exposure or were inadequately described. Tributyl phosphate has a very
low vapor pressure, reported at 25°C as 4 x 10"3 mmHg by NIOSH (2005) and 2.6 x 10"6 mmHg
by OECD (2001). Consequently, inhalation exposures originating from Superfund sites are
likely to be low and infrequent.
In the only animal inhalation study identified, Kalinina (1971) reported that rats and
rabbits (number, strain, and gender not specified) exposed to 13.6 mg/m of airborne tributyl
phosphate 5 hours/day, 5 days/week, for 4 months showed a reduction in cholinesterase activity
to 33% after 3 months of exposure. Kalinina (1971) also reported unspecified effects on
physiological and biochemical parameters of the liver. Kalinina (1971) reported that
cholinesterase activity returned to pretreatment levels in the postexposure period. In the same
-3
study, Kalinina (1971) reported exposure to lower concentrations (5.1 mg/m for rats and
4.8 mg/m3 for rabbits) for a similar period of time had no effect on cholinesterase activity. No
further details of this study were provided. The sketchy inhalation data in rabbits and rats
(Kalinina, 1971) are supported by oral data in rats that demonstrated (1) cholinergic responses at
425 mg/kg-day, the lowest dose tested by Oishi et al. (1980), and (2) clinical signs consistent
with cholinergic toxicity at doses as low as 32.5 mg/kg-day (Healy et al., 1995; Bio-Research
Laboratories, 1991). However, the Kalinina (1971) data were not peer reviewed and available
only from a secondary source (Bayer, 1994). In addition, that source did not report particle sizes,
animal gender, or strain. The Bayer (1994) source also did not specify whether these data
represented cholinesterase activity compared to that found in untreated animals, in treated
animals prior to exposure, or in some other benchmark. Because of these reporting deficiencies,
the 5 hours/day, 5 days/week, 4-month NOAELs for cholinergic effects of 4.8 mg/m3 in rabbits
-3
and 5.1 mg/m in rats are not used for derivation of a p-RfC.
Inadequate data are available to attempt to extrapolate an inhalation value using the oral
data. Consequently, no p-RfCs are derived.
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PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
TRIBUTYL PHOSPHATE
WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), tributyl
phosphate is considered "Likely to be Carcinogenic to Humans, " by the oral route of exposure,
based on a finding of increased tumor incidence in more than one species, gender, strain, and
site. Chronic (24-month) dietary administration of tributyl phosphate at a concentration of
3000 ppm produced statistically significant increased incidences of transitional cell (6/49) or
squamous cell (1/49) urinary bladder carcinomas in male rats and of urinary bladder papillomas
in male (23/49) and female (11/49) rats (Auletta et al., 1998a). Transitional cell carcinomas also
were observed in 2/49 female rats exposed to this dietary concentration, but this incidence was
not significantly different from the concurrent control incidence of 0/50. Urinary bladder
transitional cell carcinomas are an unusual type of tumor occurring in control male Sprague-
Dawley rats at incidences of 1/857 in the testing laboratory and 3/1250 in a breeding laboratory;
corresponding female incidences have been reported as 0/779 and 1/1249 (Charles River
Laboratories, 1992). The elevated incidences of urinary bladder transitional cell carcinomas in
male and female rats are evidence of carcinogenicity due to the rarity of this tumor type. Oral
exposure to tributyl phosphate also resulted in an increased incidence of hepatocellular adenomas
in male mice exposed to 3500-ppm tributyl phosphate in the diet (Auletta et al., 1998a,b).
Numerous short-term tests in both bacterial and mammalian systems have provided no evidence
for genotoxic activity of tributyl phosphate.
MODE-OF-ACTION DISCUSSION
The EPA (2005) Guidelines for Carcinogen Risk Assessment defines mode of action as a
sequence of key events and processes, starting with the 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 include mutagenic, mitogenic, antiapoptotic
(inhibition of programmed cell death), cytotoxic with reparative cell proliferation and
immunologic suppression.
Urinary Bladder Tumors
The mode of action for tributyl phosphate-induced bladder carcinogenesis has not been
conclusively identified. Results from short-term in vitro and in vivo genotoxicity tests have been
consistently negative. Evidence obtained from a study published by Arnold et al. (1997)
provided some support for a hypothesis that carcinogenic responses in the urinary bladder of rats
may arise from increased cell proliferative responses to epithelial damage caused by high doses
of tributyl phosphate or one of its metabolites. In this study, exposure of male Sprague-Dawley
rats for 10 weeks to dietary concentrations of 700 or 3000 ppm of tributyl phosphate (but not
200 ppm) induced increased incidence of urinary bladder hyperplasia that was accompanied, in
the more severe cases, by focal necrosis of the epithelium, with erosion, ulceration and
hemorrhage into the lumen in some areas (Arnold et al., 1997). Treatments to acidify the urine
did not totally inhibit the proliferative response in the bladder epithelium, but it caused the
effects of 10-weeks of exposure to 3000 ppm to be less severe (Arnold et al., 1997). Treatments
to acidify the urine inhibit the formation of calcium phosphate urinary precipitates (and
subsequent urinary bladder hyperplasia) in rats fed high levels of sodium phosphate
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(Arnold et al., 1997). Microscopic examination of filters used to sieve urine from rats in this
study did not show evidence of abnormal precipitate, microcrystals, or calculi, providing
evidence that formation of solid material was not the cause of the epithelial necrosis associated
with tributyl phosphate exposure (Arnold et al., 1997).
Key Events
Based on the limited available data, a hypothetical key event in the mode of action for
tributyl phosphate bladder carcinogenesis may be damage to the bladder epithelial cells, resulting
in regenerative hyperplasia and enhanced growth of initiated cells. Cell proliferation is believed
to increase tumor formation through one or more of the following mechanisms (Butterworth et
al., 1995; Barrett, 1993):
•	Increased number of spontaneous initiations occurring during replication.
•	Inhibition of apoptosis of initiated cells.
•	Promotion of clonal expansion of initiated cells.
•	Increased rate of neoplastic progression.
•	Selective growth advantage of initiated cells.
•	Reduced time available for DNA repair mechanisms.
Strength, Consistency, Specificity of Association
There is abundant information to support an association between tributyl phosphate
exposure and hyperplasia of the urinary bladder epithelium. This association has been observed
in both rats and mice in subchronic, chronic, and multigeneration reproductive toxicity studies
(FMC Corporation, 1985; Cascieri et al., 1985; Arnold et al., 1997; Bayer, 1996; Laham et al.,
1985; Bio/dynamics Inc., 1991a; Auletta et al., 1998a; SOCMA, 1991; Tyl et al., 1997). The
data supporting associations between cell toxicity and hyperplasia, and between hyperplasia and
neoplasia were more limited. Arnold et al. (1997) reported that the bladders of rats with
hyperplasia showed evidence of focal necrosis of the epithelium with erosion, ulceration, and
hemorrhage, with dose-related increases in severity. In contrast, another subchronic study in rats
did not report evidence of cell toxicity accompanying transitional cell hyperplasia of the bladder
(FMC Corporation, 1985; Cascieri et al., 1985). Auletta et al. (1998a) noted that rats with
bladder papillomas frequently also had hyperplasia of the bladder, providing limited evidence for
a potential link between this key event and tumor formation.
Dose-response Concordance
Table 14 shows the dose-response concordance for the available data on key events in
tributyl-phosphate-induced bladder tumors, including cell proliferation, increases in absolute and
relative bladder weight, histopathological evidence of hyperplasia, and incidence of bladder
tumors in male Sprague-Dawley rats. Overall, the data indicate that evidence of cell
proliferation (increased BRdU labeling index), increased bladder weight, and increased tumor
formation occurred at dietary concentrations of >3000 ppm and epithelial hyperplasia occurred at
dietary concentrations of >700 ppm. Arnold et al. (1997) reported evidence of focal necrosis of
the epithelium in the bladders of rats exposed to 700 ppm and higher; however, incidences were
not reported. The FMC Corporation (1985; Cascieri et al., 1985) did not report evidence of cell
toxicity accompanying transitional cell hyperplasia of the bladder in Sprague-Dawley rats
exposed to 1000 or 5000 ppm for 13 weeks. Neither lifetime nor subchronic (10-13 week)
exposure of rats to concentrations of 200 ppm induced urinary bladder hyperplasia or urinary
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Table 14. Dose-Response Concordance of Key Effects in the Urinary Bladder
of Male Sprague-Dawley Rats Treated with Tributyl Phosphate in the Diet
Reference
Exposure
Duration
Effect
Dietary Concentration (ppm)
0
200
700
1000
3000
5000
Auletta et al.,
1998a
2 years
epithelial hyperplasia (incidence)
3/50
3/50
12/49b
NT
17/49°
NT
papilloma (incidence)
0/50
0/50
2/49
NT
23/49°
NT
squamous cell carcinoma (incidence)
0/50
0/50
0/49
NT
1/49
NT
transitional cell carcinoma (incidence)
0/50
0/50
0/50
NT
6/4 9 d
NT
FMC Corporation,
1985; Cascieri et
al., 1985
13 weeks
transitional cell epithelial hyperplasia
(incidence)
0/10
0/10
NT
10/10°
NT
10/10°
Arnold etal., 1997;
Bayer, 1996
10 weeks
simple hyperplasia (incidence)
0/10
0/10
8/10°
NT
10/10°
NT
papillary/nodular hyperplasia (incidence)
0/10
0/10
2/10
NT
6/10°
NT
absolute bladder weight (g)
0.124 ±0.010a
0.129 ±0.009
0.155 ±0.011

0.218 ±0.027b

bladder/body weight (g/kg)
0.227 ±0.016
0.238 ±0.019
0.297 ± 0.020

0.446 ± 0.052b

BRdU labeling index
0.20 ±0.03
0.34 ±0.16
0.48 ±0.12
NT
1.81 ± 0.30d
NT
Arnold etal., 1997;
Bayer, 1996
10 weeks,
followed by
10 weeks
recovery
simple hyperplasia (incidence)
0/9
NT
NT
NT
2/8
NT
papillary/nodular hyperplasia (incidence)
0/9
NT
NT
NT
0/8
NT
fibrosis of submucosa (incidence)
0/9
NT
NT
NT
6/8°
NT
absolute bladder weight (g)
0.155 ±0.014
NT
NT
NT
0.227 ± 0.01 lb
NT
bladder/body weight (g/kg)
0.235 ±0.020
NT
NT
NT
0.357 ±0.027b
NT
BRdU labeling index
0.12 ±0.02
NT
NT
NT
0.08 ±0.01
NT
aMean ± standard error
bSignificantly different from control,/) < 0.05.
><0.01
dArnold et al. (1997) reported that this increase was statistically significant when compared with controls but did not report ap-value.
NT = not tested
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bladder tumors (Arnold et al., 1997; Auletta et al., 1998a; FMC Corporation, 1985), nor was
there evidence of cell proliferation after 10 weeks at this concentration (Arnold et al., 1997).
Auletta et al., 1998a described 200 ppm (9 and 12 mg/kg-day in male and female rats,
respectively) as a "NOEL for chronic toxicity", and 700 ppm (33 and 42 mg/kg-day in male and
female rats, respectively) as a "clear threshold" for oncogenic effects However, Tyl (1997;
SOCMA, 1991) reported bladder hyperplasia in 1/10 weanling male rats (see Table 9) following
maternal dietary exposure to 100 ppm (10 mg/kg-day).
Temporal Relationships
Studies of subchronic duration (10-13 weeks) have shown evidence of bladder epithelial
cell hyperplasia (Arnold et al., 1997; Bayer, 1996; FMC Corporation, 1985) without tumor
formation, indicating that hyperplasia may be a precursor to neoplastic growth. Arnold et al.
(1997) showed that tributyl phosphate-induced urinary bladder hyperplasia was reversible upon
withdrawal of the treatment compound. No urinary bladder epithelial hyperplasia was evident in
rats exposed to 3000 ppm of tributyl phosphate for 10 weeks, followed by a 10-week unexposed
recovery period, although hyperplasia was evident in all rats exposed to the same concentration
for 10 weeks and examined upon treatment termination. However, 6/8 rats in the
treated/recovery group showed fibrosis of the submucosa, compared with 0/9 in the control
group. Arnold et al. (1997) attributed the fibrosis to scarring, providing further evidence for the
role of cell toxicity in the induction of hyperplasia.
Biological Plausibility and Coherence
Dibutyl hydrogen phosphate, one of the major metabolites of tributyl phosphate in rats
(Suzuki et al., 1984), has been shown to produce similar regenerative hyperplasia of the urinary
bladder in rats (Chemicals Investigation Promoting Committee, 1995), providing potential
support for the hypothesized mode of action.
Conclusions
The key events in the hypothesized mode of action of tributyl phosphate-induced bladder
neoplasms have not been well established, although available data suggest that tributyl phosphate
may induce regenerative cell proliferation in response to epithelial damage in the bladders of
rats. It is possible that tributyl phosphate could induce cancer in either rodents or humans by a
mode of action not associated with regenerative cell proliferation.
Hepatocellular Adenomas
Very little information is available on the potential mode of action by which tributyl
phosphate increases the incidence of liver tumors in male mice. Only two studies were available
in mice: a subchronic toxicity study (Bio/dynamics Inc., 1991a) and the chronic bioassay that
reported an increased incidence of hepatocellular adenomas in male CD-I mice exposed to
3000-ppm tributyl phosphate for 18 months (Auletta et al., 1998b). No mechanistic studies
examining this endpoint were located. The subchronic study reported increased absolute and
relative liver weights and an increased incidence of centrilobular hepatocyte hypertrophy at
dietary concentrations of 2000 ppm and higher, as well as increases in circulating liver enzymes
at 8000 ppm (Bio/dynamics Inc., 1991a). The chronic study (Auletta et al., 1998b) reported
increased absolute and relative liver weights at >1000 ppm and increased hepatocellular
adenomas at 3000 ppm, but it did not observe an increased incidence of hepatocyte hypertrophy,
though liver enzymes were not analyzed. Auletta et al. (1998b) attributed the lack of
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hypertrophy to the development of tolerance to chronic tributyl phosphate administration. These
limited data are inadequate for outlining the potential key events in the mode of action for
tributyl phosphate-induced hepatocellular adenomas.
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
Oral Exposure
Oral data are sufficient to derive a quantitative estimate of cancer risk from tributyl
phosphate; this derivation is shown below. The urinary bladder response in male
Sprague-Dawley rats (Auletta et al., 1998a) is the basis of the dose-response analysis because
(1) it is the most pronounced carcinogenic response (e.g., compared with the liver tumor
response in male mice) and (2) the mechanistic understanding is inadequate to explain species
differences in tumorigenic responses to tributyl phosphate or to determine which laboratory
animal species is a better model for humans. The mode of action for bladder tumors produced by
tributyl phosphate in Sprague-Dawley rats has not been fully elucidated. Available data suggest
that development of these tumors may be related to induction of regenerative cell proliferation in
response to epithelial damage. Although the available data do not suggest mutagenic action of
tributyl phosphate, no alternative mode of action has been sufficiently characterized. Therefore,
a linear low-dose extrapolation is conducted.
Table 15 shows the dose-response data used in the quantitative cancer assessment. First,
the animal doses in the Auletta et al. (1998a) rat study are converted to human equivalent doses
(HEDs) by adjusting for differences in the body weights between humans and rats. In
accordance with EPA (2005), a factor of BW3 4 is used for cross-species scaling. Because the
test chemical was administered in the diet ad libitum for 2 years, no adjustment for discontinuous
exposure or less-than-lifetime administration is necessary. The equation used to calculate the
HEDs is shown below and Table 15 presents the HEDs.
HED = Dose x (W -70 kg)1/4
where
Dose = average daily animal dose
W = reference rat body weight for chronic study (0.523 kg) (U.S. EPA, 1988)
70 kg = reference human body weight (U.S. EPA, 1988)
Table 15. Dose-Response Data for Bladder Tumors in Male Rats3
Animal Dose
(mg/kg-day)
Human Equivalent Dose
(mg/kg-day)
Total Incidence of Bladder
Neoplasia13
0
0
0/50
8.9
2.62
0/50
32.5
9.56
2/49
143.3
42.13
30/49
"Auletta et al., 1998a
bCombined incidence of transitional cell and squamous cell carcinomas, and papillomas.
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The dose-response data in Table 15 are modeled to obtain a POD for a quantitative
assessment of cancer risk. The POD is an estimated dose, expressed in human-equivalent terms,
near the lower end of the observed range that marks the starting point for extrapolation to lower
doses. Appendix A provides details of the modeling effort. The multistage (2-degree
polynomial) model provides adequate fit to the data and calculates the lower BMDLiohed of the
best fitting models. The BMDiohed and BMDLiohed predictions by the multistage model for the
bladder tumor data are 14 and 11 mg/kg-day, respectively. The BMDLiohed for bladder tumors
(11 mg/kg-day) is used as the POD for the p-OSF. The p-OSF of 0.009 or
9 x 10"3 (mg/kg-day)"1 is calculated by dividing 0.1 (10%) by the BMDLiohed of 11 mg/kg-day.
Inhalation Exposure
No inhalation quantitative estimate (p-IUR) is derived because there were no
carcinogenicity data from inhalation exposure studies and no PBPK models for tributyl
phosphate in rats and humans that would facilitate extrapolation across routes of exposure.
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Toxicology. W.J. Hayes and E.R. Laws, Ed. Academic Press, San Diego, p. 917-1123.
Hanna, P. and K.F. Dyer. 1975. Mutagenicity of organophosphorus compounds in bacteria and
drosophila. Mutat. Res. 28:405-420.
Healy, C.E., P.C. Beyrouty and B.R. Broxup. 1995. Acute and subchronic neurotoxicity studies
with tri-n-butyl phosphate in adult Sprague-Dawley rats. Am. Ind. Hyg. Assoc. J. 56:349-355.
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1977. Evaluation of delayed neurotoxicity and dose-response relationships of phosphate esters
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Kalinina, N.I. 1971. [Toxicity of Phosphoroorganic platificators tributyl phosphate and
di(2-ethylhexyl) phenyl phosphate.] Gig. Tr. Prof. Zabol. 15(8):30—33. (Russian) (Cited in
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53:83-97.
Laham, S., J. Szabo and G. Long. 1983. Effects of tri-n-butyl phosphate on the peripheral
nervous system of the Sprague-Dawley rat. Drug Chem. Toxicol. 6:363-377.
Laham, S. and G. Long. 1984. Subacute oral toxicity of tri-n-butyl phosphate in the
Sprague-Dawley rat. J. Appl. Toxicol. 4:150-154.
Laham, S., G. Long and B. Broxup. 1985. Induction of urinary bladder hyperplasia in
Sprague-Dawley rats orally administered tri-n-butyl phosphate. Arch. Environ. Health.
40:301-306.
Lotti, M. 2001. Clinical toxicology of anticholinesterases in humans. In: Handbook of Pesticide
Toxicology, 2nd ed. R. Krieger, Ed. Academic Press, San Diego, p. 1043-1085.
Mandel, J.S., N.T. Berlinger, N. Kay et al. 1989. Organophosphate exposure inhibits non-
specific esterase staining in human blood monocytes. Am. J. Ind. Med. 15:207-212.
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assay for bacterial mutagenicity. Submitted by FMC Corporation under TSCA Section 4. OTS
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Microbiological Associates. 1990a. CHO/HGPRT mutation assay with tributyl phosphate (Final
report). TSCA Section 4 Submission. OTS Fiche # OTS0528320.
Microbiological Associates. 1990b. Chromosome aberrations in Chinese Hamster ovary (CHO)
cells with tributyl phosphate (Final report). TSCA Section 4 Submission. OTS Fiche #
OTS0528320.
Microbiological Associates. 1990c. Acute in vivo cytogenetics assay in rats (Final report).
TSCA Section 4 Submission. OTS Fiche # OTS0534089.
Mitomo, T., T. Ito, Y. Ueno et al. 1980. Toxicological studies on tributyl phosphate. (I) Acute
and subacute toxicities. J. Toxicol. Sci. 5:270-271.
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NTP (National Toxicology Program). 2008. Management Status Report. Accessed online,
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1.
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING
FOR ORAL SLOPE FACTOR
The preferred multistage cancer model in the EPA BMDS (version 1.4. lb) is fit to the
dichotomous incidence data using the extra risk option. The multistage cancer model is run for
all polynomial degrees up to n-l, where n is the number of dose groups including control; the
lowest degree polynomial providing adequate fit for comparison is used with the other models,
per EPA (2000) guidance. In accordance with EPA (2000) guidance, the benchmark doses
(BMDs) and lower bounds on the BMD (BMDLs) associated with an extra risk of 10% for all
models are calculated.
Table 15 shows the dose-response data for total neoplasms of the urinary bladder in male
rats (Auletta et al., 1998a). The incidence and human-equivalent dose data are modeled
according the procedure outlined above. As assessed by the % goodness-of-fit test, the 2- and
3-degree polynomial multistage models in the software provide adequate fits to the data for the
incidence of bladder tumors in male rats (x p> 0.1) (see Table A-l). The multistage (2-degree
polynomial) model is used because it provides adequate fit to the data with the lowest degree
polynomial. Figure A-l shows the fit of this multistage model to the data.
Table A-l. Multistage Cancer Model Predictions for Bladder Tumors in Male Rats3
Model
Degrees of
Freedom
x2
X2 Goodness of
Fit p-Value
AIC
BMDiohed
(mg/kg-day)
BMDLiohed
(mg/kg-day)
Multistage (degree = l)b
3
9.01
0.0292
96.7329
6.42
4.84
Multistage (degree = 2)b
3
0.23
0.9729
84.5594
14.17
10.81
Multistage (degree = 3)b
3
0.15
0.9273
86.4344
15.51
10.93
a Auletta et al., 1998a
bDegree of polynomial initially set to (n-1) where n = number of dose groups including control. Betas restricted
to >0.
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Multistage Model with 0.95 Confidence Level
Multistage
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
BMD
0
20
30
40
Dose
20:37 07/22 2007
Figure A-l. Fit of Multistage (2-Degree) Model to Data on Bladder Tumors in Male Rats
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APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING
FOR RAT LETHALITY
The model-fitting procedure for dichotomous data is as follows. All available
dichotomous models in the EPA BMDS (version 1.4. lb) are fit to the incidence data using the
extra risk option. The multistage model is run for all polynomial degrees up to n - 1 (where n is
the number of dose groups including controls); the lowest degree polynomial providing adequate
fit is used for comparison with the other models, per EPA (2000) guidance. Goodness-of-fit is
assessed by the % test. When several models provide adequate fit to the data (% p > 0.1), models
usually are compared using the Akaike Information Criterion (AIC). The model with the lowest
AIC would be considered to provide the best fit to the data. When several models have similar
AICs, the model resulting in the lowest BMDL is selected. Because, in this instance, lethality
data is being modeled, benchmark doses (BMDs), and lower bounds on the BMD (BMDLs)
associated with an extra risk of 5% and 10% are calculated for all models.
The Healy et al. (1995; Bio-Research Laboratories, 1991) rat lethality data are modeled
according to the procedure outlined above. As assessed by the % goodness-of-fit test, all models
in the software provide adequate fits to the data for the incidence of lethality in male and female
rats (x p > 0.1) (see Table B-l). The Probit model using log-scaled dose data, is chosen because
it is most commonly used for analyzing lethality data (Eaton and Gilbert, 2008), and it provides
the lowest scaled residual at the lowest tested dose. Figure B-l shows the fit of this model to the
data.
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Table B-l. Benchmark Dose Modeling Summary Data for Deaths Among Male and Female
Rats Treated 7 Days/Week via Gavage with Tributyl Phosphate^
Model
AIC
p-Value
Scaled
Residual—Low
Dose (Control)
Chi2
BMDS
BMDLS
BMDio
BMDL10
Multistage
1-degree poly
50.8572
0.7978
0.908 (0)
1.01
49.375
30.5272
101.42
62.7052
Quantal-Linear
50.8572
0.7978
0.908 (0)
1.01
49.375
30.5271
101.42
62.7052
Log-Probit
52.1869
0.6643
0.597 (0)
0.82
67.241
18.9491
127.802
91.0146
Log-Logistic
52.516
0.5993
0.705 (0)
1.02
65.2009
14.6164
113.973
57.196
Gamma
52.5813
0.5761
0.706 (0)
1.10
66.2781
31.2468
116.903
64.1834
Weibull
52.6241
0.5757
0.732 (0)
1.10
64.7006
31.1311
116.485
63.9458
Multistage
52.7883
0.5750
0.827 (0)
1.11
57.8964
30.7011
113.242
63.0625
Probit
54.9862
0.1829
0.814 (0.669)
3.40
120.034
82.7243
181.133
135.309
Logistic
55.3931
0.1580
0.859 (0.740)
3.69
132.05
91.4075
194.9
147.104
aHealy et al.. 1995
bNOAEL = 32.5 mg/kg-day; LOAEL (3 deaths/24 animals) = 100 mg/kg-day
Probit Model with 0.95 Confidence Level
Probit
0.5 fBlVD Lower Bound
0.4
£=
O
't3 0.2
cc
Ll_
0.1
50
100
150
200
250
300
Dose
16:34 04/09 2008
Figure B-l. Fit of Probit Model to Log Data on Male and Female Rat Lethality
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